NON-CLASSICAL T CELL DEVELOPMENT AND THEIR FUNCTIONS DURING MURINE CYTOMEGALOVIRUS Courtney K. Anderson B.S. University of Connecticut, 2009 A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Division of Biology and Medicine at Brown University Providence, Rhode Island May 2018 © May 2018 by Courtney K. Anderson Signature Page This dissertation, by Courtney K. Anderson, is accepted in its present form by the Pathobiology Graduate Program and the Department of Molecular Microbiology and Immunology as satisfying the dissertation requirement for the degree of Doctor of Philosophy. Date: Laurent Brossay, Ph.D. (Advisor) Recommended to the Graduate Council Date: Loren Fast, Ph.D. (Reader & Chair) Date: Amanda Jamieson, Ph.D. (Reader) Date: Craig Lefort, Ph.D. (Reader) Date: Sebastian Joyce, Ph.D. (Outside Reader) Approved by the Graduate Council Date: Andrew Campbell, Ph.D. Dean of the Graduate School iii CURRICULUM VITAE COURTNEY K. ANDERSON I. EDUCATION Ph.D., Pathobiology, Brown University, Providence, RI 2018 (expected) B.S., Molecular and Cell Biology, University of Connecticut, Storrs, CT 2009 II. RESEARCH EXPERIENCE Brown University Providence, RI Graduate dissertation research 2013 – 2018 Advisor: Laurent Brossay, Ph.D. Yale University New Haven, CT Post-Baccalaureate Associate 2010 – 2012 Advisor: Michael Hurwitz, M.D., Ph.D. Arch Chemicals, Inc., Cheshire, CT Summer Microbiology Technician 2009 III. PUBLICATIONS 1. Erick TK, Anderson CK, Reilly EC, Wands JR, Brossay L. NFIL3 Expression Distinguishes Tissue-Resident NK Cells and Conventional NK-like Cells in the Mouse Submandibular Glands. J Immunol. 2016 Sep 15;197(6):2485-91. 2. Anderson CK, Brossay L. The role of MHC class Ib-restricted T cells during infection. Immunogenetics. 2016 Aug;68(8):677-91. 3. Anderson CK*, Salter AI*, Toussaint LE, Reilly EC, Fugere C, Srivastava N, Kerr WG, Brossay L. Role of SHIP1 in Invariant NKT Cell Development and Functions. Journal of immunology (Baltimore, Md : 1950). 2015. Epub 2015/08/02. 4. Anderson C*, Zhou S*, Sawin E, Horvitz HR, Hurwitz ME. SLI-1 Cbl inhibits the engulfment of apoptotic cells in C. elegans through a ligase-independent function. PLoS genetics. 2012;8(12):e1003115. Epub 2012/12/29. IV. AWARDS & FELLOWSHIPS Ruth L. Kirschstein NRSA for Individual Predoctoral Fellows (F31) 2016 – 2018 NIH: National Institute of Allergy and Infectious Diseases Charles “Chick” Kuhn Graduate Award in Disease Pathogenesis 2017 27th Annual Pathobiology Graduate Program Retreat at Brown University AAI Trainee Abstract Award 2017 American Association of Immunologists, IMMUNOLOGY 2017 Meeting 2nd Place Poster Award 2016 26th Annual Pathobiology Graduate Program Retreat at Brown University Student Travel Award for CD1-MR1 Meeting 2016 International Symposium on CD1 and MR1, Lorne, Australia iv AAI Trainee Abstract Award 2015 American Association of Immunologists, IMMUNOLOGY 2015 Meeting Charles A. Janeway Memorial Poster Award 2014 40th Annual New England Immunology Conference RISP2014 Recipient 2014 RIKEN IMS Summer Program (RISP), Yokohama, Japan V. ORAL PRESENTATIONS 1. “The non-classical CD8+ T cell response to MCMV.” Washington D.C. American Association of Immunologists, IMMUNOLOGY 2017 May 2017 2. “The non-classical CD8+ T cell response to MCMV.” East Providence, RI 26th Annual Pathobiology Graduate Program Retreat Aug 2016 3. “The non-classical CD8+ T cell response to MCMV.” Lorne, Australia International Symposium on CD1 and MR1 Nov 2015 4. “Non-classical CD8+ T cell response to murine cytomegalovirus.” Woods Hole, MA 41st Annual New England Immunology Conference Oct 2015 5. “MCMV specific response of non-classical CD8+ T cells.” New Orleans, LA American Association of Immunologists, IMMUNOLOGY 2015 May 2015 6. “iNKT cell development and functions are differentially Yokohama, Japan regulated by SHIP-1.” RIKEN IMS Summer Program June 2014 VI. POSTER PRESENTATIONS 1. Anderson C.K., Reilly E.C., Lee A.Y., Vance, R.E., and Brossay L. “The non- classical CD8+ T cell response to murine cytomegalovirus.” 43rd Annual New England Immunology Conference, Woods Hole, MA. Oct 2017 2. Anderson C.K., Reilly E.C., Lee A.Y., Vance, R.E., and Brossay L. “The non- classical CD8+ T cell response to murine cytomegalovirus.” 27th Annual Pathobiology Graduate Program Retreat, East Providence, RI. Aug 2017 3. Anderson C.K., Salter A.I., Toussaint L.E., Reilly E.C., Kerr W.G, Fugère, C., and Brossay L. “Role of SHIP1 in iNKT cell development and functions.” International Symposium on CD1 and MR1, Lorne, Australia. Nov 2015 4. Anderson C.K., Reilly E.C., and Brossay L. “The non-classical CD8+ T cell response to murine cytomegalovirus.” 25th Annual Pathobiology Graduate Program Retreat, Brown University, West Greenwich, RI. Sept 2015 5. Anderson C.K., Salter A.I., Reilly E.C., Toussaint L.E., Kerr W.G, Fugère, C., and Brossay L. “Mouse iNKT cell development and functions are differentially regulated by SHIP-1.” 40th Annual New England Immunology Conference, Woods Hole, MA. Nov 2014 6. Anderson C.K., Salter A.I., Reilly E.C., Toussaint L.E., Kerr W.G, and Brossay L. “Mouse iNKT cell development and functions are differentially regulated by SHIP- v 1.” 24th Annual Pathobiology Graduate Program Retreat, Brown University, Bristol, RI. Aug 2014 7. Anderson, C.K., Salter, A.I., Reilly, E.C., Toussaint, L.E., Kerr, W.G., and Brossay, L. “Mouse iNKT cell development and functions are differentially regulated by SHIP-1.” American Association of Immunologists, IMMUNOLOGY 2014, Pittsburg, PA. May 2014 8. Anderson, C.K., Toussaint, L.E., Benoist, B., Teuscher, C., Brossay, L. “Natural killer T cell development and the influence of the Y-chromosome.” 23rd Annual Pathobiology Graduate Program Retreat, Brown University, Bristol, RI. Aug 2013 VII. TEACHING EXPERIENCE Invited University Guest Lectures Brown University Innate Immunology (BIO152) Nov, 2016, Nov 2017 Lecture Title: “Non-classical T cells: their roles during the innate immune response.” Teaching Assistantships Brown University Biology of the Eukaryotic Cell (BIO1050/2050) Fall 2013 Teaching Certificate Programs Brown University Harriet W. Sheridan Center for Teaching and Learning 2014 – 2015 Teaching Certificate I: Reflective Teaching Practices VIII. LEADERSHIP EXPERIENCE & SERVICE Undergraduate and Graduate Student Laboratory Advising Josh Pirl, Brown University, Undergraduate Student 2017 – Current Garvin Dodard, Brown University, Rotating Graduate Student 2016 Mimi Le, Brown University, Undergraduate Student 2014 – 2016 Pathobiology Graduate Program Admissions Committee 2014 – 2017 IX. PROFESSIONAL SOCIETY MEMBERSHIPS American Association of Immunologists, Trainee Member 2014 – Current AAAS, Student Member 2014 – Current vi Acknowledgements This Ph.D. would not have happened without the unwavering love and encouragement of my family and friends. Thank you to my Nana and Papa for always being my biggest champions, and my parents and siblings for having so much faith in me. Ken, thank you for always supporting me and understanding that when I said I needed to quickly pop by the lab, what I really meant was that I would be there for at least an hour. I love you. To my lab wife Maggie, if we had worked in the same lab neither of us would have gotten anything done. I suppose it was for the best. Thank you for being an amazing friend and one of the smartest women I know. I have missed you dearly since you graduated and became a grownup. Benny, I am so grateful for our friendship. I have loved every Italian cheese tasting, teatime, knit night, Patriots game, and swim practice. Thank you for being such a fierce friend, I am so glad I went through this process with you. I also want to thank my advisor, Dr. Laurent Brossay, for giving me this opportunity and helping me become a strong and successful researcher. Thank you for allowing me the independence to steer my projects, your enthusiasm for my findings, encouraging me when science was defeating, and having faith in me. I appreciate that your door was always open, literally, even though it meant you could call me back to your office when I am already halfway down the hall. Thank you for having an office with lots of snacks, although I think Celine gets the credit for that. But most of all, thank you for being an amazing mentor, for always promoting my successes, and for making it a hilarious six years. This experience would have been completely different somewhere else. Thank you to my awesome lab mates, training me, commiserating, filling our side of the lab with so much laughter, and understanding about my space heater anxiety. Michele you are wonderful. A day did not feel complete without coming upstairs to say vii hello, which was always guaranteed to put a smile on my face. Your dedication to the program is incredible, please never stop doing Grilled Cheese Day. Denise, my birthday twin. Thank you for breaking up the science, our chats about plants and crafting and restaurants were always so enjoyable. Ken and I are also forever grateful you let us park in your driveway for a year. To my committee members, Dr. Loren Fast, Dr. Amanda Jamieson, and Dr. Craig Lefort, as well as my outside reader Dr. Sebastian Joyce, I appreciate all of your thoughtful suggestions and comments. Lastly I need to acknowledge Dr. Michael Hurwitz, who gave me my first fulltime research position and started me on this path towards graduate school. viii PREFACE The work presented in this thesis was performed in the laboratory of Dr. Laurent Brossay at Brown University. I performed all of the experiments discussed, with the following exceptions: Céline Fugère performed all the tail vein injections for mixed bone marrow chimeras and adoptive transfer experiments. Kevin Carlson did all the cell sorting. Chapter 2: Alex I. Salter, Emma C. Reilly, and Leon E. Toussaint generated some of the data used for Figures 1, 3, 4, 5B, 6B,C and S1A,B. Chapter 3: Emma C. Reilly provided some of the data used in Figure 3A and Figure S4B, C. Chapter 4: IL-12Rβ2f/f mice were made at the Brown Transgenic Facility in conjunction with Shahjahan Miah. Appendix I: B6.YNKT sequencing for Figure 5 was carried out at UT Southwestern by Lindsay M. Scott. Appendix III: I assisted in generating the mixed bone marrow chimera used for Figures 3, 4, and S1, as well as small intestine preparations for Figure 6. I also made the diagrams used in Figure S1. ix TABLE OF CONTENTS Signature Page ................................................................................................................ iii Curriculum Vitae .............................................................................................................iv Acknowledgements ....................................................................................................... vii Preface .............................................................................................................................ix Table of Contents ............................................................................................................ x List of Figures and Tables ........................................................................................... xiii CHAPTER 1: INTRODUCTION ........................................................................................ 1 I. MHC class Ib-restricted T cells ................................................................................ 2 Thymic selection ........................................................................................................... 3 CD1d-restricted cells ..................................................................................................... 5 MR1-restricted T cells .................................................................................................. 10 Qa-1/HLA-E-restricted T cells ....................................................................................... 11 Qa-2-restricted T cells ................................................................................................. 13 M3-restricted T cells .................................................................................................... 15 II. Immunological regulation through the PI3K signaling pathway ....................... 16 SHIP1 phosphatase .................................................................................................... 16 The role of SHIP1 on immune cell development ............................................................. 18 III. Murine cytomegalovirus ...................................................................................... 18 NK cell and iNKT cell responses to MCMV .................................................................... 19 + CD8 T cell responses to MCMV .................................................................................. 20 Thesis Overview and Summary of Findings ........................................................... 21 References ................................................................................................................. 32 CHAPTER 2: ROLE OF SHIP1 IN INKT CELL DEVELOPMENT AND FUNCTIONS .. 62 Abstract ...................................................................................................................... 64 Introduction ................................................................................................................ 65 Materials and Methods .............................................................................................. 67 Mice ........................................................................................................................... 67 Isolation of murine lymphocytes .................................................................................... 67 Antibodies, reagents, and flow cytometric analysis ......................................................... 68 In vivo proliferation analysis ......................................................................................... 68 In vitro iNKT cell stimulation and cytokine analysis ......................................................... 69 Generation of mixed bone marrow chimera.................................................................... 69 Statistical analysis ....................................................................................................... 70 Results ........................................................................................................................ 71 Germline deletion of SHIP1 hinders iNKT cell development............................................. 71 Extrinsic expression of SHIP1 rescues T cell development .............................................. 72 Decreased number of iNKT cells in mice with germline deletion of SHIP1 is due to impaired proliferation ................................................................................................... 74 iNKT cell cytokine production is decreased in the absence of SHIP1 ................................ 75 Discussion .................................................................................................................. 76 Acknowledgements ................................................................................................... 80 Author Contributions ................................................................................................ 80 Figures ........................................................................................................................ 81 References ................................................................................................................. 91 x CHAPTER 3: MHC CLASS IB-RESTRICTED CD8+ T CELLS ARE PROTECTIVE DURING MURINE CYTOMEGALOVIRUS INFECTION ................................................ 99 Abstract .................................................................................................................... 101 Introduction .............................................................................................................. 102 Materials and Methods ............................................................................................ 105 Mice ......................................................................................................................... 105 b b-/- -/- Generation of K D Qa-1 mice via CRISPR/Cas9 ...................................................... 105 Viruses and infection protocols ................................................................................... 105 Lymphocyte isolation ................................................................................................. 106 In vivo cell proliferation experiments............................................................................ 106 Serum cytokine production ......................................................................................... 107 Secondary MCMV challenge and survival studies ........................................................ 107 + In vitro CD8 T cell stimulation and BMDC cultures ...................................................... 107 Antibodies and flow cytometry .................................................................................... 108 Statistical analysis ..................................................................................................... 109 Ethics statement ....................................................................................................... 109 Results ...................................................................................................................... 110 + b b-/- Non-classical CD8 T cells respond during acute MCMV infection in K D mice ............ 110 + MCMV-expanded non-classical CD8 T cells are distinct from innate-like T cells ............. 110 + Non-classical CD8 T cells acquire an effector phenotype during acute MCMV infection .. 111 + The expansion and activation of non-classical CD8 T cells is MCMV-dependent ............ 112 + The non-classical CD8 T cell response results in a prolonged inflammatory phenotype .. 112 + b b-/- Non-classical CD8 T cells form memory populations in long-term infected K D mice ... 113 + Non-classical CD8 T cells are sufficient to protect against MCMV-induced lethality ........ 114 + Qa-1-restricted CD8 T cells respond to MCMV ............................................................ 115 Discussion ................................................................................................................ 118 Acknowledgements ................................................................................................. 121 Author Contributions .............................................................................................. 121 Figures ...................................................................................................................... 122 References ............................................................................................................... 133 CHAPTER 4: EXTRINSIC CYTOKINE SIGNALING AFFECTS INKT CELL ACTIVATION FOLLOWING MURINE CYTOMEGALOVIRUS INFECTION ................ 142 Abstract .................................................................................................................... 144 Introduction .............................................................................................................. 145 Materials and Methods ............................................................................................ 147 Mice ......................................................................................................................... 147 Viruses and infection protocols ................................................................................... 147 α-GalCer treatment ................................................................................................... 147 Lymphocyte isolation ................................................................................................. 147 Survival studies ......................................................................................................... 148 Antibodies and flow cytometry .................................................................................... 148 Western blot ............................................................................................................. 149 Statistical analysis ..................................................................................................... 149 Results ...................................................................................................................... 150 IL-12Rβ2 and IL-18R signaling are dispensable for iNKT cell development ..................... 150 Loss of IL-12Rβ2 signaling has major effects on iNKT cell activation following MCMV infection, while IL-18R signaling only has minor effects ................................................. 151 iNKT cells are not sufficient to protect immunocompromised mice from MCMV-induced lethality .................................................................................................................... 152 xi Discussion ................................................................................................................ 153 Acknowledgements ................................................................................................. 155 Figures ...................................................................................................................... 156 References ............................................................................................................... 162 CHAPTER 5: DISCUSSION AND FUTURE DIRECTIONS ......................................... 166 The role of SHIP1 in iNKT cell development and functions ................................. 167 The MHC class Ib-restricted CD8+ T cell response to MCMV .............................. 169 What MCMV-derived antigens are being recognized? ........................................... 171 + Do MCMV-specific CD8 T cells have a biased TCR repertoire? .................................... 173 Are Qa-1-restricted T cells present in wild type mice and do they respond to MCMV in the presence of classical CD8+ T cells? ............................................................. 173 b b-/- -/- What is the participation of non-Qa-1-restricted T cells in K D Qa-1 mice? ................. 175 Extrinsic cytokine signaling affects iNKT cell activation following murine cytomegalovirus infection ...................................................................................... 176 Why are splenic iNKT cells hyporesponsive in the absence of MyD88 signaling, but hepatic iNKT cells are unaffected? ......................................................................... 177 Summary .................................................................................................................. 178 Figures ...................................................................................................................... 180 References ............................................................................................................... 184 APPENDIX I: THE ROLE OF THE Y CHROMOSOME-ENCODED ERDR1 ON INKT CELL DEVELOPMENT ................................................................................................ 188 Introduction .............................................................................................................. 190 Materials and Methods ............................................................................................ 192 Mice ......................................................................................................................... 192 Lymphocyte isolation ................................................................................................. 192 Antibodies and flow cytometry .................................................................................... 192 Sequencing of male B6.YNKT mice............................................................................... 193 Statistical analysis ..................................................................................................... 193 Results and Discussion .......................................................................................... 194 Global T cell populations are modestly affected in male B6.YNKT mice ............................ 194 Forced expression of the iNKT cell semi-invariant TCR does not rescue their defect ....... 194 The Y chromosome gene Erdr1 contains small deletions in male B6.YNKT mice .............. 195 Figures ...................................................................................................................... 197 References ............................................................................................................... 202 APPENDIX II: THE ROLE OF MHC CLASS IB-RESTRICTED T CELLS DURING INFECTION ................................................................................................................... 204 Abstract .................................................................................................................... 206 Introduction .............................................................................................................. 207 Positive selection of non-classically restricted CD8+ T cells .............................. 208 MHC class Ib-restricted CD8+ T cells and their participation during infection .. 209 Figures ...................................................................................................................... 226 References ............................................................................................................... 231 APPENDIX III: NFIL3 EXPRESSION DISTINGUISHES TISSUE-RESIDENT NK CELLS AND CONVENTIONAL NK-LIKE CELLS IN THE MOUSE SUBMANDIBULAR GLANDS ....................................................................................................................... 248 xii LIST OF FIGURES AND TABLES CHAPTER 1: INTRODUCTION Figure 1: MHC class Ib-restricted T cells recognize unique antigens ......................... 24 Figure 2: Overview of conventional and non-classical T cell development................. 25 Figure 3: The family of CD1-restricted T cells ............................................................. 26 Figure 4: Maturation of murine iNKT cell lineages ...................................................... 27 Figure 5: The mechanisms of iNKT cell activation ...................................................... 28 Figure 6: The PI3K signaling pathway and SHIP1 protein domains ........................... 29 Table 1: Mouse and human MHC class Ib molecules ................................................. 30 Table 2: MHC class Ib-restricted T cell responses ...................................................... 31 CHAPTER 2: ROLE OF SHIP1 IN INKT CELL DEVELOPMENT AND FUNCTIONS Figure 1: Loss of thymic and peripheral iNKT cells in SHIP1-/- mice ........................... 81 Figure 2: SHIP1-/- mice have a more pronounced NKT1 phenotype than littermate controls ........................................................................................................................ 82 Figure 3: SHIP1-/- mice have impaired conventional T cell development .................... 83 Figure 4: Extrinsic expression of SHIP1 regulates iNKT cell and T cell populations .. 84 Figure 5: Normal iNKT cell populations in mice conditionally deficient for SHIP1 in T cells .............................................................................................................................. 85 Figure 6: SHIP1 deficiency extrinsically affects iNKT cell proliferation and intrinsically affects cytokine production .......................................................................................... 86 Figure S1: Loss of thymic and peripheral iNKT cells in SHIP1-/- mice ......................... 87 Figure S2: Loss of thymic and peripheral iNKT cells in SHIP1-/- mice, but normal T cell populations in CD4CreSHIP1fl/fl mice ........................................................................... 88 Figure S3: SHIP1 deficiency influences the proliferative capacity and cytokine production of thymic iNKT cells from SHIP1-/- mice ..................................................... 89 Figure S4. Increased Foxp3 expression of conventional T cells, but not iNKT cells, in SHIP1-/- animals ........................................................................................................... 90 CHAPTER 3: MHC CLASS IB-RESTRICTED CD8+ T CELLS ARE PROTECTIVE DURING MURINE CYTOMEGALOVIRUS INFECTION Figure 1: Non-classical CD8+ T cells help control MCMV infection in KbDb-/- mice ... 122 Figure 2: The activation and expansion of non-classical CD8+ T cells is MCMV- dependent in KbDb-/- mice ........................................................................................... 123 Figure 3: Non-classical CD8+ T cells participate in a prolonged inflammatory phenotype in KbDb-/- mice ........................................................................................... 124 Figure 4: Non-classical CD8+ T cells in long-term MCMV-infected KbDb-/- mice form memory populations and robustly expand following secondary infection .................. 125 Figure 5: Non-classical CD8+ T cells are sufficient to protect against MCMV-induced lethality ....................................................................................................................... 126 Figure 6: The MHC class Ib molecule Qa1 participates in the CD8+ T cell response during MCMV infection .............................................................................................. 127 Figure S1: Non-classical CD8+ T cells are phenotypically similar to conventional CD8+ T cells during acute MCMV infection ......................................................................... 128 Figure S2: Following acute MCMV infection, non-classical CD8+ T cells acquire an effector phenotype ..................................................................................................... 129 Figure S3: Non-classical CD8+ T cells persist in long-term MCMV-infected KbDb-/- mice and form memory populations ................................................................................... 130 xiii Figure S4. The expansion of non-classical CD8+ T cells in KbDb-/- mice is independent of CD1d, but dependent on B2m expression.............................................................. 131 Figure S5. KbDb-/-Qa1-/- mouse generation and loss of Qa1 signaling effects ........... 132 CHAPTER 4: EXTRINSIC CYTOKINE SIGNALING AFFECTS INKT CELL ACTIATION FOLLOWING MURINE CYTOMEGALOVIRUS INFECTION Figure 1: Loss of IL-12Rβ2 signaling has major effects on iNKT cell activation following MCMV infection, while MyD88 signaling has organ-specific effects ........... 156 Figure 2: Loss of IL-12Rβ2 or MyD88 signaling does not affect iNKT cell activation following α-GalCer stimulation ................................................................................... 157 Figure 3: iNKT cells are insufficient to protect RAG1-/- mice from MCMV-induced lethality ....................................................................................................................... 158 S. Figure 1: IL-12Rβ2 signaling is dispensable for T cell development and peripheral iNKT cell populations ................................................................................................. 159 S. Figure 2: MyD88 signaling is dispensable for T cell development and peripheral iNKT cell populations ................................................................................................. 160 S. Figure 3: Loss of IL-12Rβ2 signaling, but not MyD88 signaling, affects iNKT cell activation following MCMV infection .......................................................................... 161 CHAPTER 5: DISCUSSION Figure 1: Qa-1-restricted CD8+ T cells are detectable in wild type mice using Qdm- loaded tetramers ........................................................................................................ 180 Figure 2: Generation of Qa-1-/- mice through CRISPR/Cas9-mediated editing ........ 181 Figure 3: T cell development and peripheral CD8+ T cell populations are unaffected in Qa-1-/- mice ................................................................................................................ 182 Figure 4: Two proposed roles of MHC class Ib-restricted T cells ............................. 183 APPENDIX I: THE ROLE OF THE Y CHROMOSOME-ENCODED ERDR1 ON INKT CELL DEVELOPMENT Figure 1: Male B6.YNKT mice, but not male IFNAR-/- mice, lack iNKT cells ................ 197 Figure 2: γδ and αβ T cell populations are affected in male B6.YNKT mice ................ 198 Figure 3: Overexpression of a prearranged Vα14-Jα18 TCR does not rescue the iNKT cell deficit in male B6.YNKT mice ................................................................................ 199 Figure 4: Loss of UTY does not affect T cell development or peripheral iNKT cell populations ................................................................................................................ 200 Figure 5: B6.YNKT males have small sized deletions in the Erdr1 gene on Chromosome Y .......................................................................................................... 201 APPENDIX II: THE ROLE OF MHC CLASS IB-RESTRICTED T CELLS DURING INFECTION Figure 1: The family of CD1-restriced αβ T cells ..................................................... 226 Figure 2: Examples of non-classical αβ T cell populations during microbial infection ................................................................................................................................... 227 Figure 3: Proposed roles of non-classical CD8+ αβ T cell populations in humans and mice, compared to conventional CD8+ T cells ........................................................... 228 Table 1: MHC class Ib molecules that participate in TCR-mediated responses ....... 229 Table 2: The antimicrobial response of MHC class Ib-restricted T cells in mice and humans ...................................................................................................................... 230 xiv CHAPTER 1: INTRODUCTION 1 INTRODUCTION The human body is inhabited by approximately an equal number of bacterial cells as there are human cells [1]. In addition to these commensal members of our microbiome, we also frequently come into contact with pathogenic microorganisms. For host defense to effectively protect against the myriad types of microbial pathogens, they must be distinctly recognizable from healthy tissue as foreign or ‘non-self’. Typically the immune system is loosely compartmentalized into two branches, the innate and the adaptive systems. Many forms of life contain innate immune systems, however adaptive immunity is exclusive to jawed vertebrates. The innate immune system is broad and non-specific, which allows for rapid acting responses, but rarely forms memory. This is accomplished, in part, with germ-line encoded receptors that recognize generic types of microbial ligands. The innate immune response is sufficient for protection against many infectious microorganisms due to their short lifespans. Conversely, the adaptive immune system makes a targeted response against a specific antigen, which is accomplished through humoral and cell-specific immunity. This specificity requires more time to generate and the outcome is stored in immunological memory. However, this dogma of isolated innate and adaptive immune systems is overly simplified, and they are in fact much complex and intertwined. I. MHC CLASS IB-RESTRICTED T CELLS There is increasing evidence surrounding the importance of innate-like T cells, which recognize antigens presented by non-classical major histocompatibility complex (MHC) molecules. Conventional CD8+ and CD4+ T cells are restricted by MHC class Ia and II molecules, respectively. However, there are also a number of MHC class Ib molecules, which restrict distinct T cell populations, collectively referred to as non- classical T cells. MHC class Ia molecules are highly polymorphic and present a diverse 2 array of peptide antigens. These are encoded by HLA-A, HLA-B, and HLA-C in humans and, H-2D, H-2K, and H-2L in mice. MHC class Ia genes reside in the Mhc region, along with those for MHC class Ib and MHC class II. MHC class Ib molecules are encoded by HLA-E, HLA-F, and HLA-G in humans and the H2-M, H2-Q, and H2-T regions in mice. Additionally, there are MHC class I-like molecules encoded outside of the Mhc locus, e.g. CD1, MR1 (MHC related 1), and HFE. MHC class Ib molecules are non- polymorphic and bind peptides as well as unique non-peptide antigens, though often with a more limited repertoire (Figure 1) [2, 3]. In addition, they also have lower expression at the cell surface, limited tissue distribution, and shorter cytoplasmic tails [4]. Non-classical T cells participate against a number of microbial pathogens in response to MHC class Ib-presented antigens, however not all of these molecules are capable of eliciting T cell responses (Table 1 and 2) [5]. These populations are also not limited to humans or mammals, but are evolutionarily conserved in a large number of species, including amphibians [6]. Thymic selection Conventional and non-classical T cell selection occurs in parallel within the thymus (Figure 2). T cell development begins with a CD4-CD8- double negative (DN) stage and then progresses to a CD4+CD8+ double positive (DP) stage. DP cells then undergo positive and negative selection and become CD4+, CD8+, or remain DN T cells. Conventional T cells are positively selected by thymic epithelial cells (TECs) that express both MHC class I and II molecules [7]. In contrast, hematopoietic cells (HC) participate in the positive selection of non-classical T cells. For example, natural killer T (NKT) cells and mucosal associated invariant T (MAIT) cells are selected by interactions with other DP cortical thymocytes expressing either CD1 or MR1, respectively [8-10]. Non-classical T cells frequently can have an activated or memory phenotype in naïve 3 animals, illustrated by decreased expression of CD62L and increased expression of CD44 or CD69 [11-13]. CD62L is usually highly expressed on naïve T cells, whereas CD44 and CD69 are upregulated upon activation. The unique pathway for non-classical T cell positive selection is thought to explain this unusual feature. In mice, both HCs and TECs select M3-restricted Listeria monocytogenes (LM)-specific T cells [14, 15], but the functional outcome of the resulting cells differ. When selected by HCs, LM-specific T cells have enhanced activation and effector capabilities compared to those selected by TECs [15]. On the other hand, insulin-specific Qa-1-restricted T cells are also be selected by HCs and TECs, but without differences in activation [16]. Some non-classical T cells also require unique transcription factors during development and/or additional signaling events exclusively provided by DP thymocytes. Promyelocytic leukemia zinc finger (PLZF) is a transcription factor expressed by γδ T cells [17], MAIT cells [18, 19], and NKT cells [20, 21]. It is essential for NKT cell development [20, 21], but actively inhibited in non-innate T cells [22]. Expression of a PLZF transgene during development causes conventional CD4+ T cells to gain innate- like effector functions [23-25]. However, PLZF is not expressed by all MHC class Ib- selected subsets, such as M3- and HFE-restricted T cells [15, 26]. Signaling through members of the SLAM (signaling lymphocytic activation molecule) family of receptors and SAP (SLAM-associated protein) are also important [27]. Homotypic SLAM-SLAM interactions between DP thymocytes and NKT cell precursors are essential for their developmental progression [28, 29]. M3-restricted T cells also require SAP-signaling [30]. Additionally, if MHC class II is instead expressed exclusively by HCs, CD4+ T cell positive selection becomes SAP-dependent and results in innate-like qualities [31, 32]. Interestingly, even though NKT and MAIT cells share many developmental characteristics, MAIT cells do not require SLAM signaling [18]. 4 CD1d-restricted T cells There are five members of the CD1 family: CD1a, b, and c (Group 1), CD1d (Group 2), and CD1e (Group 3). CD1a-e are all expressed in humans, however only CD1d is present in mice and rats. These MHC class I-like molecules present endogenous, exogenous, and synthetic lipid antigens (Figure 3) [33], with the exception of CD1e. Instead, CD1e assists CD1b with antigen presentation [34]. Since only CD1d is present in mice, it is the best characterized of the five isoforms. Human and murine CD1d genes share approximately 60% homology [35], and present antigens to a heterogeneous population of T cells called NKT cells. To better investigate the participation of CD1a, b and c in vivo, transgenic and humanized mice expressing group 1 CD1 molecules have been developed [36-38]. NKT cells have characteristics of natural killer (NK) cells, such as expression of the NK cell receptor NK1.1, in addition to having a T cell receptor (TCR). Originally, they were categorized into type I and type II NKT cells based on the diversity of their TCRs [39]. NKT cells rapidly produce large amounts of cytokines, including IL-4, IL-10, IL-13, IL-17, IFN-γ, and TNF-α [40]. With their rapid and robust activation potential, and broad cytokine repertoire, iNKT cells are uniquely poised to polarize the immune response. For example, NKT cells assist in the activation of NK cells, B cells, and macrophages [41-43]. On the other hand, NKT cells also suppress the immune response, which is observed during graft-versus-host disease and autoimmune disorders [44]. Interestingly, they also recognize lipid allergens in pollen and house dust extract [45, 46]. In a murine asthma model, house dust extract activates NKT cells and promotes inflammation [46]. Type I NKT cells, are also referred to as invariant NKT (iNKT) cells because they express a semi-invariant TCR. Their semi-invariant TCR is composed of a single α- chain (Vα14-Jα18 in mice and Vα24-Jα14 in humans) paired with a limited number of β- chains (Vβ2, Vβ7, and Vβ8.2 in mice and predominantly Vβ11 in humans) [47-49]. 5 Vα14-Jα18 rearrangement, and consequently iNKT cell development, requires an extended DP stage [50, 51]. Therefore RORγt-deficient mice lack iNKT cells, due to its requirement for DP cell survival [51, 52]. Recently however, an alternative pathway was discovered where functional iNKT cells bypass the DP stage and differentiate from thymocytes at the DN stage [53]. In addition to a semi-invariant TCR, iNKT cells are characterized by their ability to recognize the glycolipid alpha galactosylceramide (α- GalCer), which originates from the marine sponge Agelas mauritianus [54]. This provides an extremely powerful tool to identify and stimulate human and mouse iNKT cells in vitro and in vivo, especially since their endogenous ligands remain largely unknown [55]. iGb3 (isoglobotrihexosylceramide) is purported to be the primary endogenous ligand for iNKT cells [56], however even though it weakly activates iNKT cells, iGb3 is not found in the thymus of mice or humans [56-58]. More importantly, iNKT cell development is unaffected in iGb3 synthase-deficient animals [59]. Endogenous α-GalCer and α-GluCer (α-Glucosylceramide), both considered absent in mammals, were recently detected in thymic tissue [60]. This could explain why iNKT cells have contrasting reactivity to these two compounds [60]. β-GluCer is also asserted as a natural ligand [61-63], however multiple groups have demonstrated that its stimulatory properties are actually a result of contamination with α-linked glycolipids [60, 64]. Type II NKT cells have a more diverse TCR repertoire and antigen specificity than iNKT cells. Instead of recognizing α-GalCer, type II NKT cells are activated by lipids such as sulfatide and lysophosphatidylcholine [65]. Even though type II NKT cells do not express a semi-invariant TCR, they exhibit a proclivity for certain α- and β-chain usage (Vα3 or Vα8 paired with Vβ8 in mice) [66]. Murine NKT cells are predominantly iNKT cells, however type II NKT cells are more abundant in humans [67]. Unfortunately, the contributions of type II NKT cells remain unclear without better tools to identify them. 6 Many studies rely on comparing mouse lines that lack either iNKT cells alone (Jα18-/- mice) or both type I and type II NKT cells (CD1d-/- mice). However, the original Jα18-/- line has decreased TCR diversity, which may affect the results from these types of studies [68]. To compound this, the distinction between type I and type II NKT cells is not all encompassing, as illustrated by atypical α-Galcer-reactive human NKT cells that lack the invariant TCR [69]. Since type I NKT cells are, thus far, better characterized than type II NKT cells, my work focuses solely on iNKT cells. iNKT cells were originally classified into three subsets based on maturation, using NK1.1 and CD44 expression [44, 70]. However, it was later determined that even though iNKT cells produce numerous cytokines and chemokines [71, 72], discrete iNKT cell subsets actually have distinct responses upon activation [72]. To incorporate this, the field has recently moved away from the original linear model of NKT cell development towards four distinct lineages with divergent genetic and epigenetic programs [73]. These subsets mirror CD4+ T helper (TH) cells and regulatory T (Treg) cells [40, 74]. NKT1, NKT2, NKT17, and NKT10 cells are distinguished based on their cytokine responses and differential expression of T-bet, PLZF, GATA3, and RORγt expression (Figure 4) [75]. NKT1 cells rely on IL-15 and T-bet for development and are characterized by IFN-γ and TNF-α production [75-77]; they are shown to have anti-tumor roles [78]. NKT2 cells rely on GATA-3, produce IL-4 and IL-13 following activation, and participate in airway hyper-reactivity (AHR) [75, 79, 80]. On the other hand, NKT17 cells require RORγt expression and IL-7 for development and homeostasis, and produce IL- 17A [81-83]. This subset is indicated in exacerbating murine hepatitis [84] and AHR induced by ozone exposure [85]. As previously mentioned, iNKT cell development is PLZF-dependent [20, 21]. NKT10 cells however, were discovered to lack PLZF [86, 87]. Instead, they express E4BP4 (encoded by Nfil3), which controls their IL-10 production. 7 NKT10 cells are enriched in adipose tissue and have regulatory capabilities, including the homeostasis of Tregs and macrophages [87]. Recently, a fifth category of iNKT cells was found after α-GalCer immunization, called NKT follicular helper (NKTFH) cells. They are comparable to conventional CD4+ TFH cells in phenotype, but subordinate in function [88, 89]. The transcriptional repressor Bcl-6 is required for NKTFH formation, and their production of IL-21 aids in germinal center formation [88, 90]. However, whether they are a distinct lineage is unclear, since peripheral NKT cells proliferate and differentiate into NKTFH cells [91]. Different strains of mice are enriched for certain iNKT cell lineages. For example, C57BL/6 mice predominantly have NKT1 cells in the thymus, while BALB/c mice have robust populations of NKT1, NKT2, and NKT17 cells [75]. In mice, iNKT cells equate to roughly 1% of total lymphocytes, but this varies by tissue. They are approximately 0.5-3% of T cells in the thymus, spleen, blood, and lymph nodes, however hepatic iNKT cells make up roughly 30% of mononuclear cells in the liver [44]. Human iNKT cells (and whether or not there are equivalent NKT1, NKT2 and NKT17 subsets) are not as well defined as those in mice. However, studies indicate IFN-γ, IL-4, and IL- 17-secreting populations all exist [92-94]. Human iNKT cells are predominantly characterized using peripheral blood mononuclear cells (PBMCs). Over a 100-fold difference between individuals is observed in iNKT cell frequency, ranging from nearly zero to over 3% of circulating lymphocytes [95, 96]. In contrast, thymic iNKT cells are, on average, less than 0.002% of lymphocytes in humans [97]. iNKT cells are uniquely equipped to rapidly respond to antagonistic stimuli because they have preformed cytokine mRNAs, for example IFN-γ and IL-4 [98, 99]. iNKT cells are activated by three mechanisms: (1) TCR-dependent, (2) TCR- and cytokine-dependent, or (3) cytokine-dependent (Figure 5) [40, 100, 101]. This contrasts 8 type II NKT cells, which are mainly activated in a TCR-dependent mechanism [102, 103]. α-GalCer is considered a strong agonist and when presented by CD1d, it directly stimulates via the TCR and results in IFN-γ and IL-4 production and proliferation [104, 105]. Weaker microbial or endogenous agonists also activate through the TCR, but are insufficient to appropriately activate iNKT cells on their own. An additional signal from toll-like receptor (TLR)-activated dendritic cells (DCs) is provided through inflammatory cytokines [106]. During Salmonella typhimurium infection, iNKT cells depend on CD1d presentation of self-lipids and IL-12 for activation [107]. Similarly, Aspergillus fumigatus readily activates iNKT cells through weak self-lipid presentation by CD1d and IL-12 released following TLR recognition of β-Glucans [108]. IFN-α/β production by TLR9- stimulated dendritic cells (DCs) also acts as a second signal, alongside self-ligands [109]. The third method of iNKT cell activation is independent of lipid antigens. For example, TLR9-dependent IL-12 production, and to a lesser extent IL-18 and IFN-α/β, is important for iNKT cell activation and IFN-γ release during murine cytomegalovirus (MCMV) infection [110, 111]. The participation of cytokine-driven activation is more significant than originally perceived. TLR-dependent IL-12 production dominates iNKT cell activation, even in the presence of microbial antigens [112]. iNKT cells are rapidly activated in this manner due to their high IL-12 receptor expression [112]. Depending on the pathogen, iNKT cell participation is dispensable or physiologically relevant. Human and mouse iNKT cells both respond to Mtb-derived phosphatidylinositol mannoside (PIM) ligands, however are expendable during infection [113, 114]. In contrast, mice are more susceptible during Borrelia burgdorferi, Ehrlichia muris, Sphingomonas capsulata, and Streptococcus infections without iNKT cell participation [115-119]. 9 MR1-retricted T cells MAIT cells were discovered nearly 20 years ago in humans, however their functional capabilities remained stagnant until their antigens were better understood [49, 120]. Following the discovery of their natural ligands, antigen-loaded tetramers allowed for MAIT cell characterization and characterization in vivo [121]. Similarly to iNKT cells, MAIT cells are innate-like T cells that recognize a non-peptide antigen and are activated in TCR-dependent or -independent mechanisms. They were originally identified because of their invariant TCR. A Vα7.2-Jα33 α-chain (and less often Vα7.2-Jα12 or Vα7.2-Jα20) preferentially pairs with Vβ2 or Vβ13 in humans, while Vα19-Jα33 associates with Vβ6 or Vβ8 in mice [49, 120, 122]. MAIT cells make up between 1-4% of T cells in the circulation, but are also enriched in mucosal sites like the lamina propria of the small intestine and lungs [9]. In the liver, they make up to 45% of the lymphocytes [123]. Overall, MAIT cells are more abundant in humans than most mouse strains, which typically have very small populations in wild-type strains [18, 19]. For example, MAIT cells comprise approximately 0.1% of circulating T cells in the blood of C57BL/6 mice versus roughly 6% in humans [19]. MR1 is encoded outside of the Mhc locus, and acts as the restriction element for MAIT cells [9]. Human and murine MR1 are highly conserved and share 90% sequence homology [124, 125]. MR1 is distinct for its ability to stabilize and present bacterial products of vitamin B metabolism, such as riboflavin (vitamin B2) and folic acid (vitamin B9) [126, 127]. MR1 is only transiently expressed, but traffics to the cell surface and is stably expressed when loaded with antigen [128, 129]. Interestingly, depending on which intermediate is presented, the ligand is activating (riboflavin) or non-activating (folic acid) [126]. MAIT cell development requires bacterially-derived antigens. Hence, MAIT cells are absent in germ-free (GF) mice [9]. This is in contrast to the increase in intestinal 10 iNKT cells in GF animals [130, 131]. Colonizing GF mice with a single bacterial species, such as Enterobacter cloacae or Lactobacillus casei, reconstitutes MAIT cell populations [132]. Many bacterial and fungal species, but not viruses, rapidly activate MAIT cells in an MR1-dependent manner in vitro [132, 133]. MAIT cells secrete TH1 and TH17 cytokines, predominantly IL-17, as well as granzymes and perforin [134]. MR1-deficient mice have impaired early control or clearance to Klebsiella pneumonia, Francisella tularensis, and Mycobacterium bovis BCG [135-137]. In humans, MAIT cells respond to Mtb in an MR1-dependent manner [132, 133]. Patients with active Mtb infections have diminished MAIT cells in the blood, however the residual population is enriched to respond to BCG instead of unrelated infections, such as E. coli [138]. In contrast, murine MAIT cells respond to M. bovis BCG in an MR1-independent mechanism [132, 133, 137]. This is because MAIT cell activation, similarly to iNKT cells, also occurs through inflammatory cytokines and independently of vitamin B metabolites. This is evident by using bacteria without an intact riboflavin biosynthesis pathway, such as Enterococcus faecalis. E. faecalis activates MAIT cells through environmental IL-12/IL- 18 [139]. These findings also open up the participation of MAIT cells during viral infections in an MR1-independent mechanism [140]. In HIV positive patients, MAIT cells are decreased in the circulation and have an activated phenotype [141-143]. They are also hyporesponsive to challenge with E. coli, similarly to what is observed with MAIT cells isolated from Mtb patients [143]. The diversity observed in MAIT TCRs could aid in these selective pathogen responses [129, 144]. MAIT cells are also implicated during non-infectious disease, though with conflicting roles, such as multiple sclerosis [145, 146], Sjogren’s syndrome [147], and Celiac disease [148]. Qa-1/HLA-E restricted CD8+ T cells Human HLA-E and murine Qa-1 (encoded by H2-T23) have multiple roles, 11 spanning the innate and adaptive immune systems. Even though they share overlapping functions, they likely developed by convergent evolution and are considered functional homologues, rather than orthologs [149]. HLA-E is the least polymorphic of the MHC class Ib molecules in humans. This is illustrated by a one amino acid difference between the two primary HLA-E alleles expressed in Caucasians [150, 151]. In addition to their limited polymorphism, Qa-1/HLA-E also share low cell surface expression, wide-ranging tissue expression, and structural similarities [152]. For example, their peptide binding groves both have substitutions at positions 143 and 147 [153]. The primary peptides loaded in Qa-1/HLA-E (AMAPRTLLL), called Qa-1 determinant modifier (Qdm), are derived from the leader sequences of other MHC class I molecules [154]. As such, loading of Qdm is TAP-dependent and parallels the MHC class I antigen processing pathway [155]. Qdm originates from HLA-A, -B, -C, and -G in humans [156, 157] and H-2D and H-2L in mice [152, 154]. Qa-1/HLA-E-Qdm were first established as the ligands for the CD94/NKG2 family of receptors [158-161]. Ligation enhances or limits activity depending on whether the inhibitory receptor CD94/NKG2A or stimulatory receptor CD94/NKG2E is engaged [161- 163]. CD94/NKG2A and CD94/NKG2C contact HLA-E through mostly shared residues in a peptide-dependent manner, however CD94/NKG2A has higher binding affinity for HLA-E [164, 165]. Activated T cells and naïve NK cells express CD94/NKG2A, and loss of this inhibitory receptor results in hyperactivation. This is illustrated by excessive activation by NK cells during LCMV infection [166] and CD8+ T cells after poxvirus [167], polyoma virus [163], and Mtb [168] infection. Qdm has the ideal structure to bind Qa-1/HLA-E, but under certain conditions other peptides are bound [169] and presented to CD8+ T cells. This includes peptides from endogenous Hsp60 (heat shock protein 60, GMKFDRGYI), prokaryotic GroEL (GMQFDRGYL), and preproinsulin (ALWMRFLPL) [170-173]. Hsp60 is upregulated on 12 stressed cells and is preferentially bound in the absence of Qdm [170]. In mice with disrupted antigen processing (ERAAP1-/-, endoplasmic reticulum amino peptidase 1), the self-peptide FL9 (FYAEATPML) is recognized by a subset of Qa-1-restricted CD8+ T cells [174]. GroEL-derived peptides, the prokaryotic homolog of Hsp60, are recognized by Qa-1-restricted T cells during S. typhimurium infection, however they are also cross- reactive to stressed macrophages [171]. In addition, Qa-1 and HLA-E present bacterial antigens from LM and Mtb [168, 175-178]. The Mtb-specific HLA-E-restricted T cell response is distinct from conventional T cells and has an uncharacteristic TH2 phenotype [176, 177]. Qa-1 and HLA-E also present virally-derived antigens. HLA-E-specific T cells respond to peptides from: the BZLF-1 protein from Epstein-Barr virus (SQAPLPCVL), a core protein from hepatitis C virus (YLLPRRGPRL), and the gag protein from HIV [179- 183]. Human cytomegalovirus (HCMV) also generates HLA-E-restricted CD8+ T cells [184, 185]. In an effort to evade the NK cell response, HCMV augments CD94/NKG2A and HLA-E engagement [186]. The leader sequence of the HCMV glycoprotein UL40 (gpUL40) is identical to Qdm, or nearly identical depending on the strain. The Toledo and AD169 strains both provide identical peptides (VMAPRTLVL and VMAPRTLIL, respectively) to Qdm from certain HLA-A and HLA-C alleles [187, 188]. However HLA- E-restricted CD8+ T cells might target these infected cells if the peptide is considered non-self [185]. Interestingly, a third role for Qa-1 is as the ligand to a population of regulatory CD8+ T cells [189]. Loss of Qa-1 signaling during herpes simplex virus infection or immunization with self-peptide leads to enhanced autoimmunity because CD8+ suppressor T cells dampen CD4+ T cell autoreactivity [189]. Qa-2-restricted CD8+ T cells The Qa-2 region is encoded by four genes, H2-Q6, H2-Q7, H2-Q8, and H2-Q9 in 13 C57BL/6 mice. These genes likely evolved from paired gene duplication. H2-Q7 and H2-Q9 for example, have 99% homology [190]. BALB/c mice have differential expression of Qa-2, depending on the strain. For example, BALB/cJ mice only encode H2-Q6 and H2-Q7, and BALB/cByJ mice lack all four Qa-2 coding genes [191]. Qa-2 presents a diverse repertoire of self-peptides [192, 193] and foreign peptides [194]. This is thought to result from the shallow and hydrophobic nature of its peptide-binding groove [195]. Similarly to conventional MHC class Ia molecules, Qa-2 associates with a β2m light chain and requires TAP for peptide presentation [195, 196]. However, Qa-2 is unique for two reasons: it is attached to the cell membrane by a glycophosphatidyl- inositol (GPI) linker [197] and there are soluble forms of Qa-2 [198]. Alternative gene splicing and cleavage of membrane-bound Qa-2 both produce soluble Qa-2 [198]. Qa-2 was considered incapable of properly activating CD8+ T cells due to the structure of its α3 domain [199]. However, Q9-restricted CD8+ T cells respond during mouse polyoma virus (MPyV) infection against a nonameric peptide from the VP2 capsid protein [200]. VP2-specific T cells assist in viral clearance and are also found in wild type mice. In contrast to conventional memory CD8+ T cells, which are short-lived cells and continuously replenished with naïve T cells [201], Q9-restricted T cells form an inflationary population that proliferates in response to viral antigen [202]. Qa-2 is also important for the selection of certain CD8αα expressing intestinal intraepithelial lymphocytes (iIELs) [203]. iIELs are a heterogeneous population of αβ and γδ T cells, many of which have a CD8αα or CD8αβ co-receptor. Similarly to Qa-1/HLA-E, HLA-G is considered by some to be the functional homolog of Qa-2 in humans [204]. This is due to a number of shared characteristics, including: (1) lack of a cytoplasmic domain [205], (2) soluble alternatively spliced isoforms (HLA-G1-G4 are soluble and HLA-G5-G7 are membrane-bound) [206-208], (3) presentation of endogenous nonameric peptides [209, 210], (4) expression in placental 14 tissue and participation during embryonic development [211-213], and (5) restriction of cytotoxic CD8+ T cell responses [214]. M3-restricted CD8+ T cells M3 (encoded by H2-M3) is uniquely capable of binding N-formylated peptides, derived from bacteria and mitochondrial proteins, due to modifications in its peptide- binding groove [215-217]. This residue is necessary to initiate protein translation in prokaryotes, mitochondria, and chloroplasts. Even though N-formylated peptides are bound with greatest affinity, non-formylated peptides from influenza A are presented in vitro [218, 219]. Mice have 13 mitochondrial proteins, thus limited endogenous antigens result in low cell surface expression of M3 [220, 221]. Instead, M3 remains in the endoplasmic reticulum until it is loaded with peptides and then egresses from the ER, negotiates the Golgi apparatus, and is expressed at the cell surface [222, 223]. M3 is distinct for its ability to bind shorter peptides than classical MHC molecules, as short as two amino acids in length [224]. In addition, M3 can assemble with peptides in a TAP- dependent and -independent mechanism [222, 224-226]. M3-restricted T cells have diverse TCR repertoires [220, 225]. The best- characterized responses are observed during LM and Mtb infection. Three LM peptides are presented by M3: Attm (f-MIVTLF), Fr38 (f-MIVIL), and LemA (f-MIGWII) [227-229]. M3-restricted LM-specific CD8+ T cell populations are sufficient for protection during infection [230, 231]. However, M3-restricted T cells are not required for protection [232]. M3-restricted T cells have innate-like qualities during LM infection. This is demonstrated by their early development and rapid response, compared to conventional CD8+ T cells [233]. Additionally, they do no expand after secondary challenge [233, 234], possibly due to inhibition [235] or because they exhibit a memory phenotype during primary infection [236]. Mtb also encodes a number of formylated peptides recognized by M3- 15 restricted T cells [237, 238]. Unlike LM-specific M3-restricted T cells, Mtb-specific M3- restricted T cells are not protective [239]. Other intracellular pathogens that elicit M3- restricted responses are Chlamydia pneumonia and Salmonella enterica serovar Typhimurium [240, 241]. In addition to bacterial pathogens, M3-restricted T cells also respond to N-formylated peptides from the commensal bacteria Staphylococcus epidermidis [225]. II. IMMUNOLOGICAL REGULATION THROUGH THE PI3K SIGNALING PATHWAY The phosphoinositide 3-kinase (PI3K) signaling pathway is part of a larger PI3K/ATK/mTOR signaling axis, which helps control the cell cycle and survival [242]. Signals initiated from the cell surface (e.g. by cytokine and chemokine receptors, costimulatory receptors, or T and B cell receptors) are relayed by PI3K activation. PI3Ks transmit these messages through phospholipid second messengers, which in turn activate ATK (also referred to as protein kinase B). ATK is translocated to the nucleus and augments downstream signaling pathways for activation, growth, differentiation, metabolism, migration, and survival (Figure 6A) [242-245]. PI3K signaling is controlled by a number of mechanisms, including the phosphatases PTEN (phosphatase and tensin homolog) and SHIP1 (Src-homology 2 (SH2) domain containing inositol 5’- phosphatase 1), which act as negative regulators [246, 247]. Dysregulation of the PI3K pathway has serious effects, and components of this pathway are often mutated or amplified in many types of cancer [242]. SHIP1 phosphatase PI3Ks act by phosphorylating phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol-3,4,5-trisphosphate (PIP3). PIP3 recruits signaling molecules, such as ATK, through its Pleckstrin homology domain and aids in their membrane localization 16 [245]. SHIP1 and PTEN both dephosphorylate PIP3 back to different forms of PIP2; SHIP1 removes the phosphate from the D5 position of the inositol ring and PTEN dephosphorylates the D3 position (Figure 6A) [244, 248]. Loss of either of these molecules is thought to enhance PI3K activity [246, 247]. SHIP1 also has several homologs and isoforms from alternative splicing [249-251]. Similarly to SHIP1, SHIP-2 is a 5’ phosphatase that dephosphorylates PIP3 to PIP2 [252]. In addition to regulating PI3K signaling, SHIP-2 is also a negative regulator of glycogen synthesis induced by insulin [253]. s-SHIP is one of four murine isoforms of SHIP1 and is expressed in embryonic and hematopoietic stem cells, as well as mature hematopoietic cells [251, 254]. In contrast to SHIP-1, s-SHIP lacks an SH2 domain and is thought to participate in stem cell activation [250]. Hematopoietic cells, stromal cells, and mesenchymal cells all express SHIP1 (encoded by INPP5D) [255, 256]. The SH2 domain of SHIP1 interacts with proteins containing phosphorylated tyrosines, such as inhibitory (ITIM, immunoreceptor tyrosine- based inhibitory motif) and activating (ITAM, immunoreceptor tyrosine-based activation motif) motifs on the cytoplasmic tail of various receptors. SHIP1 is known to associate with these motifs on KLRG1 [257], the IgE receptor (FcϵRI), the CD3 complex, and TCR ζ (zeta) chain [258]. SHIP1 also has a proline rich region at its C-terminus, which allows it to associate with the plasma membrane and interact with proteins containing an SH3 domain [251]. Lastly, SHIP1 has two tyrosine (Y) motifs within its PRR which, when phosphorylated, allow it to associate with proteins containing a phosphotyrosine-binding domain (PTB): e.g. Dok-1, and Dok-1 [251]. Consequently, SHIP1 not only has catalytic activity through its phosphatase domain, but non-enzymatic scaffolding functions, which mediate protein-protein interactions (Figure 6B) [251]. 17 The role of SHIP1 on immune cell development and functions Loss of SHIP1 signaling in mice has severe pleiotropic effects. SHIP1-deficient animals have a large infiltration of myeloid cells in the lungs because of increased activation and survival [259, 260]. As a result, these mice have greatly shortened lifespans due to respiratory failure [259]. SHIP1eI20/eI20 mice, which lack phosphatase activity in both SHIP1 and s-SHIP, have even more exacerbated myeloid cell responses and earlier demise, compared to SHIP1null animals [254]. In contrast to myeloid cell hyperproliferation, SHIP1-/- mice are lymphopenic [259]. This indicates that loss of SHIP1 has different consequences on individual subsets. B cells mature more rapidly and are hyperactivated following BCR stimulation, in addition to increased proliferation and survival [261, 262]. SHIP1 has both intrinsic and extrinsic roles on NK cells. Even though SHIP1 is extrinsically necessary for their development and IFN-γ production, it is intrinsically required for NK cell differentiation [263]. The effects on T cell development and functions were originally less clear [259, 264, 265]. Thymic cellularity and peripheral T cell populations are both decreased in SHIP1-/- mice, and residual T cells adopt a Treg phenotype, with increased Foxp3 expression [259, 264, 266]. However, it was later demonstrated that T cells from mice conditionally deficient for SHIP1 in the T cell lineage were unaffected in cellularity and Treg phenotype [265]. In contrast, intrinsic SHIP1 expression negatively regulates CD4+ T cell TH1/TH2 polarization and CD8+ T cell cytotoxicity [265]. III. MURINE CYTOMEGALOVIRUS HCMV is a ubiquitous betaherpesvirus that is easily cleared following initial infection, remains latent, and reactivates asymptomatically [267]. In the United States, seroprevalence is approximately 60% in people over 6 years old, but this number continues to increase with age [268]. Multiple layers of the immune response help 18 control CMV latency and reactivation [269]. In contrast, infections in immunocompromised patients, such as neonates and transplant recipients, have greater severity. For example, HCMV is the leading, non-genetic cause of hearing loss in children [267]. However, the effects of HCMV are observed in many organs, including hepatitis, colitis, and pneumonitis [267]. Treatment includes the use of anti-viral drugs, such as ganciclovir, but HCMV is able to acquire resistance [270]. Due to the species tropism exhibited by every CMV family member, MCMV has long been used as a model for HCMV infections, owing to their similarities in genome, latency, and reactivation [269, 271, 272]. MCMV is a double stranded DNA virus, like HCMV, with a 230 kb genome that encodes 170 genes [272]. MCMV is capable of infecting macrophages, DCs, epithelial cells, and hepatocytes [273]. Within hours of intra-peritoneal inoculation, the virus travels to the liver, lymph nodes, and spleen, and then disseminates throughout the host [273]. The peak of viral replication in the spleen and liver of C57BL/6 mice is between day 2 and 3 post-infection [274]. MCMV later travels to the salivary glands (SMG), which is a privileged site of infection with delayed clearance [275]. NK cell and iNKT cell responses to MCMV The kinetics of the innate immune response to MCMV is well-characterized, with a peak of cytokines around 36 hours post-infection. MCMV is predominantly recognized in a TLR9-dependent manner, but also through TLR7 and TLR3, on plasmacytoid DCs (pDCs) [276, 277]. In the absence of pDCs, conventional DCs can also provide these TLR9-dependent immunological cues, and downstream signaling via MyD88 [278]. The inflammatory cytokines produced, e.g. IFN-α/β, IL-12, and IL-18, help to choreograph NK cell cytotoxicity, IFN-γ release, and proliferation [279-282]. NK cells are crucial for early virus control. In some mouse strains, NK cells directly target virus-infected cells through 19 their Ly49H activating receptor (also known as Cmv1 or Klra8) [283, 284]. Ly49H recognizes the viral glycoprotein m157 and renders strains resistant to MCMV (e.g. C57BL/6 mice). Strains lacking Ly49H are referred to as susceptible (e.g. BALB/c mice) [285]. Approximately half of the NK cells in C57BL/6 mice express Ly49H, and ligation with m157 leads to the expansion of Ly49H+ NK cells, cytotoxicity, and IFN-γ production [285, 286]. Consequently, the loss of Ly49H expression leads to increased susceptibility [287]. In addition to NK cells, iNKT cells are also robust producers of IFN-γ at early time points post-infection. DC-produced IL-12, and to a lesser extent IL-18 and IFN-α/β, is important for iNKT cell activation and IFN-γ production [110, 111]. In contrast to NK cells however, iNKT cells do not proliferate during infection [110]. CD8+ T cell responses to MCMV During acute infection, DC presentation of MCMV-derived peptides results in T cell proliferation, differentiation, and cytotoxicity. In C57BL/6 mice, CD8+ T cells respond to a number of MCMV epitopes, including M45 and M57 [288, 289]. Following their peak on day 7 in the spleen and liver, these populations then begin to contract and maintain a small memory (TM) population [288, 289]. CD8+ TM cells are generally divided into either central memory (TCM) and effector memory (TEM). TCM cells are self-renewing and preferentially home to secondary lymphoid organs; on the other hand, TEM cells localize to peripheral tissues and have limited proliferation, but are poised for immediate effector functions following antigen re-exposure [290]. The response to other epitopes, such as M38, m139 and IE3, results in the continued accumulation of MCMV-specific CD8+ T cells [288]. In humans, approximately 5% of circulating CD8+ T cells are CMV-specific, but this frequency continues to increase with age following seroconversion [291]. This phenomenon is referred to as memory inflation 20 and occurs due to continued antigen stimulation by latently infected cells [292]. Most inflationary CD8+ T cells are characterized as terminally differentiated effector T (TEFF) cells, but a minor population retains a TM phenotype [293]. A third subset of CD8+ TM cells is present in the salivary gland (SMG) of MCMV-infected mice, which have a tissue- resident memory (TRM) phenotype [294, 295]. The TRM population in the SMG is maintained by the recruitment of circulating inflationary cells [294]. Interestingly, in the absence of CD8+ T cells, CD4+ T cells compensate for the absence of CD8+ T cells [296]. Additionally, CD4+ T cells are primarily responsible for clearance in the SMG due to NK cell hyporesponsiveness and evasion of CD8+ T cells [297, 298]. The classical CD8+ T cell response is well-characterized following MCMV infection. However, in an effort to evade the CD8+ T cell and NK cell responses, HCMV upregulates HLA-E and loads a Qdm mimic derived from the viral gpUL40 [186-188]. This results in UL40-specific HLA-E-restricted CD8+ T cells in some patients [299]. However, the role of these HLA-E-restricted CD8+ T cells in vivo remains unclear. THESIS OVERVIEW AND SUMMARY OF FINDINGS Non-classical T cells are a heterogeneous population that responds to antigens presented by non-polymorphic MHC class Ib- and MHC class I-like molecules. Their distinct features include recognition of peptide and non-peptide antigens, rapid activation and effector functions, and unique immunological roles such as surveillance for appropriate antigen processing and stressed cells. As such, non-classical T cells do not conform to the traditional definitions of adaptive immune cells and instead, often share qualities of both the innate and adaptive immune systems. Some populations of non- classical T cells also require unique signaling events and transcription factors for their development and/or functions, e.g. SLAM or PLZF. Even though these cells are small components of an overall much larger conventional T cell family, they participate during 21 a number of infectious diseases with non-redundant roles. For these reasons, it is imperative to continue investigating the developmental requirements and anti-microbial functions of T cell populations restricted by non-classical MHC molecules. The goal of this research is twofold: to better appreciate the (1) signaling requirements for non-classical T cell development and activation and (2) the antiviral contributions of T cells restricted by MHC class Ib and MHC class I-like molecules during MCMV infection. In Chapter 2, we investigate the intrinsic and extrinsic roles of SHIP1 on iNKT cell development and functions. Given the robust activation of iNKT cells, and their polarization of downstream immune responses, it is imperative that iNKT cells are appropriately regulated. As such, iNKT cells express a number of inhibitory receptors. SHIP1 interacts with the ITIM domains on the cytoplasmic tails of these inhibitory receptors. We originally hypothesized that SHIP1-deficient iNKT cells would have altered development and enhanced functions, due to the loss of SHIP1’s negative regulation of PI3K signaling. We find that extrinsic expression of SHIP1 is sufficient for iNKT cell development, peripheral populations, and proliferation. However in contrast, SHIP1-deficient iNKT cells are hyporesponsive following TCR stimulation, and intrinsically require SHIP1. In Chapter 3, we investigate the participation of MHC class Ib-restricted CD8+ T cells during MCMV infection. Given the well-characterized role of conventional CD8+ T cells, we hypothesized that non-classical CD8+ T cells would also respond to MCMV and potentially substitute for conventional T cells. During MCMV infection, MHC class Ib-restricted CD8+ T cells are phenotypically similar to conventional T cells, rather than innate-like T cells. MCMV-expanded non-classical T cells form memory and are also sufficient to protect against MCMV-induced lethality in immunocompromised mice. These responses are TCR-dependent, rather than inflammatory cytokine-dependent. Interestingly, we determined that MCMV-specific non-classical CD8+ T cells are a heterogeneous population, and are restricted by Qa-1 22 and non-Qa-1 MHC class Ib molecules. Lastly, in Chapter 4 we revisit the response of iNKT cells during MCMV infection using new tools, to assess the requirements for IL- 12R and MyD88 signaling on their activation. MyD88 is downstream of the IL-1R/IL- 18R/TLR family. As previously observed, we confirm that IL-12R signaling is necessary for appropriate iNKT cell activation and cytokine production. Interestingly, we also discover an organ-specific requirement for MyD88 signaling by splenic iNKT cells, but not hepatic iNKT cells. Collectively, these data further our knowledge about the development and functions of non-classically-restricted T cells, in addition to recognizing distinct responses of non-classical T cell subsets during MCMV infection. 23 Figure 1. MHC class Ib-restricted T cells recognize unique antigens. Non-classical T cell populations are depicted with examples of recognized microbial antigens, presented by MHC class Ib molecules. Antigens are color-coded based on type: peptide (grey), N-formylated peptide (green), and Vitamin B metabolite (blue). APC = antigen presenting cell, MPyV = mouse polyoma virus (Adapted from Anderson and Brossay 2016 [5]). 24 Figure 2. Overview of conventional and non-classical T cell development. HSCs from the bone marrow enter the thymus as T cell precursors and go through four CD4- CD8- double negative (DN) stages (DN1-4). Commitment to the T cell lineage occurs during DN2, followed by β-chain rearrangement in DN3, and expression of a pre- TCR. The γδ T cell lineage diverges from αβ T cells early during DN3. α-chain rearrangement then occurs during DN4. Conventional CD4+ and CD8+ T cells are positively selected by TECs expressing MHC class II or I molecules. Non-classical T cells, such as MAIT and iNKT cells, recognize MHC class Ib molecules (i.e. MR1 and CD1d) presented by other DP cells. iNKT cells, but not MAIT cells, also require SLAM- SLAM interactions. DN = double negative, DP = double positive, HSC = hematopoietic stem cell, NKT = natural killer T cell, MAIT = mucosal associated invariant T cell, MHC = major histocompatibility complex, SLAM = signaling lymphocytic activation molecule, TEC = thymic epithelial cell. 25 Figure 3. The family of CD1-restricted T cells. Human group 1 CD1-restricted T cells are depicted with examples of Mtb-derived lipid antigens. (Adapted from Anderson and Brossay 2016 [5]). 26 Figure 4. Maturation of murine iNKT cell lineages. After positive selection, immature iNKT cells (PLZFhi) differentiate into at least four subsets of mature iNKT cells. These parallel CD4+ TH subsets and are distinguished based on transcription factor requirements and cytokine responses. NKT1 cells (PLZFlow) produce IFN-γ and require IL-15 and T-bet for development. NKT2 cells (PLZFhi) rely on GATA3 for development and make IL-4. NKT17 cells (PLZFint) need IL-7 and RORγt for development and produce IL-17A. On the other hand, NKT10 cells lose PLZF and gain E4BP4 expression, and produce IL-10. NKTFH cells are an inducible subset that expresses Bcl- 6 and produces IL-21. TH: T helper, FH: follicular helper. 27 Figure 5. The mechanisms of iNKT cell activation. (A) Strong agonists presented by CD1d, such as α-GalCer, activate iNKT cells in a TCR-dependent mechanism. (B) Weaker endogenous or exogenous ligands are insufficient alone, and also require inflammatory cytokine signaling for iNKT cell activation. This is achieved downstream of TLR recognition on APCs. (C) iNKT cells are activated independently of TCR recognition. This mechanism relies solely on stimulation through inflammatory cytokines from TLR stimulation. APC: antigen presenting cell, TCR: T cell receptor, TLR: toll-like receptor. 28 Figure 6. The PI3K signaling pathway and SHIP1 protein domains. (A) Following receptor activation, PI3Ks phosphorylate PI(4,5)P2 to PI(3,4,5)P3. PI(3,4,5)P3 associates with ATK, initiating a signaling cascade that leads to a number of cellular processes. The phosphatases PTEN and SHIP1 are negative regulators of this process. PTEN is a 3’-phosphatase and SHIP1 is a 5’-phosphatase, making PI(4,5)P2 and PI(3,4)P2, respectively. (B) SHIP1 has enzymatic and non-enzymatic activities. At its N-terminus is an SH2 domain, which interacts with phosphorylated tyrosines on ITIM- and ITAM-containing receptor tails. At the C-terminus is a PRR domain that interacts with SH3-containing proteins. Within the PRR are two tyrosines, which when phosphorylated, interact with proteins containing a PTB. ITAM: immunoreceptor tyrosine-based activating motif, ITIM: immunoreceptor tyrosine-based inhibitory motif, PTEN: phosphatase and tensin homolog, PIP3: phosphatidylinositol-3,4,5 trisphosphate PPR: proline rich region, PTB: phosphotyrosine-binding domain, SH: src-homology, SHIP1: src-homology 2 domain containing inositol phosphatase 1, Y: tyrosine. 29 Table 1. Mouse and human MHC class Ib molecules. MHC class Ib molecules that are known to participate in non-classical T cell responses, in addition to the types of antigens they present and whether they are encoded inside the Mhc locus. (Adapted from Anderson and Brossay 2016 [5]). 30 Table 2. MHC class Ib-restricted T cell responses. MHC class Ib-restricted T cells that participate in antimicrobial responses are listed along with: cell type(s) used for positive selection, examples of recognized antigens, whether or not they have PLZF expression, and the types of cytokines released following activation. (Adapted from Anderson and Brossay 2016 [5]). 31 REFERENCES 1. Sender, R., S. Fuchs, and R. Milo, Are We Really Vastly Outnumbered? Revisiting the Ratio of Bacterial to Host Cells in Humans. Cell, 2016. 164(3): p. 337-40. 2. Rodgers, J.R. and R.G. Cook, MHC class Ib molecules bridge innate and acquired immunity. Nat Rev Immunol, 2005. 5(6): p. 459-71. 3. Godfrey, D.I., et al., The burgeoning family of unconventional T cells. Nat Immunol, 2015. 16(11): p. 1114-23. 4. Stroynowski, I. and K.F. Lindahl, Antigen presentation by non-classical class I molecules. Curr Opin Immunol, 1994. 6(1): p. 38-44. 5. Anderson, C.K. and L. Brossay, The role of MHC class Ib-restricted T cells during infection. Immunogenetics, 2016. 68(8): p. 677-91. 6. Edholm, E.S., et al., Nonclassical MHC class I-dependent invariant T cells are evolutionarily conserved and prominent from early development in amphibians. Proc Natl Acad Sci U S A, 2013. 110(35): p. 14342-7. 7. Anderson, G., et al., Thymic epithelial cells provide unique signals for positive selection of CD4+CD8+ thymocytes in vitro. J Exp Med, 1994. 179(6): p. 2027- 31. 8. Bendelac, A., Positive selection of mouse NK1+ T cells by CD1-expressing cortical thymocytes. J Exp Med, 1995. 182(6): p. 2091-6. 9. Treiner, E., et al., Selection of evolutionarily conserved mucosal-associated invariant T cells by MR1. Nature, 2003. 422(6928): p. 164-9. 10. Seach, N., et al., Double-positive thymocytes select mucosal-associated invariant T cells. J Immunol, 2013. 191(12): p. 6002-9. 11. Jay, D.C., L.M. Reed-Loisel, and P.E. Jensen, Polyclonal MHC Ib-restricted CD8+ T cells undergo homeostatic expansion in the absence of conventional 32 MHC-restricted T cells. J Immunol, 2008. 180(5): p. 2805-14. 12. Kurepa, Z., J. Su, and J. Forman, Memory phenotype of CD8+ T cells in MHC class Ia-deficient mice. J Immunol, 2003. 170(11): p. 5414-20. 13. Macho-Fernandez, E. and M. Brigl, The Extended Family of CD1d-Restricted NKT Cells: Sifting through a Mixed Bag of TCRs, Antigens, and Functions. Front Immunol, 2015. 6: p. 362. 14. Chiu, N.M., et al., The selection of M3-restricted T cells is dependent on M3 expression and presentation of N-formylated peptides in the thymus. J Exp Med, 1999. 190(12): p. 1869-78. 15. Cho, H., et al., Positive selecting cell type determines the phenotype of MHC class Ib-restricted CD8+ T cells. Proc Natl Acad Sci U S A, 2011. 108(32): p. 13241-6. 16. Sullivan, B.A., et al., Positive selection of a Qa-1-restricted T cell receptor with specificity for insulin. Immunity, 2002. 17(1): p. 95-105. 17. Kreslavsky, T., et al., TCR-inducible PLZF transcription factor required for innate phenotype of a subset of gammadelta T cells with restricted TCR diversity. Proc Natl Acad Sci U S A, 2009. 106(30): p. 12453-8. 18. Martin, E., et al., Stepwise development of MAIT cells in mouse and human. PLoS Biol, 2009. 7(3): p. e54. 19. Rahimpour, A., et al., Identification of phenotypically and functionally heterogeneous mouse mucosal-associated invariant T cells using MR1 tetramers. J Exp Med, 2015. 212(7): p. 1095-108. 20. Kovalovsky, D., et al., The BTB-zinc finger transcriptional regulator PLZF controls the development of invariant natural killer T cell effector functions. Nat Immunol, 2008. 9(9): p. 1055-64. 21. Savage, A.K., et al., The transcription factor PLZF (Zbtb16) directs the effector 33 program of the NKT cell lineage. Immunity, 2008. 29(3): p. 391-403. 22. Zhang, S., et al., Zbtb16 (PLZF) is stably suppressed and not inducible in non- innate T cells via T cell receptor-mediated signaling. Sci Rep, 2015. 5: p. 12113. 23. Kovalovsky, D., et al., PLZF induces the spontaneous acquisition of memory/effector functions in T cells independently of NKT cell-related signals. J Immunol, 2010. 184(12): p. 6746-55. 24. Savage, A.K., M.G. Constantinides, and A. Bendelac, Promyelocytic leukemia zinc finger turns on the effector T cell program without requirement for agonist TCR signaling. J Immunol, 2011. 186(10): p. 5801-6. 25. Mao, A.P., et al., Multiple layers of transcriptional regulation by PLZF in NKT-cell development. Proc Natl Acad Sci U S A, 2016. 113(27): p. 7602-7. 26. Boucherma, R., et al., Loss of central and peripheral CD8+ T-cell tolerance to HFE in mouse models of human familial hemochromatosis. Eur J Immunol, 2012. 42(4): p. 851-62. 27. De Calisto, J., et al., SAP-Dependent and -Independent Regulation of Innate T Cell Development Involving SLAMF Receptors. Front Immunol, 2014. 5: p. 186. 28. Pasquier, B., et al., Defective NKT cell development in mice and humans lacking the adapter SAP, the X-linked lymphoproliferative syndrome gene product. J Exp Med, 2005. 201(5): p. 695-701. 29. Griewank, K., et al., Homotypic interactions mediated by Slamf1 and Slamf6 receptors control NKT cell lineage development. Immunity, 2007. 27(5): p. 751- 62. 30. Bediako, Y., et al., SAP is required for the development of innate phenotype in H2-M3--restricted Cd8(+) T cells. J Immunol, 2012. 189(10): p. 4787-96. 31. Li, W., et al., The SLAM-associated protein signaling pathway is required for development of CD4+ T cells selected by homotypic thymocyte interaction. 34 Immunity, 2007. 27(5): p. 763-74. 32. Li, W., et al., Thymic selection pathway regulates the effector function of CD4 T cells. J Exp Med, 2007. 204(9): p. 2145-57. 33. Beckman, E.M., et al., Recognition of a lipid antigen by CD1-restricted alpha beta+ T cells. Nature, 1994. 372(6507): p. 691-4. 34. de la Salle, H., et al., Assistance of microbial glycolipid antigen processing by CD1e. Science, 2005. 310(5752): p. 1321-4. 35. Hansen, T.H., et al., Patterns of nonclassical MHC antigen presentation. Nat Immunol, 2007. 8(6): p. 563-8. 36. Felio, K., et al., CD1-restricted adaptive immune responses to Mycobacteria in human group 1 CD1 transgenic mice. J Exp Med, 2009. 206(11): p. 2497-509. 37. Zhao, J., et al., Mycolic acid-specific T cells protect against Mycobacterium tuberculosis infection in a humanized transgenic mouse model. Elife, 2015. 4. 38. Lockridge, J.L., et al., Analysis of the CD1 antigen presenting system in humanized SCID mice. PLoS One, 2011. 6(6): p. e21701. 39. Cardell, S., et al., CD1-restricted CD4+ T cells in major histocompatibility complex class II-deficient mice. J Exp Med, 1995. 182(4): p. 993-1004. 40. Kumar, A., et al., Natural Killer T Cells: An Ecological Evolutionary Developmental Biology Perspective. Front Immunol, 2017. 8: p. 1858. 41. Carnaud, C., et al., Cutting edge: Cross-talk between cells of the innate immune system: NKT cells rapidly activate NK cells. J Immunol, 1999. 163(9): p. 4647-50. 42. Kitamura, H., et al., alpha-galactosylceramide induces early B-cell activation through IL-4 production by NKT cells. Cell Immunol, 2000. 199(1): p. 37-42. 43. Wesley, J.D., et al., Cutting edge: IFN-gamma signaling to macrophages is required for optimal Valpha14i NK T/NK cell cross-talk. J Immunol, 2005. 174(7): p. 3864-8. 35 44. Bendelac, A., P.B. Savage, and L. Teyton, The biology of NKT cells. Annu Rev Immunol, 2007. 25: p. 297-336. 45. Agea, E., et al., Human CD1-restricted T cell recognition of lipids from pollens. J Exp Med, 2005. 202(2): p. 295-308. 46. Wingender, G., et al., Invariant NKT cells are required for airway inflammation induced by environmental antigens. J Exp Med, 2011. 208(6): p. 1151-62. 47. Dellabona, P., et al., An invariant V alpha 24-J alpha Q/V beta 11 T cell receptor is expressed in all individuals by clonally expanded CD4-8- T cells. J Exp Med, 1994. 180(3): p. 1171-6. 48. Lantz, O. and A. Bendelac, An invariant T cell receptor alpha chain is used by a unique subset of major histocompatibility complex class I-specific CD4+ and CD4-8- T cells in mice and humans. J Exp Med, 1994. 180(3): p. 1097-106. 49. Porcelli, S., et al., Analysis of T cell antigen receptor (TCR) expression by human peripheral blood CD4-8- alpha/beta T cells demonstrates preferential use of several V beta genes and an invariant TCR alpha chain. J Exp Med, 1993. 178(1): p. 1-16. 50. Egawa, T., et al., Genetic evidence supporting selection of the Valpha14i NKT cell lineage from double-positive thymocyte precursors. Immunity, 2005. 22(6): p. 705-16. 51. Bezbradica, J.S., et al., Commitment toward the natural T (iNKT) cell lineage occurs at the CD4+8+ stage of thymic ontogeny. Proc Natl Acad Sci U S A, 2005. 102(14): p. 5114-9. 52. Guo, J., et al., Regulation of the TCRalpha repertoire by the survival window of CD4(+)CD8(+) thymocytes. Nat Immunol, 2002. 3(5): p. 469-76. 53. Dashtsoodol, N., et al., Alternative pathway for the development of Valpha14(+) NKT cells directly from CD4(-)CD8(-) thymocytes that bypasses the 36 CD4(+)CD8(+) stage. Nat Immunol, 2017. 18(3): p. 274-282. 54. Kawano, T., et al., CD1d-restricted and TCR-mediated activation of valpha14 NKT cells by glycosylceramides. Science, 1997. 278(5343): p. 1626-9. 55. Godfrey, D.I. and S.P. Berzins, Control points in NKT-cell development. Nat Rev Immunol, 2007. 7(7): p. 505-18. 56. Zhou, D., et al., Lysosomal glycosphingolipid recognition by NKT cells. Science, 2004. 306(5702): p. 1786-9. 57. Xia, C., et al., Synthesis and biological evaluation of alpha-galactosylceramide (KRN7000) and isoglobotrihexosylceramide (iGb3). Bioorg Med Chem Lett, 2006. 16(8): p. 2195-9. 58. Speak, A.O., et al., Implications for invariant natural killer T cell ligands due to the restricted presence of isoglobotrihexosylceramide in mammals. Proc Natl Acad Sci U S A, 2007. 104(14): p. 5971-6. 59. Porubsky, S., et al., Normal development and function of invariant natural killer T cells in mice with isoglobotrihexosylceramide (iGb3) deficiency. Proc Natl Acad Sci U S A, 2007. 104(14): p. 5977-82. 60. Kain, L., et al., The identification of the endogenous ligands of natural killer T cells reveals the presence of mammalian alpha-linked glycosylceramides. Immunity, 2014. 41(4): p. 543-54. 61. Ortaldo, J.R., et al., Dissociation of NKT stimulation, cytokine induction, and NK activation in vivo by the use of distinct TCR-binding ceramides. J Immunol, 2004. 172(2): p. 943-53. 62. Parekh, V.V., et al., Quantitative and qualitative differences in the in vivo response of NKT cells to distinct alpha- and beta-anomeric glycolipids. J Immunol, 2004. 173(6): p. 3693-706. 63. Brennan, P.J., et al., Invariant natural killer T cells recognize lipid self antigen 37 induced by microbial danger signals. Nat Immunol, 2011. 12(12): p. 1202-11. 64. Brennan, P.J., et al., Activation of iNKT cells by a distinct constituent of the endogenous glucosylceramide fraction. Proc Natl Acad Sci U S A, 2014. 111(37): p. 13433-8. 65. Godfrey, D.I., S. Stankovic, and A.G. Baxter, Raising the NKT cell family. Nat Immunol, 2010. 11(3): p. 197-206. 66. Park, S.H., et al., The mouse CD1d-restricted repertoire is dominated by a few autoreactive T cell receptor families. J Exp Med, 2001. 193(8): p. 893-904. 67. Exley, M.A., et al., A major fraction of human bone marrow lymphocytes are Th2- like CD1d-reactive T cells that can suppress mixed lymphocyte responses. J Immunol, 2001. 167(10): p. 5531-4. 68. Bedel, R., et al., Lower TCR repertoire diversity in Traj18-deficient mice. Nat Immunol, 2012. 13(8): p. 705-6. 69. Le Nours, J., et al., Atypical natural killer T-cell receptor recognition of CD1d-lipid antigens. Nat Commun, 2016. 7: p. 10570. 70. Benlagha, K., et al., A thymic precursor to the NK T cell lineage. Science, 2002. 296(5567): p. 553-5. 71. Godfrey, D.I. and M. Kronenberg, Going both ways: immune regulation via CD1d-dependent NKT cells. J Clin Invest, 2004. 114(10): p. 1379-88. 72. Coquet, J.M., et al., Diverse cytokine production by NKT cell subsets and identification of an IL-17-producing CD4-NK1.1- NKT cell population. Proc Natl Acad Sci U S A, 2008. 105(32): p. 11287-92. 73. Engel, I., et al., Innate-like functions of natural killer T cell subsets result from highly divergent gene programs. Nat Immunol, 2016. 17(6): p. 728-39. 74. Constantinides, M.G. and A. Bendelac, Transcriptional regulation of the NKT cell lineage. Curr Opin Immunol, 2013. 25(2): p. 161-7. 38 75. Lee, Y.J., et al., Steady-state production of IL-4 modulates immunity in mouse strains and is determined by lineage diversity of iNKT cells. Nat Immunol, 2013. 14(11): p. 1146-54. 76. Castillo, E.F., et al., Thymic and peripheral microenvironments differentially mediate development and maturation of iNKT cells by IL-15 transpresentation. Blood, 2010. 116(14): p. 2494-503. 77. Gordy, L.E., et al., IL-15 regulates homeostasis and terminal maturation of NKT cells. J Immunol, 2011. 187(12): p. 6335-45. 78. Crowe, N.Y., et al., Differential antitumor immunity mediated by NKT cell subsets in vivo. J Exp Med, 2005. 202(9): p. 1279-88. 79. Akbari, O., et al., Essential role of NKT cells producing IL-4 and IL-13 in the development of allergen-induced airway hyperreactivity. Nat Med, 2003. 9(5): p. 582-8. 80. Kim, H.Y., et al., The development of airway hyperreactivity in T-bet-deficient mice requires CD1d-restricted NKT cells. J Immunol, 2009. 182(5): p. 3252-61. 81. Watarai, H., et al., Development and function of invariant natural killer T cells producing T(h)2- and T(h)17-cytokines. PLoS Biol, 2012. 10(2): p. e1001255. 82. Webster, K.E., et al., IL-17-producing NKT cells depend exclusively on IL-7 for homeostasis and survival. Mucosal Immunol, 2014. 7(5): p. 1058-67. 83. Michel, M.L., et al., Identification of an IL-17-producing NK1.1(neg) iNKT cell population involved in airway neutrophilia. J Exp Med, 2007. 204(5): p. 995-1001. 84. Milosavljevic, N., et al., Mesenchymal stem cells attenuate acute liver injury by altering ratio between interleukin 17 producing and regulatory natural killer T cells. Liver Transpl, 2017. 23(8): p. 1040-1050. 85. Pichavant, M., et al., Ozone exposure in a mouse model induces airway hyperreactivity that requires the presence of natural killer T cells and IL-17. J Exp 39 Med, 2008. 205(2): p. 385-93. 86. Sag, D., et al., IL-10-producing NKT10 cells are a distinct regulatory invariant NKT cell subset. J Clin Invest, 2014. 124(9): p. 3725-40. 87. Lynch, L., et al., Regulatory iNKT cells lack expression of the transcription factor PLZF and control the homeostasis of T(reg) cells and macrophages in adipose tissue. Nat Immunol, 2015. 16(1): p. 85-95. 88. Chang, P.P., et al., Identification of Bcl-6-dependent follicular helper NKT cells that provide cognate help for B cell responses. Nat Immunol, 2011. 13(1): p. 35- 43. 89. Tonti, E., et al., Follicular helper NKT cells induce limited B cell responses and germinal center formation in the absence of CD4(+) T cell help. J Immunol, 2012. 188(7): p. 3217-22. 90. King, I.L., et al., Invariant natural killer T cells direct B cell responses to cognate lipid antigen in an IL-21-dependent manner. Nat Immunol, 2011. 13(1): p. 44-50. 91. Rampuria, P. and M.L. Lang, CD1d-dependent expansion of NKT follicular helper cells in vivo and in vitro is a product of cellular proliferation and differentiation. Int Immunol, 2015. 27(5): p. 253-63. 92. Chan, A.C., et al., Ex-vivo analysis of human natural killer T cells demonstrates heterogeneity between tissues and within established CD4(+) and CD4(-) subsets. Clin Exp Immunol, 2013. 172(1): p. 129-37. 93. Moreira-Teixeira, L., et al., Proinflammatory environment dictates the IL-17- producing capacity of human invariant NKT cells. J Immunol, 2011. 186(10): p. 5758-65. 94. Gumperz, J.E., et al., Functionally distinct subsets of CD1d-restricted natural killer T cells revealed by CD1d tetramer staining. J Exp Med, 2002. 195(5): p. 625-36. 40 95. Lee, P.T., et al., Testing the NKT cell hypothesis of human IDDM pathogenesis. J Clin Invest, 2002. 110(6): p. 793-800. 96. Chan, A.C., et al., Immune characterization of an individual with an exceptionally high natural killer T cell frequency and her immediate family. Clin Exp Immunol, 2009. 156(2): p. 238-45. 97. Berzins, S.P., et al., Limited correlation between human thymus and blood NKT cell content revealed by an ontogeny study of paired tissue samples. Eur J Immunol, 2005. 35(5): p. 1399-407. 98. Matsuda, J.L., et al., Mouse V alpha 14i natural killer T cells are resistant to cytokine polarization in vivo. Proc Natl Acad Sci U S A, 2003. 100(14): p. 8395- 400. 99. Stetson, D.B., et al., Constitutive cytokine mRNAs mark natural killer (NK) and NK T cells poised for rapid effector function. J Exp Med, 2003. 198(7): p. 1069- 76. 100. Leite-De-Moraes, M.C., et al., A distinct IL-18-induced pathway to fully activate NK T lymphocytes independently from TCR engagement. J Immunol, 1999. 163(11): p. 5871-6. 101. Reilly, E.C., J.R. Wands, and L. Brossay, Cytokine dependent and independent iNKT cell activation. Cytokine, 2010. 51(3): p. 227-31. 102. Jahng, A., et al., Prevention of autoimmunity by targeting a distinct, noninvariant CD1d-reactive T cell population reactive to sulfatide. J Exp Med, 2004. 199(7): p. 947-57. 103. Tatituri, R.V., et al., Recognition of microbial and mammalian phospholipid antigens by NKT cells with diverse TCRs. Proc Natl Acad Sci U S A, 2013. 110(5): p. 1827-32. 104. Crowe, N.Y., et al., Glycolipid antigen drives rapid expansion and sustained 41 cytokine production by NK T cells. J Immunol, 2003. 171(8): p. 4020-7. 105. Wilson, M.T., et al., The response of natural killer T cells to glycolipid antigens is characterized by surface receptor down-modulation and expansion. Proc Natl Acad Sci U S A, 2003. 100(19): p. 10913-8. 106. Tyznik, A.J., et al., Cutting edge: the mechanism of invariant NKT cell responses to viral danger signals. J Immunol, 2008. 181(7): p. 4452-6. 107. Brigl, M., et al., Mechanism of CD1d-restricted natural killer T cell activation during microbial infection. Nat Immunol, 2003. 4(12): p. 1230-7. 108. Cohen, N.R., et al., Innate recognition of cell wall beta-glucans drives invariant natural killer T cell responses against fungi. Cell Host Microbe, 2011. 10(5): p. 437-50. 109. Paget, C., et al., Activation of invariant NKT cells by toll-like receptor 9-stimulated dendritic cells requires type I interferon and charged glycosphingolipids. Immunity, 2007. 27(4): p. 597-609. 110. Wesley, J.D., et al., NK cell-like behavior of Valpha14i NK T cells during MCMV infection. PLoS Pathog, 2008. 4(7): p. e1000106. 111. Tyznik, A.J., et al., Distinct requirements for activation of NKT and NK cells during viral infection. J Immunol, 2014. 192(8): p. 3676-85. 112. Brigl, M., et al., Innate and cytokine-driven signals, rather than microbial antigens, dominate in natural killer T cell activation during microbial infection. J Exp Med, 2011. 208(6): p. 1163-77. 113. Behar, S.M., et al., Susceptibility of mice deficient in CD1D or TAP1 to infection with Mycobacterium tuberculosis. J Exp Med, 1999. 189(12): p. 1973-80. 114. Fischer, K., et al., Mycobacterial phosphatidylinositol mannoside is a natural antigen for CD1d-restricted T cells. Proc Natl Acad Sci U S A, 2004. 101(29): p. 10685-90. 42 115. Kumar, H., et al., Cutting edge: CD1d deficiency impairs murine host defense against the spirochete, Borrelia burgdorferi. J Immunol, 2000. 165(9): p. 4797- 801. 116. Tupin, E., et al., NKT cells prevent chronic joint inflammation after infection with Borrelia burgdorferi. Proc Natl Acad Sci U S A, 2008. 105(50): p. 19863-8. 117. Kawakami, K., et al., Critical role of Valpha14+ natural killer T cells in the innate phase of host protection against Streptococcus pneumoniae infection. Eur J Immunol, 2003. 33(12): p. 3322-30. 118. Kinjo, Y., et al., Invariant natural killer T cells recognize glycolipids from pathogenic Gram-positive bacteria. Nat Immunol, 2011. 12(10): p. 966-74. 119. Mattner, J., et al., Exogenous and endogenous glycolipid antigens activate NKT cells during microbial infections. Nature, 2005. 434(7032): p. 525-9. 120. Tilloy, F., et al., An invariant T cell receptor alpha chain defines a novel TAP- independent major histocompatibility complex class Ib-restricted alpha/beta T cell subpopulation in mammals. J Exp Med, 1999. 189(12): p. 1907-21. 121. Reantragoon, R., et al., Antigen-loaded MR1 tetramers define T cell receptor heterogeneity in mucosal-associated invariant T cells. J Exp Med, 2013. 210(11): p. 2305-20. 122. Lepore, M., et al., Parallel T-cell cloning and deep sequencing of human MAIT cells reveal stable oligoclonal TCRbeta repertoire. Nat Commun, 2014. 5: p. 3866. 123. Dusseaux, M., et al., Human MAIT cells are xenobiotic-resistant, tissue-targeted, CD161hi IL-17-secreting T cells. Blood, 2011. 117(4): p. 1250-9. 124. Yamaguchi, H., et al., A highly conserved major histocompatibility complex class I-related gene in mammals. Biochem Biophys Res Commun, 1997. 238(3): p. 697-702. 43 125. Riegert, P., V. Wanner, and S. Bahram, Genomics, isoforms, expression, and phylogeny of the MHC class I-related MR1 gene. J Immunol, 1998. 161(8): p. 4066-77. 126. Kjer-Nielsen, L., et al., MR1 presents microbial vitamin B metabolites to MAIT cells. Nature, 2012. 491(7426): p. 717-23. 127. Corbett, A.J., et al., T-cell activation by transitory neo-antigens derived from distinct microbial pathways. Nature, 2014. 509(7500): p. 361-5. 128. Chua, W.J., et al., Endogenous MHC-related protein 1 is transiently expressed on the plasma membrane in a conformation that activates mucosal-associated invariant T cells. J Immunol, 2011. 186(8): p. 4744-50. 129. Eckle, S.B., et al., A molecular basis underpinning the T cell receptor heterogeneity of mucosal-associated invariant T cells. J Exp Med, 2014. 211(8): p. 1585-600. 130. Olszak, T., et al., Microbial exposure during early life has persistent effects on natural killer T cell function. Science, 2012. 336(6080): p. 489-93. 131. Wingender, G., et al., Intestinal microbes affect phenotypes and functions of invariant natural killer T cells in mice. Gastroenterology, 2012. 143(2): p. 418-28. 132. Le Bourhis, L., et al., Antimicrobial activity of mucosal-associated invariant T cells. Nat Immunol, 2010. 11(8): p. 701-8. 133. Gold, M.C., et al., Human mucosal associated invariant T cells detect bacterially infected cells. PLoS Biol, 2010. 8(6): p. e1000407. 134. Xiao, X. and J. Cai, Mucosal-Associated Invariant T Cells: New Insights into Antigen Recognition and Activation. Front Immunol, 2017. 8: p. 1540. 135. Georgel, P., et al., The non-conventional MHC class I MR1 molecule controls infection by Klebsiella pneumoniae in mice. Mol Immunol, 2011. 48(5): p. 769-75. 136. Meierovics, A., W.J. Yankelevich, and S.C. Cowley, MAIT cells are critical for 44 optimal mucosal immune responses during in vivo pulmonary bacterial infection. Proc Natl Acad Sci U S A, 2013. 110(33): p. E3119-28. 137. Chua, W.J., et al., Polyclonal mucosa-associated invariant T cells have unique innate functions in bacterial infection. Infect Immun, 2012. 80(9): p. 3256-67. 138. Jiang, J., et al., Mucosal-associated invariant T-cell function is modulated by programmed death-1 signaling in patients with active tuberculosis. Am J Respir Crit Care Med, 2014. 190(3): p. 329-39. 139. Ussher, J.E., et al., CD161++ CD8+ T cells, including the MAIT cell subset, are specifically activated by IL-12+IL-18 in a TCR-independent manner. Eur J Immunol, 2014. 44(1): p. 195-203. 140. van Wilgenburg, B., et al., MAIT cells are activated during human viral infections. Nat Commun, 2016. 7: p. 11653. 141. Cosgrove, C., et al., Early and nonreversible decrease of CD161++ /MAIT cells in HIV infection. Blood, 2013. 121(6): p. 951-61. 142. Fernandez, C.S., et al., MAIT cells are depleted early but retain functional cytokine expression in HIV infection. Immunol Cell Biol, 2015. 93(2): p. 177-88. 143. Leeansyah, E., et al., Activation, exhaustion, and persistent decline of the antimicrobial MR1-restricted MAIT-cell population in chronic HIV-1 infection. Blood, 2013. 121(7): p. 1124-35. 144. Gold, M.C., et al., MR1-restricted MAIT cells display ligand discrimination and pathogen selectivity through distinct T cell receptor usage. J Exp Med, 2014. 211(8): p. 1601-10. 145. Annibali, V., et al., CD161(high)CD8+T cells bear pathogenetic potential in multiple sclerosis. Brain, 2011. 134(Pt 2): p. 542-54. 146. Treiner, E. and R.S. Liblau, Mucosal-Associated Invariant T Cells in Multiple Sclerosis: The Jury is Still Out. Front Immunol, 2015. 6: p. 503. 45 147. Wang, J.J., et al., Mucosal-associated invariant T cells are reduced and functionally immature in the peripheral blood of primary Sjogren's syndrome patients. Eur J Immunol, 2016. 46(10): p. 2444-2453. 148. Dunne, M.R., et al., Persistent changes in circulating and intestinal gammadelta T cell subsets, invariant natural killer T cells and mucosal-associated invariant T cells in children and adults with coeliac disease. PLoS One, 2013. 8(10): p. e76008. 149. Yeager, M., S. Kumar, and A.L. Hughes, Sequence convergence in the peptide- binding region of primate and rodent MHC class Ib molecules. Mol Biol Evol, 1997. 14(10): p. 1035-41. 150. Geraghty, D.E., et al., Polymorphism at the HLA-E locus predates most HLA-A and -B polymorphism. Hum Immunol, 1992. 33(3): p. 174-84. 151. Grimsley, C., et al., Definitive high resolution typing of HLA-E allelic polymorphisms: Identifying potential errors in existing allele data. Tissue Antigens, 2002. 60(3): p. 206-12. 152. Zeng, L., et al., A structural basis for antigen presentation by the MHC class Ib molecule, Qa-1b. J Immunol, 2012. 188(1): p. 302-10. 153. Connolly, D.J., et al., A cDNA clone encoding the mouse Qa-1a histocompatibility antigen and proposed structure of the putative peptide binding site. J Immunol, 1993. 151(11): p. 6089-98. 154. Aldrich, C.J., et al., Identification of a Tap-dependent leader peptide recognized by alloreactive T cells specific for a class Ib antigen. Cell, 1994. 79(4): p. 649-58. 155. Bai, A., J. Broen, and J. Forman, The pathway for processing leader-derived peptides that regulate the maturation and expression of Qa-1b. Immunity, 1998. 9(3): p. 413-21. 156. Braud, V., E.Y. Jones, and A. McMichael, The human major histocompatibility 46 complex class Ib molecule HLA-E binds signal sequence-derived peptides with primary anchor residues at positions 2 and 9. Eur J Immunol, 1997. 27(5): p. 1164-9. 157. Lee, N., et al., HLA-E surface expression depends on binding of TAP-dependent peptides derived from certain HLA class I signal sequences. J Immunol, 1998. 160(10): p. 4951-60. 158. Braud, V.M., et al., HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature, 1998. 391(6669): p. 795-9. 159. Lee, N., et al., HLA-E is a major ligand for the natural killer inhibitory receptor CD94/NKG2A. Proc Natl Acad Sci U S A, 1998. 95(9): p. 5199-204. 160. Vance, R.E., et al., Mouse CD94/NKG2A is a natural killer cell receptor for the nonclassical major histocompatibility complex (MHC) class I molecule Qa-1(b). J Exp Med, 1998. 188(10): p. 1841-8. 161. Vance, R.E., A.M. Jamieson, and D.H. Raulet, Recognition of the class Ib molecule Qa-1(b) by putative activating receptors CD94/NKG2C and CD94/NKG2E on mouse natural killer cells. J Exp Med, 1999. 190(12): p. 1801- 12. 162. Vance, R.E., et al., Implications of CD94 deficiency and monoallelic NKG2A expression for natural killer cell development and repertoire formation. Proc Natl Acad Sci U S A, 2002. 99(2): p. 868-73. 163. Moser, J.M., et al., CD94-NKG2A receptors regulate antiviral CD8(+) T cell responses. Nat Immunol, 2002. 3(2): p. 189-95. 164. Wada, H., et al., The inhibitory NK cell receptor CD94/NKG2A and the activating receptor CD94/NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues. Eur J Immunol, 2004. 34(1): p. 81-90. 165. Vales-Gomez, M., et al., Kinetics and peptide dependency of the binding of the 47 inhibitory NK receptor CD94/NKG2-A and the activating receptor CD94/NKG2-C to HLA-E. EMBO J, 1999. 18(15): p. 4250-60. 166. Xu, H.C., et al., Lymphocytes Negatively Regulate NK Cell Activity via Qa-1b following Viral Infection. Cell Rep, 2017. 21(9): p. 2528-2540. 167. Rapaport, A.S., et al., The Inhibitory Receptor NKG2A Sustains Virus-Specific CD8(+) T Cells in Response to a Lethal Poxvirus Infection. Immunity, 2015. 43(6): p. 1112-24. 168. Bian, Y., et al., MHC Ib molecule Qa-1 presents Mycobacterium tuberculosis peptide antigens to CD8+ T cells and contributes to protection against infection. PLoS Pathog, 2017. 13(5): p. e1006384. 169. Kraft, J.R., et al., Analysis of Qa-1(b) peptide binding specificity and the capacity of CD94/NKG2A to discriminate between Qa-1-peptide complexes. J Exp Med, 2000. 192(5): p. 613-24. 170. Davies, A., et al., A peptide from heat shock protein 60 is the dominant peptide bound to Qa-1 in the absence of the MHC class Ia leader sequence peptide Qdm. J Immunol, 2003. 170(10): p. 5027-33. 171. Lo, W.F., et al., Molecular mimicry mediated by MHC class Ib molecules after infection with gram-negative pathogens. Nat Med, 2000. 6(2): p. 215-8. 172. Chun, T., et al., Constitutive and regulated expression of the class IB molecule Qa-1 in pancreatic beta cells. Immunology, 1998. 94(1): p. 64-71. 173. Tompkins, S.M., et al., Transporters associated with antigen processing (TAP)- independent presentation of soluble insulin to alpha/beta T cells by the class Ib gene product, Qa-1(b). J Exp Med, 1998. 188(5): p. 961-71. 174. Nagarajan, N.A., F. Gonzalez, and N. Shastri, Nonclassical MHC class Ib- restricted cytotoxic T cells monitor antigen processing in the endoplasmic reticulum. Nat Immunol, 2012. 13(6): p. 579-86. 48 175. Bouwer, H.G., et al., MHC class Ib-restricted cells contribute to antilisterial immunity: evidence for Qa-1b as a key restricting element for Listeria-specific CTLs. J Immunol, 1997. 159(6): p. 2795-801. 176. Caccamo, N., et al., Human CD8 T lymphocytes recognize Mycobacterium tuberculosis antigens presented by HLA-E during active tuberculosis and express type 2 cytokines. Eur J Immunol, 2015. 45(4): p. 1069-81. 177. van Meijgaarden, K.E., et al., Human CD8+ T-cells recognizing peptides from Mycobacterium tuberculosis (Mtb) presented by HLA-E have an unorthodox Th2- like, multifunctional, Mtb inhibitory phenotype and represent a novel human T-cell subset. PLoS Pathog, 2015. 11(3): p. e1004671. 178. Harriff, M.J., et al., HLA-E Presents Glycopeptides from the Mycobacterium tuberculosis Protein MPT32 to Human CD8(+) T cells. Sci Rep, 2017. 7(1): p. 4622. 179. Garcia, P., et al., Human T cell receptor-mediated recognition of HLA-E. Eur J Immunol, 2002. 32(4): p. 936-44. 180. Jorgensen, P.B., et al., Epstein-Barr virus peptide presented by HLA-E is predominantly recognized by CD8(bright) cells in multiple sclerosis patients. PLoS One, 2012. 7(9): p. e46120. 181. Nattermann, J., et al., The HLA-A2 restricted T cell epitope HCV core 35-44 stabilizes HLA-E expression and inhibits cytolysis mediated by natural killer cells. Am J Pathol, 2005. 166(2): p. 443-53. 182. Schulte, D., et al., The HLA-E(R)/HLA-E(R) genotype affects the natural course of hepatitis C virus (HCV) infection and is associated with HLA-E-restricted recognition of an HCV-derived peptide by interferon-gamma-secreting human CD8(+) T cells. J Infect Dis, 2009. 200(9): p. 1397-401. 183. Davis, Z.B., et al., A Conserved HIV-1-Derived Peptide Presented by HLA-E 49 Renders Infected T-cells Highly Susceptible to Attack by NKG2A/CD94-Bearing Natural Killer Cells. PLoS Pathog, 2016. 12(2): p. e1005421. 184. Mazzarino, P., et al., Identification of effector-memory CMV-specific T lymphocytes that kill CMV-infected target cells in an HLA-E-restricted fashion. Eur J Immunol, 2005. 35(11): p. 3240-7. 185. Pietra, G., et al., HLA-E-restricted recognition of cytomegalovirus-derived peptides by human CD8+ cytolytic T lymphocytes. Proc Natl Acad Sci U S A, 2003. 100(19): p. 10896-901. 186. Wang, E.C., et al., UL40-mediated NK evasion during productive infection with human cytomegalovirus. Proc Natl Acad Sci U S A, 2002. 99(11): p. 7570-5. 187. Tomasec, P., et al., Surface expression of HLA-E, an inhibitor of natural killer cells, enhanced by human cytomegalovirus gpUL40. Science, 2000. 287(5455): p. 1031. 188. Ulbrecht, M., et al., Cutting edge: the human cytomegalovirus UL40 gene product contains a ligand for HLA-E and prevents NK cell-mediated lysis. J Immunol, 2000. 164(10): p. 5019-22. 189. Hu, D., et al., Analysis of regulatory CD8 T cells in Qa-1-deficient mice. Nat Immunol, 2004. 5(5): p. 516-23. 190. Devlin, J.J., et al., Duplicated gene pairs and alleles of class I genes in the Qa2 region of the murine major histocompatibility complex: a comparison. EMBO J, 1985. 4(12): p. 3203-7. 191. Mellor, A.L., J. Antoniou, and P.J. Robinson, Structure and expression of genes encoding murine Qa-2 class I antigens. Proc Natl Acad Sci U S A, 1985. 82(17): p. 5920-4. 192. Joyce, S., et al., A nonpolymorphic major histocompatibility complex class Ib molecule binds a large array of diverse self-peptides. J Exp Med, 1994. 179(2): 50 p. 579-88. 193. Rotzschke, O., et al., Qa-2 molecules are peptide receptors of higher stringency than ordinary class I molecules. Nature, 1993. 361(6413): p. 642-4. 194. Tabaczewski, P., et al., Alternative peptide binding motifs of Qa-2 class Ib molecules define rules for binding of self and nonself peptides. J Immunol, 1997. 159(6): p. 2771-81. 195. He, X., et al., Promiscuous antigen presentation by the nonclassical MHC Ib Qa- 2 is enabled by a shallow, hydrophobic groove and self-stabilized peptide conformation. Structure, 2001. 9(12): p. 1213-24. 196. Tabaczewski, P. and I. Stroynowski, Expression of secreted and glycosylphosphatidylinositol-bound Qa-2 molecules is dependent on functional TAP-2 peptide transporter. J Immunol, 1994. 152(11): p. 5268-74. 197. Stroynowski, I., et al., A single gene encodes soluble and membrane-bound forms of the major histocompatibility Qa-2 antigen: anchoring of the product by a phospholipid tail. Cell, 1987. 50(5): p. 759-68. 198. Tabaczewski, P., et al., Alternative splicing of class Ib major histocompatibility complex transcripts in vivo leads to the expression of soluble Qa-2 molecules in murine blood. Proc Natl Acad Sci U S A, 1994. 91(5): p. 1883-7. 199. Teitell, M., et al., The alpha 3 domain of the Qa-2 molecule is defective for CD8 binding and cytotoxic T lymphocyte activation. J Exp Med, 1993. 178(6): p. 2139- 45. 200. Swanson, P.A., 2nd, et al., An MHC class Ib-restricted CD8 T cell response confers antiviral immunity. J Exp Med, 2008. 205(7): p. 1647-57. 201. Vezys, V., et al., Continuous recruitment of naive T cells contributes to heterogeneity of antiviral CD8 T cells during persistent infection. J Exp Med, 2006. 203(10): p. 2263-9. 51 202. Swanson, P.A., 2nd, et al., Cutting edge: shift in antigen dependence by an antiviral MHC class Ib-restricted CD8 T cell response during persistent viral infection. J Immunol, 2009. 182(9): p. 5198-202. 203. Das, G., et al., Qa-2-dependent selection of CD8alpha/alpha T cell receptor alpha/beta(+) cells in murine intestinal intraepithelial lymphocytes. J Exp Med, 2000. 192(10): p. 1521-8. 204. Comiskey, M., et al., Evidence that HLA-G is the functional homolog of mouse Qa-2, the Ped gene product. Hum Immunol, 2003. 64(11): p. 999-1004. 205. Geraghty, D.E., B.H. Koller, and H.T. Orr, A human major histocompatibility complex class I gene that encodes a protein with a shortened cytoplasmic segment. Proc Natl Acad Sci U S A, 1987. 84(24): p. 9145-9. 206. Fujii, T., A. Ishitani, and D.E. Geraghty, A soluble form of the HLA-G antigen is encoded by a messenger ribonucleic acid containing intron 4. J Immunol, 1994. 153(12): p. 5516-24. 207. Ishitani, A. and D.E. Geraghty, Alternative splicing of HLA-G transcripts yields proteins with primary structures resembling both class I and class II antigens. Proc Natl Acad Sci U S A, 1992. 89(9): p. 3947-51. 208. Paul, P., et al., Identification of HLA-G7 as a new splice variant of the HLA-G mRNA and expression of soluble HLA-G5, -G6, and -G7 transcripts in human transfected cells. Hum Immunol, 2000. 61(11): p. 1138-49. 209. Diehl, M., et al., Nonclassical HLA-G molecules are classical peptide presenters. Curr Biol, 1996. 6(3): p. 305-14. 210. Lee, N., et al., The membrane-bound and soluble forms of HLA-G bind identical sets of endogenous peptides but differ with respect to TAP association. Immunity, 1995. 3(5): p. 591-600. 211. McElhinny, A.S., G.E. Exley, and C.M. Warner, Painting Qa-2 onto Ped slow 52 preimplantatiom embryos increases the rate of cleavage. American Journal of Reproductive Immunology, 2000. 44(1): p. 52-58. 212. Rouas-Freiss, N., et al., Direct evidence to support the role of HLA-G in protecting the fetus from maternal uterine natural killer cytolysis. Proc Natl Acad Sci U S A, 1997. 94(21): p. 11520-5. 213. Warner, C.M., et al., Analysis of Qa-2 antigen expression by preimplantation mouse embryos: possible relationship to the preimplantation-embryo- development (Ped) gene product. Biol Reprod, 1987. 36(3): p. 611-6. 214. Lenfant, F., et al., Induction of HLA-G-restricted human cytomegalovirus pp65 (UL83)-specific cytotoxic T lymphocytes in HLA-G transgenic mice. J Gen Virol, 2003. 84(Pt 2): p. 307-17. 215. Wang, C.R., B.E. Loveland, and K.F. Lindahl, H-2M3 encodes the MHC class I molecule presenting the maternally transmitted antigen of the mouse. Cell, 1991. 66(2): p. 335-45. 216. Wang, C.R., et al., Nonclassical binding of formylated peptide in crystal structure of the MHC class Ib molecule H2-M3. Cell, 1995. 82(4): p. 655-64. 217. Shawar, S.M., et al., Differential amino-terminal anchors for peptide binding to H- 2M3a or H-2Kb and H-2Db. J Immunol, 1993. 151(1): p. 201-10. 218. Smith, G.P., et al., Peptide presentation by the MHC class Ib molecule, H2-M3. Int Immunol, 1994. 6(12): p. 1917-26. 219. Byers, D.E. and K. Fischer Lindahl, H2-M3 presents a nonformylated viral epitope to CTLs generated in vitro. J Immunol, 1998. 161(1): p. 90-6. 220. Lindahl, K.F., et al., H2-M3, a full-service class Ib histocompatibility antigen. Annu Rev Immunol, 1997. 15: p. 851-79. 221. Levitt, J.M., et al., Exogenous peptides enter the endoplasmic reticulum of TAP- deficient cells and induce the maturation of nascent MHC class I molecules. Eur 53 J Immunol, 2001. 31(4): p. 1181-90. 222. Chun, T., et al., Functional roles of TAP and tapasin in the assembly of M3-N- formylated peptide complexes. J Immunol, 2001. 167(3): p. 1507-14. 223. Chiu, N.M., et al., The majority of H2-M3 is retained intracellularly in a peptide- receptive state and traffics to the cell surface in the presence of N-formylated peptides. J Exp Med, 1999. 190(3): p. 423-34. 224. Vyas, J.M., J.R. Rodgers, and R.R. Rich, H-2M3a violates the paradigm for major histocompatibility complex class I peptide binding. J Exp Med, 1995. 181(5): p. 1817-25. 225. Linehan, J.L., et al., Non-classical Immunity Controls Microbiota Impact on Skin Immunity and Tissue Repair. Cell, 2018. 172(4): p. 784-796 e18. 226. Rolph, M.S. and S.H. Kaufmann, Partially TAP-independent protection against Listeria monocytogenes by H2-M3-restricted CD8+ T cells. J Immunol, 2000. 165(8): p. 4575-80. 227. Princiotta, M.F., et al., H2-M3 restricted presentation of a Listeria-derived leader peptide. J Exp Med, 1998. 187(10): p. 1711-9. 228. Gulden, P.H., et al., A Listeria monocytogenes pentapeptide is presented to cytolytic T lymphocytes by the H2-M3 MHC class Ib molecule. Immunity, 1996. 5(1): p. 73-9. 229. Lenz, L.L., B. Dere, and M.J. Bevan, Identification of an H2-M3-restricted Listeria epitope: implications for antigen presentation by M3. Immunity, 1996. 5(1): p. 63- 72. 230. D'Orazio, S.E., et al., Class Ia MHC-deficient BALB/c mice generate CD8+ T cell- mediated protective immunity against Listeria monocytogenes infection. J Immunol, 2003. 171(1): p. 291-8. 231. Seaman, M.S., et al., Response to Listeria monocytogenes in mice lacking MHC 54 class Ia molecules. J Immunol, 1999. 162(9): p. 5429-36. 232. D'Orazio, S.E., C.A. Shaw, and M.N. Starnbach, H2-M3-restricted CD8+ T cells are not required for MHC class Ib-restricted immunity against Listeria monocytogenes. J Exp Med, 2006. 203(2): p. 383-91. 233. Kerksiek, K.M., et al., H2-M3-restricted T cells in bacterial infection: rapid primary but diminished memory responses. J Exp Med, 1999. 190(2): p. 195-204. 234. Kerksiek, K.M., et al., H2-M3-restricted memory T cells: persistence and activation without expansion. J Immunol, 2003. 170(4): p. 1862-9. 235. Hamilton, S.E., et al., MHC class Ia-restricted memory T cells inhibit expansion of a nonprotective MHC class Ib (H2-M3)-restricted memory response. Nat Immunol, 2004. 5(2): p. 159-68. 236. Lenz, L.L. and M.J. Bevan, CTL responses to H2-M3-restricted Listeria epitopes. Immunol Rev, 1997. 158: p. 115-21. 237. Chun, T., et al., Induction of M3-restricted cytotoxic T lymphocyte responses by N-formylated peptides derived from Mycobacterium tuberculosis. J Exp Med, 2001. 193(10): p. 1213-20. 238. Doi, T., et al., H2-M3-restricted CD8+ T cells induced by peptide-pulsed dendritic cells confer protection against Mycobacterium tuberculosis. J Immunol, 2007. 178(6): p. 3806-13. 239. Urdahl, K.B., D. Liggitt, and M.J. Bevan, CD8+ T cells accumulate in the lungs of Mycobacterium tuberculosis-infected Kb-/-Db-/- mice, but provide minimal protection. J Immunol, 2003. 170(4): p. 1987-94. 240. Tvinnereim, A. and B. Wizel, CD8+ T cell protective immunity against Chlamydia pneumoniae includes an H2-M3-restricted response that is largely CD4+ T cell- independent. J Immunol, 2007. 179(6): p. 3947-57. 241. Ugrinovic, S., et al., H2-M3 major histocompatibility complex class Ib-restricted 55 CD8 T cells induced by Salmonella enterica serovar Typhimurium infection recognize proteins released by Salmonella serovar Typhimurium. Infect Immun, 2005. 73(12): p. 8002-8. 242. Liu, P., et al., Targeting the phosphoinositide 3-kinase pathway in cancer. Nat Rev Drug Discov, 2009. 8(8): p. 627-44. 243. Wymann, M.P. and L. Pirola, Structure and function of phosphoinositide 3- kinases. Biochim Biophys Acta, 1998. 1436(1-2): p. 127-50. 244. Okkenhaug, K. and B. Vanhaesebroeck, PI3K in lymphocyte development, differentiation and activation. Nat Rev Immunol, 2003. 3(4): p. 317-30. 245. Anderson, K.E. and S.P. Jackson, Class I phosphoinositide 3-kinases. Int J Biochem Cell Biol, 2003. 35(7): p. 1028-33. 246. Aman, M.J., et al., The inositol phosphatase SHIP inhibits Akt/PKB activation in B cells. J Biol Chem, 1998. 273(51): p. 33922-8. 247. Stambolic, V., et al., Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell, 1998. 95(1): p. 29-39. 248. Ravetch, J.V. and L.L. Lanier, Immune inhibitory receptors. Science, 2000. 290(5489): p. 84-9. 249. Pesesse, X., et al., The SH2 domain containing inositol 5-phosphatase SHIP2 displays phosphatidylinositol 3,4,5-trisphosphate and inositol 1,3,4,5- tetrakisphosphate 5-phosphatase activity. FEBS Lett, 1998. 437(3): p. 301-3. 250. Tu, Z., et al., Embryonic and hematopoietic stem cells express a novel SH2- containing inositol 5'-phosphatase isoform that partners with the Grb2 adapter protein. Blood, 2001. 98(7): p. 2028-38. 251. Conde, C., G. Gloire, and J. Piette, Enzymatic and non-enzymatic activities of SHIP-1 in signal transduction and cancer. Biochem Pharmacol, 2011. 82(10): p. 1320-34. 56 252. Erneux, C., et al., SHIP2 multiple functions: a balance between a negative control of PtdIns(3,4,5)P(3) level, a positive control of PtdIns(3,4)P(2) production, and intrinsic docking properties. J Cell Biochem, 2011. 112(9): p. 2203-9. 253. Sasaoka, T., et al., SH2-containing inositol phosphatase 2 negatively regulates insulin-induced glycogen synthesis in L6 myotubes. Diabetologia, 2001. 44(10): p. 1258-67. 254. Nguyen, N.Y., et al., An ENU-induced mouse mutant of SHIP1 reveals a critical role of the stem cell isoform for suppression of macrophage activation. Blood, 2011. 117(20): p. 5362-71. 255. Hazen, A.L., et al., SHIP is required for a functional hematopoietic stem cell niche. Blood, 2009. 113(13): p. 2924-33. 256. Iyer, S., et al., SHIP1-expressing mesenchymal stem cells regulate hematopoietic stem cell homeostasis and lineage commitment during aging. Stem Cells Dev, 2015. 24(9): p. 1073-81. 257. Tessmer, M.S., et al., KLRG1 binds cadherins and preferentially associates with SHIP-1. Int Immunol, 2007. 19(4): p. 391-400. 258. Osborne, M.A., et al., The inositol 5'-phosphatase SHIP binds to immunoreceptor signaling motifs and responds to high affinity IgE receptor aggregation. J Biol Chem, 1996. 271(46): p. 29271-8. 259. Helgason, C.D., et al., Targeted disruption of SHIP leads to hemopoietic perturbations, lung pathology, and a shortened life span. Genes Dev, 1998. 12(11): p. 1610-20. 260. Liu, Q., et al., SHIP is a negative regulator of growth factor receptor-mediated PKB/Akt activation and myeloid cell survival. Genes Dev, 1999. 13(7): p. 786-91. 261. Brauweiler, A., et al., Differential regulation of B cell development, activation, and death by the src homology 2 domain-containing 5' inositol phosphatase (SHIP). J 57 Exp Med, 2000. 191(9): p. 1545-54. 262. Helgason, C.D., et al., A dual role for Src homology 2 domain-containing inositol- 5-phosphatase (SHIP) in immunity: aberrant development and enhanced function of b lymphocytes in ship -/- mice. J Exp Med, 2000. 191(5): p. 781-94. 263. Banh, C., et al., Mouse natural killer cell development and maturation are differentially regulated by SHIP-1. Blood, 2012. 120(23): p. 4583-90. 264. Kashiwada, M., et al., Downstream of tyrosine kinases-1 and Src homology 2- containing inositol 5'-phosphatase are required for regulation of CD4+CD25+ T cell development. J Immunol, 2006. 176(7): p. 3958-65. 265. Tarasenko, T., et al., T cell-specific deletion of the inositol phosphatase SHIP reveals its role in regulating Th1/Th2 and cytotoxic responses. Proc Natl Acad Sci U S A, 2007. 104(27): p. 11382-7. 266. Collazo, M.M., et al., SHIP limits immunoregulatory capacity in the T-cell compartment. Blood, 2009. 113(13): p. 2934-44. 267. Crough, T. and R. Khanna, Immunobiology of human cytomegalovirus: from bench to bedside. Clin Microbiol Rev, 2009. 22(1): p. 76-98, Table of Contents. 268. Staras, S.A., et al., Seroprevalence of cytomegalovirus infection in the United States, 1988-1994. Clin Infect Dis, 2006. 43(9): p. 1143-51. 269. Polic, B., et al., Hierarchical and redundant lymphocyte subset control precludes cytomegalovirus replication during latent infection. J Exp Med, 1998. 188(6): p. 1047-54. 270. Campos, A.B., et al., Human cytomegalovirus antiviral drug resistance in hematopoietic stem cell transplantation: current state of the art. Rev Med Virol, 2016. 26(3): p. 161-82. 271. Jordan, M.C., J.D. Shanley, and J.G. Stevens, Immunosuppression reactivates and disseminates latent murine cytomegalovirus. J Gen Virol, 1977. 37(2): p. 58 419-23. 272. Rawlinson, W.D., H.E. Farrell, and B.G. Barrell, Analysis of the complete DNA sequence of murine cytomegalovirus. J Virol, 1996. 70(12): p. 8833-49. 273. Hsu, K.M., et al., Murine cytomegalovirus displays selective infection of cells within hours after systemic administration. J Gen Virol, 2009. 90(Pt 1): p. 33-43. 274. Allan, J.E. and G.R. Shellam, Genetic control of murine cytomegalovirus infection: virus titres in resistant and susceptible strains of mice. Arch Virol, 1984. 81(1-2): p. 139-50. 275. Campbell, A.E., V.J. Cavanaugh, and J.S. Slater, The salivary glands as a privileged site of cytomegalovirus immune evasion and persistence. Med Microbiol Immunol, 2008. 197(2): p. 205-13. 276. Zucchini, N., et al., Cutting edge: Overlapping functions of TLR7 and TLR9 for innate defense against a herpesvirus infection. J Immunol, 2008. 180(9): p. 5799- 803. 277. Tabeta, K., et al., Toll-like receptors 9 and 3 as essential components of innate immune defense against mouse cytomegalovirus infection. Proc Natl Acad Sci U S A, 2004. 101(10): p. 3516-21. 278. Puttur, F., et al., Conventional Dendritic Cells Confer Protection against Mouse Cytomegalovirus Infection via TLR9 and MyD88 Signaling. Cell Rep, 2016. 17(4): p. 1113-1127. 279. Orange, J.S. and C.A. Biron, An absolute and restricted requirement for IL-12 in natural killer cell IFN-gamma production and antiviral defense. Studies of natural killer and T cell responses in contrasting viral infections. J Immunol, 1996. 156(3): p. 1138-42. 280. Dalod, M., et al., Interferon alpha/beta and interleukin 12 responses to viral infections: pathways regulating dendritic cell cytokine expression in vivo. J Exp 59 Med, 2002. 195(4): p. 517-28. 281. Nguyen, K.B., et al., Coordinated and distinct roles for IFN-alpha beta, IL-12, and IL-15 regulation of NK cell responses to viral infection. J Immunol, 2002. 169(8): p. 4279-87. 282. Pien, G.C., et al., Cutting edge: selective IL-18 requirements for induction of compartmental IFN-gamma responses during viral infection. J Immunol, 2000. 165(9): p. 4787-91. 283. Daniels, K.A., et al., Murine cytomegalovirus is regulated by a discrete subset of natural killer cells reactive with monoclonal antibody to Ly49H. J Exp Med, 2001. 194(1): p. 29-44. 284. Brown, M.G., et al., Vital involvement of a natural killer cell activation receptor in resistance to viral infection. Science, 2001. 292(5518): p. 934-7. 285. Arase, H., et al., Direct recognition of cytomegalovirus by activating and inhibitory NK cell receptors. Science, 2002. 296(5571): p. 1323-6. 286. Smith, H.R., et al., Recognition of a virus-encoded ligand by a natural killer cell activation receptor. Proc Natl Acad Sci U S A, 2002. 99(13): p. 8826-31. 287. Fodil-Cornu, N., et al., Ly49h-deficient C57BL/6 mice: a new mouse cytomegalovirus-susceptible model remains resistant to unrelated pathogens controlled by the NK gene complex. J Immunol, 2008. 181(9): p. 6394-405. 288. Munks, M.W., et al., Four distinct patterns of memory CD8 T cell responses to chronic murine cytomegalovirus infection. J Immunol, 2006. 177(1): p. 450-8. 289. Schlub, T.E., et al., Comparing the kinetics of NK cells, CD4, and CD8 T cells in murine cytomegalovirus infection. J Immunol, 2011. 187(3): p. 1385-92. 290. White, J.T., E.W. Cross, and R.M. Kedl, Antigen-inexperienced memory CD8(+) T cells: where they come from and why we need them. Nat Rev Immunol, 2017. 17(6): p. 391-400. 60 291. Sylwester, A.W., et al., Broadly targeted human cytomegalovirus-specific CD4+ and CD8+ T cells dominate the memory compartments of exposed subjects. J Exp Med, 2005. 202(5): p. 673-85. 292. Smith, C.J., H. Turula, and C.M. Snyder, Systemic hematogenous maintenance of memory inflation by MCMV infection. PLoS Pathog, 2014. 10(7): p. e1004233. 293. Quinn, M., et al., Memory T cells specific for murine cytomegalovirus re-emerge after multiple challenges and recapitulate immunity in various adoptive transfer scenarios. J Immunol, 2015. 194(4): p. 1726-36. 294. Smith, C.J., et al., Murine CMV Infection Induces the Continuous Production of Mucosal Resident T Cells. Cell Rep, 2015. 13(6): p. 1137-1148. 295. Thom, J.T., et al., The Salivary Gland Acts as a Sink for Tissue-Resident Memory CD8(+) T Cells, Facilitating Protection from Local Cytomegalovirus Infection. Cell Rep, 2015. 13(6): p. 1125-1136. 296. Jonjic, S., et al., Efficacious control of cytomegalovirus infection after long-term depletion of CD8+ T lymphocytes. J Virol, 1990. 64(11): p. 5457-64. 297. Walton, S.M., et al., Absence of cross-presenting cells in the salivary gland and viral immune evasion confine cytomegalovirus immune control to effector CD4 T cells. PLoS Pathog, 2011. 7(8): p. e1002214. 298. Tessmer, M.S., E.C. Reilly, and L. Brossay, Salivary gland NK cells are phenotypically and functionally unique. PLoS Pathog, 2011. 7(1): p. e1001254. 299. Romagnani, C., et al., HLA-E-restricted recognition of human cytomegalovirus by a subset of cytolytic T lymphocytes. Hum Immunol, 2004. 65(5): p. 437-45. 61 CHAPTER 2: ROLE OF SHIP1 IN INKT CELL DEVELOPMENT AND FUNCTIONS Originally published in The Journal of Immunology, September 1, 2015 Volume 195, Number 5, Pages 2149 – 2156 Copyright © 2015 The American Association of Immunologists, Inc. 62 Role of SHIP1 in iNKT cell development and functions Courtney K. Anderson,*,1 Alexander I. Salter,*,1 Leon E. Toussaint,* Emma C. Reilly,* Céline Fugère,* Neetu Srivastava,†‡ William G. Kerr, †‡§ and Laurent Brossasy* 1 C.K.A. and A.I.S. contributed equally to this work * Division of Biology and Medicine, Department of Molecular Microbiology and Immunology, Brown University, Providence, RI 02912 † Department of Pediatrics, SUNY Upstate Medical University, Syracuse, NY 13210 ‡ Department of Microbiology and Immunology, SUNY Upstate Medical University, Syracuse, NY 13210 § Department of Chemistry, Syracuse University, Syracuse, NY 13210 This work was supported by an NIH research grant (AI46709) to LB and NIH research grants (RO1 HL072523, R01 HL085580, R01 HL107127) and the Paige Arnold Butterfly Run to WGK. 63 ABSTRACT SH2-containing inositol phosphatase-1 (SHIP1) is a 5' inositol phosphatase known to negatively regulate the signaling product of the phosphoinositide-3 kinase (PI3K) pathway, phosphatidylinositol (3,4,5)-trisphosphate (PIP3). SHIP1 is recruited to a large number of inhibitory receptors expressed on invariant natural killer (iNKT) cells. We hypothesized that SHIP1 deletion would have major effects on iNKT cell development by altering the thresholds for positive and negative selection. Germline SHIP1 deletion has been shown to affect T cells, as well as other immune cell populations. However, the role of SHIP1 on T cell function has been controversial and its participation on iNKT cell development and function has not been examined. We evaluated the consequences of SHIP1 deletion on iNKT cells using germline-deficient mice, chimeric mice, and conditionally deficient mice. We found that T cell and iNKT cell development are impaired in germline-deficient animals. However, this phenotype can be rescued by extrinsic expression of SHIP1. In contrast, SHIP1 is required cell autonomously for optimal iNKT cell cytokine secretion. This suggests that SHIP1 calibrates the threshold of iNKT cell reactivity. These data further our understanding of how iNKT cell activation is regulated and provide insights into the biology of this unique cell lineage. 64 INTRODUCTION Natural Killer T cells (NKT) are a heterogeneous subset of innate lymphocytes that express NK cell markers, in addition to a TCR. There are multiple functionally distinct categories of NKT cells, including invariant NKT (iNKT) cells, also known as type I NKT cells (1, 2). iNKT cells represent a small fraction of mature T cells within the thymus, spleen, and lymph nodes. However, iNKT cells also accumulate in non- lymphoid organs, including the blood, liver, and gut. In mice, iNKT cells make up a robust population within the liver, ranging between 25–40% of the lymphocytes (3). iNKT cell development occurs in the thymus from the same precursors as conventional T cells, but diverges during positive selection (1, 2, 4). While conventional T cells are selected and restricted by classical MHC peptide antigens presented by thymic cortical epithelial cells, iNKT cells are selected by CD4+CD8+ double positive (DP) cortical thymocytes that express CD1d (1, 2). CD1d is a non-classical MHC class I-like molecule that preferentially binds glycolipid antigens (1, 2). iNKT cells are able to recognize presented glycolipid antigens due to their unique semi-invariant TCR, which consists of an invariant Vα14-Jα18 chain that preferentially dimerizes with a limited number of β- chains, mainly Vβ8.2, Vβ7, and Vβ2 (1, 2, 4). In addition to their unique TCR repertoire, iNKT cells are characterized by their ability to rapidly secrete a wide array of cytokines upon stimulation, either through direct TCR activation or indirectly through cytokine signaling. This can include the production of large amounts of IFN-γ and IL-4 (1, 5), allowing iNKT cells to participate in either TH1- or TH2-polarized responses. Due to their rapid and diverse responses, iNKT cells are multifunctional and capable of augmenting the participation of other immune cells, including B cells, NK cells, macrophages, and other T cells (6–10). The PI3K signaling pathway participates in a number of cellular processes, not limited to cellular activation, development, migration, proliferation, and survival (11, 12). 65 PI3Ks phosphorylate PI(4,5)P2 to PI(3,4,5)P3. PI(3,4,5)P3 is a second messenger that attracts effector proteins containing a Pleckstrin-homology domain and assists in their attachment to the inside of the plasma membrane, leading to downstream cellular responses (11, 13). Together with PTEN (phosphatase and tensin homologue deleted on chromosome 10), SHIP1 is an important negative regulator of PI3K signaling. SHIP1 is expressed predominantly in hematopoietic cells, as well as mesenchymal stem cells and stromal cells (14, 15), and acts by dephosphorylating PI(3,4,5)P3 into PI(3,4)P2 (16). The Src homology 2 (SH2) domain of SHIP1 allows it to associate with both ITAM- and ITIM-containing receptor tails, including SLAM family receptors and TCR associated CD3 chains (17–19). Recently, our lab has shown that SHIP1 is recruited to the ITIM of KLRG1 receptors to negatively regulate intracellular signaling (20). Global loss of SHIP1 results in a pleiotropic phenotype, due to its role in the development and function of a number of immune cells. Germline-deficient SHIP1 animals have increased myeloid cell number, attributed to heightened proliferation and survival, but are conversely lymphopenic (21). B cell development and survival are also affected by SHIP1 regulation and BCR signaling is hypersensitive (22, 23). However, the role of SHIP1 in T cell development and functions is less clear. Some studies claim that deletion of SHIP1 affects T cell development, while others report no major developmental issues (21, 24, 25). Using germline-deficient mice, chimeric mice, and conditionally-deficient mice, we revisited these studies with a special emphasis on iNKT cells, which are known to express inhibitory receptors capable of associating with SHIP1. We found that iNKT cell and T cell development are significantly impaired in mice with germline deletion of SHIP1. However, this phenotype could be rescued by extrinsic expression of SHIP1. In contrast, SHIP1 was required cell autonomously for optimal iNKT cell cytokine secretion, suggesting that SHIP1 calibrates the threshold of iNKT cell reactivity. 66 MATERIALS AND METHODS Mice. Inbred C57BL/6 mice and BALB/c mice were purchased from Taconic Farms, Inc. (Germantown, NY). SHIP1 mice were described previously and bred to acquire SHIP1+/+, SHIP1+/−, and SHIP1−/− animals (26). C57BL/6 Jα18 heterozygous mice were kindly provided by Dr. M Taniguchi (RIKEN Research Center for Allergy and Immunology, Yokohama, Japan) and bred to obtain Jα18−/− mice in our facility. B6 Thy1.1 mice were acquired from Jackson Laboratories (Bar Harbor, Maine). CD4CreSHIPfl/fl (referred to as CD4cre) and SHIPfl/fl (referred to as Control) samples were described previously (27). All mice were maintained at Brown University in pathogen free facilities. Isolation of Murine Lymphocytes. Mice were sacrificed by isofluorane treatment and a cardiac puncture was performed before harvesting organs. Thymic lymphocytes were obtained by dissociating thymi with the plunger of a 3mL syringe, passing the sample through mesh, and washing one time in 1% PBS-serum. Spleens were minced with the plunger of a 3mL syringe and samples were passed through mesh. Red blood cells were removed to enrich for splenic lymphocytes using a Lympholyte-M gradient (Cedarlane Laboratories Ltd., Canada) or ammonium chloride treatment. Livers were perfused with 1% PBS-serum prior to harvesting and hepatic lymphocytes were obtained by homogenizing with a gentleMACS Dissociator (Miltenyi Biotec). Liver samples were then washed three times in 1% PBS-serum and layered onto a two-step discontinuous Percoll gradient (Pharmacia Fine Chemicals, Piscataway, NJ). The fat layer was removed and lymphocytes were harvested from the gradient interface and washed one time in 1% PBS-serum. Bone marrow lymphocytes were obtained from the femur and tibia of mice by flushing the interior of the bone with 1% PBS-Serum. Red blood cells were lysed using ammonium chloride to enrich for lymphocytes. To obtain blood lymphocytes, samples from cardiac puncture were treated with ammonium chloride. Lymph nodes 67 were dissociated with the plunger of a 3 mL syringe, passed through mess, and washed once. Cell counts were performed using either a hemocytometer and Trypan Blue (GIBCO) or on an MACSQuant (Miltenyi Biotec) with Propidium Iodide (Miltenyi Biotec) to exclude dead cells. Antibodies, Reagents, and Flow Cytometric Analysis. Samples were stained with a 2.4G2 blocking antibody and monoclonal antibodies in 1% PBS-serum for 20 minutes on ice in the dark. For samples stained with CD1 tetramer, cells were incubated for 15 minutes at room temperature in the dark, followed by 15 minutes on ice in the dark. Samples requiring intracellular staining were then fixed and permeabilized using CytoFix/CytoPerm (BD Biosciences) and stained with intracellular antibodies for 15 minutes on ice in the dark. Events were collected on either a FACSAria (BD Biosciences) or a MACSQuant (Miltenyi Biotec). Data were analyzed using FlowJo (Tree Star Inc.). IL-17A-AlexaFluor488, CD45.2-FITC, HSA-FITC, TCRβ-FITC, TCRVβ2-FITC, CD45.1-PE, PLZF-PE, TCRβ-PE, Foxp3-PE-Cy5.5, NK1.1-PerCPCy5.5, TCRβ- PerCPCy5.5, RORγt-PerCPeFluor710, TCRβ8.1/2-PerCPeFluor710, Ly49G2- PerCPeFluor710, IFN-γ-PE-Cy7, NK1.1-PE-Cy7, T-Bet-PE-Cy7, CD45.1-APC, CD25- APC, IL-4-APC, CD4-APCeFluor780, CD44-APCeFluor780, CD45.2-APCeFluor780, CD90.1-APCeFluor780, CD8α-eFluor450, CD3-eFluor450, Ki67-eFluor450, and TNF-α- eFluor540 were purchased from eBioscience (San Diego, CA). Ly49C/I-FITC, Ly49A/D- PE, and CD4-PerCP were purchased from BD Pharmingen. TCRVβ7-PE was purchased from Biolegend. CD1d tetramer loaded with α-GalCer, CD1d tetramer loaded with PBS- 57 as well as unloaded controls were provided by the NIH Tetramer Facility. In vivo Proliferation Analysis. Recipient mice were sub-lethally irradiated with 7.5 Gy and placed on oral Sulfamethoxazole and Trimethoprim (Hi-tech Pharmacal) treatment. 68 On day three post-irradiation, donor mice were sacrificed, thymi were harvested, and thymocytes were isolated. iNKT cells were enriched by labeling samples with anti-CD8 magnetic beads and depleting CD8+ cells using an AutoMACS (Miltenyi Biotec). Cells were then stained for 10 minutes at 37°C in the dark with 10µM Cell Proliferation Dye eFuor450 (eBioscience) in PBS. Irradiated recipients were intravenously injected with 5– 10 million cells. iNKT cell proliferation was analyzed in recipient spleen, liver, and blood samples on day 7 post-injection. In vitro iNKT Cell Simulation and Cytokine Analysis. A 96-well plate was coated with 0.5 µg/well of α-mouse CD3ε and CD28 antibodies (eBioscience). Thymic iNKT cells were sorted using a FACSAria (BD Biosciences) and plated in triplicate at a concentration of 10,000 or 30,000 cells/well, depending on the experiment. The supernatant was collected 24 hours post-stimulation and cytokines were quantified using a Cytometric Bead Array Flex Sets (BD Biosciences) and analyzed with FCAP Array Software (BD Biosciences). For cytokine stimulation, 2 × 106 thymocytes were treated with PMA (200 ng/mL) and Ionomycin (5 µg/mL) for 2.5 hours at 37 °C in RPMI cell culture media with 10% FBS. Samples were stained with cell surface markers, fixed and permeabilized, and stained with anti-cytokine antibodies. Generation of Mixed Bone Marrow Chimera. Jα18−/− recipient mice (CD45.2+) were lethally irradiated with 10.5 Gy and placed on oral Sulfamethoxazole and Trimethoprim (Hi-tech Pharmacal) treatment for two weeks. One day post-irradiation, donor bone marrow cells were harvested under sterile conditions from SHIP1+/− and SHIP1−/− animals (CD45.1+) and control C57BL/6 animals (CD45.2+). Anti-CD5 and anti-DX5 magnetic beads were used to deplete mature T and NK cells using an AutoMACS (Miltenyi Biotec). Mixed bone marrow chimeric mice were generated by pooling cells 69 from SHIP1+/− or SHIP1−/− animals with cells from C57BL/6 animals in a 1:1 ratio. 1×106 cells were injected intravenously into recipients and allowed to reconstitute for 8– 10 weeks. Statistical Analysis. All statistical analyses were accomplished with Prism Version 5.0 (GraphPad Software) or Excel (Microsoft) using unpaired two-tailed Student's t tests. A paired two-tailed Student's t test was used for samples from mixed bone marrow chimera. P value < 0.0001 ****, P value 0.0001 to 0.001 ***, P value 0.001 to 0.01 **, P value 0.01 to 0.05 *, and P value > 0.05 is not significant. 70 RESULTS Germline deletion of SHIP1 hinders iNKT cell development SHIP1 (encoded by INPP5D) expression is an important regulator of PI3K activity. Loss of SHIP1 in mice leads to a number of hematopoietic defects, such as myeloid cell infiltration in the lungs and a severe inflammation of the small intestine that resembles human Crohn's disease (21, 27). Given its proposed role as a negative regulator of TCR signaling (28, 29), and because iNKT cell development is critically dependent on unique signals emanating from its semi-invariant TCR (30), we hypothesized that SHIP1 may play a role in iNKT cell development and functions. To minimize any pleiotropic effects, SHIP1−/− animals were sacrificed prior to seven weeks of age. Characterization of SHIP1-deficient mice revealed an increased frequency of iNKT cells in the thymus, yet decreased frequencies in the spleen and liver, compared to SHIP1+/+ and SHIP1+/− animals (Fig. 1A, Supplementary Fig. 1A). In contrast, the total number of iNKT cells was significantly decreased in the thymus and spleen (Fig. 1B). To exclude this being due to a relocation to other organs, we determined that the frequency of iNKT cells in the bone marrow, blood, inguinal lymph nodes, and mesenteric lymph nodes was also decreased (Supplementary Fig. 2A&B). We next wanted to determine whether SHIP1 deletion was causing iNKT cells to arrest at a specific stage of maturation. However, there was no difference in iNKT cell maturation between Stages 1–3, distinguished by NK1.1 and CD44 expression (Fig. 1C, Supplementary Fig. 1B). Notably, the frequency of the Stage 0 iNKT cells in SHIP1 deficient animals was modestly increased, while the total number was comparable to littermate controls (Supplementary Fig. 1C). These results indicated that residual iNKT cells are capable of undergoing normal development. We also examined if the absence of SHIP1 influenced the ratio of NKT1, NKT2 and NKT17 cell lineages. iNKT cells have recently been phenotypically classified according to their function and transcription factor expression, 71 and are distinguished using anti-PLZF, anti-RORγt and anti-T-bet mAbs (31). When comparing thymic iNKT cells from SHIP1-deficient mice and littermate controls, we found a modest skew toward the NKT1 lineage (Fig. 2). A BALB/c positive control was used to define the gating strategy for these experiments, since they have robust populations of the three NKT lineages. Interestingly, when we examined the thymic T cell compartment, it revealed that T cell development was being affected globally. SHIP1−/− mice had increased frequencies of double negative (DN) and CD4 and CD8 single positive (SP) T cell populations, and a decreased frequency of CD4+CD8+ DP T cells, compared to SHIP1+/− and SHIP1+/+ controls (Fig. 3). However, although the mice had been crossed more than 15 times onto the B6 background, there was a spectrum in the severity of the impairment observed in the T cell compartment, most likely due to the pleiotropic effects of global SHIP1 loss (Fig. 3B). Similarly to T cells, iNKT cell frequency was also quite variable in the thymus of SHIP1-deficient animals (Fig. 1A). Interestingly, SHIP1−/− mice with less severely affected T cell development were also those with less serious defects in their iNKT cell population (not shown). Overall, the data demonstrated that germline deletion of SHIP1 hindered both T cell and iNKT cell development. Extrinsic expression of SHIP1 rescues T cell development As mentioned above, germline deletion of SHIP1 results in massive myeloid infiltration of the lung, making it difficult to study the role of SHIP1 in lymphocytes without the influence of an inflammatory environment. In addition, the intrinsic role of SHIP1 in iNKT cell development cannot be examined in SHIP1-deficient animals. To circumvent these two issues, we generated a series of mixed bone marrow chimeras. SHIP1−/− bone marrow, which is on a congenic background, and wild-type C57BL/6 competitor bone marrow were transferred at a 1:1 ratio into lethally irradiated Jα18−/− recipients (Fig. 4A). 72 Jα18−/− animals lack the Jα18 gene segment necessary for generation of the iNKT cell invariant α-chain. Therefore, all iNKT cells present in mixed bone marrow chimeric mice were derived from the two donor populations. Analysis of the chimeric mice indicated that conventional T cell development was globally intact. In contrast to the germline- deficient mice, the thymic T cell compartment of the chimera did not reveal significant differences in the DP, DN, or CD4 and CD8 SP populations (Fig. 4B). These data demonstrated that SHIP1 was required extrinsically during the development of conventional T cells. Similarly to conventional T cells, iNKT cells derived from C57BL/6 and SHIP1−/− donor populations had comparable frequencies in all the organs tested, suggesting that extrinsic expression of SHIP1 was sufficient for normal development of the iNKT cell compartment as well (Fig. 4C). To confirm these results, we also analyzed mice that were conditionally deficient for SHIP1. In these mice (CD4CreSHIP1fl/fl, referred to as CD4Cre), a transgene for Cre recombinase driven by the cd4 enhancer/promoter/silencer (32, 33) allows specific gene deletion at the double negative 4 (DN4)/DP stages of T cell development. To verify that SHIP1 was efficiently deleted from T cells, splenic T cells and non T cell populations from CD4Cre and SHIP1fl/fl control animals (referred to as Control) were sorted for protein analysis by Western blot using an α-mouse SHIP1 primary antibody. Expression of SHIP1 in T cells was strongly reduced, indicating as expected, that INPP5D had been efficiently deleted from the T cell lineage in CD4Cre animals (Supplementary Fig. 2C). In agreement with the data from our chimeric mice, we found that conventional T cell development was globally intact, confirming that SHIP1 was not required intrinsically for T cell development (Supplementary Fig. 2D). The iNKT cell compartment was also intact in the thymus, spleen, and liver of CD4Cre and Control animals (Fig. 5A). In addition, analysis of the different developmental thymic iNKT cell stages did not show any abnormalities (Fig. 5B). Taken together, these data further illustrated that extrinsic 73 expression of SHIP1 was sufficient to rescue iNKT and T cell development. Decreased number of iNKT cells in mice with germline deletion of SHIP1 is due to impaired proliferation SHIP1 has been shown to have opposing roles on myeloid and lymphoid cell proliferation (21, 34). Therefore, the reduced number of iNKT cells in the thymus and the spleen could potentially be the result of impaired proliferation in SHIP1-deficient animals. To evaluate the role of SHIP1 on iNKT cell proliferation, thymic iNKT cells from SHIP1−/− and wild-type C57BL/6 animals were labeled with Cell Proliferation Dye eFluor450 and injected intravenously into sublethally irradiated recipients. This lymphopenia-induced in vivo proliferation assay has shown that iNKT cells undergo extensive proliferation (35). We found that iNKT cells from SHIP1−/− animals had a decreased proliferative capacity on Day 7 compared to control wild-type cells in the liver, spleen, and blood (Fig. 6A). We also investigated steady state iNKT cell proliferation on Day 0 using intracellular Ki67 expression. In agreement with the impaired proliferation, we observed that SHIP1−/− thymic iNKT cells had significantly decreased Ki67 expression compared to C57BL/6 thymic iNKT cells (Supplementary Fig. 3A). Interestingly, SHIP1−/− animals with less affected T cell development and iNKT cell populations had comparable Ki67 expression to C57BL/6 controls (Supplementary Fig. 3B). We also observed thymic iNKT cells from CD4Cre and Control animals to have comparable Ki67 levels (Supplementary Fig. 3C). Notably, CD69 expression, a marker of early activation, was not affected in SHIP1-deficient iNKT cells (data not shown). Taken together, these data indicated that the lower number of iNKT cells in SHIP1−/− mice was due to a decreased proliferative potential, which could be rescued with extrinsic expression of SHIP1. 74 iNKT cell cytokine production is decreased in the absence of SHIP1 We next sought to determine whether deletion of SHIP1 affected iNKT cell functions. iNKT cells are unusually poised to produce cytokines and can secrete a large amount ex vivo without priming. Thymic iNKT cells were sorted from SHIP1−/− and SHIP1+/− animals and stimulated with a combination of anti-CD3 and anti-CD28 mAbs for 24 hours and culture supernatants were then collected to measure cytokine production. Ligation of CD3 or CD28 on T cells has been shown to induce SHIP1 tyrosine phosphorylation (36). Using cytometric bead array flex sets, we measured the amount of cytokines produced by activated thymic iNKT cells. We found that IL-4, IFN-γ, IL-17A and TNF-α cytokine production from SHIP1−/−iNKT cells were significantly decreased compared to iNKT cells from littermate controls (Fig. 6B). To circumvent the developmental issues observed in mice with germline deletion of SHIP1, and to compare iNKT cells that develop in the same environment, we also stimulated iNKT cells that were sorted from mixed bone marrow chimeric mice. Similarly to iNKT cells from germline-deficient mice, we found that cytokine production was significantly decreased in SHIP1−/− samples, compared to C57BL/6 samples from the same mixed chimera (Fig. 6C). Thus, although SHIP1 was not required intrinsically for iNKT cell development, it was required for optimal iNKT cell cytokine production (37). We next wanted to determine whether SHIP1-deficient iNKT cells were capable of producing cytokines irrespective of TCR stimulation. To accomplish this, we stimulated thymocytes from SHIP1−/− animals and littermate controls for 2.5 hours with PMA and Ionomycin, which act downstream of SHIP1. Interestingly, there were also significant decreases in the frequency of iNKT cells producing IFN-γ and TNF-α (Supplementary Fig. 3D&E). Altogether, these data demonstrate that SHIP1-deficient iNKT cells are hyporesponsive. 75 DISCUSSION The PI3K signaling pathway participates in a number of cellular processes, including activation, development, proliferation, and survival (11, 12). PI3K phosphorylates the D3 position of PI(4,5)P2 molecules to yield PI(3,4,5)P3 (38). PTEN and SHIP1 can prevent the initiation of these signals by converting PI(3,4,5)P3 to PI(4,5)P2 and PI(3,4)P2, respectively (39, 40). It has been shown that the absence of PTEN in T cells impairs the development of iNKT cells (41). Loss of regulation by SHIP1 has been shown to have differential effects on myeloid and lymphoid cells, including both NK and T cells (24, 26, 37, 39, 42, 43). However, the role of SHIP1 on iNKT cells is not well characterized. We found that SHIP1 is not required intrinsically for iNKT cell development, but is essential for optimal iNKT cell cytokine production. We also report that SHIP1 expression in trans is sufficient for the normal development of conventional T cells, at least in non-mucosal tissues (27). Regarding conventional T cells, there is some debate in the literature about the role of SHIP1. Some studies have reported that germline deletion of SHIP1 affects T cell development, while others found no major developmental issues (21, 24). In one study using a T cell-specific deletion model of SHIP1, no T cell developmental issues were observed (25). However, there was still expression of a near full-length version of SHIP1 that only lacked the enzyme domain (44), complicating the interpretation of these data. The findings presented here clarify these inconsistencies. iNKT cells express a variety of inhibitory receptors, such as Ly49 molecules, CD94/NKG2A, and PD-1 (45–47). In addition, mice conditionally deficient in PTEN express higher levels of Ly49C/I, which appears to be PIP3 dependent (41). Therefore, we hypothesized that SHIP1 deletion would affect iNKT cell development more severely than conventional T cells. Instead, we observed that germline deletion of SHIP1 had a variable effect on thymocyte development, ranging between a modest defect and a 76 major alteration in the frequency of DN and DP cells (see Figure 2C). However, we found that the intensity of the iNKT cell developmental defect was linked to the strength of the T cell development defect (data not shown). Thus, the absence of SHIP1 did not appear to have a greater effect on iNKT cell development than conventional T cells. Notably, cell surface expression of Ly49 receptors on iNKT cells was not influenced by absence of SHIP1 (data not shown). Altogether, our data indicate that extrinsic expression of SHIP1 indirectly influences iNKT cell development. The reduction in the iNKT compartment is analogous to the impaired B and NK cell cellularity that is also observed in SHIP1−/− animals (42, 48). However, there are discordant findings regarding an autonomous role for SHIP1 in peripheral NK cell homeostasis using NCR1CreSHIPflox/flox mice (37) and mixed bone marrow chimeras (42). SHIP1 has been shown to participate in negatively regulating T cell signaling by affecting localization of Tec Kinase at the membrane, which is involved in cell proliferation following stimulation of the TCR (28). It has also been shown that T cells from SHIP1−/− animals have a poor proliferative response to TCR stimulation ex vivo (24). We found that SHIP1−/− thymic iNKT cells (or a subset of iNKT cells) have a decreased proliferative capacity in vivo and decreased Ki67 expression. However, animals with the least affected iNKT cell compartment had comparable Ki67 expression to C57BL/6 controls. Additionally, CD4Cre and Control animals had comparable Ki67 expression. Therefore, SHIP1 is required in trans for normal iNKT cell proliferation, similarly to conventional T cells (25). This explains the decreased number of iNKT cells found in SHIP1−/− animals. Although less likely, the decreased number of total DP cells could additionally result in poor positive selection of NKT cells. Although iNKT cell development was unaffected in chimeric mice, we found that intrinsic absence of SHIP1 had an impact on iNKT cell cytokine secretion. The poor cytokine response of SHIP1-deficient iNKT cells was somewhat unexpected since 77 SHIP1 is a negative regulator of intracellular signaling, but in line with work showing that it promotes, rather than inhibits IFN-γ production by NK cells (37, 49). However, there are several non-mutually exclusive possibilities that could explain this phenotype. First, it is conceivable that inhibitory receptor signaling via SHIP1 calibrates the threshold of iNKT cell reactivity. In fact, NK cells were also shown to be hyporesponsive in the absence of the phosphatases SHIP1 (42) and SHP-1 (50). Similarly to NK cells (26, 37, 51), the absence of SHIP1 may render iNKT cells hyporesponsive. Alternatively, the lower cytokine release from stimulated SHIP1-deficient iNKT cells could be due to a different differentiation program. For instance, it has been shown that a large proportion of SHIP1-deficient conventional T cells become phenotypically similar to regulatory T cells (Tregs) by expressing Foxp3 (24, 43). We first confirmed the previously observed increase of the Treg population in SHIP1-deficient animals (Supplementary Fig. 4A&C). However, even though other conditions can cause regulatory iNKT cells (52, 53), we found that SHIP1-deficient iNKT cells do not acquire Foxp3 expression (Supplementary Fig. 4B&C). Another possibility is that the impaired iNKT cell cytokine production is the result of the expansion of a specific iNKT cell subset. However, this is unlikely since we found that SHIP1 deficient iNKT cells only skew slightly toward the NKT1 lineage. Taken together, our data indicate that deletion of the negative regulator SHIP1 leads to a decrease of iNKT cell cytokine secretion potential due to an alteration in the threshold for iNKT cell reactivity. The roles of other phosphatases, such as tyrosine phosphatases SHP-1 and SHP-2, may also contribute to iNKT cell activation. Additionally, isoforms of SHIP1 such as s-SHIP and SHIP-2, may participate in immune cell development and function. SHIP- 2 is also expressed on hematopoietic cells and contains a 5-phosphatase activity (54), whereas embryonic stem cells and progenitor cells express s-SHIP (55). ENU-induced SHIP1 mutants (SHIP1el20) also affect s-SHIP and result in an enhanced impairment, 78 compared to SHIP1−/− animals, including reduced T cell populations (56). The contribution of SHP-1, SHP-2, SHIP-2, and other phosphatases on iNKT cell development and functions still warrant further investigation. 79 ACKNOWLEDGEMENTS We thank Kevin Carlson for cell sorting. AUTHOR CONTRIBUTIONS C.K.A conceived, performed, analyzed the experiments and wrote the paper. A.I.S. conceived, performed, analyzed the experiments and wrote the paper. L.E.T. conceived, performed, and analyzed the experiments. E.C.R. conceived, performed, and analyzed the experiments. C.F. conceived, performed, and analyzed the experiments. N.S performed the experiments. W.G.K provided reagents. L.B. conceived, analyzed the experiments, and wrote the paper. The authors have no conflict of interest to declare. 80 Figure 1. Loss of thymic and peripheral iNKT cells in SHIP1−/− mice. (A) Frequencies of iNKT cell populations in the thymus, spleen, and liver from SHIP1+/+, SHIP1+/−, and SHIP1−/− mice. (B) Total iNKT cell numbers from each indicated organ. (C) Frequencies of thymic iNKT cells within the three stages of maturation, CD44loNK1.1− (Stage I), CD44hiNK1.1− (Stage II), and CD44hiNK1.1+ (Stage III). iNKT cells are defined as HSAloCD1tet+ in the thymus and TCRβ+CD1tet+ in the spleen and liver within the lymphoid gate. Black circles and bars: SHIP1+/+ (n=14), gray circles & bars: SHIP1+/− (n=18), and white circles & bars: SHIP1−/− mice (n=16). Data are pooled from at least 3 independent experiments and each dot is representative of 1 mouse. Error bars indicate SEM. 81 Figure 2. SHIP1−/− mice have a more pronounced NKT1 phenotype than littermate controls. (A) Representative staining of thymic NKT1, NKT2, and NKT17 populations from BALB/c control, SHIP1+/−, and SHIP1−/− mice using mAb for PLZF, ROR-γt, and T- Bet. (B) Frequency of thymic NKT1, NKT2, and NKT17 populations based on PLZF and ROR-γt expression. Black bars: BALB/c (n=3), grey bars: SHIP1+/− (n=9), and white bars: SHIP1−/− mice (n=9). iNKT cells are defined as TCRβ+CD1tet+ within the lymphoid gate. Data are pooled from 3 independent experiments and each dot is representative of 1 mouse. Error bars indicate SEM. 82 Figure 3. SHIP1−/− mice have impaired conventional T cell development. (A) and (B) Frequencies of thymic lymphocyte populations that are DN (CD4−CD8−), DP (CD4+CD8+), CD4 SP, and CD8 SP from SHIP1+/+, SHIP1+/−, and SHIP1−/− mice. (C) Representative staining of thymic T cell development, illustrating DN (CD4−CD8−), DP (CD4+CD8+), CD4 SP, and CD8 SP populations after the lymphoid gate in SHIP1+/+, SHIP1+/−, and SHIP1−/− mice. Black circles & bars: SHIP1+/+ (n=14), gray circles & bars: SHIP1+/− (n=18), and white circles & bars: SHIP1−/− mice (n=16). Data are pooled from at least 3 independent experiments and each dot is representative of 1 mouse. Error bars indicate SEM. 83 Figure 4. Extrinsic expression of SHIP1 regulates iNKT cell and T cell populations. (A) Diagram representing mixed bone marrow chimera generation. SHIP1−/− (CD45.1+) and C57BL/6 (CD45.2+) bone marrow cells were mixed at an equal ratio and intravenously injected into lethally irradiated Jα18−/− recipients. Recipients were analyzed 6 to 8 weeks following reconstitution. (B) Frequencies of thymic lymphocyte populations that are DN (CD4−CD8−), DP (CD4+CD8+), CD4 SP, and CD8 SP from mixed chimeric mice. (C) Frequencies of total iNKT cell populations in the thymus, spleen, and liver from C57BL/6 and SHIP1−/−donors in mixed chimeras. iNKT cells are defined as HSAloCD1tet+ in the thymus and TCRβ+CD1tet+ in the spleen and liver within the lymphoid gate. Black circles & bars: C57BL/6 competitor (n=9) and white circles & bars: SHIP1+/− (n=9). Data are pooled from at least 3 independent experiments and each dot is representative of 1 mouse. Error bars indicate SEM. 84 Figure 5. Normal iNKT cell populations in mice conditionally deficient for SHIP1 in T cells. (A) Representative staining of iNKT cells from the thymus, spleen, and liver of CD4Cre mice and littermate controls. (B) Frequencies of thymic iNKT cells within the three stages of maturation, CD44loNK1.1− (Stage I), CD44hiNK1.1− (Stage II), and CD44hiNK1.1+ (Stage III) from CD4Cre mice (n=8) and littermate controls (n=9). Data are pooled from 3 independent experiments and each dot is representative of 1 mouse. Black bars: Control mice and gray bars: CD4Cre mice. iNKT cells are defined as HSAloCD1tet+ in the thymus and TCRβ+CD1tet+in the spleen and liver within the lymphoid gate. Error bars indicate SEM. 85 Figure 6. SHIP1 deficiency extrinsically affects iNKT cell proliferation and intrinsically affects cytokine production. (A) Day 7 labeling of pooled donor iNKT cells found in the liver, spleen, and blood of recipient animals from SHIP1−/− (CD45.1+) and C57BL/6 (CD45.2+) mice. iNKT cells are defined TCRβ+CD1tet+ in the liver, spleen, and blood in the lymphoid gate. Data are representative of 3 independent experiments. (B) Cytokine production was assessed in vitro using sorted thymic iNKT cells from SHIP1+/−and SHIP1−/− animals. Represents pooled data from four independent experiments, normalized to heterozygous control samples. (C) Cytokine production was assessed in vitro using sorted thymic iNKT cells from SHIP1−/− and competitor C57BL/6 populations from mixed chimera. Represents pooled data from two independent experiments, normalized to C57BL/6 control samples. Error bars indicate SEM. 86 Supplementary Figure 1. Loss of thymic and peripheral iNKT cells in SHIP1-/- mice. (A) Representative staining of iNKT cell populations in the thymus, spleen, and liver from SHIP+/+, SHIP+/-, and SHIP-/- mice. (B) Representative staining of thymic iNKT cell maturation: CD44loNK1.1- (Stage I), CD44hiNK1.1- (Stage II), and CD44hiNK1.1+ (Stage III). iNKT cells are defined as HSAloCD1tet+ in the thymus and TCRβ+CD1tet+ in the spleen and liver within the lymphoid gate. (C) Frequency and absolute number of thymic Stage 0 iNKT cells from SHIP+/+, SHIP+/-, and SHIP-/- mice. Data are pooled from 2 independent experiments (n=5). Black circles: SHIP+/+, gray circles: SHIP+/-, white circles: SHIP-/- mice, dotted line: unloaded CD1 tetramer control. Error bars indicate SEM. 87 Supplementary Figure 2. Loss of thymic and peripheral iNKT cells in SHIP1-/- mice, but normal T cell populations in CD4CreSHIP1fl/fl mice. (A) Representative staining of iNKT cell populations in the bone marrow, blood, inguinal lymph nodes (iLN), and mesenteric lymph nodes (mLN) from SHIP+/+, SHIP+/-, and SHIP-/- mice. (B) Frequency of iNKT cells in each indicated organ from SHIP+/+, SHIP+/-, and SHIP-/- mice. Data are pooled from 3 independent experiments (n=6-8). (C) SHIP1 protein expression from splenocytes was determined using Western blot. T cells (TCRβ+CD4+) and Non T cells (TCRβ-CD4-) were sorted from CD4Cre mice and littermate Controls. Cell lysate was blotted with α-SHIP1 and β-actin antibodies. (D) Frequencies of thymic lymphocyte populations that are DN (CD4-CD8-), DP (CD4+CD8+), CD4 SP, and CD8 SP from CD4Cre mice (n=10) and littermate controls (n=10). Data are pooled from 3 independent experiments and each dot is representative of 1 mouse. iNKT cells are defined as TCRβ+CD1tet+ within the lymphoid gate. Black circles: SHIP+/+, gray circles: SHIP+/-, and white circles: SHIP-/- mice. Black bars: Control mice and grey bars: CD4Cre. Error bars indicate SEM. 88 Supplementary Figure 3. SHIP1 deficiency influences the proliferative capacity and cytokine production of thymic iNKT cells from SHIP1-/- mice. Intracellular Ki67 expression was used to determine steady state proliferative capacity of thymic iNKT cells. (A) Frequencies of Ki67+ thymic iNKT cells from C57BL/6 (n=8) and SHIP1-/- mice (n=14). Data are representative of 4 independent experiments. (B) Representative staining of Ki67+ thymic iNKT cells from C57BL/6 control mice and SHIP1-/- mice with impaired iNKT cell frequency and normal iNKT cell frequency. (C) Frequencies of Ki67+ thymic iNKT cells from CD4Cre mice (n=5) and littermate controls (n=5) and representative staining. Data are representative of 2 independent experiments. iNKT cells are defined as HSAloCD1tet+ in the thymus within the lymphoid gate. Error bars indicate SEM. (D) Cytokine production of thymic iNKT cells from SHIP1+/+ (n=6) and SHIP1-/- (n=6) mice following 2.5 hours of PMA and Ionomycin stimulation, compared to unstimulated controls (n=4). Data are pooled from two independent experiments. (E) Representative TCRβ MFI of thymic iNKT cells following PMA and Ionomycin stimulation from SHIP1+/+ and SHIP1-/- mice and unstimulated control. Numbers in upper right corner indicate MFI for each histogram. iNKT cells are defined as TCRβ+CD1tet+ within the lymphoid gate. Black bars: SHIP+/+, gray bars: SHIP-/-, and white bars: unstimulated control. Error bars indicate SEM. 89 Supplementary Figure 4. Increased Foxp3 expression of conventional T cells, but not iNKT cells, in SHIP1-/- animals. (A) Regulatory T cells in the thymus, spleen, and liver of SHIP1+/- and SHIP1-/- mice. (B) Frequency of Foxp3+ iNKT cells in the thymus, spleen, and liver of SHIP1+/- and SHIP1-/- mice. (C) Representative staining of Foxp3 and CD25 expression of splenic CD4+ T cell and iNKT cell populations in SHIP1+/- (n=7) and SHIP1-/- mice (n=7). Data are representative of 3 independent experiments. Regulatory T cells are defined as Foxp3+CD4+TCRβ+ and iNKT cells are defined as TCRβ+CD1tet+ within the lymphoid gate. Error bars indicate SEM. 90 REFERENCES 1. Kronenberg M, Gapin L. The unconventional lifestyle of NKT cells. Nature reviews. Immunology. 2002;2:557–568. 2. Godfrey DI, Stankovic S, Baxter AG. Raising the NKT cell family. Nature immunology. 2010;11:197–206. 3. Klugewitz K, Adams DH, Emoto M, Eulenburg K, Hamann A. The composition of intrahepatic lymphocytes: shaped by selective recruitment? Trends in immunology. 2004;25:590–594. 4. Carpenter AC, Bosselut R. Decision checkpoints in the thymus. Nature immunology. 2010;11:666–673. 5. Reilly EC, Wands JR, Brossay L. Cytokine dependent and independent iNKT cell activation. Cytokine. 2010;51:227–231. 6. Carnaud C, Lee D, Donnars O, Park SH, Beavis A, Koezuka Y, Bendelac A. Cutting edge: Cross-talk between cells of the innate immune system: NKT cells rapidly activate NK cells. Journal of immunology (Baltimore, Md. : 1950) 1999;163:4647–4650. 7. Eberl G, MacDonald HR. Selective induction of NK cell proliferation and cytotoxicity by activated NKT cells. European journal of immunology. 2000;30:985–992. 8. Wesley JD, Robbins SH, Sidobre S, Kronenberg M, Terrizzi S, Brossay L. Cutting edge: IFN-gamma signaling to macrophages is required for optimal Valpha14i NK T/NK cell cross-talk. Journal of immunology (Baltimore, Md. : 1950) 2005;174:3864–3868. 9. Hermans IF, Silk JD, Gileadi U, Salio M, Mathew B, Ritter G, Schmidt R, Harris AL, Old L, Cerundolo V. NKT cells enhance CD4+ and CD8+ T cell responses to soluble antigen in vivo through direct interaction with dendritic cells. Journal of 91 immunology (Baltimore, Md. : 1950) 2003;171:5140–5147. 10. Reilly EC, Thompson EA, Aspeslagh S, Wands JR, Elewaut D, Brossay L. Activated iNKT cells promote memory CD8+ T cell differentiation during viral infection. PloS one. 2012;7:e37991. 11. Wymann MP, Pirola L. Structure and function of phosphoinositide 3-kinases. Biochimica et biophysica acta. 1998;1436:127–150. 12. Anderson KE, Jackson SP. Class I phosphoinositide 3-kinases. The international journal of biochemistry & cell biology. 2003;35:1028–1033. 13. Ooms LM, Horan KA, Rahman P, Seaton G, Gurung R, Kethesparan DS, Mitchell CA. The role of the inositol polyphosphate 5-phosphatases in cellular function and human disease. The Biochemical journal. 2009;419:29–49. 14. Hazen AL, Smith MJ, Desponts C, Winter O, Moser K, Kerr WG. SHIP is required for a functional hematopoietic stem cell niche. Blood. 2009;113:2924–2933. 15. Iyer S, Brooks R, Gumbleton M, Kerr WG. SHIP1-Expressing Mesenchymal Stem Cells Regulate Hematopoietic Stem Cell Homeostasis and Lineage Commitment During Aging. Stem cells and development. 2014. 16. Ravetch JV, Lanier LL. Immune inhibitory receptors. Science (New York, N.Y.) 2000;290:84–89. 17. Latour S, Gish G, Helgason CD, Humphries RK, Pawson T, Veillette A. Regulation of SLAM-mediated signal transduction by SAP, the X-linked lymphoproliferative gene product. Nature immunology. 2001;2:681–690. 18. Pesesse X, Backers K, Moreau C, Zhang J, Blero D, Paternotte N, Erneux C. SHIP1/2 interaction with tyrosine phosphorylated peptides mimicking an immunoreceptor signalling motif. Advances in enzyme regulation. 2006;46:142– 153. 19. Peng Q, Malhotra S, Torchia JA, Kerr WG, Coggeshall KM, Humphrey MB. 92 TREM2- and DAP12-dependent activation of PI3K requires DAP10 and is inhibited by SHIP1. Science signaling. 2010;3:ra38. 20. Tessmer MS, Fugere C, Stevenaert F, Naidenko OV, Chong HJ, Leclercq G, Brossay L. KLRG1 binds cadherins and preferentially associates with SHIP- 1. International immunology. 2007;19:391–400. 21. Helgason CD, Damen JE, Rosten P, Grewal R, Sorensen P, Chappel SM, Borowski A, Jirik F, Krystal G, Humphries RK. Targeted disruption of SHIP leads to hemopoietic perturbations, lung pathology, and a shortened life span. Genes & development. 1998;12:1610–1620. 22. Brauweiler A, Tamir I, Dal Porto J, Benschop RJ, Helgason CD, Humphries RK, Freed JH, Cambier JC. Differential regulation of B cell development, activation, and death by the src homology 2 domain-containing 5' inositol phosphatase (SHIP) The Journal of experimental medicine. 2000;191:1545–1554. 23. Helgason CD, Kalberer CP, Damen JE, Chappel SM, Pineault N, Krystal G, Humphries RK. A dual role for Src homology 2 domain-containing inositol-5- phosphatase (SHIP) in immunity: aberrant development and enhanced function of b lymphocytes in ship −/− mice. The Journal of experimental medicine. 2000;191:781–794. 24. Kashiwada M, Cattoretti G, McKeag L, Rouse T, Showalter BM, Al-Alem U, Niki M, Pandolfi PP, Field EH, Rothman PB. Downstream of tyrosine kinases-1 and Src homology 2-containing inositol 5'-phosphatase are required for regulation of CD4+CD25+ T cell development. Journal of immunology (Baltimore, Md. : 1950) 2006;176:3958–3965. 25. Tarasenko T, Kole HK, Chi AW, Mentink-Kane MM, Wynn TA, Bolland S. T cell- specific deletion of the inositol phosphatase SHIP reveals its role in regulating Th1/Th2 and cytotoxic responses. Proceedings of the National Academy of 93 Sciences of the United States of America. 2007;104:11382–11387. 26. Wang JW, Howson JM, Ghansah T, Desponts C, Ninos JM, May SL, Nguyen KH, Toyama-Sorimachi N, Kerr WG. Influence of SHIP on the NK repertoire and allogeneic bone marrow transplantation. Science (New York, N.Y.) 2002;295:2094–2097. 27. Park MY, Srivastava N, Sudan R, Viernes DR, Chisholm JD, Engelman RW, Kerr WG. Impaired T-cell survival promotes mucosal inflammatory disease in SHIP1- deficient mice. Mucosal immunology. 2014;7:1429–1439. 28. Tomlinson MG, Heath VL, Turck CW, Watson SP, Weiss A. SHIP family inositol phosphatases interact with and negatively regulate the Tec tyrosine kinase. The Journal of biological chemistry. 2004;279:55089–55096. 29. Dong S, Corre B, Foulon E, Dufour E, Veillette A, Acuto O, Michel F. T cell receptor for antigen induces linker for activation of T cell-dependent activation of a negative signaling complex involving Dok-2, SHIP-1, and Grb-2. The Journal of experimental medicine. 2006;203:2509–2518. 30. Bendelac A, Savage PB, Teyton L. The biology of NKT cells. Annual review of immunology. 2007;25:297–336. 31. Lee YJ, Holzapfel KL, Zhu J, Jameson SC, Hogquist KA. Steady-state production of IL-4 modulates immunity in mouse strains and is determined by lineage diversity of iNKT cells. Nature immunology. 2013;14:1146–1154. 32. Sawada S, Scarborough JD, Killeen N, Littman DR. A lineage-specific transcriptional silencer regulates CD4 gene expression during T lymphocyte development. Cell. 1994;77:917–929. 33. Lee PP, Fitzpatrick DR, Beard C, Jessup HK, Lehar S, Makar KW, Perez- Melgosa M, Sweetser MT, Schlissel MS, Nguyen S, Cherry SR, Tsai JH, Tucker SM, Weaver WM, Kelso A, Jaenisch R, Wilson CB. A critical role for Dnmt1 and 94 DNA methylation in T cell development, function, and survival. Immunity. 2001;15:763–774. 34. Gloire G, Erneux C, Piette J. The role of SHIP1 in T-lymphocyte life and death. Biochemical Society transactions. 2007;35:277–280. 35. Elewaut D, Brossay L, Santee SM, Naidenko OV, Burdin N, De Winter H, Matsuda J, Ware CF, Cheroutre H, Kronenberg M. Membrane lymphotoxin is required for the development of different subpopulations of NK T cells. Journal of immunology (Baltimore, Md. : 1950) 2000;165:671–679. 36. Edmunds C, Parry RV, Burgess SJ, Reaves B, Ward SG. CD28 stimulates tyrosine phosphorylation, cellular redistribution and catalytic activity of the inositol lipid 5-phosphatase SHIP. European journal of immunology. 1999;29:3507– 3515. 37. Gumbleton M, Vivier E, Kerr WG. SHIP1 Intrinsically Regulates NK Cell Signaling and Education, Resulting in Tolerance of an MHC Class I-Mismatched Bone Marrow Graft in Mice. Journal of immunology (Baltimore, Md. : 1950) 2015;194:2847–2854. 38. Kerr WG, Colucci F. Inositol phospholipid signaling and the biology of natural killer cells. Journal of innate immunity. 2011;3:249–257. 39. Srivastava N, Sudan R, Kerr WG. Role of Inositol Poly-Phosphatases and Their Targets in T Cell Biology. Frontiers in immunology. 2013;4:288. 40. Newton RH, Turka LA. Regulation of T cell homeostasis and responses by pten. Frontiers in immunology. 2012;3:151. 41. Kishimoto H, Ohteki T, Yajima N, Kawahara K, Natsui M, Kawarasaki S, Hamada K, Horie Y, Kubo Y, Arase S, Taniguchi M, Vanhaesebroeck B, Mak TW, Nakano T, Koyasu S, Sasaki T, Suzuki A. The Pten/PI3K pathway governs the homeostasis of Valpha14iNKT cells. Blood. 2007;109:3316–3324. 95 42. Banh C, Miah SM, Kerr WG, Brossay L. Mouse natural killer cell development and maturation are differentially regulated by SHIP-1. Blood. 2012;120:4583– 4590. 43. Collazo MM, Wood D, Paraiso KH, Lund E, Engelman RW, Le CT, Stauch D, Kotsch K, Kerr WG. SHIP limits immunoregulatory capacity in the T-cell compartment. Blood. 2009;113:2934–2944. 44. Maxwell MJ, Duan M, Armes JE, Anderson GP, Tarlinton DM, Hibbs ML. Genetic segregation of inflammatory lung disease and autoimmune disease severity in SHIP-1−/− mice. Journal of immunology (Baltimore, Md. : 1950) 2011;186:7164– 7175. 45. Hayakawa Y, Berzins SP, Crowe NY, Godfrey DI, Smyth MJ. Antigen-induced tolerance by intrathymic modulation of self-recognizing inhibitory receptors. Nature immunology. 2004;5:590–596. 46. Griewank K, Borowski C, Rietdijk S, Wang N, Julien A, Wei DG, Mamchak AA, Terhorst C, Bendelac A. Homotypic interactions mediated by Slamf1 and Slamf6 receptors control NKT cell lineage development. Immunity. 2007;27:751–762. 47. Parekh VV, Lalani S, Kim S, Halder R, Azuma M, Yagita H, Kumar V, Wu L, Van Kaer L. PD-1/PD-L Blockade Prevents Anergy Induction and Enhances the Anti- Tumor Activities of Glycolipid-Activated Invariant NKT Cells. The Journal of Immunology. 2009;182:2816–2826. 48. Karlsson MC, Guinamard R, Bolland S, Sankala M, Steinman RM, Ravetch JV. Macrophages control the retention and trafficking of B lymphocytes in the splenic marginal zone. The Journal of experimental medicine. 2003;198:333–340. 49. Fortenbery NR, Paraiso KH, Taniguchi M, Brooks C, Ibrahim L, Kerr WG. SHIP influences signals from CD48 and MHC class I ligands that regulate NK cell homeostasis, effector function, and repertoire formation. Journal of immunology 96 (Baltimore, Md. : 1950) 2010;184:5065–5074. 50. Viant C, Fenis A, Chicanne G, Payrastre B, Ugolini S, Vivier E. SHP-1-mediated inhibitory signals promote responsiveness and anti-tumour functions of natural killer cells. Nature communications. 2014;5:5108. 51. Wahle JA, Paraiso KH, Costello AL, Goll EL, Sentman CL, Kerr WG. Cutting edge: dominance by an MHC-independent inhibitory receptor compromises NK killing of complex targets. Journal of immunology (Baltimore, Md. : 1950) 2006;176:7165–7169. 52. Monteiro M, Almeida CF, Caridade M, Ribot JC, Duarte J, Agua-Doce A, Wollenberg I, Silva-Santos B, Graca L. Identification of regulatory Foxp3+ invariant NKT cells induced by TGF-beta. Journal of immunology (Baltimore, Md. : 1950) 2010;185:2157–2163. 53. Moreira-Teixeira L, Resende M, Coffre M, Devergne O, Herbeuval JP, Hermine O, Schneider E, Rogge L, Ruemmele FM, Dy M, Cordeiro-da-Silva A, Leite-de- Moraes MC. Proinflammatory environment dictates the IL-17-producing capacity of human invariant NKT cells. Journal of immunology (Baltimore, Md. : 1950) 2011;186:5758–5765. 54. Pesesse X, Moreau C, Drayer AL, Woscholski R, Parker P, Erneux C. The SH2 domain containing inositol 5-phosphatase SHIP2 displays phosphatidylinositol 3,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate 5-phosphatase activity. FEBS letters. 1998;437:301–303. 55. Tu Z, Ninos JM, Ma Z, Wang JW, Lemos MP, Desponts C, Ghansah T, Howson JM, Kerr WG. Embryonic and hematopoietic stem cells express a novel SH2- containing inositol 5'-phosphatase isoform that partners with the Grb2 adapter protein. Blood. 2001;98:2028–2038. 56. Nguyen NY, Maxwell MJ, Ooms LM, Davies EM, Hilton AA, Collinge JE, Hilton 97 DJ, Kile BT, Mitchell CA, Hibbs ML, Jane SM, Curtis DJ. An ENU-induced mouse mutant of SHIP1 reveals a critical role of the stem cell isoform for suppression of macrophage activation. Blood. 2011;117:5362–5371. 98 CHAPTER 3: MHC CLASS IB-RESTRICTED CD8+ T CELLS ARE PROTECTIVE DURING MURINE CYTOMEGALOVIRUS INFECTION 99 MHC class Ib-restricted CD8+ T cells are protective during murine cytomegalovirus infection Courtney K. Anderson1, Emma C. Reilly1, Angus Y. Lee2, and Laurent Brossay1* 1 Department of Molecular Microbiology & Immunology, Division of Biology and 2 Medicine, Brown University, Providence, RI 02912. Cancer Research Laboratory, University of California, Berkeley, CA 94720. This work was supported by the NIH research grants R01 AI46709 (LB) and F31 AI124556 (CA) and an American Association of Immunologists Career in Immunology Fellowship (LB). 100 ABSTRACT The CD8+ T cell response during human cytomegalovirus (HCMV) infection has almost exclusively been limited to examining conventional T cells. However, there is now growing evidence informing on the participation of non-classical CD8+ T cells, which are restricted by MHC class Ib molecules rather than MHC class Ia, during both bacterial and viral infections. Using the well-established murine model of CMV infection, we investigate the role of MHC class Ib-restricted T cells. We are able to examine their response in the absence of classical CD8+ T cells using KbDb-/- mice, which lack MHC class Ia molecules. Non-classical CD8+ T cells robustly expand after MCMV challenge, become highly activated effector cells, and are reminiscent of conventional T cells rather than other innate-like non-classical T cells; these phenotypes are virus-dependent, rather than inflammatory cytokine-dependent. MCMV-expanded non-classical CD8+ T cells are also capable of forming durable memory and robustly proliferate upon secondary infection. Significantly, when acting as the sole component of the adaptive immune response, this population is sufficient to protect KbDbRAG1-/- mice from MCMV-induced lethality. Using in vitro blocking experiments and newly generated KbDb-/-Qa-1-/- mice, we determine that the MHC class Ib molecule Qa-1 (encoded by H2-T23) restricts a large component of this population. Together these studies indicate that non-classical CD8+ T cells are an, as of yet, unappreciated component of the adaptive immune response towards CMV. This population is uniquely restricted by a non-classical MHC, yet behaves in a conventional manner. This may allow them to compensate for the viral evasion of classical T cells by CMV. 101 INTRODUCTION HCMV is a ubiquitous pathogen that is relatively easily resolved in healthy individuals. In the United States, approximately half of the population is seropositive for CMV [1]. As a member of the herpes virus family, it remains latent and can then periodically reactivate, asymptomatically. CMV is superbly adept at evading components of the host’s innate and adaptive immune systems, however a multilayered immune response maintains suppression [2]. This allows for a balance between viral escape mechanisms and host immune control [3]. However, primary and reactivated infections are both potential sources of morbidity and mortality for immunocompromised patients. CMV is also a serious congenital infection, and the leading non-genetic cause of hearing loss in children [4]. Each member of the CMV family displays species-specific tropism, requiring animal models to better understand the multifaceted immune response it elicits. Murine CMV (MCMV) is well-established to have similarities in genome [5], latency [2] and reactivation [6]. There are also parallels in immune response, with NK cells and T cells both being important components during CMV infection. Impairment of the NK cell response in mice and humans both cause more severe disease [7, 8], and there are positive correlations between the presence of an intact CD8+ T cell response and outcome [9-12]. In fact, a large fraction of the T cell compartment is devoted to CMV. In seropositive individuals, approximately 10% of circulating CD4+ and CD8+ T cells are CMV-specific [13]. Compared to the well-characterized roles of classical CD8+ T cells, the immunologic contributions of non-classical CD8+ T cells remain poorly understood. Non-classical CD8+ T cells are restricted by MHC class Ib molecules (e.g. HLA-E, -F, and -G in humans), rather than MHC class Ia (i.e. HLA-A, -B, and -C). These are structurally similar to MHC class Ia and usually paired with a β2-microglobulin light 102 chain, but are generally non-polymorphic and have lower cell surface expression [14]. Some MHC class Ib molecules present unique types of antigens. For example, MR1 presents vitamin B metabolites to mucosal associated invariant T (MAIT) cells [15], CD1 presents lipids to natural killer T (NKT) cells [16], and M3 presents N-formylated peptides [17]. Non-classical CD8+ T cells also participate in host defenses against a number of bacterial and viral pathogens [18]. There is evidence that HLA-E-restricted CD8+ T cells are present following HCMV infection [19, 20], however it is unclear whether they have a definitive role during the host immune response. HLA-E is considered the least polymorphic of the non-classical MHC molecules; two proteins are predominantly expressed out of three variants, which only differ by one amino acid [21, 22]. Qa-1 (encoded by H2-T23) is the functional homologue of HLA-E in mice. HLA-E/Qa-1 are normally loaded with peptides, known as Qa-1 determinant modifier (Qdm, AMAPRTLLL), derived from the leader sequences of classical MHC molecules [23-25]. HLA-E- and Qa-1-bound Qdm were first found to interact with members of the CD94/NKG2 family of receptors [26- 29]. NK cells and CD8+ T cells are repressed upon engagement with the inhibitory receptor CD94/NKG2A [30, 31]. In an attempt to avoid the NK cell response via CD94/NKG2A [32], certain HCMV strains encode their own ligand for HLA-E within the signal sequence of the glycoprotein UL40 (gpUL40) [33, 34]. The leader sequences from strains such as Toledo and AD169 are identical to those within certain HLA haplotypes (VMAPRTLVL and VMAPRTLIL, respectively). However others are not, which could open up virally infected cells to the activity of HLA-E-restricted CD8+ T cells [20]. The role of conventional CD8+ T cells during CMV infection is well characterized. However, CMV employs a number of mechanisms to avoid the classical T cell response, including down-regulation of conventional MHC molecules and 103 upregulation of HLA-E [33, 35, 36]. In this study, we sought to determine the contribution and importance of non-classical CD8+ T cells during MCMV infection. To explore their role in the absence of conventional CD8+ T cells, we utilize KbDb-/- mice, which lack MHC class Ia molecules. We found that non-classical CD8+ T cells participate during MCMV infection in a virus-dependent manner, and that they are sufficient to protect against MCMV-induced lethality. This population is phenotypically similar to conventional CD8+ T cells, rather than other innate-like MHC class Ib- restricted T cells, and capable of robust secondary responses. Using CRISPR/Cas9- generated KbDb-/-Qa-1-/- mice and blocking with monoclonal antibodies (mAbs), we determine that a large component of this novel population is comprised of Qa-1- restricted T cells. Together, these studies add another contributing immunologic layer in the attrition against CMV. The unique properties of non-classical T cells make them attractive targets to examine during infections, as well as for vaccine development. 104 MATERIALS AND METHODS Mice. KbDb-/- mice were obtained from Taconic Biosciences and maintained in-house. B6.SJL (CD45.1+) mice from Taconic were crossed with KbDb-/- mice to obtain congenic KbDb-/- (KbDb-/-.SJL) animals. CD1d-/- mice from Jackson Laboratory were crossed with KbDb-/- mice to obtain KbDbCD1d-/-. RAG1-/- mice from Jackson Laboratory were crossed with KbDb-/- mice to obtain RAG1KbDb-/- animals. C57BL/6 and β2m-/- mice were obtained from Jackson Laboratory. Generation of KbDb-/-Qa-1-/- mice via CRISPR/Cas9. Two guideRNAs (gRNA) were used to target H2-T23 and create a deletion in exon 3 and its flanking intron: 5'- GGCTATGTCATTCGCGGTCC-3' (gRNA 1) and 5'-GGATTTCCCCCAAACCGCAG-3 (gRNA 2). They were selected using CHOPCHOP (http://chopchop.cbu.uib.no) and CRISPR design (http://crispr.mit.edu) online tools [37-39]. Cas9/gRNA injection was performed on KbDb+/- zygotes. Founders were genotyped and backcrossed to C57BL/6 mice for one generation and then KbDb-/- mice for one generation before generating the triple knockout. Two primers sets were used for genotyping KbDbQa-1-/- mice. (1) Qa- 1-External-F: TCTGCTTAGGTTTGGGGTTG and Qa-1-External-R: CTACAGGGGAAAAGCAGTTTTG produce a 524 bp WT band and a 340 bp mutant band. (2) Qa-1-Mutant-F: CATCCAAACGCCTACCCAGA and Qa-1-WT-R: TGAGGCTATGTCATTCGCGG produce a 303 bp WT band and no mutant product. Viruses and infection protocols. MCMV-RVG102 (referred to as MCMV WT) was a gift from Dr. John Hamilton (Duke University), and expresses recombinant EGFP under the immediate early-1 promoter [40]. Infections were performed with 5 x 104 or 1 x 105 PFU i.p. Stocks were prepared in vivo from salivary gland homogenate [41] and viral titers were determined via standard plaque assay using mouse embryonic fibroblast 105 (MEF) cells. Mice were considered latently infected at 8 weeks post-MCMV infection. Lymphocyte isolation. Spleens were dissociated in 1% PBS-serum, filtered through nylon mesh, and underlayed with lympholyte-M (Cedarlane Laboratories). Alternatively, spleens were dissociated in 150 mM NH4Cl for 10 minutes, filtered through nylon mesh, and washed once with 1% PBS-serum. Livers were profused with 1% PBS-serum, dissociated using GentleMACS program E0.1 (Miltenyi Biotech) and passed through nylon mesh. Samples were washed three times in 1% PBS-serum and overlaid onto a two-step discontinuous Percoll gradient (GE Healthcare Bio-Sciences). Salivary glands were cleaned of connective tissue and lymph nodes and dissociated in collagenase IV (Sigma-Aldrich) using GentleMACS program Heart 01.01. Samples were incubated at 37 °C with shaking for 5 minutes, followed by the Heart 01.01 program; five-minute incubation and Heart 01.01 program were then repeated. Samples were filtered through nylon mesh, washed once in 1% PBS-serum, and underlaid with Lympholyte-M. All gradients were centrifuged at 2500 RPM for 20 minutes at room temperature. Blood was collected from a cardiac puncture into heparin-containing tubes. Red blood cells were lysed in 150 mM NH4Cl for 10 minutes and washed twice in 1% PBS-serum. To obtain serum, blood was allowed to clot at room temperature, in the absence of heparin, for 4 hours and stored at 4 °C overnight. Serum was collected following centrifugation at 4 °C. In vivo cell proliferation dye experiments. KbDb-/- splenocytes were labeled with cell proliferation dye eF450 (eBioscience) per the manufacturer’s instructions. Briefly, 10 x 106 cells/ml were labeled with 10 µM dye and incubated for 10 minutes at 37 °C in the dark, followed by 5 minutes on ice. Samples were washed 3X with 10% RPMI-serum and 1X with 1% PBS-serum. CD8+ T cells were enriched using an AutoMACS (Miltenyi 106 Biotech) and CD8α MicroBeads (Miltenyi Biotech). Samples were i.v. injected into KbDb-/-.SJL recipients. Two hours post-injection, recipients were left naïve, stimulated with 50 µg CPG ODN + 100 µg Poly(I:C), or infected with MCMV. On day 2, mice received second dose of 50 µg CPG ODN + 100 µg Poly(I:C). Donor CD8+ T cell proliferation was analyzed on day 4. Serum cytokine production. Serum cytokines from time-course experiments were measured by LEGNEDPlex bead-based immunoassay using the Mouse Th Cytokine Panel (13-plex), per the manufacturer’s instructions (Biolegend). Serum IFN-γ levels were also determined via sandwich ELISA on day 7 post-MCMV using the following: purified IFN-γ (clone R4-6A2, eBioscience), Biotin-IFN-γ (clone XMG1.2, eBioscience), and peroxidase-conjugated streptavidin (Jackson ImmunoResearch Labs) [42]. Secondary MCMV challenge and survival studies. Approximately 50,000 sorted memory CD8+ T cells (KLRG1-CD27+) from long-term infected KbDb-/- spleens were adoptively transferred per recipient. For secondary MCMV infections, cells were injected i.v. into KbDb-/-.SJL recipients. For survival studies, cells were injected into RAG1KbDb-/- recipients. Two hours post-injection, recipients were infected with MCMV. In vitro CD8+ T cell stimulation and BMDC cultures. KbDb-/- BMDCs were prepared in vitro by flushing fibulas and tibias with 1% PBS-serum. 1 x 107 cells were grown in 10% DMEM-serum + 10 ng/ml GM-CSF (eBioscience) in a T75 flask. Media was replaced on day 3. On day 6, suspended and loosely adherent cells were harvested and plated 1 x 105 cells/well of a 96-well plate. BMDC were infected with 1 x 105 PFU MCMV (MOI 1), plates were centrifuged at 2000 RPM for 20 minutes at room 107 temperature, and then incubated 2 hours at 37 °C. Cells were washed one time with 10% DMEM-serum and fresh 10% DMEM-serum + 10 ng/ml GM-CSF (eBioscience) was added. BMDCs were used on day 2-post infection for in vitro stimulation experiments. CD8+ T cells were enriched from individual KbDb-/- spleens using an AutoMACS (Miltenyi Biotech) and CD8α MicroBeads (Miltenyi Biotech). 1 x 105 cells were plated in triplicate onto uninfected or MCMV-infected BMDCs for 6 hours at 37 °C in 10% DMEM-serum + GolgiPlug Protein Transport Inhibitor (BD Biosciences), per the manufacturer’s instructions. Infected BMDCs were pretreated for at least 30 minutes with 20 µg/ml of either mouse anti-mouse Qa-1 (BD Biosciences), mouse anti-mouse Qa-2 (BioLegend), or mouse anti-mouse IgG2a isotype control (eBioscience). Antibodies were also present throughout the 6-hour incubation. Following incubation, triplicate samples were pooled and stained for intracellular IFN-γ and TNF-α production. Antibodies and flow cytometry. Cells were stained in 1% PBS-serum containing 2.4G2 and cell surface antibodies for 20 minutes on ice in the dark. Staining with CD1d tetramer (NIH Tetramer Facility) was performed for 15 minutes at room temperature and 15 minutes on ice in the dark. For intracellular staining, cells were fixed using Cytofix/Cytoperm (BD Biosciences) for 30 minutes and stained in Perm/Wash Buffer (BD Biosciences) for 30 minutes. For intranuclear staining, cells were fixed using Fixation/Permeabilization Solution (eBioscience) for 30 minutes and then stained in Permeabilization Buffer (eBioscience) for 30 minutes. Samples were run on a FACSAria III (BD Biosciences) or MACSQuant (Miltenyi Biotech) and analyzed using FlowJo (Tree Star Inc.). The mAbs listed below were used for flow 108 cytometry and purchased from BioLegend, eBioscience, Thermo Fischer Scientific, or BD Biosciences: FITC-CD69, FITC-TCRβ, PE-CD8β.2, PE-CD19, PE-CD27, PE-CD69, PE-CX3CR1, PE-IFN-γ, PE-PLZF, PE-TCRβ, PerCP-Cy5.5-CD69, PerCP-Cy5.5- TCRβ, PerCP-eF710-CD127, PE-Cy7-KLRG1, PE-Cy7-T-bet, APC-CD45.2, APC- CD103, APC-CX3CR1, APC-Streptavidin, APC-eF780-CD44, APC-eF780-CD45.1, APC-eF780-CD45.2, APC-eF780-KLRG1, eF450-CD8, eF450-TNF-α, BV421-CD127, BV421-CD1d tetramer, BV510-TCRβ, BV605-CD8, BV711-CD62L, BV785-NK1.1, Biotin-Qa-1. Statistical analysis. Statistical analyses were performed with Prism 7.0 (Graph-Pad Software, Inc.). Unpaired two-tailed Student’s t-tests were used to compare two individual groups. Log-rank (Mantel Cox) tests were used for survival studies. Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. Ethics statement. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals, defined by the NIH (PHS Assurance #A3284-01). Animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Brown University. All animals were housed in a centralized and AAALAC-accredited research animal facility that is fully staffed with trained husbandry, technical, and veterinary personnel. 109 RESULTS Non-classical CD8+ T cells respond during acute MCMV infection in KbDb-/- mice To study the non-classical CD8+ T cell response in the absence of classical CD8+ T cells, we took advantage of KbDb-/- mice [43]. H-2Kb and H-2Db encode MHC class Ia molecules in mice on the C57BL/6 background, thus any residual CD8+ T cells present in KbDb-/- animals are selected by MHC class Ib molecules. As previously reported, we find greatly diminished populations of CD8+ T cells in naïve KbDb-/- animals (Fig. 1A) [43, 44]. However, both the frequency and absolute number of these cells robustly increases in the spleen, liver (Fig. 1A, S1A), and blood (data not shown) on day 7 post-MCMV infection. This response peaks on day 14, and equates to an approximate 5-fold and 17-fold expansion in the spleen and liver, respectively (Fig. 1B). MCMV-expanded non-classical CD8+ T cells then begin to contract by day 21 (Fig. 1B). MCMV-expanded non-classical CD8+ T cells are distinct from innate-like T cells Many non-classical T cells have a unique innate-like phenotype and do not require clonal expansion following stimulation, which allows for more rapid effector functions [45]. Based on the kinetics that we observe for MCMV-expanded non- classical CD8+ T cells, we wondered whether they were more similar to conventional T cells or maintained innate-like characteristics. The transcription factor promyelocytic leukemia zinc finger (PLZF) is thought to act as a major regulator for innate-like T cells. For example, γδ T cells, MAIT cells, and NKT cells all express PLZF [46-49]. Although PLZF-expressing CD8+ T cells are present in naïve KbDb-/- mice, we find that most CD8+ T cells were PLZFneg and T-bethi on day 7 post-MCMV infection (Fig. S1B). Non- classical T cells also express NK1.1, such as NKT cells. Others have a CD8αα 110 homodimer rather than a CD8αβ heterodimer as their co-receptor, for example Qa-2- restricted intestinal intraepithelial lymphocytes [50]. NK1.1+ and CD8αα+ T cells are both enriched in naïve KbDb-/- animals, however the population responding to MCMV expresses a CD8αβ heterodimer and are NK1.1- (Fig. S1C, D). Together these data indicate that non-classical CD8+ T cells are phenotypically more similar to conventional T cells than innate-like T cells, following MCMV infection. Non-classical CD8+ T cells acquire an effector phenotype during acute MCMV infection Conventional CD8+ T cells down-regulate CD62L and upregulate CD44 expression following activation during acute infection and become cytotoxic T lymphocytes (CTLs, CD44hiCD62Llo). In KbDb-/- mice on day 7 post-MCMV infection, there is a significant increase in CTLs and a decrease in naïve (CD44loCD62Lhi) CD8+ T cells, compared to uninfected controls (Fig. S2A, C). However, many non-classical CD8+ T cells from naive KbDb-/- animals are already CD44hiCD62Llo, potentially misconstruing interpretation (Figure S2A, C) [51]. To better evaluate the activation status of MCMV-expanded non-classical CD8+ T cells we monitored KLRG1 expression, which is upregulated on short-lived effector CD8+ T cells (TEFF, KLRG1+CD127-) [52]. Non-classical CD8+ T cells do not express KLRG1 in naïve animals, however KLRG1 becomes upregulated on day 5 post-infection and is highly expressed on day 7 (Fig. 1C, S2B, D). We also find that they upregulate CD94/NKG2A, which is commonly acquired in response to infection [53], and became CX3CR1high, recently shown to associate with terminal effector cell differentiation following MCMV challenge (Fig. S2E-G) [54]. 111 The expansion and activation of non-classical CD8+ T cells is MCMV-dependent Non-classical T cells do not always require antigen stimulation and some respond to non-cognate activation through environmental cytokines. For example, iNKT cell activation during MCMV infection is a result of infection-induced inflammatory cytokines and independent of lipid Ag presentation by CD1d [55-57]. This led us to ask whether the non-classical CD8+ T cell response in KbDb-/- mice is cytokine-dependent or MCMV-dependent. Non-classical CD8+ T cells were adoptively transferred into KbDb-/- .SJL recipients and left naïve, treated with CPG ODN and Poly(I:C), or infected with MCMV. CPG ODN and Poly(I:C) are TLR9 and TLR3 agonists, respectively. They were used to simulate MCMV infection, which signals through TLR2, TLR3, and TLR9 [58, 59]. Donor cells in the spleen and liver of naïve recipients do not undergo detectable homeostatic proliferation on day 4, and CPG ODN/Poly(I:C) treatment only results in minimal proliferation (Fig. 2A, B). In contrast, a significant number of donor cells from MCMV-infected mice proliferate, particularly in the liver (Fig. 2A, B). In agreement with these findings, only donor cells from virally-infected recipients express both CD69 and KLRG1 in the spleen and liver (Fig. 2C, D). Altogether, these data suggest that environmental inflammation is not sufficient for non-classical CD8+ T cell proliferation or activation following MCMV infection. The non-classical CD8+ T cell response results in a prolonged inflammatory phenotype NK cells are an important component of the innate immune response to MCMV, producing a peak of IFN-γ at 38 hours post-infection, even in MHC class I-deficient animals [60]. Interestingly, although there is only residual IFN-γ detected in the serum of wild type animals on day 7 post-infection, it remains elevated in KbDb-/- mice (Fig. 112 3A). This phenotype is striking when compared to β2m-/- animals at the same time- point, which also have minimal IFN-γ. The high level of serum IFN-γ in KbDb-/- mice returns to normal levels by day 14 post-infection (Fig. 3B). These data indicate that non-classical CD8+ T cells contribute to elevated serum IFN-γ levels, since β2m-/- animals lack both MHC class Ia- and Ib-restricted CD8+ T cells. Non-classical CD8+ T cells form memory populations in long-term infected KbDb-/- mice We observe that KbDb-/- animals are protected from MCMV for upwards of 11 months without outward signs of illness. This coincides with elevated populations of non-classical CD8+ T cells in the spleen and liver of long-term infected animals, compared to naïve controls (Fig. S3A, B). We next wanted to determine whether non- classical CD8+ T cells are actually capable of establishing durable memory. Memory CD8+ T cell (TM) populations are differentiated into central memory (TCM, KLRG!- CD127+CD44+CD62L+) and effector memory (TEM, KLRG1-CD127+CD44+CD62L-) cells (Fig. S3C). Similarly to conventional CD8+ T cells [61], non-classical TM cells have differential expression of CX3CR1. KLRG1+ non-classical CD8+ T cells express the highest CX3CR1 levels, TCM cells express minimal CX3CR1, and TEM cells have intermediate expression (Fig. S3D). We next investigated whether non-classical CD8+ T cells are capable of developing a tissue-resident memory (TRM, CD69+CD103+) phenotype in the salivary gland (SMG), a privileged site of infection for CMV. We find that non-classical CD8+ T cells are recruited to the SMG and persist in this tissue (Fig. 4A, S3E). Although there are fewer total CD8+ T cells compared to C57BL/6 mice (Fig. 4B), the proportion of CD8+ T cells that are TRM is comparable in KbDb-/- mice (Fig. 4C, S3F). 113 Using an adoptive transfer approach, we then examined non-classical CD8+ T cell memory properties. TM cells (KLRG1-CD27+) were isolated from long-term infected KbDb-/- mice and 5 x 104 cells were transferred into KbDb-/-.SJL recipients, which were then challenged with MCMV. Donor cells undergo significant expansion, comprising up to of 25% of total splenic CD8+ T cells (Fig. 4D), and become highly activated (KLRG1+) upon secondary challenge (Fig. 4D, E). This is greater than recipient non- classical CD8+ T cells, which are only experiencing their primary MCMV infection (Fig. 4E). Assuming 100% engraftment in the spleen, this accounts for roughly a four-fold increase (Fig. 4F). However, this is a substantial underrepresentation because donor cells are also found in other organs, such as the liver and blood. In contrast, naïve non-classical CD8+ T cell donors make up a very minor portion of recipient organs, and their activation is equivalent to that of recipient cells (data not shown). Together these data illustrate that non-classical CD8+ T cells are capable of forming memory populations and behave with the characteristics of memory cells during secondary infection. Non-classical CD8+ T cells are sufficient to protect against MCMV-induced lethality We next investigated the protective potential of non-classical CD8+ T cells against MCMV. In MCMV-infected RAG1-/- mice, NK cells initially control infection but eventually succumb due to the outgrowth of MCMV mutants [62]. To avoid rejection of KbDb-deficient CD8+ T cells in RAG1-/- mice by NK cells, we generated KbDbRAG1-/- animals. These mice received approximately 5 x 104 CD8+ TM cells from long-term infected KbDb-/- mice, and were subsequently infected. As expected, KbDbRAG1-/- controls succumb to infection on average within 20 days. In contrast, mice that receive TM donor cells are protected against MCMV-induced lethality for upwards of 100 days 114 (Fig. 5A). The antiviral effects of non-classical CD8+ T cells were confirmed by an absence of contaminating lymphocyte populations at the end of each study (data not shown). Taken together, these data demonstrate that non-classical CD8+ T cells are able to successfully control MCMV infection as the sole component of the adaptive immune system. Qa-1-restricted CD8+ T cells respond to MCMV MCMV-expanded non-classical CD8+ T cells could be recognizing antigens presented by a number of MHC class Ib molecules. MHC class II-restricted CD8+ T cells are also found under certain conditions, including following SIV vaccination in Rhesus macaques [63] and in HIV-infected individuals [64]. We excluded MHC class II by comparing the expansion of non-classical CD8+ T cells in KbDb-/- and β2m-/- mice. Unlike KbDb-/- animals, β2m-/- mice have negligible non-classical CD8+ T cells present in the spleen or liver on day 7 post-infection (Fig. S4A, B). This indicates that they are restricted by MHC class Ib molecules. We next investigated which MHC class Ib molecule is required for the observed phenotypes. Mice encode over 30 MHC class Ib genes, many due to gene duplications, and 21 are thought to be transcribed [65]. However, not all of these are capable of binding peptides, such as HFE and TL (encoded by H2-T3 and H2-T18) [66, 67]. Others present unique antigens that are unlikely in the context of a viral infection. For example, MR1 presents vitamin B metabolites and M3 binds N-formylated peptides of mitochondrial or prokaryotic origin [15, 17]. We therefore focused on three non-classical molecules, which present pathogen-derived antigens: CD1d, Qa-1, and Qa-2 [68-70]. The majority of MHC class Ib molecules are encoded in the same chromosome as MHC class Ia, however CD1d outside of the Mhc locus. This allowed us to produce KbDb-/-CD1d-/- mice. We found that the magnitude of the CD8+ T cell response to MCMV is comparable to KbDb-/- 115 animals (Fig. S4A, C), indicating CD1d is dispensable. To test the roles of Qa-1 and Qa-2, we next performed an in vitro restimulation assay using blocking mAbs and BMDCs derived from KbDb-/- mice. MCMV-infected BMDCs are capable of stimulating non-classical CD8+ T cells from long-term infected KbDb-/- mice to produce significantly more IFN-γ and TNF-α than uninfected BMDCs (Fig. 6A, B). Importantly, although anti-Qa-2 treatment does not have any detectable effects, there is a significant decrease in TNF-α production following anti-Qa-1 treatment and a similar trend for IFN-γ (Fig 6A, B). To validate these data, we next generated KbDb-/-Qa-1-/- mice. Since H2-T23 is located approximately 0.8 megabases from H-2D, we targeted it directly on KbDb+/- zygotes using CRISPR/Cas9 technology (Fig. S5A, B). The resultant line has a 184 bp deletion in the third exon and flanking intron of H2-T23 (Fig. S5C) and completely lacks Qa-1 expression at the cell surface, compared to KbDb-/-Qa-1+/+ and KbDb-/-Qa-1+/- littermate controls (Fig. S5D). As expected, Qa-1 expression in heterozygous mice is approximately half of wild type levels (Fig. S5E). We next examined the non-classical CD8+ T cell response in the resultant triple knockout and littermates on day 7 post-infection. In agreement with our blocking studies, KbDb-/-Qa-1-/- mice have significantly dampened CD8+ T cell responses (Fig. 6C, D). There are decreased frequencies in the spleen, liver, and blood and decreased absolute numbers in the spleen and blood, with a similar trend in the liver (Fig. 6C, D). These data imply that a large component of the non-classical CD8+ T cell response is comprised of Qa-1-restricted cells. The CD8+ T cell response is also compromised in KbDb-/-Qa-1+/- mice, which suggests a critical role for the level of Qa-1 expression. Remarkably, non-Qa-1-restricted CD8+ T cells are poorly activated by MCMV. This is evidenced by the frequency of TEFF (Fig. S5F) and terminally differentiated TEFF cells (Fig. S5G, KLRG1+CX3CR1+). However, this is not a global 116 defect because the NK cell response is unaffected by the loss of Qa-1 signaling at this time-point (Fig. S5H). These data suggest that Qa-1-restricted cells comprise a significant portion of the non-classical CD8+ T cells responding to MCMV in KbDb-/- animals, and are more poised to respond than other MHC class Ib-restricted T cell populations. 117 DISCUSSION Increasing evidence demonstrates that MHC class Ib-restricted T cells are capable of responding to several bacterial and viral pathogens (reviewed in [18]). Thus far, the non-classical T cell response to CMV has largely gone unappreciated. HLA-E- restricted T cells are detectable in a subset of HCMV seropositive individuals that respond to gpUL40 peptides ex vivo [19], however their participation and protective capabilities remain ambiguous. In this report, we provide evidence that non-classical CD8+ T cells can form TM cells and provide long-term protection against MCMV. We also establish that the MHC class Ib molecule Qa-1 restricts a large component of this population. Non-classical T cells, such as iNKT and MAIT cells, are predisposed to rapid activation in the presence of agonist antigens and/or inflammatory cytokines, but are unable to form memory [45]. For example, M3-restricted T cells develop and respond before conventional CD8+ T cells during L. monocytogenes infection [71], but do not expand after secondary challenge [71, 72]. Surprisingly, we discovered that MCMV- specific non-classical CD8+ T cells behave similarly to conventional TEFF cells, rather than innate-like T cells. This is illustrated by a lack of PLZF expression and the formation of memory populations, including a comparable proportion of TRM cells to that observed in wild type SMGs. More importantly, we demonstrate that non-classical CD8+ TM cells prevent lethal infection when adoptively transferred into immunocompromised mice. Others have shown that γδ T cells can also prevent lethality [73, 74]. However, in this study, we achieved protection with the transfer of 5 to 20 times fewer cells. This number is more physiological, and comparable to what is reported for conventional CD8+ TM cells [9]. Non-classical T cells are believed to represent an ancient and evolutionarily conserved branch of the adaptive system, so their memory phenotype is somewhat surprising [75, 76]. However, in agreement with 118 our findings, a recent study characterizes three Qa-1-restricted CD8+ T cell clones that also behave similarly to conventional T cells [77]. Using neutralizing mAbs, we found that blocking Qa-1 inhibits the cytokine response of non-classical CD8+ TM cells after restimulation. The essential role of Qa-1 is further established using KbDb-/-Qa-1-/- mice, which exhibit an impaired CD8+ T cell response on day 7 post-innoculation. This reduced CD8+ T cell response could be the consequence of increased NK cell-mediated regulation of anti-viral T cells, as recently shown with LCMV-infected Qa-1-deficient animals [78]. However, we consider this unlikely since NK cells from infected KbDbQa-1-/- animals are not hyperactivated, possibly due to lack of MHC class I-mediated licensing. Interestingly, the level of Qa-1 expression tightly regulates the CD8+ T cell response, since it is affected in both KbDb-/- Qa-1+/- and KbDb-/-Qa-1-/- mice. In addition to its interaction with the TCR, Qa-1 is the ligand for the inhibitory receptor CD94/NKG2A on NK and CD8+ T cells [28, 29]. This inhibitory signal restricts the magnitude of activation for virus-specific CD8+ T cells following poxvirus [79] and polyoma virus infection [31], as well as NK cell-mediated killing of HCMV-infected cells [33]. Loss of CD94/NKG2A signaling also results in abnormal T cell activation during M. tuberculosis infection [69]. In several of these studies, the absence of the CD94/NKG2A inhibitory signals is presumably amplified by the lack of Qa-1-restricted regulatory CD8+ T cells (Treg) [80]. Accordingly, we predicted that the residual CD8+ T cells in KbDb-/-Qa-1-/- mice would be hyper-responsive. Unexpectedly, the remaining CD8+ T cells are poorly activated after MCMV infection. Therefore, among the non- classical CD8+ T cell subsets, Qa-1-restricted CD8+ T cells may deliver the dominant response during MCMV infection. In addition to Qdm, Qa-1/HLA-E presents a diverse peptide repertoire. In the absence of Qdm, Qa-1 preferentially binds a peptide derived from heat shock protein 119 60 (Hsp60) [81]. Hsp60 is upregulated on stressed cells and is the homologue of prokaryotic GroEL. Qa-1 also presents GroEL-derived antigens from S. typhimurium however, GroEL-specific CD8+ T cells are cross-reactive and can lyse stressed macrophages [82]. When ERAAP1 (endoplasmic reticulum amino peptidase 1) is dysfunctional, Qa-1 can load the self-peptide FL9; this allows FL9-specific CD8+ T cells to survey for, and eliminate, ERAAP1-deficient cells [83]. In allograft recipients, HLA- E-restricted T cells specific for the HCMV gpUL40 [33, 34] also lead to alloreactivity, depending on a person’s HLA genotype [84]. However, MCMV does not encode a UL40 homologue. Therefore, the MCMV-derived antigen recognized by Qa-1- restricted CD8+ T cells is unlikely to induce alloreactivity; this is substantiated by MHC class Ib-restricted CD8+ T cells protecting against lethal infection for upwards of 100 days without apparent signs of autoimmunity. The characterization of MCMV-derived antigens bound to Qa-1 will consequently, be of particular therapeutic interest. However, identification of such molecules is beyond the scope of this study and requires further experimentation. To conclude, even though no one immune component is required during CMV infection, a multilayered response is necessary to keep this highly immunoevasive virus in check. CMV employs multiple mechanisms to elude individual cell types, with at least seven genes to avoid the NK cell response [85]. Here, we provide the first evidence of a protective MHC class Ib-restricted CD8+ T cell response to MCMV, including Qa-1-restricted and non-Qa-1-restricted cells. Recent evidence suggests that Qa-1/HLA-E-restricted T cells have the potential to exploit immunoevasion in a CMV- based vaccine [86]. We show here that they can form memory and substitute for conventional T cells. This suggests that Qa-1-restricted cells may represent a category of T cells positioned between innate-like and adaptive T cells, whose activity might be only revealed when the conventional T cell response is absent or impeded. 120 ACKNOWLEDGMENTS We thank Kevin Carlson for cell sorting and Céline Fugère for i.v. injections. We also thank Dr. John Hamilton for MCMV-RVG102. AUTHORSHIP CONTRIBUTIONS C.K.A. designed, performed, and analyzed the experiments, in addition to writing the paper. E.C.R. designed, performed, and analyzed the experiments. A.Y.L. performed Cas9 mRNA/gRNA injections. L.B. designed experiments and wrote the paper. The authors have no conflict of interest to declare. 121 Figure 1. Non-classical CD8+ T cells help control MCMV infection in KbDb-/- mice. (A) Representative staining of CD8+ T cells in the spleen and liver of KbDb-/- mice on day 0 and day 7 post-MCMV infection. (B) Frequency (⎯) and number (- -) of CD8+ T cells in the spleen and liver of KbDb-/- mice on indicated days post-MCMV infection (n=9). Numbers on graphs indicate fold-change of cell number compared to day zero. (C) Frequency of CD8+ T cells with an effector phenotype (KLRG1+CD127-) in the spleen (⎯) and liver (- -) of KbDb-/- mice on indicated days post-MCMV infection (n=9). Data are pooled from three (B, C) and error bars indicate SEM. 122 Figure 2. The activation and expansion of non-classical CD8+ T cells is MCMV- dependent in KbDb-/- mice. (A) Representative labeling of donor non-classical CD8+ T cells (CD45.2+) with CPD in the spleen and liver of KbDb-/-.SJL (CD45.1+) recipients on day 4 post-transfer. Recipients were left naïve (black), treated with Poly(I:C) + CpG (grey), or infected with MCMV (white). Histograms are gated on total donor CD8+ T cells. Frequencies of donor CD8+ T cells that (B) have undergone more than one cycle of proliferation, (C) are CD69+, or (D) are KLRG1+ (n=5-6). Data are pooled from two independent experiments and error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. 123 Figure 3. Non-classical CD8+ T cells participate in a prolonged inflammatory phenotype in KbDb-/- mice. (A) Serum IFN-γ levels from B6, KbDb-/-, and Β2m-/- mice on day 7 post-MCMV infection were determined by sandwich ELISA (n=7). (B) Serum IFN-γ levels from KbDb-/- mice on indicated days-post MCMV infection were determined via bead-based immunoassay (n=8-9). Dotted line is limit of detection. Data are pooled from two (A) or three (B) independent experiments and error bars indicate SEM. **p<0.01, ***p<0.001. 124 Figure 4. Non-classical CD8+ T cells in long-term MCMV-infected KbDb-/- mice form memory populations and robustly expand following secondary infection. (A) Frequency and (B) number of CD8+ T cells in the SMG from long-term infected KbDb-/- and C57BL/6 mice (n=12). (C) Frequency of TRM CD8+ T cells (CD103+CD69+) in the SMG from long-term infected KbDb-/- and C57BL/6 mice (n=12). (D) Representative expansion and activation (KLRG1+CD127-) of donor non- classical CD8+ T cells (CD45.2+) transferred into KbDb-/-.SJL (CD45.1+) recipients, on day 7 post-MCMV infection. Donor TM (KLRG1-CD27+) cells were sorted from long- term infected KbDb-/- mice >8 weeks post-infection. (E) Frequency of donor and recipient non-classical CD8+ T cells that became TEFF cells (KLRG1+CD127-) on day 7 post-infection (n=7). (F) Fold-change of donor CD8+ T cell number, assuming 100% engraftment in indicated organ, on day 7 post-infection (n=7). Data are pooled from two independent experiments and error bars indicate SEM. **p<0.01 and ****p<0.0001. 125 Figure 5. Non-classical CD8+ T cells are sufficient to protect against MCMV- induced lethality. (A) Survival curve of RAG1KbDb-/- mice controls (- -) or those that received 50,000 TM CD8+ T cells (KLRG1-CD27+) from long-term infected KbDb-/- mice (⎯) (n=6). Cells were sorted from KbDb-/- mice >8 weeks post-infection. Data are one representative of three independent experiments. ***p<0.001, Mantel-Cox test. 126 Figure 6. The MHC class Ib molecule Qa1 participates in the CD8+ T cell response during MCMV infection. Frequency of (A) IFN-γ+ and (B) TNF-α+ non- classical CD8+ T cells in response to ex vivo stimulation with uninfected or MCMV- infected KbDb-/- BMDCs. CD8+ T cells were enriched >8 weeks post-MCMV infection from KbDb-/- mice. BMDCs were pretreated in the presence of anti-Qa-1, anti-Qa-2, or IgG isotype control antibodies prior to co-culture (n=16). (C) Frequency, (D) absolute number, and (E) representative staining of CD8+ T cells in the spleen, liver, and blood of KbDb-/-Qa-1+/+, KbDb-/-Qa-1+/-, and KbDb-/-Qa-1-/- mice on day 7 post-MCMV infection (n=9-11). Data are pooled from three independent experiments and error bars indicate SEM. *p<0.05 and **p<0.01. 127 Supplementary Figure 1. Non-classical CD8+ T cells are phenotypically similar to conventional CD8+ T cells during acute MCMV infection. (A) Frequency and number of CD8+ T cells in the spleen and liver of KbDb-/- mice on day 0 and day 7 post- MCMV infection (n=12-15). (B) Representative histograms of PLZF and T-bet expression of CD8+ T cells from the spleen and liver of KbDb-/- mice on day 0 (black line) and 7 (grey line) post-MCMV infection. iNKT cells were used as positive control for PLZF (dotted line). Frequency of CD8+ T cells that are (C) CD8αα+ (n=3) or (D) NK1.1+ (n=9) from the spleen and liver of KbDb-/-on day 0 and 7 post-MCMV infection. Data are pooled from at least three (A, D) or one (C) independent experiments. Error bars indicate SEM. *p<0.05 and ****p<0.0001. 128 Supplementary Figure 2. Following acute MCMV infection, non-classical CD8+ T cells acquire an effector phenotype. (A) CD44 and CD62L expression and (B) CD127 and KLRG1 expression of CD8+ T cells from the spleen and liver of KbDb-/- mice on day 0 and 7 post-MCMV infection. (C) Frequency of naïve (CD62L+CD44-), CTL (CD62L-CD44+), and memory (CD62L+CD44+) CD8+ T cells in the spleen and liver of KbDb-/- mice on day 0 and day 7 post-MCMV infection (n=12). (D) Frequency of effector CD8+ T cells (KLRG1+CD127-) in the spleen and liver of KbDb-/- mice on day 0 and day 7 post-MCMV infection (n=15). (E) Frequency of CD8+ T cells that are NKG2A/C/E+ in the spleen and liver of KbDb-/- mice on day 0 and day 7 post-MCMV infection (n=9). Data are pooled from at least three independent experiments. (F) Expression and (G) frequency of CD8+ T cells that are CX3CR1+ and KLRG1+ from the spleen and liver of KbDb-/- mice on indicated day post-MCMV infection (n=3). Data are pooled from at least three (C-E) and one (G) independent experiment. Error bars indicate SEM. **p<0.01 and ****p<0.0001. 129 Supplemental Figure 3. Non-classical CD8+ T cells persist in long-term MCMV- infected KbDb-/- mice and form memory populations. (A) Frequency and (B) number of CD8+ T cells in the spleen and liver from naïve (n=8) and long-term infected KbDb-/- mice (n=15-16). Long-term infected mice were used >8 weeks post-infection (wpi). (C) Representative gating strategy for CD8+ TEFF (KLRG1+CD127-), TEM + + - + + + (CD127 CD44 CD62L ), and TCM (CD127 CD44 CD62L ) cells. (D) Representative CX3CR1 expression from splenic CD8+ TEFF, TEM, and TCM cells from long-term infected KbDb-/- mice. (E) Representative CD8+ T cells in the SMG of long-term infected KbDb-/- and B6 mice. (F) CD103 and CD69 expression of SMG CD8+ T cells from long-term infected KbDb-/- and C57BL/6 mice. Data are representative of two (C-F) or pooled from three (A, B) independent experiments and error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. 130 Supplementary Figure 4. The expansion of non-classical CD8+ T cells in KbDb-/- mice is independent of CD1d, but dependent on B2m expression. (A) Representative staining of CD8+ T cells in the spleen and liver of KbDb-/-.SJL, KbDbCD1d-/-, and B2m-/- mice on day 7 post-MCMV infection. (B) Frequency of CD8+ T cells in the spleen and liver of KbDb-/- or KbDb-/-.SJL and KbDbCD1d-/- mice on day 7 post-MCMV infection (n=7-9). (C) Frequency of CD8+ T cells in the spleen and liver of KbDb-/-or KbDb-/-.SJL and B2m-/- mice on day 7 post-MCMV infection (n=6-12). Data are representative of at least two independent experiments and error bars indicate SEM. ***p<0.001 and ****p<0.0001. 131 Supplementary Figure 5. KbDb-/-Qa1-/- mouse generation and loss of Qa1 signaling effects. (A) Generation of KbDb-/-Qa1-/- mice. (B) GuideRNA (gRNA, blue) targeting to H2-T23 exon 3 and flanking intron. Protospacer adjacent motifs (PAM) are in green, arrows indicate beginning/end of deleted region. (C) Sequence of H2-T23 in KbDb-/-Qa-1-/- mice, compared to wild type. (D) Representative histograms and (E) mean fluorescent intensity (MFI) of Qa1 on CD19+ lymphocytes from the spleen on day 7 post-infection (n=3-4). Data are one of two independent experiments. (F) KLRG1+CD127-, (G) KLRG1+CX3CR1+ non-classical CD8+ T cells, and (H) KLRG1+ NK cells from indicated organs of KbDb-/-Qa-1+/+, KbDb-/-Qa-1+/-, and KbDb-/-Qa-1-/- mice on day 7 post-infection (n=9-11). Data are pooled from three independent experiments. 132 REFERENCES 1. Bate, S.L., S.C. Dollard, and M.J. Cannon, Cytomegalovirus seroprevalence in the United States: the national health and nutrition examination surveys, 1988- 2004. Clin Infect Dis, 2010. 50(11): p. 1439-47. 2. Polic, B., et al., Hierarchical and redundant lymphocyte subset control precludes cytomegalovirus replication during latent infection. J Exp Med, 1998. 188(6): p. 1047-54. 3. Jackson, S.E., G.M. Mason, and M.R. Wills, Human cytomegalovirus immunity and immune evasion. Virus Res, 2011. 157(2): p. 151-60. 4. Grosse, S.D., D.S. Ross, and S.C. Dollard, Congenital cytomegalovirus (CMV) infection as a cause of permanent bilateral hearing loss: a quantitative assessment. J Clin Virol, 2008. 41(2): p. 57-62. 5. Rawlinson, W.D., H.E. Farrell, and B.G. Barrell, Analysis of the complete DNA sequence of murine cytomegalovirus. J Virol, 1996. 70(12): p. 8833-49. 6. Jordan, M.C., J.D. Shanley, and J.G. Stevens, Immunosuppression reactivates and disseminates latent murine cytomegalovirus. J Gen Virol, 1977. 37(2): p. 419-23. 7. Bukowski, J.F., B.A. Woda, and R.M. Welsh, Pathogenesis of murine cytomegalovirus infection in natural killer cell-depleted mice. J Virol, 1984. 52(1): p. 119-28. 8. Biron, C.A., K.S. Byron, and J.L. Sullivan, Severe herpesvirus infections in an adolescent without natural killer cells. N Engl J Med, 1989. 320(26): p. 1731-5. 9. Quinn, M., et al., Memory T cells specific for murine cytomegalovirus re-emerge after multiple challenges and recapitulate immunity in various adoptive transfer scenarios. J Immunol, 2015. 194(4): p. 1726-36. 10. Reddehase, M.J., et al., CD8-positive T lymphocytes specific for murine 133 cytomegalovirus immediate-early antigens mediate protective immunity. J Virol, 1987. 61(10): p. 3102-8. 11. Quinnan, G.V., Jr., et al., Cytotoxic t cells in cytomegalovirus infection: HLA- restricted T-lymphocyte and non-T-lymphocyte cytotoxic responses correlate with recovery from cytomegalovirus infection in bone-marrow-transplant recipients. N Engl J Med, 1982. 307(1): p. 7-13. 12. Walter, E.A., et al., Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor. N Engl J Med, 1995. 333(16): p. 1038-44. 13. Sylwester, A.W., et al., Broadly targeted human cytomegalovirus-specific CD4+ and CD8+ T cells dominate the memory compartments of exposed subjects. J Exp Med, 2005. 202(5): p. 673-85. 14. Stroynowski, I. and K.F. Lindahl, Antigen presentation by non-classical class I molecules. Curr Opin Immunol, 1994. 6(1): p. 38-44. 15. Kjer-Nielsen, L., et al., MR1 presents microbial vitamin B metabolites to MAIT cells. Nature, 2012. 491(7426): p. 717-23. 16. Beckman, E.M., et al., Recognition of a lipid antigen by CD1-restricted alpha beta+ T cells. Nature, 1994. 372(6507): p. 691-4. 17. Wang, C.R., B.E. Loveland, and K.F. Lindahl, H-2M3 encodes the MHC class I molecule presenting the maternally transmitted antigen of the mouse. Cell, 1991. 66(2): p. 335-45. 18. Anderson, C.K. and L. Brossay, The role of MHC class Ib-restricted T cells during infection. Immunogenetics, 2016. 68(8): p. 677-91. 19. Mazzarino, P., et al., Identification of effector-memory CMV-specific T lymphocytes that kill CMV-infected target cells in an HLA-E-restricted fashion. Eur J Immunol, 2005. 35(11): p. 3240-7. 134 20. Pietra, G., et al., HLA-E-restricted recognition of cytomegalovirus-derived peptides by human CD8+ cytolytic T lymphocytes. Proc Natl Acad Sci U S A, 2003. 100(19): p. 10896-901. 21. Geraghty, D.E., et al., Polymorphism at the HLA-E locus predates most HLA-A and -B polymorphism. Hum Immunol, 1992. 33(3): p. 174-84. 22. Grimsley, C., et al., Definitive high resolution typing of HLA-E allelic polymorphisms: Identifying potential errors in existing allele data. Tissue Antigens, 2002. 60(3): p. 206-12. 23. Aldrich, C.J., et al., Identification of a Tap-dependent leader peptide recognized by alloreactive T cells specific for a class Ib antigen. Cell, 1994. 79(4): p. 649- 58. 24. Braud, V., E.Y. Jones, and A. McMichael, The human major histocompatibility complex class Ib molecule HLA-E binds signal sequence-derived peptides with primary anchor residues at positions 2 and 9. Eur J Immunol, 1997. 27(5): p. 1164-9. 25. Lee, N., et al., HLA-E surface expression depends on binding of TAP- dependent peptides derived from certain HLA class I signal sequences. J Immunol, 1998. 160(10): p. 4951-60. 26. Braud, V.M., et al., HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature, 1998. 391(6669): p. 795-9. 27. Lee, N., et al., HLA-E is a major ligand for the natural killer inhibitory receptor CD94/NKG2A. Proc Natl Acad Sci U S A, 1998. 95(9): p. 5199-204. 28. Vance, R.E., et al., Mouse CD94/NKG2A is a natural killer cell receptor for the nonclassical major histocompatibility complex (MHC) class I molecule Qa-1(b). J Exp Med, 1998. 188(10): p. 1841-8. 29. Vance, R.E., A.M. Jamieson, and D.H. Raulet, Recognition of the class Ib 135 molecule Qa-1(b) by putative activating receptors CD94/NKG2C and CD94/NKG2E on mouse natural killer cells. J Exp Med, 1999. 190(12): p. 1801- 12. 30. Vance, R.E., et al., Implications of CD94 deficiency and monoallelic NKG2A expression for natural killer cell development and repertoire formation. Proc Natl Acad Sci U S A, 2002. 99(2): p. 868-73. 31. Moser, J.M., et al., CD94-NKG2A receptors regulate antiviral CD8(+) T cell responses. Nat Immunol, 2002. 3(2): p. 189-95. 32. Wang, E.C., et al., UL40-mediated NK evasion during productive infection with human cytomegalovirus. Proc Natl Acad Sci U S A, 2002. 99(11): p. 7570-5. 33. Tomasec, P., et al., Surface expression of HLA-E, an inhibitor of natural killer cells, enhanced by human cytomegalovirus gpUL40. Science, 2000. 287(5455): p. 1031. 34. Ulbrecht, M., et al., Cutting edge: the human cytomegalovirus UL40 gene product contains a ligand for HLA-E and prevents NK cell-mediated lysis. J Immunol, 2000. 164(10): p. 5019-22. 35. Halenius, A., C. Gerke, and H. Hengel, Classical and non-classical MHC I molecule manipulation by human cytomegalovirus: so many targets-but how many arrows in the quiver? Cell Mol Immunol, 2015. 12(2): p. 139-53. 36. Yunis, J., et al., Murine cytomegalovirus degrades MHC class II to colonize the salivary glands. PLoS Pathog, 2018. 14(2): p. e1006905. 37. Labun, K., et al., CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering. Nucleic Acids Res, 2016. 44(W1): p. W272-6. 38. Montague, T.G., et al., CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res, 2014. 42(Web Server issue): p. W401-7. 39. Hsu, P.D., et al., DNA targeting specificity of RNA-guided Cas9 nucleases. Nat 136 Biotechnol, 2013. 31(9): p. 827-32. 40. Henry, S.C., et al., Enhanced green fluorescent protein as a marker for localizing murine cytomegalovirus in acute and latent infection. J Virol Methods, 2000. 89(1-2): p. 61-73. 41. Orange, J.S., et al., Requirement for natural killer cell-produced interferon gamma in defense against murine cytomegalovirus infection and enhancement of this defense pathway by interleukin 12 administration. J Exp Med, 1995. 182(4): p. 1045-56. 42. Wesley, J.D., et al., Cutting edge: IFN-gamma signaling to macrophages is required for optimal Valpha14i NK T/NK cell cross-talk. J Immunol, 2005. 174(7): p. 3864-8. 43. Perarnau, B., et al., Single H2Kb, H2Db and double H2KbDb knockout mice: peripheral CD8+ T cell repertoire and anti-lymphocytic choriomeningitis virus cytolytic responses. Eur J Immunol, 1999. 29(4): p. 1243-52. 44. Vugmeyster, Y., et al., Major histocompatibility complex (MHC) class I KbDb -/- deficient mice possess functional CD8+ T cells and natural killer cells. Proc Natl Acad Sci U S A, 1998. 95(21): p. 12492-7. 45. Godfrey, D.I., et al., The burgeoning family of unconventional T cells. Nat Immunol, 2015. 16(11): p. 1114-23. 46. Kreslavsky, T., et al., TCR-inducible PLZF transcription factor required for innate phenotype of a subset of gammadelta T cells with restricted TCR diversity. Proc Natl Acad Sci U S A, 2009. 106(30): p. 12453-8. 47. Rahimpour, A., et al., Identification of phenotypically and functionally heterogeneous mouse mucosal-associated invariant T cells using MR1 tetramers. J Exp Med, 2015. 212(7): p. 1095-108. 48. Kovalovsky, D., et al., The BTB-zinc finger transcriptional regulator PLZF 137 controls the development of invariant natural killer T cell effector functions. Nat Immunol, 2008. 9(9): p. 1055-64. 49. Savage, A.K., et al., The transcription factor PLZF (Zbtb16) directs the effector program of the NKT cell lineage. Immunity, 2008. 29(3): p. 391-403. 50. Das, G. and C.A. Janeway, Development of Cd8α/α and Cd8α/β T Cells in Major Histocompatibility Complex Class I–Deficient Mice. The Journal of Experimental Medicine, 1999. 190(6): p. 881-884. 51. Kurepa, Z., J. Su, and J. Forman, Memory phenotype of CD8+ T cells in MHC class Ia-deficient mice. J Immunol, 2003. 170(11): p. 5414-20. 52. Robbins, S.H., et al., Differential regulation of killer cell lectin-like receptor G1 expression on T cells. J Immunol, 2003. 170(12): p. 5876-85. 53. McMahon, C.W., et al., Viral and bacterial infections induce expression of multiple NK cell receptors in responding CD8(+) T cells. J Immunol, 2002. 169(3): p. 1444-52. 54. Gerlach, C., et al., The Chemokine Receptor CX3CR1 Defines Three Antigen- Experienced CD8 T Cell Subsets with Distinct Roles in Immune Surveillance and Homeostasis. Immunity, 2016. 45(6): p. 1270-1284. 55. Tyznik, A.J., et al., Cutting edge: the mechanism of invariant NKT cell responses to viral danger signals. J Immunol, 2008. 181(7): p. 4452-6. 56. Wesley, J.D., et al., NK cell-like behavior of Valpha14i NK T cells during MCMV infection. PLoS Pathog, 2008. 4(7): p. e1000106. 57. Holzapfel, K.L., et al., Antigen-dependent versus -independent activation of invariant NKT cells during infection. J Immunol, 2014. 192(12): p. 5490-8. 58. Szomolanyi-Tsuda, E., et al., Role for TLR2 in NK cell-mediated control of murine cytomegalovirus in vivo. J Virol, 2006. 80(9): p. 4286-91. 59. Tabeta, K., et al., Toll-like receptors 9 and 3 as essential components of innate 138 immune defense against mouse cytomegalovirus infection. Proc Natl Acad Sci U S A, 2004. 101(10): p. 3516-21. 60. Orr, M.T., W.J. Murphy, and L.L. Lanier, 'Unlicensed' natural killer cells dominate the response to cytomegalovirus infection. Nat Immunol, 2010. 11(4): p. 321-7. 61. Bottcher, J.P., et al., Functional classification of memory CD8(+) T cells by CX3CR1 expression. Nat Commun, 2015. 6: p. 8306. 62. French, A.R., et al., Escape of mutant double-stranded DNA virus from innate immune control. Immunity, 2004. 20(6): p. 747-56. 63. Hansen, S.G., et al., Cytomegalovirus vectors violate CD8+ T cell epitope recognition paradigms. Science, 2013. 340(6135): p. 1237874. 64. Ranasinghe, S., et al., Antiviral CD8+ T Cells Restricted by Human Leukocyte Antigen Class II Exist during Natural HIV Infection and Exhibit Clonal Expansion. Immunity, 2016. 45(4): p. 917-930. 65. Ohtsuka, M., et al., Major histocompatibility complex (Mhc) class Ib gene duplications, organization and expression patterns in mouse strain C57BL/6. BMC Genomics, 2008. 9: p. 178. 66. Lebron, J.A., et al., Crystal structure of the hemochromatosis protein HFE and characterization of its interaction with transferrin receptor. Cell, 1998. 93(1): p. 111-23. 67. Liu, Y., et al., The crystal structure of a TL/CD8alphaalpha complex at 2.1 A resolution: implications for modulation of T cell activation and memory. Immunity, 2003. 18(2): p. 205-15. 68. Fischer, K., et al., Mycobacterial phosphatidylinositol mannoside is a natural antigen for CD1d-restricted T cells. Proc Natl Acad Sci U S A, 2004. 101(29): p. 10685-90. 139 69. Bian, Y., et al., MHC Ib molecule Qa-1 presents Mycobacterium tuberculosis peptide antigens to CD8+ T cells and contributes to protection against infection. PLoS Pathog, 2017. 13(5): p. e1006384. 70. Swanson, P.A., 2nd, et al., An MHC class Ib-restricted CD8 T cell response confers antiviral immunity. J Exp Med, 2008. 205(7): p. 1647-57. 71. Kerksiek, K.M., et al., H2-M3-restricted T cells in bacterial infection: rapid primary but diminished memory responses. J Exp Med, 1999. 190(2): p. 195- 204. 72. Kerksiek, K.M., et al., H2-M3-restricted memory T cells: persistence and activation without expansion. J Immunol, 2003. 170(4): p. 1862-9. 73. Khairallah, C., et al., gammadelta T cells confer protection against murine cytomegalovirus (MCMV). PLoS Pathog, 2015. 11(3): p. e1004702. 74. Sell, S., et al., Control of murine cytomegalovirus infection by gammadelta T cells. PLoS Pathog, 2015. 11(2): p. e1004481. 75. Joly, E. and V. Rouillon, The orthology of HLA-E and H2-Qa1 is hidden by their concerted evolution with other MHC class I molecules. Biol Direct, 2006. 1: p. 2. 76. Linehan, J.L., et al., Non-classical Immunity Controls Microbiota Impact on Skin Immunity and Tissue Repair. Cell, 2018. 172(4): p. 784-796 e18. 77. Doorduijn, E.M., et al., T Cells Engaging the Conserved MHC Class Ib Molecule Qa-1(b) with TAP-Independent Peptides Are Semi-Invariant Lymphocytes. Front Immunol, 2018. 9: p. 60. 78. Xu, H.C., et al., Lymphocytes Negatively Regulate NK Cell Activity via Qa-1b following Viral Infection. Cell Rep, 2017. 21(9): p. 2528-2540. 79. Rapaport, A.S., et al., The Inhibitory Receptor NKG2A Sustains Virus-Specific CD8(+) T Cells in Response to a Lethal Poxvirus Infection. Immunity, 2015. 43(6): p. 1112-24. 140 80. Hu, D., et al., Analysis of regulatory CD8 T cells in Qa-1-deficient mice. Nat Immunol, 2004. 5(5): p. 516-23. 81. Davies, A., et al., A peptide from heat shock protein 60 is the dominant peptide bound to Qa-1 in the absence of the MHC class Ia leader sequence peptide Qdm. J Immunol, 2003. 170(10): p. 5027-33. 82. Lo, W.F., et al., Molecular mimicry mediated by MHC class Ib molecules after infection with gram-negative pathogens. Nat Med, 2000. 6(2): p. 215-8. 83. Nagarajan, N.A., F. Gonzalez, and N. Shastri, Nonclassical MHC class Ib- restricted cytotoxic T cells monitor antigen processing in the endoplasmic reticulum. Nat Immunol, 2012. 13(6): p. 579-86. 84. Romagnani, C., et al., HLA-E-restricted recognition of human cytomegalovirus by a subset of cytolytic T lymphocytes. Hum Immunol, 2004. 65(5): p. 437-45. 85. Wilkinson, G.W., et al., Modulation of natural killer cells by human cytomegalovirus. J Clin Virol, 2008. 41(3): p. 206-12. 86. Hansen, S.G., et al., Broadly targeted CD8(+) T cell responses restricted by major histocompatibility complex E. Science, 2016. 351(6274): p. 714-20. 141 CHAPTER 4: EXTRINSIC CYTOKINE SIGNALING AFFECTS INKT CELL ACTIVATION FOLLOWING MURINE CYTOMEGALOVIRUS INFECTION 142 Extrinsic cytokine signaling affects iNKT cell activation following murine cytomegalovirus infection Courtney K. Anderson1 , S.M. Shahjahan Miah1, and Laurent Brossasy1 1 Division of Biology and Medicine, Department of Molecular Microbiology and Immunology, Brown University, Providence, RI 02912 143 ABSTRACT Natural killer T (NKT) cells are an innate-like population characterized by their recognition of lipid antigens and rapid cytokine production following activation. Unlike conventional T cell populations, which require TCR ligation, iNKT cells are also stimulated through inflammatory cytokines. This feature allows iNKT cells to respond during viral infections in the absence of virally-derived antigens, through intermediary activation of dendritic cells (DCs). Using the well-characterized murine model of cytomegalovirus infection (MCMV), we sought to definitively examine the cytokine signaling required for iNKT cell activation. Using conditionally deficient mouse lines, we investigate how engagement of the IL-12R and MyD88 signaling affect the iNKT cell response to MCMV. MyD88 is a downstream adaptor of toll-like receptors (TLRs) and the IL-1R/IL-18R family. Previous work illustrates the importance of IL-12, however there are contrasting results for the involvement of IL-18 and type I interferons (IFN). We find that IL-12 signaling is necessary, however there is also an organ-specific requirement of MyD88 signaling. MyD88-deficient hepatic iNKT cells are unaffected by loss of TLR and IL-1R/IL-18R stimulation, whereas splenic iNKT cells are hyporesponsive. We also find that iNKT cells are insufficient to protect against MCMV- induced lethality. 144 Introduction CD1d-restricted T cells are a unique innate-like family of T cells activated by lipid antigens, rather than traditional peptides [1]. They are referred to as natural killer T (NKT) cells, owing to their expression of NK cell markers, such as NK1.1, and a T cell receptor (TCR). Type I NKT cells, are also referred to as iNKT cells because they have a limited TCR repertoire with a single α-chain paired to a limited number of β-chains. In mice, Vα14-Jα18 preferentially pairs with Vβ2, Vβ7, or Vβ8.2 and in humans, Vα24-Jα18 pairs with Vβ11 [2-4]. Type II NKT cells express a more diverse TCR repertoire [5]. iNKT cells undergo rapid activation and cytokine production upon interaction with a strong agonist, such as the glycolipid α-galactosylceramide (α-GalCer) [6, 7]. This is partly due to expression of preformed IFN-γ and IL-4 transcripts [8, 9]. iNKT cells are also uniquely activated independently of the TCR by inflammatory cytokines (e.g. IFN- α/β, IL-12, and IL-18) or in combination with weak antigens [10, 11]. This cytokine- dependent activation requires DCs stimulated through TLR engagement [12-14]. The ability to get activated by cytokines without TCR-ligand engagement allows iNKT cells to participate during viral infections [15, 16]. Murine cytomegalovirus (MCMV) is a well-characterized model of viral infection that remains latent in cells following clearance. The acute cytotoxic response against infected cells is led by natural killer (NK) cells, which are large producers of IFN-γ, perforin, and granzymes [17, 18]. Previous work has also investigated the factors necessary for iNKT cell activation during MCMV infection, but relied on global knockout mouse lines and in vitro studies [15, 16]. We previously found that IL-12, and to a lesser extent IFN-α/β, is necessary for iNKT cell activation and cytokine production, but that CD1d was dispensable [15]. Others showed that IL-12 and IL-18 were required for in vitro activation of iNKT cells, but only IL-12 in vivo [16]. We wanted to more accurately establish the importance and contributions of iNKT cells during MCMV infection. To 145 elucidate this, we used mice conditionally deficient for either IL-12Rβ2 or MyD88 signaling in the T cell lineage. In addition to the required role of IL-12, we find that there is organ specificity in the requirement of MyD88. Loss of signaling through MyD88 is necessary for the optimal cytokine response of splenic iNKT cells. However, this was not a result of an intrinsic defect in cytokine production. Furthermore, unlike conventional T cells, iNKT cells are not sufficient to protect immunocompromised mice from MCMV-induced lethality. 146 MATERIALS & METHODS Mice. IL-12Rβ2fl/fl mice were generated at the Brown Transgenic Facility using embryonic stem cells purchased from the KOMP Repository. MyD88fl/fl, eYFP, CD4cre, and RAG1-/- mice were purchased from Jackson Laboratory. RAG1-/- mice were maintained in-house. IL-12Rβ2fl/flCD4cre+/- and MyD88fl/fleYFP+/-CD4cre+/- mice were generated and maintained in-house. Balb/c mice were purchased from Taconic. Viruses and infection protocols. MCMV-RVG102 (referred to as MCMV) was a gift from Dr. John Hamilton (Duke University), and expresses recombinant EGFP under the immediate early-1 promoter [19]. Infections were performed with 5 x 104 or 1 x 105 PFU i.p. Virus stocks were prepared from salivary gland homogenate [17] and viral titers were determined via standard plaque assay using mouse embryonic fibroblast (MEF) cells. α-GalCer treatment. Mice were treated with 2µg of α-GalCer (KRN7000, Avanti Polar Lipids, Inc.) in 0.5% PBS-Tween i.p. for two hours. Controls were treated with 0.5% PBS-Tween i.p. Lymphocyte isolation. Spleens were dissociated in 1% PBS-serum, filtered through nylon mesh, and underlayed with lympholyte-M (Cedarlane Laboratories). Alternatively, spleens were dissociated in 150 mM NH4Cl for 10 minutes, filtered through nylon mesh, and washed once with 1% PBS-serum. Livers were profused with 1% PBS-serum, dissociated using GentleMACS program E0.1 (Miltenyi Biotech) and passed through nylon mesh. Samples were washed three times in 1% PBS-serum and overlayed onto a two-step discontinuous Percoll gradient (GE Healthcare Bio-Sciences). Samples were filtered through nylon mesh, washed once in 1% PBS-serum, and underlayed with 147 Lympholyte-M. Gradients were centrifuged at 2500 RPM for 20 minutes at room temperature. Blood was collected from a cardiac puncture into heparin-containing tubes. Red blood cells were lysed in 150 mM NH4Cl for 10 minutes and washed twice in 1% PBS-serum. Survival studies. Approximately 50,000 NKT cells were sorted from naïve or long-term infected B6 spleens and adoptively transferred per RAG1-/- recipient. Prior to sorting, pooled spleens were enriched for iNKT cells using an AutoMACS (Miltenyi Biotech) and CD8α and CD19 MicroBeads (Miltenyi Biotech) to deplete CD8+ cells and B cells. Two hours post-injection, recipients were infected with MCMV. Antibodies and flow cytometry. Cells were stained in 1% PBS-serum containing 2.4G2 and cell surface antibodies for 20 minutes on ice in the dark. Staining involving CD1d tetramer (NIH Tetramer Facility) was performed for 15 minutes at room temperature and 15 minutes on ice in the dark. For intracellular staining, cells were fixed using Cytofix/Cytoperm (BD Biosciences) for 30 minutes and stained in Perm/Wash Buffer (BD Biosciences) for 30 minutes. For intranuclear staining, cells were fixed using Fixation/Permeabilization Solution (eBioscience) for 30 minutes and then stained in Permeabilization Buffer (eBioscience) for 30 minutes. To maintain eYFP expression during intranuclear staining, samples were prefixed with fresh 4% PFA (Electron Microscopy Science) for 15 minutes, fixed using Fixation/Permeabilization Solution 2 (Miltenyi Biotech) for 40 minutes, incubated with 1% Triton X-100 for 15 minutes, and then stained for 30 minutes in PBS. Samples were run on a FACSAria III (BD Biosciences) or MACSQuant (Miltenyi Biotech) and analyzed using FlowJo (Tree Star Inc.). The antibodies listed below were used for flow cytometry and purchased from BioLegend, eBioscience (now Thermo Fischer Scientific) or BD Biosciences: CD4 – 148 APC, CD4 – BV570, CD8 – BV605, CD19 – PE, CD69 – PerCP-Cy5.5, IFN-γ – PE, IL-4 – PE-Cy7, PLZF – PE, RORγt – PerCP-eF710, T-bet – PE-Cy7, TCRβ – BV510, TCRβ – FITC, TCRβ – PerCP-Cy5.5. Western blot. Protein lysate was run on a 4-20% Mini-Protean TGX gel (BioRad) and transferred onto a nitrocellulose membrane (BioRad). Incubated overnight at 4 °C with purified anti-MyD88 antibody (ProSci) followed by peroxidase-conjugated donkey anti- rabbit (Jackson ImmunoReseach) and Precision Protein StrepTactin-HRP Conjugate (BioRad). Imaged following incubation with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Membrane was stripped and incubated overnight at 4 °C with purified anti-β-actin followed by peroxidase-conjugated donkey anti-mouse (Jackson ImmunoResearch) and Precision Protein StrepTactin-HRP Conjugate (BioRad). Statistical analysis. Statistical analyses were performed with Prism 7.0 (Graph-Pad Software, Inc.). Unpaired two-tailed Student’s t-tests were used to compare two individual groups. Log-rank (Mantel Cox) tests were used for survival studies. Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. 149 RESULTS IL-12Rβ2 and MyD88 signaling are dispensable for iNKT cell development To better characterize the roles of IL-12 and MyD88 on iNKT cell activation during MCMV infection, we used two conditionally deficient mouse lines. We generated IL-12Rβ2 conditional knockouts (cKO) by crossing newly created IL-12Rβ2fl/fl mice with a line expressing Cre recombinase driven by the CD4 promoter (CD4cre); this allows for deletion at the DN4/DP stage of T cell development [20, 21]. IL-12Rβ2fl/fl CD4cre+/- mice (referred to as IL-12Rβ2 cKO) have comparable T cell development and peripheral iNKT cell populations to littermate controls (IL-12Rβ2fl/+ CD4cre+/-, referred to as IL-12Rβ2 control) (Fig. S1A, B). iNKT cells are further differentiated into four main subsets, based on their transcription factor requirements and cytokine responses [22]. Different strains of mice are enriched for certain iNKT cell lineages, for example C57BL/6 mice predominantly have NKT1 cells in the spleen and liver, while BALB/c mice also have robust populations of NKT2 and NKT17 cells [22]. IL-15 and IL-7 are important for differentiation into NKT1 and NKT17, respectively [23-26]. Loss of IL-12 signaling does not affect the ratio of NKT1, NKT2, and NKT17 cells in IL-12Rβ2 cKO mice (Fig. S1C, D). A BALB/c control was used to establish the gating for these experiments. To investigate the role of TLR, IL-1R, and IL-18R signaling we generated mice conditionally deficient for MyD88. MyD88 is an adaptor protein that signals downstream of all TLRs (except TLR3), and the IL-1R/IL-18R superfamily [27]. MyD88 cKOs were made by crossing MyD88fl/fl mice with CD4Cre expressing mice. In addition, we also crossed to a line expressing enhanced yellow fluorescent protein (eYFP) in the Rosa locus. Rosa-eYFP is surrounded by loxP-flanked stop sequences, so it acts as a reporter of Cre recombinase activity [28]. IL12rb2 and Rosa-eYFP are both on Chromosome 6, which prohibits the use of this tool for IL-12Rβ2 cKO mice. As anticipated, western blot confirms T cell-specific loss of Myd88 protein expression in 150 MyD88 cKOs, compared to littermate controls (Fig. S2A). eYFP expression also effectively labels over 95% of T cells and iNKT cells (Fig. S2B). Similarly to what we find for IL-12Rβ2 cKO mice, MyD88 cKOs have normal T cell development and iNKT cell populations in the thymus, as well as the periphery (Fig. S2C-F). Loss of IL-12Rβ2 signaling has major effects on iNKT cell activation following MCMV infection, while MyD88 signaling has organ-specific effects. We next wanted to investigate the iNKT cell-specific response to MCMV in the absence of the IL-12R and MyD88. Our lab and others have shown that IL-12 in particular, but also IFN-α/β and IL-18, are important for optimal iNKT cell activation [15, 16]. The peak of the iNKT cell response is day 1.5 post-infection, characterized by robust IFN-γ production in the spleen and liver, but not IL-4 [15]. At 36 hours post- infection, there are comparable frequencies of iNKT cells in the spleen and liver of IL- 12Rβ2 cKO and control mice, however the IFN-γ response is completely abolished (Fig. 1A, B). This is further substantiated by a decrease in CD69 expression (Fig. 1C). The NK cell response however, is unaffected at this time-point (Fig. 1D). Loss of MyD88 signaling also does not affect iNKT cell frequencies in the spleen or liver 36 hours post-infection (Fig. 1E). However, splenic iNKT cells are hyporesponsive in MyD88 cKO mice (Fig. 1F). This impairment results in approximately half the amount of IFN-γ produced by littermate controls, but does not coincide with decreased CD69 expression (Fig. 1G). In contrast, hepatic iNKT cells are unaffected in MyD88 cKOs (Fig. 1F-G). NK cells also exhibit normal IFN-γ production (Fig. 1H). However, iNKT cells from IL-12Rβ2 cKO and MyD88 cKO mice produce robust IFN-γ and IL-4 production following a 2 hour stimulation with α-GalCer (Fig. 2A, B). This illustrates that the decreased cytokine responses of iNKT cells from IL-12Rβ2 cKO and MyD88 cKO mice are not a result of an intrinsic defect. 151 iNKT cells are not sufficient to protect immunocompromised mice from MCMV- induced lethality MCMV is highly adept at immune evasion, and a multilayered immune response is necessary for maintaining latency and keeping virus infection in check. In the absence of the adaptive immune system, NK cells are unable to sufficiently control viral replication, due to viral mutants escaping Ly49H recognition [29]. As a result, RAG1-/- mice all succumb to MCMV infection around two to four weeks post-infection [29]. However, adoptive transfer of memory CD8+ T cells, or γδ T cell from previously infected mice, reverses lethality [30-32]. γδ T cells, similarly to iNKT cells, are generally considered an innate-like T cell population. However, only γδ T cells from previously infected animals elicit this protective response [31, 32]. We were interested to see whether iNKT cells are also able to reverse MCMV-induced lethality. iNKT cells were obtained from either naïve or long-term infected C57BL/6 mice and 5 x 104 were adoptively transferred per RAG1-/- recipient. However in contrast to γδ T cells, neither naïve nor activated iNKT cells are sufficient for protection (Fig. 3A). 152 DISCUSSION Our results illustrate that IL-12 signaling is absolutely necessary for iNKT cell activation during MCMV infection, which is consistent with work using IL-12-/- mice [15, 16]. We also find that iNKT cell IFN-γ production is differentially affected in MyD88 cKO mice. Splenic iNKT cells are hyporesponsive, while hepatic iNKT cells have normal IFN- γ. These data illustrate that iNKT cells in the liver, do not require TLR or IL-1R/IL-18R engagement. In contrast, a component of this pathway is necessary for appropriate cytokine production in the spleen. Previous work using IL-18-deficient mice saw no effect on IFN-γ production by splenic iNKT cells [16]. However, in addition to the loss of IL-18 signaling, iNKT cells from MyD88 cKO mice also lack IL-1R and TLR signaling (with the exception of TLR3), which could explain these discordant results. Whether or not iNKT cells actually express TLRs, or are directly stimulated by their ligands, remains controversial. Human iNKT cells express all TLRs, except TLR8, but are not activated in the presence of any TLR ligands [33]. Others find that murine iNKT cells express TLR2 and TLR4, and are capable of activation through their ligands [34, 35]. The expression of TLRs by murine iNKT cells is also upregulated following TCR-activation, which enhances stimulation [36]. Myd88 is also downstream of the IL-33R (another member of the IL-1R superfamily), which has relatively recently been better characterized. IL-33 signaling alone, and more so alongside IL-12, activates iNKT cells to produce IFN-γ [37]. Interestingly, IL-33 also amplifies NK cell expansion following MCMV infection [38]. Whether or not loss of IL-1, IL-33, or TLR signaling by splenic iNKT cells is the cause of their hyporesponsive phenotype in MyD88 cKOs requires further elucidation. After α-GalCer stimulation, iNKT cells produce large amounts IFN-γ and IL-4, and rapidly expand [6, 7]. However, even though iNKT cells become activated and have robust IFN-γ production following MCMV infection, they do not proliferate [15]. This could potentially affect the number of cells required for protection. In fact, when γδ T 153 cells were shown to reverse MCMV-induced lethality, 5 to 20 times more cells were used [31, 32]. γδ T cells also proliferate following MCMV infection [31]. In contrast, we utilized a comparable number to what was reported for conventional CD8+ T cells [30]. iNKT cells do contribute to the overall immune response following MCMV infection. For instance, NK cells are impaired in the absence of iNKT cells [15]. However, whether or not iNKT cells are sufficient to protect immunocompromised mice from virus-induced lethality, or substitute for classical members of the adaptive immune system, is unclear. 154 ACKNOWLEDGEMENTS We thank Kevin Carlson for cell sorting and Céline Fugère for i.v. injections. In addition, we would like to acknowledge Dr. Christian Vosshenrich (The Pasteur Institute) for sharing his protocol for preserving fluorescent protein expression during intranuclear staining. 155 Figure 1. Loss of IL-12Rβ2 signaling has major effects on iNKT cell activation following MCMV infection, while MyD88 signaling MyD88 signaling has organ- specific effects. (A) Frequency of iNKT cells, (B) IFN-γ+ iNKT cells, (C) CD69+ iNKT cells, and (D) IFN-γ+ NK cells from the spleen and liver of IL-12Rβ2 control and cKO mice at 36 hours post-infection with MCMV. Data are pooled from one (C; n=3-5) or two independent experiments (A, B, D; n=6-8). (F) Frequency of iNKT cells, (F) IFN-γ+ iNKT cells, (G) CD69 MFI of iNKT cells, and (H) IFN-γ+ NK cells from the spleen and liver of MyD88 control and cKO mice at 36 hours post-infection with MCMV. Data are pooled from three independent experiments (E, F, H; n=11-12) or one representative of three independent experiments (G; n=4-6). Error bars indicate SEM and *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. 156 Figure 2. Loss of IL-12Rβ2 or MyD88 signaling does not affect iNKT cell activation following α-GalCer stimulation. Frequency of IFN- γ+ and IL-4+ iNKT cells from the spleen and liver of (A) IL-12Rβ2 control and cKO mice and (B) MyD88 control and cKO mice 2 hours post-stimulation with α-GalCer. Data are pooled from two independent experiments (n=5-6). Error bars indicate SEM. 157 Figure 3. iNKT cells are insufficient to protect RAG1-/- mice from MCMV-induced lethality. (A) Survival curve of RAG1-/- mice controls (---) or those that received 50,000 iNKT cells from naïve (- -) or long-term infected (L.T.I) C57BL/6 mice (⎯). Cells were sorted from C57BL/6 mice >8 weeks post-infection. Data are representative of one experiment (n=5). 158 Supplementary Figure 1. IL-12Rβ2 signaling is dispensable for T cell development and peripheral iNKT cell populations. (A) The CD4-CD8- double negative (DN), CD4+CD8+ double positive (DP), and CD4+ or CD8 + single positive (SP) stages of T cell development in the thymus of IL-12Rβ2 control and cKO mice (n=8). (B) Frequency of iNKT cells in indicated organs from IL-12Rβ2 control and cKO mice (n=3-8). (C) Frequency and (D) representative staining of NKT1, NKT2, and NKT17 lineages from the thymus of IL-12Rβ2 control and cKO mice, compared to a BALB/c control (n=8). Data are representative of two independent experiments. Error bars indicate SEM and *p<0.05. 159 Supplementary Figure 2. MyD88 signaling is dispensable for T cell development and peripheral iNKT cell populations. (A) Western blot indicating MyD88 cKO mice lack expression of MyD88 in the T cell lineage only, compared to control animals. (B) Representative eYFP expression of total splenocytes and iNKT cells. (C) The CD4-CD8- double negative (DN), CD4+CD8+ double positive (DP), and CD4+ or CD8 + single positive (SP) stages of T cell development in the thymus of MyD88 control and cKO mice (n=9). (D) Frequency of iNKT cells in indicated organs from MyD88 control and cKO mice (n=4). (C) Frequency and (D) representative staining of NKT1, NKT2, and NKT17 lineages from the thymus of MyD88 control and cKO mice, compared to a BALB/c control. Gated on eYFP+ iNKT cells (n=4). Data are representative of at least two independent experiments (A-C) or one experiment (D-F). Error bars indicate SEM. 160 Supplementary Figure 3. Loss of IL-12Rβ2 signaling, but not MyD88 signaling, affects iNKT cell activation following MCMV infection. Representative staining of (A) IFN-γ+ iNKT cells and (B) IFN-γ+ NK cells from the spleen and liver of of IL-12Rβ2 control and cKO mice at 36 hours post-infection with MCMV. Representative staining of (C) IFN- γ+ iNKT cells and (D) IFN-γ+ NK cells from the spleen and liver of MyD88 control and cKO mice at 36 hours post-infection with MCMV. Data are representative of two (A, B) or three (C, D) independent experiments. 161 REFERENCES 1. Beckman, E.M., et al., Recognition of a lipid antigen by CD1-restricted alpha beta+ T cells. Nature, 1994. 372(6507): p. 691-4. 2. Dellabona, P., et al., An invariant V alpha 24-J alpha Q/V beta 11 T cell receptor is expressed in all individuals by clonally expanded CD4-8- T cells. J Exp Med, 1994. 180(3): p. 1171-6. 3. Lantz, O. and A. Bendelac, An invariant T cell receptor alpha chain is used by a unique subset of major histocompatibility complex class I-specific CD4+ and CD4-8- T cells in mice and humans. J Exp Med, 1994. 180(3): p. 1097-106. 4. Porcelli, S., et al., Analysis of T cell antigen receptor (TCR) expression by human peripheral blood CD4-8- alpha/beta T cells demonstrates preferential use of several V beta genes and an invariant TCR alpha chain. J Exp Med, 1993. 178(1): p. 1-16. 5. Macho-Fernandez, E. and M. Brigl, The Extended Family of CD1d-Restricted NKT Cells: Sifting through a Mixed Bag of TCRs, Antigens, and Functions. Front Immunol, 2015. 6: p. 362. 6. Crowe, N.Y., et al., Glycolipid antigen drives rapid expansion and sustained cytokine production by NK T cells. J Immunol, 2003. 171(8): p. 4020-7. 7. Wilson, M.T., et al., The response of natural killer T cells to glycolipid antigens is characterized by surface receptor down-modulation and expansion. Proc Natl Acad Sci U S A, 2003. 100(19): p. 10913-8. 8. Matsuda, J.L., et al., Mouse V alpha 14i natural killer T cells are resistant to cytokine polarization in vivo. Proc Natl Acad Sci U S A, 2003. 100(14): p. 8395- 400. 9. Stetson, D.B., et al., Constitutive cytokine mRNAs mark natural killer (NK) and NK T cells poised for rapid effector function. J Exp Med, 2003. 198(7): p. 1069- 162 76. 10. Reilly, E.C., J.R. Wands, and L. Brossay, Cytokine dependent and independent iNKT cell activation. Cytokine, 2010. 51(3): p. 227-31. 11. Kumar, A., et al., Natural Killer T Cells: An Ecological Evolutionary Developmental Biology Perspective. Front Immunol, 2017. 8: p. 1858. 12. Paget, C., et al., Activation of invariant NKT cells by toll-like receptor 9-stimulated dendritic cells requires type I interferon and charged glycosphingolipids. Immunity, 2007. 27(4): p. 597-609. 13. Cohen, N.R., et al., Innate recognition of cell wall beta-glucans drives invariant natural killer T cell responses against fungi. Cell Host Microbe, 2011. 10(5): p. 437-50. 14. Tyznik, A.J., et al., Cutting edge: the mechanism of invariant NKT cell responses to viral danger signals. J Immunol, 2008. 181(7): p. 4452-6. 15. Wesley, J.D., et al., NK cell-like behavior of Valpha14i NK T cells during MCMV infection. PLoS Pathog, 2008. 4(7): p. e1000106. 16. Tyznik, A.J., et al., Distinct requirements for activation of NKT and NK cells during viral infection. J Immunol, 2014. 192(8): p. 3676-85. 17. Orange, J.S., et al., Requirement for natural killer cell-produced interferon gamma in defense against murine cytomegalovirus infection and enhancement of this defense pathway by interleukin 12 administration. J Exp Med, 1995. 182(4): p. 1045-56. 18. Loh, J., et al., Natural killer cells utilize both perforin and gamma interferon to regulate murine cytomegalovirus infection in the spleen and liver. J Virol, 2005. 79(1): p. 661-7. 19. Henry, S.C., et al., Enhanced green fluorescent protein as a marker for localizing murine cytomegalovirus in acute and latent infection. J Virol Methods, 2000. 163 89(1-2): p. 61-73. 20. Sawada, S., et al., A lineage-specific transcriptional silencer regulates CD4 gene expression during T lymphocyte development. Cell, 1994. 77(6): p. 917-29. 21. Lee, P.P., et al., A critical role for Dnmt1 and DNA methylation in T cell development, function, and survival. Immunity, 2001. 15(5): p. 763-74. 22. Lee, Y.J., et al., Steady-state production of IL-4 modulates immunity in mouse strains and is determined by lineage diversity of iNKT cells. Nat Immunol, 2013. 14(11): p. 1146-54. 23. Castillo, E.F., et al., Thymic and peripheral microenvironments differentially mediate development and maturation of iNKT cells by IL-15 transpresentation. Blood, 2010. 116(14): p. 2494-503. 24. Gordy, L.E., et al., IL-15 regulates homeostasis and terminal maturation of NKT cells. J Immunol, 2011. 187(12): p. 6335-45. 25. Watarai, H., et al., Development and function of invariant natural killer T cells producing T(h)2- and T(h)17-cytokines. PLoS Biol, 2012. 10(2): p. e1001255. 26. Webster, K.E., et al., IL-17-producing NKT cells depend exclusively on IL-7 for homeostasis and survival. Mucosal Immunol, 2014. 7(5): p. 1058-67. 27. Adachi, O., et al., Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity, 1998. 9(1): p. 143-50. 28. Srinivas, S., et al., Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev Biol, 2001. 1: p. 4. 29. French, A.R., et al., Escape of mutant double-stranded DNA virus from innate immune control. Immunity, 2004. 20(6): p. 747-56. 30. Quinn, M., et al., Memory T cells specific for murine cytomegalovirus re-emerge after multiple challenges and recapitulate immunity in various adoptive transfer scenarios. J Immunol, 2015. 194(4): p. 1726-36. 164 31. Sell, S., et al., Control of murine cytomegalovirus infection by gammadelta T cells. PLoS Pathog, 2015. 11(2): p. e1004481. 32. Khairallah, C., et al., gammadelta T cells confer protection against murine cytomegalovirus (MCMV). PLoS Pathog, 2015. 11(3): p. e1004702. 33. Moreno, M., et al., Differential indirect activation of human invariant natural killer T cells by Toll-like receptor agonists. Immunotherapy, 2009. 1(4): p. 557-70. 34. Kim, J.H., et al., Direct engagement of TLR4 in invariant NKT cells regulates immune diseases by differential IL-4 and IFN-gamma production in mice. PLoS One, 2012. 7(9): p. e45348. 35. Askenase, P.W., et al., TLR-dependent IL-4 production by invariant Valpha14+Jalpha18+ NKT cells to initiate contact sensitivity in vivo. J Immunol, 2005. 175(10): p. 6390-401. 36. Kulkarni, R.R., et al., Costimulatory activation of murine invariant natural killer T cells by toll-like receptor agonists. Cell Immunol, 2012. 277(1-2): p. 33-43. 37. Bourgeois, E., et al., The pro-Th2 cytokine IL-33 directly interacts with invariant NKT and NK cells to induce IFN-gamma production. Eur J Immunol, 2009. 39(4): p. 1046-55. 38. Nabekura, T., J.P. Girard, and L.L. Lanier, IL-33 receptor ST2 amplifies the expansion of NK cells and enhances host defense during mouse cytomegalovirus infection. J Immunol, 2015. 194(12): p. 5948-52. 165 CHAPTER 5: DISCUSSION AND FUTURE DIRECTIONS 166 The line that separates the innate and adaptive branches of the immune system is continuously blurring. This is in part, due to discoveries like adaptive features of NK cells and innate-like capabilities of certain T cells [1-4]. Non-classical T cells are restricted by MHC class Ib molecules, which often present restricted peptide repertoires and/or atypical antigens, e.g. glycolipids, vitamin metabolites, and formylated peptides. It is evident that their development and functions often require unique intrinsic signaling events or transcription factors from conventional T cells. The protective capabilities of non-classical T cells are also becoming more appreciated. Since MHC class Ib molecules are non-polymorphic, compared to the highly polymorphic MHC class Ia molecules, non-classical T cells represent novel targets for more effective vaccine coverage. Despite current progress, overall MHC class Ib-restricted T cells remain poorly characterized. This is in part due to the difficulty of identifying different non- classical T cell populations amidst conventional T cells. The work described in this thesis makes strides in adding to our understanding of MHC class Ib-restricted T cells. First we evaluate how loss of SHIP1, a 5’-phosphatase that negatively regulates the PI3K pathway, affects the development and functions of iNKT cells. In addition, we investigate the roles of non-classical T cells restricted by MHC class Ib and MHC class I-like molecules during murine cytomegalovirus (MCMV) infection. MCMV is a well-characterized animal model to study the immune response of human CMV (HCMV), a ubiquitous pathogen with serious morbidity and mortality in immunocompromised patients and neonates. The conclusions, current experiments, and future directions for these studies are individually discussed below: THE ROLE OF SHIP1 IN INKT CELL DEVELOPMENT AND FUNCTIONS Previous work illustrates both intrinsic and extrinsic requirements for SHIP1 by T cells and NK cells. SHIP1 is necessary for appropriate CD4+ T cell TH1/TH2 167 differentiation and CD8+ T cell cytotoxicity, and for the terminal maturation and cytokine production of NK cells [5, 6]. iNKT cells have characteristics of both T cells and NK cells, including a number of inhibitory receptors found on NK cells, such as CD94/NKG2A and Ly49A, C, I, and G2 [7]. Since SHIP1 acts downstream of ITIM (immunoreceptor tyrosine-based inhibitory motif) containing receptor tails, we originally hypothesized that loss of SHIP1 would severely impact iNKT cell development, and result in their over activation to stimulation. Even thought iNKT cell populations in the thymus and periphery are decreased in SHIP1-/- mice, this is likely a result of the pleiotropic effects created by myeloid cell hyperproliferation and activation. Experiments using mixed bone marrow chimera and T cell-specific loss of SHIP1 (SHIP1fl/flCD4cre mice), revealed unaffected T cell development and normal iNKT cell numbers. This shows that SHIP1 is not required for iNKT cells progressing through development in the thymus, but also not on the CD1d-expressing CD4+CD8+ double positive cells that positively select iNKT cells. These data parallel the extrinsic role of SHIP1 on NK and T cells [5, 6]. Interestingly, we also establish that iNKT cells are hyporesponsive in the absence of SHIP1, rather than hyperactivated as originally theorized. iNKT cells from SHIP1-/- mice have decreased IFN-γ, TNF-α, IL-4, and IL-17A production following TCR cross-linking with CD3/CD28. PMA and Ionomycin treatment, which bypass the TCR, also results in decreased IFN-γ and TNF-α. Using iNKT cells isolated from mixed bone marrow chimeras, we still observe decreased IFN-γ, TNF-α, and IL-4 levels following TCR cross-linking, but IL-17A is restored. Taken together these data indicate that SHIP1-deficient iNKT cells are defective in their production of TH1 and TH2 cytokines, but not TH17. This is in contrast to CD4+ T cells, which only have decreased TH2 cytokine polarization in the absence of SHIP1 [5]. Unfortunately, these studies are limited by the severe lung disease and early demise of SHIP1-/- mice. This makes it impossible to 168 investigate how loss of SHIP1 signaling affects the iNKT cell response during infection or α-GalCer stimulation. The defects observed in iNKT cells are counterintuitive, considering SHIP1 is a negative regulator of PI3K signaling. Our results however, are reminiscent of the defects observed by SHIP1-deficient NK cells and T cells, which demonstrate SHIP1 enhances cytokine production. The role of SHIP1 may be masked or rescued by other phosphatases, such as PTEN, SHIP-2, or s-SHIP. s-SHIP was originally thought to only be expressed in embryonic tissues, but actually has much wider expression, including hematopoietic cells [8]. Loss of both SHIP-1 and s-SHIP exacerbates myeloid cell defects [8]. It would be interesting to determine whether loss of both forms of SHIP1 have additional intrinsic roles on iNKT cells or if their activation and cytokine production are more affected. The tyrosine phosphatases SHP-1 and SHP-2 (SH2 domain- containing phosphatase-1 and -2) are also recruited to the ITIMs of inhibitory receptors and negatively regulate signaling events from the cell surface [9]. Our lab has recently shown that SHP-2, as well as SHP-1, is dispensable for iNKT cell development and effector functions [10]. Since none of these phosphatases on their own dramatically influences iNKT cell development or activation, it is likely that they have redundant roles. Mice that are deficient in more than one phosphatase are required to elucidate these compensatory roles. THE MHC CLASS IB-RESTRICTED CD8+ T CELL RESPONSE TO MCMV Aside from iNKT cells, non-classical T cells are not known to respond to MCMV. Here, we describe a novel population of Qa-1-restricted and non-Qa-1-restricted non- classical CD8+ T cells participating during infection. By using KbDb-/- mice, which lack MHC class Ia-restricted CD8+ T cells, we are able to exclusively examine non-classical T cells without them being masked by the conventional T cell response. Instead of 169 behaving like innate-like T cells, MCMV-expanded non-classical CD8+ T cells act like conventional T cells; the main difference being their recognition of MHC class Ib- presented antigens, rather than MHC class Ia. This is supported by the kinetics of their acute response and ability to form memory T (TM) cell populations, which is highly unusual for MHC class Ib-restricted T cells. Non-classical TM cells also have more robust responses than naïve cells during secondary infection, including enhanced proliferation and KLRG1 expression. Strikingly, TM cells are also sufficient to protect immunocompromised mice (RAG1KbDb-/-) from MCMV-induced lethality when adoptively transferred prior to infection. This perhaps best illustrates that non-classical CD8+ T cells possess protective capabilities. MCMV mutates to avoid the Ly49H-mediated NK cell response, which is ultimately what leads to lethality in RAG-deficient animals [11]. However, these data illustrate that non-classical CD8+ TM cells offer protection in the absence of NK cells and other adaptive immune cells. Due to the exquisite immune evasion capabilities of CMV, we believe non-classical CD8+ T cells could substitute for the classical CD8+ T cell response. We also determine that Qa-1 acts as a primary restriction molecule for this population, using KbDb-/-Qa-1-/- mice. KbDb-/-Qa-1-/- mice were generated through CRISPR/Cas9 gene editing of KbDb+/- zygotes. Injections with CRISPR/Cas9 and two selected guideRNAs (gRNA) were performed at UC Berkeley, however we performed the gRNA design and founder genotyping. Qa-1 signaling is complicated by its ability to interact with multiple types of immune cell receptors. KbDb-/-Qa-1-/- mice lack Qa-1- restricted cytotoxic and suppressor CD8+ T cells, as well as CD94/NKG2A engagement on NK cells and activated CD8+ T cells. Loss of the inhibitory signal from Qa-1 and CD94/NKG2A engagement leads to enhanced NK and CD8+ T cell activation and cytotoxicity during multiple infection models [12-14]. Interestingly, KbDb-/-Qa-1-/- mice have severely reduced CD8+ T cell numbers during acute MCMV infection and the 170 remaining population is less activated. Since NK cells are unaffected at this time-point, we believe that loss of Qa-1-TCR signaling is the cause, rather than decreased CD94/NKG2A engagement. This is further supported by the absence of Qdm (Qa-1 determinant modifier) in mice on the KbDb-/- background. Qdm is the ligand for CD94/NKG2A, which is provided by the leader sequence of H2-D. We hypothesize that Qa-1-restricted non-classical CD8+ T cells are more effective than other MHC class Ib- restricted T cells during MCMV infection, given their significant differences in activation. These findings are extremely enlightening and expand our current knowledge of CMV- specific T cells. However, they also open up a number of pressing follow-up questions. What MCMV-derived antigens are being recognized? Non-classical CD8+ T cells are responding in an MCMV-dependent manner, rather than a result of inflammatory cytokine signaling. We hypothesize that the antigen being recognized is also MCMV-derived, and not due to upregulation of endogenous peptides. HLA-E/Qa-1 present a diverse peptide repertoire and lack a well-defined peptide binding motif, like Qa-2 [15], so algorithms to predict MCMV-derived peptides bound to Qa-1 are not available. To compensate for this, we have three proposed methods to identify Qa-1-presented antigens: MCMV-deletion mutants, an MCMV open reading frame (ORF) library, and LC-MS (liquid chromatography-mass spectrometry). There are advantages and disadvantages with all three methods, so a combination of these techniques will likely be required. These studies will all use bone marrow dendritic cells (BMDCs) or mouse embryonic fibroblast (MEF) cells derived from KbDb-/- mice because we are limited to MHC class Ia-deficient cell lines that are permissive to MCMV. MEFs are the cell line used for MCMV plaque assays, thus we know they become well infected. We have obtained several MCMV mutants with large sized deletions in their 171 genomes. If the peptide recognized by non-classical CD8+ T cells is absent, we anticipate that they will have decreased expansion and activation in vivo. This would allow us to focus on a specific region of the viral genome. However, together these mutants do not span the entire 230 kb of the MCMV genome, and the antigen could reside outside of the deleted regions. Additionally, the overall fitness of these viruses could be affected, such as their proliferation or infectivity. To offset these types of consequences, we can also use the mutants to infect KbDb-/- BMDCs and incubate with non-classical CD8+ TM cells ex vivo. We have successfully shown that MCMV-infected BMDCs stimulate non-classical CD8+ TM cells to produce significant amounts of IFN-γ and TNF-α, unlike uninfected cells. The MCMV ORF library contains the 170 known MCMV ORFs and was successfully used to determine epitopes recognized by classical CD8+ and CD4+ cells [16-18]. Each ORF is expressed on an individual plasmid, which will be used to transfect KbDb-/- BMDCs. Transfected cells will then act as antigen presenting cells for non-classical CD8+ T cells from day 7 infected KbDb-/- mice. An empty vector and lacZ control to determine the background and MCMV-infected BMDCs will act as a positive control. To verify that the assay is effective, we will first use C57BL/6 BMDCs and classical CD8+ T cells to recapitulate known antigens [16]. If any ORFs successfully stimulate non-classical CD8+ T cells, overlapping peptide libraries will be used to define the specific epitopes being recognized. The most direct method to determine the MCMV-derived antigen(s) is through LC-MS, however since we are not proficient at this technique we will seek outside expertise. Briefly, peptides bound to MHC class Ib molecules will be isolated, separated using LC, and subjected to MS sequencing and analysis. To define peptides that are presented specifically by Qa-1 during MCMV infection, we well compare the repertoires of BMDCs from uninfected KbDb-/-, infected KbDb-/-, and infected KbDb-/-Qa-1-/- mice. 172 Peptides observed in infected KbDb-/- and KbDb-/-Qa-1-/- groups can be eliminated as non- Qa-1-presented, while those from uninfected and infected KbDb-/- groups will be eliminated as endogenous. This kind of subtractive approach was successfully used to determine the peptide repertoire of Qa-1 in the absence of functional ERAAP (endoplasmic reticulum aminopeptidase associated with antigen processing) [19]. Qa-1 presents a diverse peptide repertoire in the absence of Qdm, so we anticipate that non- classical T cells are recognizing multiple MCMV-derived epitopes. Do MCMV-specific non-classical CD8+ T cells have a biased TCR repertoire? Some non-classical T cells are well known for their preferential α- and β-chain usage, such as iNKT cells and MAIT cells [20, 21]. Additionally, after Plasmodium berghei infection, a rodent malaria model, classical CD8+ T cells that respond to a GAP50 epitope are almost exclusively Vβ8.1+ [22]. We are interested in determining whether there is any bias in the TCRs expressed by MCMV-expanded non-classical CD8+ T cells. To accomplish this we are collaborating with Dr. Paul Thomas at St. Jude Children’s Research Hospital to do single-cell, paired alpha and beta chain sequencing [23, 24]. We will compare the repertoire of CD8+ TM cells responding to their second MCMV infection and naive cells. By using cells responding to their second infection, we hope to enrich for MCMV-specific cells. Are Qa-1-restricted T cells present in wild type mice and do they respond to MCMV in the presence of classical CD8+ T cells? Without the ability to visualize Qa-1-restricted CD8+ T cells during MCMV infection, it is difficult to address whether they are present in wild type (WT) animals. Once we determine the peptide(s) being recognized (see above), we can make Qa-1 tetramers loaded with these epitopes. This population can then be examined in vivo in 173 KbDb-/- mice, but will be absent from KbDb-/-Qa-1-/- animals. Qa-1 tetramers can also be used to investigate the occurrence of Qa-1-restricted T cells in C57BL/6 mice. In the presence of the conventional CD8+ T cell response, Qa-1 restricted T cells are a minor subset. However, we hypothesize that small populations of MCMV-expanded Qa-1- specific CD8+ T cells will be present in WT mice and be visible with tetramers. Both Qa- 1-Qdm and Qa-1-FL9-loaded tetramers effectively label these Qa-1-specific CD8+ T cell populations in naïve WT mice [25]; FL9 is preferentially loaded in the absence of functional ERAAP [25]. Qa-1 expression is upregulated following MCMV infection (data not shown), and we detect a population of Qdm-specific CD8+ T cells on day 7 post- infection in C57BL/6 mice (Figure 1). However, whether there are MCMV-specific Qa-1- restricted CD8+ T cells in C57BL/6 mice will require further investigation. Another tool we have developed to help answer this question is Qa-1-/- mice, which we generated at the same time as KbDb-/-Qa-1-/- mice by CRISPR/Cas9. In this case, genetic editing occurred on the WT allele of KbDb+/- zygotes (Figure 2A). These mice have a 183 bp deletion in exon 3 and the flanking intron of H2-T23, which results in a premature stop codon (Figure 2B-C). There is complete loss of Qa-1 expression at the cell surface too (Figure 2D). Our Qa-1-/- line is different from the one currently available because we generated it on the C57BL/6 background, rather than the 129 background [26]. These mice will simultaneously allow us to investigate how loss of Qa- 1 affects cytotoxic CD8+ T cells, but also CD94/NKG2A engagement. Mouse NK cells do not express CD94/NKG2C and CD94/NKG2E at the cell surface [12, 27]. Thus far, preliminary data indicates that Qa-1 signaling in naïve animals does not affect T cell development in the thymus or peripheral CD8+ T cell populations (Figure 3A-D). We also observe that loss of Qa-1 does not alter the activation of CD8+ T cells in the spleen or liver of naïve animals (Figure 3E). To distinguish loss of TCR signaling from a lack CD94/NKG2A inhibitory signaling on CD8+ T cells, we would need to compare any 174 findings to NKG2A-/- or CD94-/- animals [12, 28]. What is the participation of non-Qa-1-restricted T cells in KbDb-/-Qa1-/- mice? KbDb-/-Qa-1-/- mice have dramatically reduced populations of CD8+ T cells during acute MCMV infection, however it is not a 100% reduction; another MHC class Ib molecule(s) must restrict the remaining CD8+ T cells. Whether or not this residual population is responding directly to MCMV or to inflammatory cytokines, and is able to form immunological memory, requires further investigation. To begin answering these questions we are generating long-term infected KbDb-/-Qa-1-/- mice to determine whether non-classical CD8+ T cells form TCM, TEM, and TRM populations in the spleen, liver, and SMG. Additionally we will sort CD8+ TM cells from the spleen and adoptively transfer them into RAG1KbDb-/- mice. Given their decreased activation, we want to examine whether non-Qa-1-restricted CD8+ T cells are capable of protecting immunocompromised mice from MCMV-induced lethality. Since MHC class Ib and Ia are both encoded in the Mhc locus, we originally blocked individual MHC class Ib molecules during CD8+ TM cell restimulation. Blocking with anti-Qa-1 antibodies indicates a potential role of Qa-1, later confirmed with KbDb-/- Qa-1-/- mice, however anti-Qa-2 antibodies have no visible effect. It is difficult to determine whether this negative result is due to poor blocking of the antibody versus no role for Qa-2. Moving forward, we plan to use BMDCs lacking different MHC class Ib molecules to restimulate CD8+ TM cells from KbDb-/-Qa-1-/- mice. For example, Qa-2-/- (currently being generated at the Brown Transgenic Facility) and M3-/- [29]. B2m-/- BMDCs will act as a negative control and KbDb-/-Qa-1-/- BMDCs will be the positive control. This type of method successfully identified Qa-2-restricted cells responding to Mtb [30]. Thus, we do not anticipate recognition of Kb and Db on Qa-2-/- and M3-/- BMDCs is a concern due to the short incubation length. 175 EXTRINSIC CYTOKINE SIGNALING AFFECTS INKT CELL ACTIVATION FOLLOWING MCMV iNKT cells are well recognized for their TCR-independent activation, which allows them to participate during viral infections in the absence of glycolipid antigens. During acute MCMV infection, iNKT cells are robust producers of IFN-γ [31]. Their response is thought to be predominantly IL-12-dependent, but also to a lesser extent due to IL-18 and type I IFNs [31, 32]. Previous studies have largely relied on knockout mouse lines to determine these findings, including those from our own lab. We sought to revisit the signaling required for the iNKT cell response by using mice conditionally deficient (cKO) for different signaling pathways in the T cell lineage. This includes IL- 12Rβ2 and MyD88. MyD88 is a downstream adaptor molecule of toll-like receptor (TLR) signaling (except TLR3), in addition to the IL-1R/IL-18R superfamily. This not only allows us to investigate the roles of IL-1 and IL-18, but also whether there is a direct effect of TLR ligands on iNKT cells. Using IL-12Rβ2 cKO mice we show that the iNKT cell IFN-γ response is completely abrogated. This illustrates that during MCMV, IL-12 is a necessary signal for iNKT cell activation. In contrast, we observe organ specificity for the requirement of MyD88 signaling. MyD88-deficient iNKT cells have approximately half the IFN-γ production of those from littermate controls in the spleen, however they are unaffected in the liver. These data illustrate that signaling through TLRs and the IL-1R/IL-18R are both dispensable in the liver. In contrast, a component of this signaling pathway is necessary for splenic iNKT cells. These phenotypes are not a result of an intrinsic defect in iNKT cell cytokine production though. Both IL-12Rβ2- and MyD88-deficient iNKT cells have comparable IFN-γ and IL-4 production to their littermate controls following α-GalCer stimulation. This also illustrates that cytokine signaling is not required in addition to CD1d-presentation of α-GalCer. 176 Why are splenic iNKT cells hyporesponsive in the absence of MyD88 signaling, but hepatic iNKT cells are unaffected? Our results clearly show that loss of IL-12 signaling abolishes iNKT cell cytokine production and results in decreased activation (CD69 expression) at 36 hours post- infection. Why then, in the absence of MyD88 signaling do iNKT cells have dampened IFN-γ production in the spleen, but not the liver? If IL-12 is necessary, but not sufficient, for iNKT cell cytokine production one would think this would be true in both the spleen and liver. Splenic and hepatic iNKT cells could have differential requirements for IL-18, however previous studies using IL-18-/- mice observed no defects in the splenic iNKT cell response to MCMV [32]. In C57BL/6 mice there are almost exclusively NKT1 cells in both the spleen and liver [33]. However, iNKT cells could be physiologically different in these two organs, similarly to NK cells. For example, the liver is populated by tissue- resident NK cells and conventional NK (cNK) cells, while the spleen only has cNK cells [34]. A more likely explanation is that the loss of MyD88 signaling is in turn affecting IL- 12 availability, which subsequently dampens the iNKT cell response. Since the Cd4 promoter drives Cre recombinase expression in MyD88 cKO mice, this insinuates that another T cell is likely the culprit. For example, CD4+ T cells lacking IL-1-induced MyD88 signaling are unable to overcome Treg suppression [35]. To determine whether IL-1R/IL-18R/TLR signaling is conditioning the IL-12- dependent response of iNKT cells, we will adoptively transfer MyD88 cKO or MyD88 control iNKT cells into wild type mice, and then infect with MCMV. Donor cells are traceable because of their YFP expression, which allows us to distinguish them even though they begin to down-regulate TCR expression at 36 hours post-infection. If MyD88-deficient iNKT cells have a normal response in a wild type environment, this would indicate they are extrinsically affected in the spleen of MyD88 cKO mice, rather than through an intrinsic loss of TLR or IL-1R/IL-18R signaling. To determine if this is a 177 result of decreased IL-12, we can investigate IL-12 levels in the spleen and liver of MyD88 cKO and control animals during acute infection. Once these studies are completed, additional work will need to be done to determine which cell type is instigating this phenotype. Even though CD4cre is predominantly used as a T cell- specific Cre, other subsets also express CD4, such as lymphoid tissue-inducer (Lti) cells [36]. We have previously observed this type of off-target effect using CD4cre to delete SHP-2 (Ptpn11), which causes mice to develop cartilage tumors independently of T cell activity [10]. Summary The work presented in this thesis expands our current knowledge of two different non-classical T cell subsets, CD1d- and Qa-1-restricted T cells. Non-classical T cells restricted by MHC class I-like and MHC class Ib molecules have novel requirements for their development and activation. However, the ability of these populations to form immunological memory is generally not well understood. Each individual non-classical T cell subset is distinct, including the types of antigens they respond to, and whether or not they are protective during certain infections. There are a number of questions remaining about the contributions of non-classical T cells within the overall scope of the immune system. Here we present work examining the iNKT cell response in the absence of SHIP1 signaling, which is necessary for optimal cytokine production, but not for development or proliferation. We also illustrate that iNKT cells rely on inflammatory cytokines for their activation, and that IL-12 is necessary for this response. However, loss of MyD88 signaling also exclusively affects splenic iNKT cells. In contrast we determine that the MHC class Ib-restricted CD8+ T cell response is dependent on MCMV-derived antigens, rather than a result of inflammatory cytokines. These non- classical CD8+ T cells are populated by both Qa-1-restricted cells and non-Qa-1- 178 restricted cells. However, Qa-1-restricted CD8+ T cells may be more adept at responding to MCMV, than other MHC class Ib-restricted populations. The functions of non-classical T cells may be masked unless the conventional T cell response is impaired or evaded, which is a characteristic of many chronic infections, including CMV. Thus, non-classical CD8+ T cells can be separated into two broad categories, innate-like T cells with early effector responses and those more similar to conventional T cells, which may participate during immune evasion (Figure 4). For these reasons, it is imperative to continue investigating the developmental requirements and anti-microbial functions of MHC class Ib-restricted T cell populations. Non-classical T cells are unique targets for immune therapy and vaccine development because they recognize peptide and non-peptide antigens presented by nonpolymoprhic MHC molecules. 179 Figure 1: Qa-1-restricted CD8+ T cells are detectable in wild type mice using Qdm- loaded tetramers. (A) Qa1-Qdm tetramer staining of C57BL/6 splenocytes on Day 0 and 7 post-MCMV infection. Samples are first gated on total CD8+ T cells and also stained with NKG2A/C/E to inhibit binding of the tetramer. 180 Figure 2. Generation of Qa-1-/- mice through CRISPR/Cas9-mediated editing. (A) Generation of Qa1-/- mice on KbDb+/- zygotes using CRISPR/Cas9. (B) Schematic of guideRNA (gRNA) targeting to H2-T23 exon 3 and flanking intron. gRNAs are illustrated in blue and protospacer adjacent motifs (PAM) are in green. Blue arrows indicate beginning and end of deleted region. (C) Sequence of H2-T23 in Qa-1-/- mice, compared to wild-type control, indicating a 183 bp deletion. (D) Representative histograms of Qa-1 expression on CD19+ lymphocytes from the spleen of naïve Qa-1+/+ (black), Qa-1+/- (grey), and Qa-1-/- mice (white), compared to secondary control (---). 181 Figure 3. T cell development and peripheral CD8+ T cell populations are unaffected in Qa-1-/- mice. (A) The CD4-CD8- double negative (DN), CD4+CD8+ double positive (DP), and CD4+ or CD8+ single positive (SP) stages of T cell development in the thymus. (B) The CD44+, CD44+ CD25+, CD25+, and CD44-CD25- (DN1-4) stages of T cell development. (C) CD8+ T cell frequency and (D) number in the spleen and liver. (E) KLRG1 expression of CD8+ T cells in the spleen and liver of naïve Qa-1+/+, Qa-1+/-, and Qa1-/- mice. Data are representative of three independent experiments (n=7-11). Error bars indicate SEM and *p<0.05. 182 Figure 4. Two proposed roles of MHC class Ib-restricted T cells. (A) The responses of innate-like non-classical T cells, such as iNKT cells or MAIT cells, have earlier kinetics than conventional T cells. (B) During chronic infections, MHC class Ib-restricted T cell populations might respond in the absence or immune evasion of the classical T cell response. Graphs are not to scale (Adapted from Anderson and Brossay 2016 [37]). 183 REFERENCES 1. Sun, J.C., et al., NK cells and immune "memory". J Immunol, 2011. 186(4): p. 1891-7. 2. O'Sullivan, T.E., J.C. Sun, and L.L. Lanier, Natural Killer Cell Memory. Immunity, 2015. 43(4): p. 634-45. 3. Godfrey, D.I., S. Stankovic, and A.G. Baxter, Raising the NKT cell family. Nat Immunol, 2010. 11(3): p. 197-206. 4. Godfrey, D.I., et al., The burgeoning family of unconventional T cells. Nat Immunol, 2015. 16(11): p. 1114-23. 5. Tarasenko, T., et al., T cell-specific deletion of the inositol phosphatase SHIP reveals its role in regulating Th1/Th2 and cytotoxic responses. Proc Natl Acad Sci U S A, 2007. 104(27): p. 11382-7. 6. Banh, C., et al., Mouse natural killer cell development and maturation are differentially regulated by SHIP-1. Blood, 2012. 120(23): p. 4583-90. 7. Bendelac, A., P.B. Savage, and L. Teyton, The biology of NKT cells. Annu Rev Immunol, 2007. 25: p. 297-336. 8. Nguyen, N.Y., et al., An ENU-induced mouse mutant of SHIP1 reveals a critical role of the stem cell isoform for suppression of macrophage activation. Blood, 2011. 117(20): p. 5362-71. 9. Lorenz, U., SHP-1 and SHP-2 in T cells: two phosphatases functioning at many levels. Immunol Rev, 2009. 228(1): p. 342-59. 10. Miah, S.M.S., et al., Ptpn11 Deletion in CD4(+) Cells Does Not Affect T Cell Development and Functions but Causes Cartilage Tumors in a T Cell- Independent Manner. Front Immunol, 2017. 8: p. 1326. 11. French, A.R., et al., Escape of mutant double-stranded DNA virus from innate immune control. Immunity, 2004. 20(6): p. 747-56. 184 12. Rapaport, A.S., et al., The Inhibitory Receptor NKG2A Sustains Virus-Specific CD8(+) T Cells in Response to a Lethal Poxvirus Infection. Immunity, 2015. 43(6): p. 1112-24. 13. Moser, J.M., et al., CD94-NKG2A receptors regulate antiviral CD8(+) T cell responses. Nat Immunol, 2002. 3(2): p. 189-95. 14. Bian, Y., et al., MHC Ib molecule Qa-1 presents Mycobacterium tuberculosis peptide antigens to CD8+ T cells and contributes to protection against infection. PLoS Pathog, 2017. 13(5): p. e1006384. 15. Chiang, E.Y. and I. Stroynowski, The role of structurally conserved class I MHC in tumor rejection: contribution of the Q8 locus. J Immunol, 2006. 177(4): p. 2123-30. 16. Munks, M.W., et al., Genome-wide analysis reveals a highly diverse CD8 T cell response to murine cytomegalovirus. J Immunol, 2006. 176(6): p. 3760-6. 17. Walton, S.M., et al., The dynamics of mouse cytomegalovirus-specific CD4 T cell responses during acute and latent infection. J Immunol, 2008. 181(2): p. 1128- 34. 18. Munks, M.W., et al., Four distinct patterns of memory CD8 T cell responses to chronic murine cytomegalovirus infection. J Immunol, 2006. 177(1): p. 450-8. 19. Nagarajan, N.A., et al., ERAAP Shapes the Peptidome Associated with Classical and Nonclassical MHC Class I Molecules. J Immunol, 2016. 197(4): p. 1035-43. 20. Kumar, A., et al., Natural Killer T Cells: An Ecological Evolutionary Developmental Biology Perspective. Front Immunol, 2017. 8: p. 1858. 21. Treiner, E., et al., Mucosal-associated invariant T (MAIT) cells: an evolutionarily conserved T cell subset. Microbes Infect, 2005. 7(3): p. 552-9. 22. Van Braeckel-Budimir, N., et al., A T Cell Receptor Locus Harbors a Malaria- Specific Immune Response Gene. Immunity, 2017. 47(5): p. 835-847 e4. 185 23. Dash, P., G.C. Wang, and P.G. Thomas, Single-Cell Analysis of T-Cell Receptor alphabeta Repertoire. Methods Mol Biol, 2015. 1343: p. 181-97. 24. Greene, J.M., et al., MR1-restricted mucosal-associated invariant T (MAIT) cells respond to mycobacterial vaccination and infection in nonhuman primates. Mucosal Immunol, 2017. 10(3): p. 802-813. 25. Nagarajan, N.A., F. Gonzalez, and N. Shastri, Nonclassical MHC class Ib- restricted cytotoxic T cells monitor antigen processing in the endoplasmic reticulum. Nat Immunol, 2012. 13(6): p. 579-86. 26. Hu, D., et al., Analysis of regulatory CD8 T cells in Qa-1-deficient mice. Nat Immunol, 2004. 5(5): p. 516-23. 27. Vance, R.E., et al., Implications of CD94 deficiency and monoallelic NKG2A expression for natural killer cell development and repertoire formation. Proc Natl Acad Sci U S A, 2002. 99(2): p. 868-73. 28. Orr, M.T., et al., Development and function of CD94-deficient natural killer cells. PLoS One, 2010. 5(12): p. e15184. 29. Xu, H., et al., Impaired response to Listeria in H2-M3-deficient mice reveals a nonredundant role of MHC class Ib-specific T cells in host defense. J Exp Med, 2006. 203(2): p. 449-59. 30. Shang, S., et al., Nonclassical MHC Ib-restricted CD8+ T Cells Recognize Mycobacterium tuberculosis-Derived Protein Antigens and Contribute to Protection Against Infection. PLoS Pathog, 2016. 12(6): p. e1005688. 31. Wesley, J.D., et al., NK cell-like behavior of Valpha14i NK T cells during MCMV infection. PLoS Pathog, 2008. 4(7): p. e1000106. 32. Tyznik, A.J., et al., Distinct requirements for activation of NKT and NK cells during viral infection. J Immunol, 2014. 192(8): p. 3676-85. 33. Lee, Y.J., et al., Steady-state production of IL-4 modulates immunity in mouse 186 strains and is determined by lineage diversity of iNKT cells. Nat Immunol, 2013. 14(11): p. 1146-54. 34. Erick, T.K. and L. Brossay, Phenotype and functions of conventional and non- conventional NK cells. Curr Opin Immunol, 2016. 38: p. 67-74. 35. Schenten, D., et al., Signaling through the adaptor molecule MyD88 in CD4+ T cells is required to overcome suppression by regulatory T cells. Immunity, 2014. 40(1): p. 78-90. 36. Sawa, S., et al., Lineage relationship analysis of RORgammat+ innate lymphoid cells. Science, 2010. 330(6004): p. 665-9. 37. Anderson, C.K. and L. Brossay, The role of MHC class Ib-restricted T cells during infection. Immunogenetics, 2016. 68(8): p. 677-91. 187 APPENDIX I: THE ROLE OF THE Y CHROMOSOME-ENCODED ERDR1 ON INKT CELL DEVELOPMENT 188 The role of the Y chromosome-encoded Erdr1 on iNKT cell development Courtney K. Anderson1, Leon Toussaint1, Lindsay M. Scott2, Christophe Benoist3, Cory Teuscher4, Bruce Beutler2, Laurent Brossay1 1 Division of Biology and Medicine, Department of Molecular Microbiology and Immunology, Brown University, Providence, RI 02912 2 Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA 3 Division of Immunology, Department of Microbiology and Immunobiology, Harvard Medical School, and Evergrande Center for Immunologic Diseases, Harvard Medical School and Brigham and Women's Hospital, Boston, MA, USA 4 Department of Medicine, University of Vermont, Burlington, VT 05405 189 INTRODUCTION The Y chromosome is approximately 60 Mb in size, making up only ~2-3% of the entire human genome [1]. It contains unique features, including two pseudoautosomal (PAR) regions (PAR1 and PAR2) at the end of each arm. PAR1 and PAR2 are homologous to areas on the X chromosome. During meiosis in males, genetic material is exchanged between the X and Y chromosomes through recombination within these regions. The remainder of the Y chromosome is termed the non-recombining Y (NRY) and is made up of areas of euchromatin and heterochromatin [1]. There are a number of immunodeficiencies linked to the X chromosome, including X-linked severe combined immunodeficiency (SCID). Patients with X-linked SCID have deficiencies in T cells and NK cells due to mutations in the common cytokine receptor γ-chain (IL-2Rγ) [2]. However, very little is known about the immunological role of genes encoded on the Y chromosome. To date, there are three studies in mice that describe a potential connection. Yaa (y-linked autoimmune accelerating) resembles systemic lupus erythematosus and results from duplication of the TLR7 gene from the X chromosome to the Y chromosome [3]. Another group characterized a Y-linked B and NK cell deficiency, without effects on T cell development [4]. Thirdly, our lab shows that iNKT cells are absent in male B6.IFN-αβR1−/− mice with over 99% penetrance, compared to their female littermates [5]. The genetic link for this phenotype is unknown, however it is independent of IFN-α/β signaling, and a result of a factor on the Y chromosome of B6.IFN-αβR1−/− males [5]. B6.IFN-αβR1−/− mice were originally created on the 129Sv/Ev background, however 129.IFN-αβR1−/− and 129Sv/Ev males have similar populations of iNKT cells to their female littermates [5]. We have since crossed the Y chromosome from the B6.IFN- αβR1−/− line completely onto the C57BL/6 background, denoted B6.YNKT. In addition to severe iNKT cell deficits, T cell development is also affected and there are increased 190 populations of γδ T cells in the thymus and periphery. Interestingly, forced expression of a prearranged invariant Vα14-Jα18 TCR is not sufficient to rescue the iNKT cell defect. Sequencing of the B6.YNKT Y chromosome reveals small deletions (~3000 and 5000 bps) present in the Erdr1 (erythroid differentiation regulator 1) gene, which are not observed in control samples. Erdr1 is located in the PAR regions of the X and Y chromosomes. The immunological role of Erdr1 is unclear, however recombinant Erdr1 enhances NK cell cytotoxicity by exocytosis of lytic granules [6]. Here, we provide evidence for a role of Erdr1 encoded on the murine Y chromosome on iNKT cell development. 191 MATERIALS AND METHODS Mice. B6.YNKT mice were generated by crossing the Y chromosome from B6.IFN- αβR1−/− mice onto the C57BL/6 background and were maintained in-house. IFN-αβR-/- and B6.SJL mice were purchased from The Jackson Laboratories. Female Vα14-Jα18 transgenic mice (Stock number: 014639) were purchased from The Jackson Laboratory, crossed with male B6.YNKT mice, and maintained in-house. Dr. Terry Magnuson from the University of North Carolina at Chapel Hill provided male YUTY- mice and female littermate controls [7]. 129S2/Sv mice were purchased from Charles River. Lymphocyte isolation. Spleens were dissociated in 1% PBS-serum, filtered through nylon mesh, and underlayed with lympholyte-M (Cedarlane Laboratories). Alternatively, spleens were dissociated in 150 mM NH4Cl for 10 minutes, filtered through nylon mesh, and washed once with 1% PBS-serum. Livers were profused with 1% PBS-serum, dissociated using GentleMACS program E0.1 (Miltenyi Biotech) and passed through nylon mesh. Samples were washed three times in 1% PBS-serum and overlayed onto a two-step discontinuous Percoll gradient (GE Healthcare Bio-Sciences). Samples were filtered through nylon mesh, washed once in 1% PBS-serum, and underlayed with Lympholyte-M. Gradients were centrifuged at 2500 RPM for 20 minutes at room temperature. Thymi were dissociated in 1% PBS-serum, filtered through nylon mesh, and washed one time in 1% PBS-serum. Antibodies and flow cytometry. Cells were stained in 1% PBS-serum containing 2.4G2, CD1d tetramer loaded with KRN7000 (Avanti Polar Lipids, Inc.), and cell surface antibodies for 15 minutes at room temperature and 15 minutes on ice in the dark. CD1d tetramer was loaded in-house and conjugated to either PE or APC. Samples were run on a FACSAria III (BD Biosciences) or MACSQuant (Miltenyi Biotech) and analyzed 192 using FlowJo (Tree Star Inc.). The antibodies listed below were used for flow cytometry and purchased from eBioscience (now Thermo Fischer Scientific) or BD Biosciences: CD4 – APC, CD4 – APC-eF780, CD4 – PerCP, CD8 – CD8α, HSA – FITC, TCRβ – FITC, TCRβ – PE, TCRγδ – PE. Sequencing of male B6.YNKT mice. gDNA was extracted from 100 µL of whole blood using the Gentra Puregene Whole Blood kit (Qiagen). Samples were first added to tubes containing 0.03M EDTA, pH 8.0. gDNA quality was checked by running through an agarose gel. The concentration of gDNA was determined using the Quanti-iT Picogreen dsDNA kit (Life Technologies). Sequencing was performed on a HiSeq 2500 System (Illumina) at UT Southwestern Medical Center. Statistical analysis. Statistical analyses were performed with Prism 7.0 (Graph-Pad Software, Inc.). Unpaired two-tailed Student’s t-tests were used to compare two individual groups. Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. 193 RESULTS AND DISCUSSION Global T cell populations are modestly affected in male B6.YNKT mice B6.YNKT males lack iNKT cells, compared to female littermates (Fig. 1A), similarly to what was previously observed with B6.IFN-αβR1−/− mice [5]. B6.IFN-αβR1−/− mice are still available from Jackson Laboratories, but no longer carry this Y-linked defect (Fig. 1B). Interestingly, we observe that total cellularity is affected in the thymus of B6.YNKT males (Fig. 2A). In addition to diminished iNKT cell populations, γδ T cells and conventional αβ T cells also appear modestly affected in frequency and total cell number in the thymus and spleen (Fig. 2B-E). This indicates that the Y chromosome-linked factor is affecting T cells more globally, than iNKT cells alone. Forced expression of the iNKT cell semi-invariant T cell receptor does not rescue their defect Similarly to conventional T cells, iNKT cells become committed to the T cell lineage during a double negative (CD4-CD8-, DN) stage, enter a double positive (DP) stage (CD4+CD8+), and are then selected by CD1d-expressing DP thymocytes [8]. In contrast, MHC class I- or II-expressing thymic epithelial cells select conventional T cells. We previously found an increase in DN stage thymocytes and decrease in DP stage thymocytes in B6.IFN-αβR1−/− males [5], which is also observed in B6.YNKT males (Fig. 3A). β-chain selection occurs during the late DN stage/immature CD8+ single positive stage (ISP8), while α-chain rearrangement takes place in the ISP8 [9, 10]. iNKT cell development requires extended DP stage survival for Vα14-Jα18 α-chain rearrangement [11]. For example, RORγt is essential for DP cell survival [12], and RORγt-deficient mice lack iNKT cells [13]. We wondered whether expression of a prearranged Vα14- Jα18 α-chain would rescue the iNKT cell defect in B6.YNKT males. Female Vα14-Jα18 transgenic mice were crossed with B6.YNKT males to yield B6.YNKT males and females 194 with or without the transgene (Tg). As expected, B6.YNKTTg+ females have large increases in iNKT cell populations in the spleen, liver, and thymus. For example, up to 70% of hepatic lymphocytes are iNKT cells (Fig. 3B). However, iNKT cell populations from male B6.YNKT and B6.YNKTTg+ animals are comparable, with only a minimal increase in frequency in the spleen (Fig. 3B). These data indicate that inadequate rearrangement of the iNKT cell α-chain is unlikely to be affecting iNKT cell development. The Y chromosome gene Erdr1 contains small deletions in male B6.YNKT mice Previous microarray data indicates that four genes (Ddx3y, Eif2s3y, UTY, and Kdm5d/Jarid1d) on the Y chromosome of B6.YNKT mice are down-regulated in iNKT cells, but not CD4+ T cells from the same animals (Dr. Leon Toussaint, data not shown). UTY, and its homologue on the X chromosome UTX, are histone demethylases. Interestingly, UTX epigenetically regulates iNKT cell development [14]. To investigate the role of these genes, we obtained samples from mice lacking expression of UTY, designated YUTY- [7]. Interestingly, we observe no differences in the iNKT cell populations from YUTY- mice in the spleen or thymus, as well as normal T cell development, compared to their WT female littermates (Fig.4A, B). To better explore the potential abnormalities present in B6.YNKT mice, we sequenced the Y chromosome. Two samples from male B6.YNKT mice were individually sequenced and compared to the original parental 129 strain and a C57BL/6 control. Interestingly, this reveals that Ddx3y, Eif2s3y, UTY, and Kdm5d/Jarid1d are unaffected at the genetic level. Instead, we find two candidate deletions of approximately 2800 and 5400 bp in Erdr1 that are present in the B6.YNKT samples, but not either control sample (Fig. 5A, B). Erdr1 was not included in the original microarray. Attempted deletion of Erdr1 on either the X or Y chromosome results in embryonic lethality [15], indicating we likely still have a functional copy of Erdr1 present in B6.YNKT animals. Erdr1 plays a role 195 in hematopoietic progenitor cell growth and survival [16] and NK cell cytotoxicity [6]. Further investigations are necessary to determine how these two Erdr1 deletions affect Ddx3y, Eif2s3y, Uty, and Kdm5d/Jarid1d expression levels and iNKT cell development. 196 Figure 1. Male B6.YNKT mice, but not male IFN-αβR1−/− mice, lack iNKT cells. (A) iNKT cell frequencies in the spleen, liver, and thymus of female and male B6.YNKT mice (n=9-13). Data are pooled from at least four independent experiments. (B) Representative staining of female and male B6.YNKT and IFN-αβR1−/− mice. Error bars indicate SEM and ***p<0.001 and ****p<0.0001. 197 Figure 2. γδ and αβ T cell populations are affected in male B6.YNKT mice. (A) Total cells in the spleen and thymus of female and male B6.YNKT cell mice (n=5-6). (B) Frequency and (D) absolute number of T cells (CD3+) in the spleen and thymus of male B6.SJL controls and B6.YNKT males (n=2-5). (C) Frequency and (E) absolute number of TCRγδ+ and TCRαβ+ cells in the spleen and thymus of male B6.SJL controls and B6.YNKT males (n=2-5). Samples were first gated on CD3+ cells. Data are pooled from three (A) and one (B-E) independent experiments. Error bars indicate SEM and *p<0.05, **p<0.01, and ***p<0.001. 198 Figure 3. Overexpression of a prearranged Vα14-Jα18 TCR does not rescue the iNKT cell deficit in male B6.YNKT mice. (A) The CD4-CD8- double negative (DN), CD4+CD8+ double positive (DP), and CD4+ or CD8 + single positive (SP) stages of T cell development in the thymus of female and male B6.YNKT mice (n=2-5). (B) iNKT cell frequencies in the spleen, liver, and thymus of female and male B6.YNKT mice, with and without expression of a Vα14-Jα18 transgene (Tg) (n=7-12). Data are pooled from at least four (B) and one (A) independent experiments. Error bars indicate SEM and **p<0.01, ***p<0.001 and ****p<0.0001. 199 Figure 4. Loss of UTY does not affect T cell development or peripheral iNKT cell populations. (A) iNKT cell frequencies in the spleen and thymus of WT female and male YUTY- mice (n=2-3). (B) The CD4-CD8- double negative (DN), CD4+CD8+ double positive (DP), and CD4+ or CD8 + single positive (SP) stages of T cell development in the thymus of WT female and male YUTY- mice (n=2-3). Data are from one independent experiment and error bars indicate SEM. 200 Figure 5. B6.YNKT males have small sized deletions in the Erdr1 gene on Chromosome Y. (A, B) Sequencing data of genomic DNA prepared from the blood of two individual B6.YNKT male mice, compared to male 129S2/SV control (parental strain) and C57BL/6 control. Color-coding indicates genomic alterations shared between B6.YNKT mouse and indicated samples. Small deletions shared between both B6.YNKT samples are green and outlined in bold. GSD = genomic duplicated region. 201 REFERENCES 1. Quintana-Murci, L. and M. Fellous, The Human Y Chromosome: The Biological Role of a "Functional Wasteland". J Biomed Biotechnol, 2001. 1(1): p. 18-24. 2. Noguchi, M., et al., Interleukin-2 receptor gamma chain mutation results in X- linked severe combined immunodeficiency in humans. Cell, 1993. 73(1): p. 147- 57. 3. Pisitkun, P., et al., Autoreactive B cell responses to RNA-related antigens due to TLR7 gene duplication. Science, 2006. 312(5780): p. 1669-72. 4. Sun, S.L., et al., Y chromosome-linked B and NK cell deficiency in mice. J Immunol, 2013. 190(12): p. 6209-20. 5. Wesley, J.D., et al., A Y chromosome-linked factor impairs NK T development. J Immunol, 2007. 179(6): p. 3480-7. 6. Lee, H.R., et al., ERDR1 enhances human NK cell cytotoxicity through an actin- regulated degranulation-dependent pathway. Cell Immunol, 2014. 292(1-2): p. 78-84. 7. Shpargel, K.B., et al., UTX and UTY demonstrate histone demethylase- independent function in mouse embryonic development. PLoS Genet, 2012. 8(9): p. e1002964. 8. Bendelac, A., Positive selection of mouse NK1+ T cells by CD1-expressing cortical thymocytes. J Exp Med, 1995. 182(6): p. 2091-6. 9. Godfrey, D.I. and S.P. Berzins, Control points in NKT-cell development. Nat Rev Immunol, 2007. 7(7): p. 505-18. 10. Godfrey, D.I., S. Stankovic, and A.G. Baxter, Raising the NKT cell family. Nat Immunol, 2010. 11(3): p. 197-206. 11. Egawa, T., et al., Genetic evidence supporting selection of the Valpha14i NKT cell lineage from double-positive thymocyte precursors. Immunity, 2005. 22(6): p. 202 705-16. 12. Guo, J., et al., Regulation of the TCRalpha repertoire by the survival window of CD4(+)CD8(+) thymocytes. Nat Immunol, 2002. 3(5): p. 469-76. 13. Bezbradica, J.S., et al., Commitment toward the natural T (iNKT) cell lineage occurs at the CD4+8+ stage of thymic ontogeny. Proc Natl Acad Sci U S A, 2005. 102(14): p. 5114-9. 14. Beyaz, S., et al., The histone demethylase UTX regulates the lineage-specific epigenetic program of invariant natural killer T cells. Nat Immunol, 2017. 18(2): p. 184-195. 15. Zuo, E., et al., One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs. Cell Res, 2017. 27(7): p. 933-945. 16. Dormer, P., E. Spitzer, and W. Moller, EDR is a stress-related survival factor from stroma and other tissues acting on early haematopoietic progenitors (E- Mix). Cytokine, 2004. 27(2-3): p. 47-57. 203 APPENDIX II: THE ROLE OF MHC CLASS IB-RESTRICTED T CELLS DURING INFECTION Originally published in Immunogenetics, July 1, 2016 Volume 68, Issue 8, Pages 677 – 691 https://link.springer.com/article/10.1007%2Fs00251-016-0932-z Reprinted by permission from Nature Springer: Immunogenetics. The role of MHC class Ib-restricted T cells during infection. Anderson, CK & Brossay, L. © Springer-Verlag Berlin Heidelberg 2016 204 The role of MHC class Ib-restricted T cells during infection Courtney K. Anderson* and Laurent Brossay* * Department of Molecular Microbiology & Immunology, Division of Biology and Medicine, Brown University, Providence, RI 02912 Acknowledgements: We would like to thank Timothy Erick for critical reading of the manuscript. This work was supported by National Institutes of Health Research Grant RO1 AI46709 and AAI Careers in Immunology Fellowship (L.B.) and National Institutes of Health Fellowship F31 AI124556 (C. K. A.) 205 Abstract Even though MHC class Ia and many Ib molecules have similarities in structure, MHC class Ib molecules tend to have more specialized functions, which include the presentation of non-peptidic antigens to non-classical T cells. Likewise, non-classical T cells also have unique characteristics, including an innate-like phenotype in naïve animals and rapid effector functions. In this review, we discuss the role of MAIT and NKT cells during infection, but also the contribution of less studied MHC class Ib-restricted T cells such as Qa-1-, Qa-2-, and M3-restricted T cells. We focus on describing the types of antigens presented to non-classical T cells, their response and cytokine profile following infection, as well as the overall impact of these T cells to the immune system. 206 A. Introduction The innate and adaptive branches of the immune system are not mutually exclusive, and there is a growing interest in innate-like T cells, many of which are restricted by non-classical MHC molecules. Cytotoxic CD8+ T cells are restricted by MHC class I molecules and categorized into two groups: classical MHC class Ia and non-classical MHC class Ib. MHC class Ia molecules are highly polymorphic and encoded in the Mhc locus by H-2K, H-2D, and H-2L in mice and HLA-A, HLA-B, and HLA-C in humans. The MHC class Ib family has evolved more diverse, and specialized, functions than their classical counterparts. These molecules are predominantly found in the H2-Q, H2-T, and H2-M regions in mice and HLA-E, HLA-F, and HLA-G in humans within the Mhc locus. Many are capable of presenting antigens, while others are incapable of antigen binding or participate in responses outside of the immune system. For example, murine M1 and M10 bind to V2R G protein-coupled receptors and play a role in pheromone detection (Loconto et al. 2003), while ZAG in humans and mice binds fatty acids and polyethylene glycol, contributing to lipid metabolism (Delker et al. 2004; Hirai et al. 1998). MHC class Ib molecules are also well known to interact with NK cell receptors (Braud et al. 1998; Lee et al. 1998b; Vance et al. 1999; Vance et al. 1998). Some have proposed to classify MHC class Ib molecules according to their age, such as ‘Young,’ ‘Middle-aged,’ and ‘Old’ (Rodgers and Cook 2005). For example, ‘Old’ genes diverged during early vertebrate evolution and many members of this subset fall outside the Mhc gene locus in humans and rodents, such as CD1, MR1, and HFE (Rodgers and Cook 2005). Generally, non-classical MHC molecules present a more diverse array of antigens, e.g. CD1 presents glycolipid antigens (Beckman et al. 1994), MR1 presents Vitamin B metabolites (Kjer-Nielsen et al. 2012), and M3 presents formylated peptides (Smith et al. 1994) (Table 1). These molecules also tend to have more restricted tissue localization, lower expression at the cell surface, limited polymorphism, and shorter 207 cytoplasmic tails (Stroynowski and Lindahl 1994). In this review, we will discuss MHC class Ib-restricted T cell responses in humans and mice in the context of infection. We will focus on 1) the antigens (or lack thereof) presented by this family of molecules, 2) the cytokine profile of MHC class Ib-restricted T cells, and 3) the overall contribution of non-classical T cells to the immune response (Table 2). B. Positive selection of non-classically restricted CD8+ T cells: Non-classical CD8+ T cells often have an innate-like phenotype, which includes increased expression of CD44 and decreased CD62L expression (Jay et al. 2008; Kurepa et al. 2003). It has been proposed that this results from unusual positive selection. For example, the conditions that T10- and T22-restricted γδ T cells undergo positive selection affect their effector phenotype. Cells that develop in the presence of T22 are able to produce IFN-γ, whereas antigen-naïve T10- and T22-reactive γδ T cells during development produce IL-17 (Jensen et al. 2008). Thymic epithelial cells (TECs) are essential for the positive selection of conventional T cells (Anderson et al. 1994). On the other hand, invariant natural killer T (iNKT) cells are selected by CD4+CD8+ double positive (DP) cortical thymocytes that present CD1d (Bendelac 1995). Similarly to iNKT cells, MAIT cells are also selected by hematopoietic cells (HCs) (Treiner et al. 2003), which are DP thymocytes expressing MR1 (Seach et al. 2013). MHC class Ib-restricted CD8+ T cells that are specific for Listeria monocytogenes antigens are also selected for by HCs, whereas MHC class Ia-restricted T cells are inadequately selected (Urdahl et al. 2002). Interestingly, it was recently shown that M3-restricted T cells could be selected by TECs or HCs, but that the selecting cell type played a role in their phenotype (Chiu et al. 1999b; Cho et al. 2011). Cells selected by HCs acquired enhanced effector functions (Cho et al. 2011). Similarly, using transgenic mice possessing a TCR specific for a Qa- 1-presented insulin-derived peptide, it was determined that either TECs or HCs selected 208 Qa-1-restricted CD8+ T cells (Sullivan et al. 2002). However, in contrast to M3’s role during positive selection, there was no observable difference in phenotype between these two differentially selected populations (Sullivan et al. 2002). This demonstrates that individual MHC class Ib-restricted T cell populations have different requirements for positive selection. C. MHC class Ib-restricted CD8+ T cells and their participation during infection The CD1-restricted family of T cells The CD1 locus is not linked to the Mhc locus. CD1 presents self and foreign lipid antigens to multiple CD1-restricted T cell populations, rather than peptides (Figure 1) (Beckman et al. 1994). Five isoforms of human CD1 are expressed, which are organized into three groups: Group 1 CD1 (CD1a, b, c), Group 2 CD1 (CD1d), and Group 3 CD1 (CD1e). T cells are able to directly recognize lipids presented by all CD1 isoforms except CD1e, which is thought to aid CD1b in ligand processing and presentation (de la Salle et al. 2005). Unlike MHC class Ia molecules, the transporter associated with antigen processing (TAP) is not required for CD1 antigen loading (Brutkiewicz et al. 1995; Hanau et al. 1994). CD1d is the only isoform expressed in rodents. There are two types of Group 2 CD1-restricted T cells, type I iNKT cells and type II NKT cells, which were initially classified based on TCR diversity (Cardell et al. 1995). iNKT cells participate during a variety of infectious diseases and can be activated in a TCR-dependent or TCR-independent manner by responding to environmental cytokines, like IL-12 and IL-18, rather than ligand stimulation (Leite-De-Moraes et al. 1999). This is evident following murine cytomegalovirus (MCMV) infection, where iNKT cells become activated in an IL-12-dependent manner, and is partially contingent on type I interferons (Holzapfel et al. 2014; Tyznik et al. 2014; Wesley et al. 2008). On the other hand, type II NKT cell activation mainly occurs in a TCR-dependent manner to self- 209 glycolipids of self-phospholipids, whose antigen repertoire can be either exclusive or promiscuous (Jahng et al. 2004; Tatituri et al. 2013). Type II NKT cells appear to have opposing roles, capable of participating in protective responses or promoting pathology. However, the information about this subset has remained limited because type II NKT cells cannot be labeled as a single population with CD1d tetramers like iNKT cells. In addition, early studies to determine the functions of type II NKT cells using Jα18-/- mice may need to be revisited, due to an impaired TCR repertoire of the original mice (Bedel et al. 2012). iNKT cells respond to CD1d-presented ligands derived from a number of bacteria and even protozoa, including: bacteroides fragilis-derived sphingolipid α- galactosylceramide (An et al. 2014; Wieland Brown et al. 2013); borrelia burgdorferi- derived galactosyl diacylglycerol (Kinjo et al. 2006); helicobacter pylori-derived cholesteryl α-glucoside (Ito et al. 2013); Sphingomonas bacteria-derived α-linked galacturonic acid (Kinjo et al. 2005); Streptococus pneumoniae-derived glycolipids containing diacylglycerol (Kinjo et al. 2011); and lipopeptidophosphoglycan derivatives from the protozoan entamoeba histolytica (Lotter et al. 2009). Human and murine iNKT cells also respond to M. tuberculosis (Mtb) phosphatidylinositol mannoside (PIM) ligands (Fischer et al. 2004). However, iNKT cells are dispensable during mycobacterial infection, as illustrated using CD1d-/- animals (Behar et al. 1999). In contrast, iNKT cells are physiologically relevant for clearance and protection against other pathogenic microorganisms. iNKT cells produce IFN-γ in response to B. burgdorferi infection in vivo and both CD1d-deficient animals (Kumar et al. 2000) and Jα18-/-BALB/c mice were more susceptible to infection, developing chronic joint inflammation and arthritis (Tupin et al. 2008). iNKT cell participation is also implied during E. histolytica infection, which causes increased amebic liver abscesses in the absence of iNKT cells in Jα18-/- mice (Lotter et al. 2006) and CD1d-/- mice, (Lotter et al. 2009); CD1d is necessary for iNKT cell IFN-γ 210 production, as well as IL-12 from TLR signaling (Lotter et al. 2009). Jα18-/- animals have also been reported to be susceptible to Streptococcus infections (Kawakami et al. 2003). Predictably, due to expression of CD1d ligands the CD1d-restricted response was shown to be necessary for protection against S. pneumoniae and Group B Streptococcus through IFN-γ and IL-17 production in the lung (Kinjo et al. 2011). This CD1d restricted response was validated using Nur77GFP transgenic mice, which upregulate GFP following TCR engagement (Holzapfel et al. 2014). Unexpectedly however, although it was shown that iNKT cells respond to S. typhimurium infection in a CD1d restricted manner (Brigl et al. 2003), iNKT cells produced IFN-γ without TCR engagement (Holzapfel et al. 2014). In contrast to type I and II NKT cells, investigations into Group 1 CD1-restricted T cell responses have mainly focused on mycobacterial infection. CD1a, CD1b, and CD1c molecules present different types of glycolipids, owing to structural differences in their antigen-binding grooves (Gadola et al. 2002; Scharf et al. 2010; Zajonc et al. 2005). Circulating CD1-restricted T cells are observed in patients previously infected with Mtb or immunized with Mycobacterium bovis bacillus Calmette-Guerin (BCG) (Kawashima et al. 2003; Ulrichs et al. 2003). These Mycobacterium-specific T cells are capable of producing IFN-γ ex vivo and recognize M. bovis BCG-infected cells (Kawashima et al. 2003). Interestingly, there is also a small population of CD1b-restricted germline- encoded, mycolyl lipid-reactive (GEM) T cells present in uninfected patients (Van Rhijn et al. 2013). To counteract the lack of an animal model to study Group 1 CD1 molecules in vivo, human group 1 CD1 transgenic (hCD1Tg) mice were generated, which express all Group 1 CD1 isoforms (Felio et al. 2009). Mtb infection and immunization of hCD1Tg mice are both capable of inducing a CD1-restricted T cell response, characteristic of classical T cells; this includes a slow primary response to immunization and rapid secondary response (Felio et al. 2009). Importantly, in hCD1Tg mice expressing a 211 mycolic acid-specific TCR transgene, immune protection against Mtb was observed (Zhao et al. 2015). A second group investigated the CD1 repertoire in a humanized mouse model using NSG mice engrafted with human fetal thymus and fetal liver, as well as CD34+ hematopoietic cells (Lockridge et al. 2011). CD1a, CD1b, CD1c, and CD1d were all expressed in these animals, and Group 1 CD1-restricted T cells were present (Lockridge et al. 2011), though their response following Mtb infection still remains to be seen. To date, Group 1 CD1 molecules have been shown to present eight Mycobacterium-derived ligands, the majority of which are loaded in CD1b (Siddiqui et al. 2015). However, a role for Group 1-restricted T cell populations during other infections has not been reported. MR1-restricted mucosal associated invariant T (MAIT) cells MAIT cells were first described in 1993 (Porcelli et al. 1993), but it was not until six years later that they were recognized as a distinct population (Figure 2) (Tilloy et al. 1999). MAIT cells are unique innate-like T cells found at mucosal sites and in the circulation of humans and mammals. MAIT cells are restricted by MR1 (MHC-related protein 1) (Treiner et al. 2003), which is encoded outside the Mhc gene locus. There is 90% sequence homology between MR1 in humans and mice (Riegert et al. 1998). Wild- type mice generally have low frequencies of MAIT cells (Rahimpour et al. 2015), but they are more abundant in humans and make up approximately 1-4% of circulating T cells (Martin et al. 2009). The antimicrobial role of MAIT cells was first alluded to based on their absence in germ-free (GF) mice (Treiner et al. 2003), however they can successfully expand in GF mice following inoculation with a single bacterial species, i.e. Bacteroides thetaiotaomicron, Bifidobacterium animalis, Enterobacter cloacae, or Lactobacillus casei (Le Bourhis et al. 2010). Two groups then found that MAIT cells are able to respond to a number of bacterial and fungal species in vitro using infected 212 PMDCs or BMDCs, but not to viruses, e.g. Lactobacillus acidophilus, Mtb, Pseudomonas aeroginosa, Salmonella typhimurium, Staphylococcus aureus, Saccharomyces cerevisiae, and Candida albicans (Gold et al. 2010; Le Bourhis et al. 2010). A major breakthrough in this field was the discovery that MR1 presents Vitamin B metabolites, such as Vitamin B2 (riboflavin) and Vitamin B9 (folic acid) derivatives (Kjer-Nielsen et al. 2012). MR1 is able to bind and stabilize the unstable intermediates of the riboflavin biosynthesis pathway for presentation (Corbett et al. 2014). Interestingly, these ligands can be activating or non-activating in nature by presenting riboflavin or folic acid derivatives, respectively (Kjer-Nielsen et al. 2012). MAIT cells are now thought to be involved in the early control of a number of bacterial pathogens. Following up on the observation that β2m-/- mice are more vulnerable to Klebsiella pneumonia than wild-type mice (Cogen and Moore 2009), Georgel et al. determined that MR1-/- mice also had increased susceptibility compared to MR1-sufficient animals (Georgel et al. 2011). MAIT cells also robustly expand in the lungs of mice infected with the live vaccine strain (LVS) of Francisella tularensis in an MR1- and IL-12p40-dependent manner (Meierovics et al. 2013). Their expansion inversely correlated with bacterial burden, and was accompanied by the production of IFN-γ, TNF-α, and IL-17A (Meierovics et al. 2013). Interestingly, MAIT cells were also observed to contribute during chronic infection of F. tularensis LVS, even when classical CD4+ and CD8+ T cells were recruited, but were insufficient for bacterial clearance alone (Meierovics et al. 2013). MR1-/- mice also have increased bacterial burden on Day 10 following M. bovis BCG infection, compared to wild-type mice (Chua et al. 2012). This disparity was no longer observed at later time points (Day 30), illustrating the importance of MAIT cells during early immunological control (Chua et al. 2012). Interestingly, this was suggested to be an MR1-independent, but IL-12-dependent response (Chua et al. 2012). In contrast, it appears that human MAIT cells require MR1 for appropriate IFN-γ 213 production in response to Mtb infected APCs (Gold et al. 2010; Le Bourhis et al. 2010). Many studies have observed decreased numbers of circulating MAIT cells in tuberculosis (TB) patients (Gold et al. 2010; Le Bourhis et al. 2010). The remaining MAIT cell population in patients with active TB produced significantly more IFN-γ and TNF-α in response to BCG and decreased cytokine production following E. coli infection (Jiang et al. 2014). This suggests that MAIT cells are enriched in this environment to respond to Mtb (Jiang et al. 2014). In support of this concept, it was shown that the heterogeneity of the MAIT cell TCR repertoire might allow for pathogen specificity (Eckle et al. 2014; Gold et al. 2014). Cell lines expressing MAIT cell TCRs also become activated in an MR1-dependent manner following S. enterica serovar Typhimurium infection (Reantragoon et al. 2012). Additionally, human MAIT cells produce IFN-γ, TNF-α, and IL-2 following incubation with either S. typhimurium supernatant or the synthetic riboflavin derivative 6-hydroxymethyl-8-D-ribityllumazine (rRL-6-CH2OH) in the presence of MR1-expressing APCs (Reantragoon et al. 2013). Overall however, the functional role of MAIT cells is more ambiguous in humans than mice. The MAIT cell field is relatively young, but has recently burgeoned due to the development of MR1 tetramers (Reantragoon et al. 2013). Nevertheless, a number of lingering questions remain. For instance, the role of MAIT cells during bacterial infection has been relatively well documented, however the in vivo role of MR1-restricted T cells has not been well defined for yeast. There is also the potential that MR1 could bind and present additional ligands to Vitamin B derivatives. Finally, it appears that MAIT cell activation can occur in a TCR-independent manner, similarly to iNKT cells, irrespective of bacterial/fungal riboflavin metabolism (Chua et al. 2012; Meierovics et al. 2013; Ussher et al. 2014). This opens up potential avenues to study MAIT cell responses during viral infections and autoimmune disorders, such as HIV (Fernandez et al. 2015; Leeansyah et al. 2013), multiple sclerosis (Treiner and Liblau 2015), inflammatory bowel 214 disease (Treiner 2015), and Celiac disease (Dunne et al. 2013). HFE-specific CD8+ T cells HFE, or human hemochromatosis protein, was first discovered due to its association with hereditary hemochromatosis (HH) patients, a genetic disorder that results in iron overload (Feder et al. 1996). The HFE heavy chain forms a noncovalent bond with an associated β2m light chain (Feder et al. 1996; Feder et al. 1997), similarly to many other MHC class Ib molecules. However, in one common HFE mutation seen in HH patients, the C282Y mutation, the ability to bind β2m is disrupted. This prevents HFE expression at the cell surface by perturbing a critical disulfide bridge in the α3 domain (Feder et al. 1997). HFE-deficient and β2m-deficient mice both recapitulate the HH phenotype (Santos et al. 1996; Zhou et al. 1998). Even though HFE is structurally similar to other MHC class I molecules, its peptide-binding groove does not support antigen binding (similarly to TL, see below). This is due to the α1 and α2 domains being in closer proximity, since the α1 helix has a 4Å translocation towards the α2 domain this results in a narrower groove (Lebron et al. 1998). Rather than antigen binding, HFE is predominantly known for associating with the transferrin receptors to regulate iron homeostasis (Goswami and Andrews 2006; Lebron et al. 1998; Parkkila et al. 1997). However, there is evidence that suggests a potential immunological role for HFE as well. For example, the iron overload phenotype is even more pronounced in mice that are deficient for both β2m and RAG1, compared to β2m-/- animals (Santos et al. 2000). HFE-deficient animals on a RAG1 background also have increased iron overload, compared to HFE-/- animals (Miranda et al. 2004). A role for CD8+ T cells was initially proposed because many HH patients have unusually small CD8+ T cell populations in circulation (Macedo et al. 2010; Porto et al. 1994). However, the exact nature of this deficiency is unclear, as it could be an indirect 215 result of iron overload or a direct result of HFE regulating CD8+ T cells (Costa et al. 2015; Reuben et al. 2014). Interestingly, HFE is thought to impede activation via its α1 and α2 helixes by influencing MHC class I antigen processing and presentation (Reuben et al. 2014). Rohrlich et al. also showed that HFE influences the TCR repertoire, as evidenced by a decreased number of Vα6 TCRs in HFE-deficient mice (Rohrlich et al. 2005). Importantly, a subset of CD8+ T cells directly recognize HFE via their TCR and produce IL-6, IL-10, and hepcidin (Boucherma et al. 2012; Rohrlich et al. 2005). Overall, although HFE is not capable of binding and presenting antigens, there is evidence for an immunological role of HFE. Additional studies will be necessary to investigate the functions of HFE-reactive CD8+ T cells, as well as the immune system’s role in iron metabolism. TL-restricted CD8+ T cells Thymus leukemia antigen (TL) was first discovered during the development of spontaneous or radiation induced leukemia (Old and Boyse 1963) and subsequently mapped to the Mhc locus (Boyse et al. 1964). TL is encoded by the H2-T3 and H2-T18 genes in mice and is considered an ancient MHC class Ib gene that diverged over 100 million years ago (Davis et al. 2002). There is no human homologue for TL, but it has been suggested that HLA-G is a functional homologue (Attinger et al. 2005; Huang et al. 2011). TL is expressed on intestinal epithelial cells (IELs) (Hershberg et al. 1990; Wu et al. 1991) and immature thymocytes of certain mouse strains (Chen et al. 1985). Following activation, T cells and APCs also express TL (Cook and Landolfi 1983; Madakamutil et al. 2004). T cells can also “snatch” TL from intestinal epithelial cells to present it on their cell surface (Pardigon et al. 2006). The expression of TL at the cell surface is dependent on β2m (Yokoyama et al. 1982), but not on peptide binding (Weber et al. 2002). Even though TL exhibits approximately 70% identity with MHC class Ia 216 molecules, its peptide-binding groove is closed due to the α1 helix being 7Å closer to the α2 helix (Liu et al. 2003). TL cell surface expression is also TAP-independent, which distinguishes it from classical MHC class Ia molecules (Holcombe et al. 1995; Rodgers et al. 1995). TL binds the CD8αα homodimer with higher affinity than to CD8αβ (Leishman et al. 2001; Tsujimura et al. 2001). This is a result of three exposed amino acids in the α3 helix of TL (Attinger et al. 2005). In contrast, MHC class Ia molecules have comparable affinities to CD8αβ and CD8αα (Kern et al. 1999). Unlike CD8αβ heterodimers (Bosselut et al. 1999), the CD8αα homodimer does not act as a co- receptor, rather it inhibits activation by acting as a co-repressor (Cheroutre and Lambolez 2008). CD8αβ T cells that co-express CD8αα are abundant in the intestinal mucosa. However, TL is not required for the formation of CD8+ T cell memory (Williams and Bevan 2005). It has also been shown that TL is important for controlling IEL function, for example inhibiting IEL proliferation (Olivares-Villagomez et al. 2008). Both αβ (Morita et al. 1994) and γδ TCRs (Tsujimura et al. 1996) can recognize TL. These TL-specific cytotoxic CD8+ T cells recognize the α1 and α2 domains of TL with CD8αα helping to stabilize the TL/TCR interaction (Tsujimura et al. 2003) in a TAP-independent mechanism (Tsujimura et al. 2000). Perhaps not surprisingly, due to its closed binding groove, the role of TL-restricted T cells during infectious disease clearance is limited. M3-specific CD8+ T cells In contrast to MHC class Ia molecules and many MHC class Ib molecules, M3 binds N-formylated peptides (Shawar et al. 1993; Smith et al. 1994). Thus, M3 is able to present peptides of prokaryotic or mitochondrial origin (Wang et al. 1991). There is also evidence that M3 can bind non-formylated peptides (Byers and Fischer Lindahl 1998), however M3 binds N-formylated peptides with much higher affinity than unformylated ones (Smith et al. 1994). The crystal structure of M3 showed that the specificity for N- 217 formylated peptides was a result of alterations in its peptide-binding groove (Wang et al. 1995). There are very few endogenous N-formylated peptides available, which results in low M3 expression at the cell surface (Levitt et al. 2001), and sequesters M3 in the endoplasmic reticulum. TAP is required for M3 stabilization in the ER, while tapasin is necessary for intracellular peptide loading (Chun et al. 2001a). Dependency on TAP further differentiates M3 from other MHC class Ib molecules like CD1 and TL (Brutkiewicz et al. 1995; Hanau et al. 1994; Holcombe et al. 1995; Rodgers et al. 1995). However, both TAP-dependent and -independent presentation have been observed for L. monocytogenes peptides (Rolph and Kaufmann 2000). Exogenous antigens are capable of inducing M3 expression at the cell surface, unlike lowered temperatures (Chiu et al. 1999a), which occurs with MHC class Ia molecules (Ljunggren et al. 1990). M3-restricted CD8+ T cells specific to a number of intracellular pathogens have been characterized, e.g. Mtb, L. monocytogenes, Chlamydia pneumonia, and S. enterica serovar Typhimurium (Chun et al. 2001b; Gulden et al. 1996; Lenz et al. 1996; Princiotta et al. 1998; Tvinnereim and Wizel 2007; Ugrinovic et al. 2005). The most well studied M3-restricted CD8+ T cells are specific to L. monocytogenes. Using MHC class Ia- deficient (KbDb-/-) mice, it has been shown that M3-restricted CD8+ T cells are sufficient to protect against L. monocytogenes infection (D'Orazio et al. 2003; Seaman et al. 1999). In this context, M3 presents three L. monocytogenes-derived peptides: Attm (f- MIVTLF) (Princiotta et al. 1998), Fr38 (f-MIVIL) (Gulden et al. 1996), and LemA (f- MIGWII) (Lenz et al. 1996). However, M3-restricted CD8+ T cells are not required for protection against L. monocytogenes (D'Orazio et al. 2006). Nevertheless, using M3- deficient animals it was determined that the M3-restricted and MHC class Ia-restricted immune responses are not redundant (Xu et al. 2006). The significance of M3-restricted memory CD8+ T cells during L. monocytogenes infection is more ambiguous. In contrast to primary infection, after secondary infection M3-restricted CD8+ T cells do not 218 significantly expand (Kerksiek et al. 1999). It has been suggested that rather than contributing to a memory phenotype during a secondary infection, M3-restricted CD8+ T cells are already in a memory state in naïve animals, due to interactions with cross- reactive antigens from commensal bacteria (Lenz and Bevan 1997). Alternatively, it has been proposed that M3-restricted memory CD8+ T cells are constrained in the presence of MHC class Ia-restricted memory cells (Hamilton et al. 2004). M3 also appears to contribute to the immune response against Mtb infection. Chun et al. showed that the Mtb genome contained a number of N-formylated peptides capable of binding to M3, and that M3-restricted CD8+ T cells can recognize a number of these peptides in mice (Chun et al. 2001b). Immunization of mice with dendritic cells pulsed with an N-formylated Mtb peptide are also able to elicit an H2-M3-mediated response (Doi et al. 2007). However, although MHC class Ib-restricted T cells accumulate in the lung, they provide minimal protection against Mtb infection (Urdahl et al. 2003). Further investigations are necessary to determine whether N-formylated peptides from other bacterial species elicit an M3-specific response. CD8+ T cells-restricted by Qa-2 and HLA-G The H2-Q6, -Q7, -Q8, and -Q9 genes in mice encode Qa-2. It is thought that the Qa-2 region resulted from a series of gene pair duplications, for example H2-Q7 and H2- Q9 are nearly identical, with over 99% homology (Devlin et al. 1985). Originally, Qa-2 was thought to have a restricted peptide repertoire (Rotzschke et al. 1993), however it is capable of binding a diverse array of endogenous and foreign peptides (Joyce et al. 1994; Tabaczewski et al. 1997). The crystal structure of Q9 revealed that it associates with β2m, and the peptide-binding groove is more hydrophobic and shallower than classical MHC molecules, which could play a role in its promiscuous peptide repertoire (He et al. 2001). Qa-2 is unique among MHC molecules for being anchored to the cell 219 membrane by a glycophosphatidyl inositol (GPI) linker (Stroynowski et al. 1987), which is necessary for T cell activation (Robinson et al. 1989). In addition, there are soluble and membrane-linked forms of Qa-2, both of which require TAP (Tabaczewski and Stroynowski 1994), that arise because of alternative splicing or cleavage post-translation (Tabaczewski et al. 1994). However, the α3 domain of Qa-2 is unable to effectively interact with CD8 to appropriately activate cytotoxic T cells (Teitell et al. 1993). There is evidence that Qa-2 participates in resistance to the murine parasite Taenia crassiceps. This was based on the observation that BALB/cAnN mice (Qa-2null) are susceptible to T. crassiceps, while BALB/cJ mice (Qa-2+) are resistant (Fragoso et al. 1996). In support of these findings, it was later shown that Qa-2 transgenic mice have increased clearance of the parasite (Fragoso et al. 1998). In addition, Q9-specific CD8+ T cells have been extensively characterized during the response to mouse polyoma virus (MPyV). Q9-restricted T cells from KbDb-/- mice are able to control MPyV infection, impede tumor formation, and recognize a nonameric peptide derived from the virus’s VP2 capsid protein (termed VP2.139) (Swanson et al. 2008). This population is present in wild-type mice as well. However, Q9-restricted T cells are somewhat different from classical CD8+ T cells because they form an inflationary population during persistent infection for approximately 12 weeks (Swanson et al. 2008). Further characterization showed that immunization with a VP2.139 peptide in mice carrying different MHC haplotypes could generate an MPyV-specific non-classical CD8+ T cell response (Hofstetter et al. 2013). However, the authors were not able to determine whether this population provided enhanced control during MPyV infection (Hofstetter et al. 2013). Importantly, human HLA-G is proposed to be the functional homologue of Qa-2 (Comiskey et al. 2003). The expression of HLA-G is primarily limited to placental tissues such as cytotrophoblasts (Kovats et al. 1990), whereas classical MHC molecules are 220 believed to be poorly expressed (Hunt et al. 1987). There are four membrane-bound isoforms of HLA-G, HLA-G1-G4, and three soluble isoforms, HLA-G5-G7, which occur via alternative splicing (Fujii et al. 1994; Ishitani and Geraghty 1992; Kirszenbaum et al. 1994; Paul et al. 2000). Secreted and membrane-bound forms can both present endogenous nonameric peptides (Diehl et al. 1996; Lee et al. 1995), however truncated isoforms do not associate with β2m (Morales et al. 2007). HLA-G has a number of immunomodulatory effects (Amiot et al. 2014; Guleria and Sayegh 2007), presumably mediated via interaction with the inhibitory receptors ILT2, ILT4, and KIR2DL4 (CD158d) (LeMaoult et al. 2005; Rajagopalan and Long 2012). HLA-G may be critical for immune tolerance during pregnancy to protect the fetus from rejection by maternal effector cells (Rouas-Freiss et al. 1997). In mice, Qa-2 is thought to participate during embryonic cleavage division and survival following preimplantation (McElhinny et al. 2000; Warner et al. 1987). Similarly to Qa-2, HLA-G molecules are capable of invoking a cytotoxic T cell response that is specific to HLA-G. This was first illustrated using HLA-G transgenic mice and skin graft experiments (Horuzsko et al. 1997; Schmidt et al. 1997). In a follow up experiment utilizing HLA-G tetramers for the predominant human cytomegalovirus (HCMV) peptide pp65, some HCMV-specific CD8+ T cells restricted by HLA-G were observed (Lenfant et al. 2003). However, the relevance of these HLA-G-restricted T cells, and their presence in humans, remains to be seen. Qa-1 and HLA-E-restricted CD8+ T cells Before HLA-E was known to present antigenic peptides to HLA-E-restricted T cell populations, it was first shown to be a ligand for the CD94/NKG2 family of NK cell receptors (NKG2A, NKG2B, and NKG2C) (Braud et al. 1998; Lee et al. 1998b). Qa-1 in mice (encoded by H2-T23) can similarly bind to CD94/NKG2 receptors (Vance et al. 1999; Vance et al. 1998). Expression of HLA-E at the cell surface is dependent on TAP 221 (Lee et al. 1998a) and loading of nonamer peptides derived from the leader sequences of HLA-A, HLA-B, HLA-C, or HLA-G molecules (Braud et al. 1997; Lee et al. 1998a). Likewise, Qa-1 also binds signal sequence-derived peptides (AMAPRTLLL) from MHC class Ia molecules in a TAP-dependent manner (Aldrich et al. 1994). The primary ligands for Qa-1 and HLA-E are denoted Qdm, or Qa-1 determinant modifier, however other peptides can be loaded under different circumstances. For instance, the self- peptide FL9 (FYAEATPML) was recently identified in ERAAP-deficient mice (Nagarajan et al. 2012). However, these are not the only similarities between Qa-1 and HLA-E, which are considered functional homologues that arose by convergent evolution (Yeager et al. 1997). Both have low cell surface expression and broad tissue distribution, limited polymorphism, and structural homology (Zeng et al. 2012). This also includes similarities within their peptide-binding groove, such as unique substitutions at positions 143 and 147 (Connolly et al. 1993). HLA-E, for example, is the least polymorphic of the human non-classical MHC molecules. Caucasians only have two alleles, designated HLA-E*0101 and HLA-E*0103, which differ from each other at one amino acid position (Geraghty et al. 1992; Grimsley et al. 2002). In addition to the role of HLA-E and Qa-1 during innate immunity, these MHC class Ib molecules can also present microbial antigens to CD8+ T cells. HLA-E and Qa- 1 bind peptides from bacteria, such as S. typhimurium, S. enterica serovar Typhi, L. monocytogenes, and Mtb (Bouwer et al. 1997; Caccamo et al. 2015; Lo et al. 2000; Salerno-Goncalves et al. 2004; van Meijgaarden et al. 2015). Interestingly, in the absence of Qdm, a peptide derived from heat shock protein 60 (Hsp60) is primarily loaded into Qa-1 (GMKFDRGYI) (Davies et al. 2003). Hsp60 is well conserved in prokaryotes, whose homologue is GroEL in bacteria. In agreement with these observations, it was found that Qa-1 presents a S. typhimurium-derived peptide from GroEL (GMQFDRGYL) to CD8+ cytotoxic T cells (Lo et al. 2000). These GroEL-specific 222 CD8+ T cells were also cross-reactive with Hsp60 and lyse stressed macrophages (Lo et al. 2000). Similarly, HLA-E presentation of GroEL-derived antigens from S. enterica caused targeted cell lysis of infected cells, as well as IFN-γ production (Salerno- Goncalves et al. 2004). HLA-E also participates during Mtb infection. This was first proposed when it was determined that the predominant CD8+ T cell response in latently infected patients was MHC class Ia and CD1 independent (Heinzel et al. 2002; Lewinsohn et al. 2000). The recognition of Mtb-derived peptides presented by HLA-E (Caccamo et al. 2015; van Meijgaarden et al. 2015) differs from the classically restricted CD8+ T cell response. HLA-E-restricted T cells appear to acquire a Th2 phenotype, producing TNF-α, IL-4, IL-5, IL-10, and IL-13, but have poor cytotoxicity in response to stimulation (Caccamo et al. 2015; van Meijgaarden et al. 2015). HLA-E and Qa-1 are also capable of presenting virally derived peptides. For example, HLA-E-reactive T cells to Epstein-Barr virus recognize a peptide from its BZLF-1 protein (SQAPLPCVL) (Garcia et al. 2002; Jorgensen et al. 2012). A peptide derived from a Hepatitis C virus (HCV) core protein (YLLPRRGPRL) was also shown to bind HLA-E, stabilize its cell surface expression, and protect cells from NK cell lysis (Nattermann et al. 2005). Additionally, 40% of an HCV infected cohort was determined to have an HLA-E-reactive T cell response, resulting in IFN-γ production, which was not observed from healthy control samples (Schulte et al. 2009). Interestingly, there was an increased incidence of HLA-E- specific CD8+ T cells to HCV in patients with the HLA-E*0101 allele, compared to the HLA-E*0103 allele (Schulte et al. 2009). Perhaps the most intriguing HLA-E-restricted response occurs during HCMV infection. HCMV employs a variety of mechanisms to downregulate the expression of conventional HLA molecules at the cell surface and avoid the classical CD8+ T cell response. One immunoevasive mechanism HCMV employs is to inhibit ERAP1 function, the human homolog of murine ERAAP, through miR-US4-1 (Kim et al. 2011). 223 This miRNA obstructs cytotoxic CD8+ T cells from lysing infected cells by inhibiting HCMV antigen derivation (Kim et al. 2011). HCMV also provides its own peptide, derived from the signal sequence of its glycoprotein UL40 (gpUL40) to load in HLA-E molecules, increasing cell surface expression independently of TAP (Tomasec et al. 2000; Ulbrecht et al. 2000). The UL40 leader sequence from the AD169 and Toledo HCMV strains are both able to provide peptides (VMAPRTLIL and VMAPRTLVL, respectively) that bind HLA-E. This is thought to be a mechanism for escaping the NK cell response because UL40 deletion mutants are unable to evade NK cells through CD94/NKG2A inhibition (Wang et al. 2002). However, HLA-E-restricted CD8+ T cells can also recognize these gpUL40-derived peptides via their TCR (Pietra et al. 2003). HLA-E-restricted HCMV-specific T cells have an effector memory phenotype, can kill HCMV infected target cells, and produce IFN-γ in response to contact with UL40 leader peptides (Mazzarino et al. 2005). Interestingly, the gpUL40 leader sequence of AD169 and Toledo HCMV strains is identical to certain HLA-A and HLA-Cw leader peptide alleles, possibly leading to a non-classical T cell evasion mechanism (Pietra et al. 2010; Pietra et al. 2003). In addition to increased expression of HLA-E, HCMV also disrupts ERAP1 function (Kim et al. 2011). As mentioned previously, Qa-1-restricted cells kill ERAAP-deficient cells in naïve mice (Nagarajan et al. 2012). Therefore, it would be interesting to determine whether HLA-E-restricted CD8+ T cells play a similar role during HCMV infection. Overall, the role of Qa-1/HLA-E-restricted T cells may only be revealed when the MHC class I and/or conventional CD8+ T cell response is failing (Figure 3, see proposed model below). T cell responses restricted by unidentified MHC class Ib molecules and other non- classical T cell responses Certain non-classical T cell responses are a result of, as of yet, undetermined 224 MHC class Ib molecules. For example, non-classically restricted CD8+ T cells from KbDb- /- mice are sufficient to control chronic γ-herpesvirus 68 (Braaten et al. 2006). While the exact restriction of this population is currently unknown, it is dependent on β2m and CD1d is dispensable (Braaten et al. 2006). In addition, MHC class Ib-restricted T cells respond following lymphocytic choriomeningitis virus (LCMV) infection in both KbDb-/- and KbDbCIITA-/- mice, which also lack MHC class II molecules, however they were inadequate to fully clear the virus (Chen et al. 2011). Non-classical T cells are also present in a wide range of species. For instance, in the amphibian Xenopus laevis, iVα6 T cells are restricted by the MHC class Ib molecule XNC10 and resemble iNKT cells found mice and humans (Edholm et al. 2013). XNC10-restricted T cells were essential for an appropriate antiviral response, and successful viral clearance, following infection with frog virus 3 (Edholm et al. 2015). Studies such as these illustrate the importance of non-classical MHC class Ib-restricted T cells, and their biological relevance, in a wide range of species. Concluding remarks and proposed models Non-classical T cells are unique in many ways – strategic localization at barrier sites, recognition of a wide array of unique microbial pathogens, and rapid effector responses. These features led to the hypothesis that the primary function of non- classical T cells such as iNKT and MAIT cells is to rapidly respond to infections. We propose that another, non-mutually exclusive, function for these cells may be revealed during chronic infection when classical T cell and NK cell responses are impaired (Figure 3). This has been recently documented in the case of Qa-1/HLA-E-restricted T cells, which exploit pathogen immunoevasion adaptations (Hansen et al. 2016). Together with the low polymorphism of MHC class Ib molecules, the unique characteristics of MHC class Ib-restricted T cells render them attractive targets for vaccine development, especially when the immune system is compromised. 225 Fig. 1 The family of CD1-restricted αβ T cells. Group 1 CD1-specific T cells are depicted with examples of Mtb glycolipid antigens. 226 Fig. 2 Examples of non-classical αβ T cell populations during microbial infection. 227 Fig. 3 Proposed roles of non-classical CD8+ αβ T cell populations in humans and mice, compared to conventional CD8+ T cells. A) MHC class Ib-restricted CD8+ T cells, e.g. iNKT cells and MAIT cells, have a faster effector response following infection, compared to MHC class Ia-restricted T cells. B) During chronic infection, microorganisms can employ immunoevasion mechanisms to dampen the classical T cell response and/or NK cell response. MHC class Ib-specific responses could represent a backup system that responds following MHC class Ib upregulation, for example HLA-E. 228 Table 1 MHC class Ib molecules that participate in TCR-mediated responses 229 Table 2 The antimicrobial response of MHC class Ib-restricted T cells in mice and humans 230 REFERENCES Aldrich CJ, DeCloux A, Woods AS, Cotter RJ, Soloski MJ, Forman J (1994) Identification of a Tap-dependent leader peptide recognized by alloreactive T cells specific for a class Ib antigen Cell 79:649-658 Amiot L, Vu N, Samson M (2014) Immunomodulatory properties of HLA-G in infectious diseases J Immunol Res 2014:298569 doi:10.1155/2014/298569 An D et al. (2014) Sphingolipids from a symbiotic microbe regulate homeostasis of host intestinal natural killer T cells Cell 156:123-133 doi:10.1016/j.cell.2013.11.042 Anderson G, Owen JJ, Moore NC, Jenkinson EJ (1994) Thymic epithelial cells provide unique signals for positive selection of CD4+CD8+ thymocytes in vitro The Journal of experimental medicine 179:2027-2031 Attinger A et al. (2005) Molecular basis for the high affinity interaction between the thymic leukemia antigen and the CD8alphaalpha molecule Journal of immunology (Baltimore, Md : 1950) 174:3501-3507 Beckman EM, Porcelli SA, Morita CT, Behar SM, Furlong ST, Brenner MB (1994) Recognition of a lipid antigen by CD1-restricted alpha beta+ T cells Nature 372:691-694 doi:10.1038/372691a0 Bedel R, Matsuda JL, Brigl M, White J, Kappler J, Marrack P, Gapin L (2012) Lower TCR repertoire diversity in Traj18-deficient mice Nature immunology 13:705-706 doi:10.1038/ni.2347 Behar SM, Dascher CC, Grusby MJ, Wang CR, Brenner MB (1999) Susceptibility of mice deficient in CD1D or TAP1 to infection with Mycobacterium tuberculosis The Journal of experimental medicine 189:1973-1980 Bendelac A (1995) Positive selection of mouse NK1+ T cells by CD1-expressing cortical thymocytes The Journal of experimental medicine 182:2091-2096 Bosselut R, Zhang W, Ashe JM, Kopacz JL, Samelson LE, Singer A (1999) Association of the adaptor molecule LAT with CD4 and CD8 coreceptors identifies a new coreceptor function in T cell receptor signal transduction The Journal of experimental medicine 190:1517-1526 Boucherma R et al. (2012) Loss of central and peripheral CD8+ T-cell tolerance to HFE in mouse models of human familial hemochromatosis European journal of immunology 42:851-862 doi:10.1002/eji.201141664 Bouwer HG, Seaman MS, Forman J, Hinrichs DJ (1997) MHC class Ib-restricted cells contribute to antilisterial immunity: evidence for Qa-1b as a key restricting element for Listeria-specific CTLs Journal of immunology (Baltimore, Md : 1950) 159:2795-2801 Boyse EA, Old LJ, Luell S (1964) Genetic Determination of the Tl (Thymusleukaemia) Antigen in the Mouse Nature 201:779 Braaten DC, McClellan JS, Messaoudi I, Tibbetts SA, McClellan KB, Nikolich-Zugich J, Virgin HW (2006) Effective control of chronic gamma-herpesvirus infection by unconventional MHC Class Ia-independent CD8 T cells PLoS pathogens 2:e37 doi:10.1371/journal.ppat.0020037 Braud V, Jones EY, McMichael A (1997) The human major histocompatibility complex class Ib molecule HLA-E binds signal sequence-derived peptides with primary 231 anchor residues at positions 2 and 9 European journal of immunology 27:1164- 1169 doi:10.1002/eji.1830270517 Braud VM et al. (1998) HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C Nature 391:795-799 doi:10.1038/35869 Brigl M, Bry L, Kent SC, Gumperz JE, Brenner MB (2003) Mechanism of CD1d-restricted natural killer T cell activation during microbial infection Nature immunology 4:1230-1237 doi:10.1038/ni1002 Brutkiewicz RR, Bennink JR, Yewdell JW, Bendelac A (1995) TAP-independent, beta 2- microglobulin-dependent surface expression of functional mouse CD1.1 The Journal of experimental medicine 182:1913-1919 Byers DE, Fischer Lindahl K (1998) H2-M3 presents a nonformylated viral epitope to CTLs generated in vitro Journal of immunology (Baltimore, Md : 1950) 161:90-96 Caccamo N et al. (2015) Human CD8 T lymphocytes recognize Mycobacterium tuberculosis antigens presented by HLA-E during active tuberculosis and express type 2 cytokines European journal of immunology 45:1069-1081 doi:10.1002/eji.201445193 Cardell S, Tangri S, Chan S, Kronenberg M, Benoist C, Mathis D (1995) CD1-restricted CD4+ T cells in major histocompatibility complex class II-deficient mice The Journal of experimental medicine 182:993-1004 Chen L, Jay DC, Fairbanks JD, He X, Jensen PE (2011) An MHC class Ib-restricted CD8+ T cell response to lymphocytic choriomeningitis virus Journal of immunology (Baltimore, Md : 1950) 187:6463-6472 doi:10.4049/jimmunol.1101171 Chen YT, Obata Y, Stockert E, Old LJ (1985) Thymus-leukemia (TL) antigens of the mouse. Analysis of TL mRNA and TL cDNA TL+ and TL- strains The Journal of experimental medicine 162:1134-1148 Cheroutre H, Lambolez F (2008) Doubting the TCR coreceptor function of CD8alphaalpha Immunity 28:149-159 doi:10.1016/j.immuni.2008.01.005 Chiu NM, Chun T, Fay M, Mandal M, Wang CR (1999a) The majority of H2-M3 is retained intracellularly in a peptide-receptive state and traffics to the cell surface in the presence of N-formylated peptides The Journal of experimental medicine 190:423-434 Chiu NM, Wang B, Kerksiek KM, Kurlander R, Pamer EG, Wang CR (1999b) The selection of M3-restricted T cells is dependent on M3 expression and presentation of N-formylated peptides in the thymus The Journal of experimental medicine 190:1869-1878 Cho H, Bediako Y, Xu H, Choi HJ, Wang CR (2011) Positive selecting cell type determines the phenotype of MHC class Ib-restricted CD8+ T cells Proceedings of the National Academy of Sciences of the United States of America 108:13241- 13246 doi:10.1073/pnas.1105118108 Chua WJ, Truscott SM, Eickhoff CS, Blazevic A, Hoft DF, Hansen TH (2012) Polyclonal mucosa-associated invariant T cells have unique innate functions in bacterial infection Infect Immun 80:3256-3267 doi:10.1128/IAI.00279-12 Chun T, Grandea AG, 3rd, Lybarger L, Forman J, Van Kaer L, Wang CR (2001a) 232 Functional roles of TAP and tapasin in the assembly of M3-N-formylated peptide complexes Journal of immunology (Baltimore, Md : 1950) 167:1507-1514 Chun T, Serbina NV, Nolt D, Wang B, Chiu NM, Flynn JL, Wang CR (2001b) Induction of M3-restricted cytotoxic T lymphocyte responses by N-formylated peptides derived from Mycobacterium tuberculosis The Journal of experimental medicine 193:1213-1220 Cogen AL, Moore TA (2009) Beta2-microglobulin-dependent bacterial clearance and survival during murine Klebsiella pneumoniae bacteremia Infect Immun 77:360- 366 doi:10.1128/IAI.00909-08 Comiskey M, Goldstein CY, De Fazio SR, Mammolenti M, Newmark JA, Warner CM (2003) Evidence that HLA-G is the functional homolog of mouse Qa-2, the Ped gene product Hum Immunol 64:999-1004 Connolly DJ et al. (1993) A cDNA clone encoding the mouse Qa-1a histocompatibility antigen and proposed structure of the putative peptide binding site Journal of immunology (Baltimore, Md : 1950) 151:6089-6098 Cook RG, Landolfi NF (1983) Expression of the thymus leukemia antigen by activated peripheral T lymphocytes The Journal of experimental medicine 158:1012-1017 Corbett AJ et al. (2014) T-cell activation by transitory neo-antigens derived from distinct microbial pathways Nature 509:361-365 doi:10.1038/nature13160 Costa M et al. (2015) Lymphocyte gene expression signatures from patients and mouse models of hereditary hemochromatosis reveal a function of HFE as a negative regulator of CD8+ T-lymphocyte activation and differentiation in vivo PloS one 10:e0124246 doi:10.1371/journal.pone.0124246 D'Orazio SE, Halme DG, Ploegh HL, Starnbach MN (2003) Class Ia MHC-deficient BALB/c mice generate CD8+ T cell-mediated protective immunity against Listeria monocytogenes infection Journal of immunology (Baltimore, Md : 1950) 171:291- 298 D'Orazio SE, Shaw CA, Starnbach MN (2006) H2-M3-restricted CD8+ T cells are not required for MHC class Ib-restricted immunity against Listeria monocytogenes The Journal of experimental medicine 203:383-391 doi:10.1084/jem.20052256 Davies A et al. (2003) A peptide from heat shock protein 60 is the dominant peptide bound to Qa-1 in the absence of the MHC class Ia leader sequence peptide Qdm Journal of immunology (Baltimore, Md : 1950) 170:5027-5033 Davis BK, Cook RG, Rich RR, Rodgers JR (2002) Hyperconservation of the putative antigen recognition site of the MHC class I-b molecule TL in the subfamily Murinae: evidence that thymus leukemia antigen is an ancient mammalian gene Journal of immunology (Baltimore, Md : 1950) 169:6890-6899 de la Salle H et al. (2005) Assistance of microbial glycolipid antigen processing by CD1e Science 310:1321-1324 doi:10.1126/science.1115301 Delker SL, West AP, Jr., McDermott L, Kennedy MW, Bjorkman PJ (2004) Crystallographic studies of ligand binding by Zn-alpha2-glycoprotein J Struct Biol 148:205-213 doi:10.1016/j.jsb.2004.04.009 Devlin JJ, Weiss EH, Paulson M, Flavell RA (1985) Duplicated gene pairs and alleles of class I genes in the Qa2 region of the murine major histocompatibility complex: a 233 comparison The EMBO journal 4:3203-3207 Diehl M, Munz C, Keilholz W, Stevanovic S, Holmes N, Loke YW, Rammensee HG (1996) Nonclassical HLA-G molecules are classical peptide presenters Curr Biol 6:305-314 Doi T, Yamada H, Yajima T, Wajjwalku W, Hara T, Yoshikai Y (2007) H2-M3-restricted CD8+ T cells induced by peptide-pulsed dendritic cells confer protection against Mycobacterium tuberculosis Journal of immunology (Baltimore, Md : 1950) 178:3806-3813 Dunne MR, Elliott L, Hussey S, Mahmud N, Kelly J, Doherty DG, Feighery CF (2013) Persistent changes in circulating and intestinal gammadelta T cell subsets, invariant natural killer T cells and mucosal-associated invariant T cells in children and adults with coeliac disease PloS one 8:e76008 doi:10.1371/journal.pone.0076008 Eckle SB et al. (2014) A molecular basis underpinning the T cell receptor heterogeneity of mucosal-associated invariant T cells The Journal of experimental medicine 211:1585-1600 doi:10.1084/jem.20140484 Edholm ES et al. (2013) Nonclassical MHC class I-dependent invariant T cells are evolutionarily conserved and prominent from early development in amphibians Proceedings of the National Academy of Sciences of the United States of America 110:14342-14347 doi:10.1073/pnas.1309840110 Edholm ES, Grayfer L, De Jesus Andino F, Robert J (2015) Nonclassical MHC- Restricted Invariant Valpha6 T Cells Are Critical for Efficient Early Innate Antiviral Immunity in the Amphibian Xenopus laevis Journal of immunology (Baltimore, Md : 1950) 195:576-586 doi:10.4049/jimmunol.1500458 Feder JN et al. (1996) A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis Nat Genet 13:399-408 doi:10.1038/ng0896-399 Feder JN et al. (1997) The hemochromatosis founder mutation in HLA-H disrupts beta2- microglobulin interaction and cell surface expression The Journal of biological chemistry 272:14025-14028 Felio K et al. (2009) CD1-restricted adaptive immune responses to Mycobacteria in human group 1 CD1 transgenic mice The Journal of experimental medicine 206:2497-2509 doi:10.1084/jem.20090898 Fernandez CS, Amarasena T, Kelleher AD, Rossjohn J, McCluskey J, Godfrey DI, Kent SJ (2015) MAIT cells are depleted early but retain functional cytokine expression in HIV infection Immunol Cell Biol 93:177-188 doi:10.1038/icb.2014.91 Fischer K et al. (2004) Mycobacterial phosphatidylinositol mannoside is a natural antigen for CD1d-restricted T cells Proceedings of the National Academy of Sciences of the United States of America 101:10685-10690 doi:10.1073/pnas.0403787101 Fragoso G, Lamoyi E, Mellor A, Lomeli C, Govezensky T, Sciutto E (1996) Genetic control of susceptibility to Taenia crassiceps cysticercosis Parasitology 112 ( Pt 1):119-124 Fragoso G, Lamoyi E, Mellor A, Lomeli C, Hernandez M, Sciutto E (1998) Increased resistance to Taenia crassiceps murine cysticercosis in Qa-2 transgenic mice Infect Immun 66:760-764 234 Fujii T, Ishitani A, Geraghty DE (1994) A soluble form of the HLA-G antigen is encoded by a messenger ribonucleic acid containing intron 4 Journal of immunology (Baltimore, Md : 1950) 153:5516-5524 Gadola SD et al. (2002) Structure of human CD1b with bound ligands at 2.3 A, a maze for alkyl chains Nature immunology 3:721-726 doi:10.1038/ni821 Garcia P et al. (2002) Human T cell receptor-mediated recognition of HLA-E European journal of immunology 32:936-944 doi:10.1002/1521- 4141(200204)32:4<936::AID-IMMU936>3.0.CO;2-M Georgel P, Radosavljevic M, Macquin C, Bahram S (2011) The non-conventional MHC class I MR1 molecule controls infection by Klebsiella pneumoniae in mice Mol Immunol 48:769-775 doi:10.1016/j.molimm.2010.12.002 Geraghty DE, Stockschleader M, Ishitani A, Hansen JA (1992) Polymorphism at the HLA-E locus predates most HLA-A and -B polymorphism Hum Immunol 33:174- 184 Gold MC et al. (2010) Human mucosal associated invariant T cells detect bacterially infected cells PLoS Biol 8:e1000407 doi:10.1371/journal.pbio.1000407 Gold MC et al. (2014) MR1-restricted MAIT cells display ligand discrimination and pathogen selectivity through distinct T cell receptor usage The Journal of experimental medicine 211:1601-1610 doi:10.1084/jem.20140507 Goswami T, Andrews NC (2006) Hereditary hemochromatosis protein, HFE, interaction with transferrin receptor 2 suggests a molecular mechanism for mammalian iron sensing The Journal of biological chemistry 281:28494-28498 doi:10.1074/jbc.C600197200 Grimsley C et al. (2002) Definitive high resolution typing of HLA-E allelic polymorphisms: Identifying potential errors in existing allele data Tissue Antigens 60:206-212 Gulden PH et al. (1996) A Listeria monocytogenes pentapeptide is presented to cytolytic T lymphocytes by the H2-M3 MHC class Ib molecule Immunity 5:73-79 Guleria I, Sayegh MH (2007) Maternal acceptance of the fetus: true human tolerance Journal of immunology (Baltimore, Md : 1950) 178:3345-3351 Hamilton SE, Porter BB, Messingham KA, Badovinac VP, Harty JT (2004) MHC class Ia- restricted memory T cells inhibit expansion of a nonprotective MHC class Ib (H2- M3)-restricted memory response Nature immunology 5:159-168 doi:10.1038/ni1026 Hanau D et al. (1994) CD1 expression is not affected by human peptide transporter deficiency Hum Immunol 41:61-68 Hansen SG et al. (2016) Broadly targeted CD8(+) T cell responses restricted by major histocompatibility complex E Science 351:714-720 doi:10.1126/science.aac9475 He X, Tabaczewski P, Ho J, Stroynowski I, Garcia KC (2001) Promiscuous antigen presentation by the nonclassical MHC Ib Qa-2 is enabled by a shallow, hydrophobic groove and self-stabilized peptide conformation Structure 9:1213- 1224 Heinzel AS et al. (2002) HLA-E-dependent presentation of Mtb-derived antigen to human CD8+ T cells The Journal of experimental medicine 196:1473-1481 235 Hershberg R, Eghtesady P, Sydora B, Brorson K, Cheroutre H, Modlin R, Kronenberg M (1990) Expression of the thymus leukemia antigen in mouse intestinal epithelium Proceedings of the National Academy of Sciences of the United States of America 87:9727-9731 Hirai K, Hussey HJ, Barber MD, Price SA, Tisdale MJ (1998) Biological evaluation of a lipid-mobilizing factor isolated from the urine of cancer patients Cancer Res 58:2359-2365 Hofstetter AR, Evavold BD, Lukacher AE (2013) Peptide immunization elicits polyomavirus-specific MHC class ib-restricted CD8 T cells in MHC class ia allogeneic mice Viral Immunol 26:109-113 doi:10.1089/vim.2012.0052 Holcombe HR, Castano AR, Cheroutre H, Teitell M, Maher JK, Peterson PA, Kronenberg M (1995) Nonclassical behavior of the thymus leukemia antigen: peptide transporter-independent expression of a nonclassical class I molecule The Journal of experimental medicine 181:1433-1443 Holzapfel KL, Tyznik AJ, Kronenberg M, Hogquist KA (2014) Antigen-dependent versus -independent activation of invariant NKT cells during infection Journal of immunology (Baltimore, Md : 1950) 192:5490-5498 doi:10.4049/jimmunol.1400722 Horuzsko A, Antoniou J, Tomlinson P, Portik-Dobos V, Mellor AL (1997) HLA-G functions as a restriction element and a transplantation antigen in mice International immunology 9:645-653 Huang Y et al. (2011) Mucosal memory CD8(+) T cells are selected in the periphery by an MHC class I molecule Nature immunology 12:1086-1095 doi:10.1038/ni.2106 Hunt JS, Andrews GK, Wood GW (1987) Normal trophoblasts resist induction of class I HLA Journal of immunology (Baltimore, Md : 1950) 138:2481-2487 Ishitani A, Geraghty DE (1992) Alternative splicing of HLA-G transcripts yields proteins with primary structures resembling both class I and class II antigens Proceedings of the National Academy of Sciences of the United States of America 89:3947- 3951 Ito Y et al. (2013) Helicobacter pylori cholesteryl alpha-glucosides contribute to its pathogenicity and immune response by natural killer T cells PloS one 8:e78191 doi:10.1371/journal.pone.0078191 Jahng A, Maricic I, Aguilera C, Cardell S, Halder RC, Kumar V (2004) Prevention of autoimmunity by targeting a distinct, noninvariant CD1d-reactive T cell population reactive to sulfatide The Journal of experimental medicine 199:947-957 doi:10.1084/jem.20031389 Jay DC, Reed-Loisel LM, Jensen PE (2008) Polyclonal MHC Ib-restricted CD8+ T cells undergo homeostatic expansion in the absence of conventional MHC-restricted T cells Journal of immunology (Baltimore, Md : 1950) 180:2805-2814 Jensen KD et al. (2008) Thymic selection determines gammadelta T cell effector fate: antigen-naive cells make interleukin-17 and antigen-experienced cells make interferon gamma Immunity 29:90-100 doi:10.1016/j.immuni.2008.04.022 Jiang J et al. (2014) Mucosal-associated invariant T-cell function is modulated by programmed death-1 signaling in patients with active tuberculosis Am J Respir Crit Care Med 190:329-339 doi:10.1164/rccm.201401-0106OC 236 Jorgensen PB, Livbjerg AH, Hansen HJ, Petersen T, Hollsberg P (2012) Epstein-Barr virus peptide presented by HLA-E is predominantly recognized by CD8(bright) cells in multiple sclerosis patients PloS one 7:e46120 doi:10.1371/journal.pone.0046120 Joyce S, Tabaczewski P, Angeletti RH, Nathenson SG, Stroynowski I (1994) A nonpolymorphic major histocompatibility complex class Ib molecule binds a large array of diverse self-peptides The Journal of experimental medicine 179:579-588 Kawakami K et al. (2003) Critical role of Valpha14+ natural killer T cells in the innate phase of host protection against Streptococcus pneumoniae infection European journal of immunology 33:3322-3330 doi:10.1002/eji.200324254 Kawashima T et al. (2003) Cutting edge: major CD8 T cell response to live bacillus Calmette-Guerin is mediated by CD1 molecules Journal of immunology (Baltimore, Md : 1950) 170:5345-5348 Kerksiek KM, Busch DH, Pilip IM, Allen SE, Pamer EG (1999) H2-M3-restricted T cells in bacterial infection: rapid primary but diminished memory responses The Journal of experimental medicine 190:195-204 Kern P, Hussey RE, Spoerl R, Reinherz EL, Chang HC (1999) Expression, purification, and functional analysis of murine ectodomain fragments of CD8alphaalpha and CD8alphabeta dimers The Journal of biological chemistry 274:27237-27243 Kim S et al. (2011) Human cytomegalovirus microRNA miR-US4-1 inhibits CD8(+) T cell responses by targeting the aminopeptidase ERAP1 Nature immunology 12:984- 991 doi:10.1038/ni.2097 Kinjo Y et al. (2011) Invariant natural killer T cells recognize glycolipids from pathogenic Gram-positive bacteria Nature immunology 12:966-974 doi:10.1038/ni.2096 Kinjo Y et al. (2006) Natural killer T cells recognize diacylglycerol antigens from pathogenic bacteria Nature immunology 7:978-986 doi:10.1038/ni1380 Kinjo Y et al. (2005) Recognition of bacterial glycosphingolipids by natural killer T cells Nature 434:520-525 doi:10.1038/nature03407 Kirszenbaum M, Moreau P, Gluckman E, Dausset J, Carosella E (1994) An alternatively spliced form of HLA-G mRNA in human trophoblasts and evidence for the presence of HLA-G transcript in adult lymphocytes Proceedings of the National Academy of Sciences of the United States of America 91:4209-4213 Kjer-Nielsen L et al. (2012) MR1 presents microbial vitamin B metabolites to MAIT cells Nature 491:717-723 doi:10.1038/nature11605 Kovats S, Main EK, Librach C, Stubblebine M, Fisher SJ, DeMars R (1990) A class I antigen, HLA-G, expressed in human trophoblasts Science 248:220-223 Kumar H, Belperron A, Barthold SW, Bockenstedt LK (2000) Cutting edge: CD1d deficiency impairs murine host defense against the spirochete, Borrelia burgdorferi Journal of immunology (Baltimore, Md : 1950) 165:4797-4801 Kurepa Z, Su J, Forman J (2003) Memory phenotype of CD8+ T cells in MHC class Ia- deficient mice Journal of immunology (Baltimore, Md : 1950) 170:5414-5420 Le Bourhis L et al. (2010) Antimicrobial activity of mucosal-associated invariant T cells Nature immunology 11:701-708 doi:10.1038/ni.1890 237 Lebron JA et al. (1998) Crystal structure of the hemochromatosis protein HFE and characterization of its interaction with transferrin receptor Cell 93:111-123 Lee N, Goodlett DR, Ishitani A, Marquardt H, Geraghty DE (1998a) HLA-E surface expression depends on binding of TAP-dependent peptides derived from certain HLA class I signal sequences Journal of immunology (Baltimore, Md : 1950) 160:4951-4960 Lee N, Llano M, Carretero M, Ishitani A, Navarro F, Lopez-Botet M, Geraghty DE (1998b) HLA-E is a major ligand for the natural killer inhibitory receptor CD94/NKG2A Proceedings of the National Academy of Sciences of the United States of America 95:5199-5204 Lee N, Malacko AR, Ishitani A, Chen MC, Bajorath J, Marquardt H, Geraghty DE (1995) The membrane-bound and soluble forms of HLA-G bind identical sets of endogenous peptides but differ with respect to TAP association Immunity 3:591- 600 Leeansyah E et al. (2013) Activation, exhaustion, and persistent decline of the antimicrobial MR1-restricted MAIT-cell population in chronic HIV-1 infection Blood 121:1124-1135 doi:10.1182/blood-2012-07-445429 Leishman AJ et al. (2001) T cell responses modulated through interaction between CD8alphaalpha and the nonclassical MHC class I molecule, TL Science 294:1936-1939 doi:10.1126/science.1063564 Leite-De-Moraes MC et al. (1999) A distinct IL-18-induced pathway to fully activate NK T lymphocytes independently from TCR engagement Journal of immunology (Baltimore, Md : 1950) 163:5871-5876 LeMaoult J, Zafaranloo K, Le Danff C, Carosella ED (2005) HLA-G up-regulates ILT2, ILT3, ILT4, and KIR2DL4 in antigen presenting cells, NK cells, and T cells FASEB J 19:662-664 doi:10.1096/fj.04-1617fje Lenfant F, Pizzato N, Liang S, Davrinche C, Le Bouteiller P, Horuzsko A (2003) Induction of HLA-G-restricted human cytomegalovirus pp65 (UL83)-specific cytotoxic T lymphocytes in HLA-G transgenic mice The Journal of general virology 84:307-317 doi:10.1099/vir.0.18735-0 Lenz LL, Bevan MJ (1997) CTL responses to H2-M3-restricted Listeria epitopes Immunological reviews 158:115-121 Lenz LL, Dere B, Bevan MJ (1996) Identification of an H2-M3-restricted Listeria epitope: implications for antigen presentation by M3 Immunity 5:63-72 Levitt JM, Howell DD, Rodgers JR, Rich RR (2001) Exogenous peptides enter the endoplasmic reticulum of TAP-deficient cells and induce the maturation of nascent MHC class I molecules European journal of immunology 31:1181-1190 doi:10.1002/1521-4141(200104)31:4<1181::AID-IMMU1181>3.0.CO;2-J Lewinsohn DM, Briden AL, Reed SG, Grabstein KH, Alderson MR (2000) Mycobacterium tuberculosis-reactive CD8+ T lymphocytes: the relative contribution of classical versus nonclassical HLA restriction Journal of immunology (Baltimore, Md : 1950) 165:925-930 Liu Y et al. (2003) The crystal structure of a TL/CD8alphaalpha complex at 2.1 A resolution: implications for modulation of T cell activation and memory Immunity 18:205-215 238 Ljunggren HG et al. (1990) Empty MHC class I molecules come out in the cold Nature 346:476-480 doi:10.1038/346476a0 Lo WF, Woods AS, DeCloux A, Cotter RJ, Metcalf ES, Soloski MJ (2000) Molecular mimicry mediated by MHC class Ib molecules after infection with gram-negative pathogens Nat Med 6:215-218 doi:10.1038/72329 Lockridge JL et al. (2011) Analysis of the CD1 antigen presenting system in humanized SCID mice PloS one 6:e21701 doi:10.1371/journal.pone.0021701 Loconto J et al. (2003) Functional expression of murine V2R pheromone receptors involves selective association with the M10 and M1 families of MHC class Ib molecules Cell 112:607-618 Lotter H et al. (2009) Natural killer T cells activated by a lipopeptidophosphoglycan from Entamoeba histolytica are critically important to control amebic liver abscess PLoS pathogens 5:e1000434 doi:10.1371/journal.ppat.1000434 Lotter H, Jacobs T, Gaworski I, Tannich E (2006) Sexual dimorphism in the control of amebic liver abscess in a mouse model of disease Infect Immun 74:118-124 doi:10.1128/IAI.74.1.118-124.2006 Macedo MF, Porto G, Costa M, Vieira CP, Rocha B, Cruz E (2010) Low numbers of CD8+ T lymphocytes in hereditary haemochromatosis are explained by a decrease of the most mature CD8+ effector memory T cells Clin Exp Immunol 159:363-371 doi:10.1111/j.1365-2249.2009.04066.x Madakamutil LT et al. (2004) CD8alphaalpha-mediated survival and differentiation of CD8 memory T cell precursors Science 304:590-593 doi:10.1126/science.1092316 Martin E et al. (2009) Stepwise development of MAIT cells in mouse and human PLoS Biol 7:e54 doi:10.1371/journal.pbio.1000054 Mazzarino P et al. (2005) Identification of effector-memory CMV-specific T lymphocytes that kill CMV-infected target cells in an HLA-E-restricted fashion European journal of immunology 35:3240-3247 doi:10.1002/eji.200535343 McElhinny AS, Exley GE, Warner CM (2000) Painting Qa-2 onto Ped slow preimplantatiom embryos increases the rate of cleavage Am J Reprod Immunol 44:52-58 doi:DOI 10.1111/j.8755-8920.2000.440108.x Meierovics A, Yankelevich WJ, Cowley SC (2013) MAIT cells are critical for optimal mucosal immune responses during in vivo pulmonary bacterial infection Proceedings of the National Academy of Sciences of the United States of America 110:E3119-3128 doi:10.1073/pnas.1302799110 Miranda CJ, Makui H, Andrews NC, Santos MM (2004) Contributions of beta2- microglobulin-dependent molecules and lymphocytes to iron regulation: insights from HfeRag1(-/-) and beta2mRag1(-/-) double knock-out mice Blood 103:2847- 2849 doi:10.1182/blood-2003-09-3300 Morales PJ, Pace JL, Platt JS, Langat DK, Hunt JS (2007) Synthesis of beta(2)- microglobulin-free, disulphide-linked HLA-G5 homodimers in human placental villous cytotrophoblast cells Immunology 122:179-188 doi:10.1111/j.1365- 2567.2007.02623.x Morita A et al. (1994) TL antigen as a transplantation antigen recognized by TL- 239 restricted cytotoxic T cells The Journal of experimental medicine 179:777-784 Nagarajan NA, Gonzalez F, Shastri N (2012) Nonclassical MHC class Ib-restricted cytotoxic T cells monitor antigen processing in the endoplasmic reticulum Nature immunology 13:579-586 doi:10.1038/ni.2282 Nattermann J et al. (2005) The HLA-A2 restricted T cell epitope HCV core 35-44 stabilizes HLA-E expression and inhibits cytolysis mediated by natural killer cells The American journal of pathology 166:443-453 doi:10.1016/S0002- 9440(10)62267-5 Old LJ, Boyse EA (1963) Antigenic Properties of Experimental Leukemias. I. Serological Studies in Vitro with Spontaneous and Radiation-Induced Leukemias J Natl Cancer Inst 31:977-995 Olivares-Villagomez D, Mendez-Fernandez YV, Parekh VV, Lalani S, Vincent TL, Cheroutre H, Van Kaer L (2008) Thymus leukemia antigen controls intraepithelial lymphocyte function and inflammatory bowel disease Proceedings of the National Academy of Sciences of the United States of America 105:17931-17936 doi:10.1073/pnas.0808242105 Pardigon N et al. (2006) CD8 alpha alpha-mediated intraepithelial lymphocyte snatching of thymic leukemia MHC class Ib molecules in vitro and in vivo Journal of immunology (Baltimore, Md : 1950) 177:1590-1598 Parkkila S et al. (1997) Association of the transferrin receptor in human placenta with HFE, the protein defective in hereditary hemochromatosis Proceedings of the National Academy of Sciences of the United States of America 94:13198-13202 Paul P et al. (2000) Identification of HLA-G7 as a new splice variant of the HLA-G mRNA and expression of soluble HLA-G5, -G6, and -G7 transcripts in human transfected cells Hum Immunol 61:1138-1149 Pietra G, Romagnani C, Manzini C, Moretta L, Mingari MC (2010) The emerging role of HLA-E-restricted CD8+ T lymphocytes in the adaptive immune response to pathogens and tumors Journal of biomedicine & biotechnology 2010:907092 doi:10.1155/2010/907092 Pietra G et al. (2003) HLA-E-restricted recognition of cytomegalovirus-derived peptides by human CD8+ cytolytic T lymphocytes Proceedings of the National Academy of Sciences of the United States of America 100:10896-10901 doi:10.1073/pnas.1834449100 Porcelli S, Yockey CE, Brenner MB, Balk SP (1993) Analysis of T cell antigen receptor (TCR) expression by human peripheral blood CD4-8- alpha/beta T cells demonstrates preferential use of several V beta genes and an invariant TCR alpha chain The Journal of experimental medicine 178:1-16 Porto G, Reimao R, Goncalves C, Vicente C, Justica B, de Sousa M (1994) Haemochromatosis as a window into the study of the immunological system: a novel correlation between CD8+ lymphocytes and iron overload Eur J Haematol 52:283-290 Princiotta MF, Lenz LL, Bevan MJ, Staerz UD (1998) H2-M3 restricted presentation of a Listeria-derived leader peptide The Journal of experimental medicine 187:1711- 1719 Rahimpour A et al. (2015) Identification of phenotypically and functionally heterogeneous 240 mouse mucosal-associated invariant T cells using MR1 tetramers The Journal of experimental medicine 212:1095-1108 doi:10.1084/jem.20142110 Rajagopalan S, Long EO (2012) KIR2DL4 (CD158d): An activation receptor for HLA-G Frontiers in immunology 3:258 doi:10.3389/fimmu.2012.00258 Reantragoon R et al. (2013) Antigen-loaded MR1 tetramers define T cell receptor heterogeneity in mucosal-associated invariant T cells The Journal of experimental medicine 210:2305-2320 doi:10.1084/jem.20130958 Reantragoon R et al. (2012) Structural insight into MR1-mediated recognition of the mucosal associated invariant T cell receptor The Journal of experimental medicine 209:761-774 doi:10.1084/jem.20112095 Reuben A, Phenix M, Santos MM, Lapointe R (2014) The WT hemochromatosis protein HFE inhibits CD8(+) T-lymphocyte activation European journal of immunology 44:1604-1614 doi:10.1002/eji.201343955 Riegert P, Wanner V, Bahram S (1998) Genomics, isoforms, expression, and phylogeny of the MHC class I-related MR1 gene Journal of immunology (Baltimore, Md : 1950) 161:4066-4077 Robinson PJ, Millrain M, Antoniou J, Simpson E, Mellor AL (1989) A glycophospholipid anchor is required for Qa-2-mediated T cell activation Nature 342:85-87 doi:10.1038/342085a0 Rodgers JR, Cook RG (2005) MHC class Ib molecules bridge innate and acquired immunity Nature reviews Immunology 5:459-471 doi:10.1038/nri1635 Rodgers JR, Mehta V, Cook RG (1995) Surface expression of beta 2-microglobulin- associated thymus-leukemia antigen is independent of TAP2 European journal of immunology 25:1001-1007 doi:10.1002/eji.1830250421 Rohrlich PS et al. (2005) Direct recognition by alphabeta cytolytic T cells of Hfe, a MHC class Ib molecule without antigen-presenting function Proceedings of the National Academy of Sciences of the United States of America 102:12855-12860 doi:10.1073/pnas.0502309102 Rolph MS, Kaufmann SH (2000) Partially TAP-independent protection against Listeria monocytogenes by H2-M3-restricted CD8+ T cells Journal of immunology (Baltimore, Md : 1950) 165:4575-4580 Rotzschke O, Falk K, Stevanovic S, Grahovac B, Soloski MJ, Jung G, Rammensee HG (1993) Qa-2 molecules are peptide receptors of higher stringency than ordinary class I molecules Nature 361:642-644 doi:10.1038/361642a0 Rouas-Freiss N, Goncalves RM, Menier C, Dausset J, Carosella ED (1997) Direct evidence to support the role of HLA-G in protecting the fetus from maternal uterine natural killer cytolysis Proceedings of the National Academy of Sciences of the United States of America 94:11520-11525 Salerno-Goncalves R, Fernandez-Vina M, Lewinsohn DM, Sztein MB (2004) Identification of a human HLA-E-restricted CD8+ T cell subset in volunteers immunized with Salmonella enterica serovar Typhi strain Ty21a typhoid vaccine Journal of immunology (Baltimore, Md : 1950) 173:5852-5862 Santos M, Schilham MW, Rademakers LH, Marx JJ, de Sousa M, Clevers H (1996) Defective iron homeostasis in beta 2-microglobulin knockout mice recapitulates 241 hereditary hemochromatosis in man The Journal of experimental medicine 184:1975-1985 Santos MM, de Sousa M, Rademakers LH, Clevers H, Marx JJ, Schilham MW (2000) Iron overload and heart fibrosis in mice deficient for both beta2-microglobulin and Rag1 The American journal of pathology 157:1883-1892 Scharf L et al. (2010) The 2.5 A structure of CD1c in complex with a mycobacterial lipid reveals an open groove ideally suited for diverse antigen presentation Immunity 33:853-862 doi:10.1016/j.immuni.2010.11.026 Schmidt CM, Garrett E, Orr HT (1997) Cytotoxic T lymphocyte recognition of HLA-G in mice Hum Immunol 55:127-139 Schulte D et al. (2009) The HLA-E(R)/HLA-E(R) genotype affects the natural course of hepatitis C virus (HCV) infection and is associated with HLA-E-restricted recognition of an HCV-derived peptide by interferon-gamma-secreting human CD8(+) T cells The Journal of infectious diseases 200:1397-1401 doi:10.1086/605889 Seach N et al. (2013) Double-positive thymocytes select mucosal-associated invariant T cells Journal of immunology (Baltimore, Md : 1950) 191:6002-6009 doi:10.4049/jimmunol.1301212 Seaman MS, Perarnau B, Lindahl KF, Lemonnier FA, Forman J (1999) Response to Listeria monocytogenes in mice lacking MHC class Ia molecules Journal of immunology (Baltimore, Md : 1950) 162:5429-5436 Shawar SM, Vyas JM, Shen E, Rodgers JR, Rich RR (1993) Differential amino-terminal anchors for peptide binding to H-2M3a or H-2Kb and H-2Db Journal of immunology (Baltimore, Md : 1950) 151:201-210 Siddiqui S, Visvabharathy L, Wang CR (2015) Role of Group 1 CD1-Restricted T Cells in Infectious Disease Frontiers in immunology 6:337 doi:10.3389/fimmu.2015.00337 Smith GP, Dabhi VM, Pamer EG, Lindahl KF (1994) Peptide presentation by the MHC class Ib molecule, H2-M3 International immunology 6:1917-1926 Stroynowski I, Lindahl KF (1994) Antigen presentation by non-classical class I molecules Current opinion in immunology 6:38-44 Stroynowski I, Soloski M, Low MG, Hood L (1987) A single gene encodes soluble and membrane-bound forms of the major histocompatibility Qa-2 antigen: anchoring of the product by a phospholipid tail Cell 50:759-768 Sullivan BA, Kraj P, Weber DA, Ignatowicz L, Jensen PE (2002) Positive selection of a Qa-1-restricted T cell receptor with specificity for insulin Immunity 17:95-105 Swanson PA, 2nd, Pack CD, Hadley A, Wang CR, Stroynowski I, Jensen PE, Lukacher AE (2008) An MHC class Ib-restricted CD8 T cell response confers antiviral immunity The Journal of experimental medicine 205:1647-1657 doi:10.1084/jem.20080570 Tabaczewski P, Chiang E, Henson M, Stroynowski I (1997) Alternative peptide binding motifs of Qa-2 class Ib molecules define rules for binding of self and nonself peptides Journal of immunology (Baltimore, Md : 1950) 159:2771-2781 Tabaczewski P, Shirwan H, Lewis K, Stroynowski I (1994) Alternative splicing of class Ib major histocompatibility complex transcripts in vivo leads to the expression of 242 soluble Qa-2 molecules in murine blood Proceedings of the National Academy of Sciences of the United States of America 91:1883-1887 Tabaczewski P, Stroynowski I (1994) Expression of secreted and glycosylphosphatidylinositol-bound Qa-2 molecules is dependent on functional TAP-2 peptide transporter Journal of immunology (Baltimore, Md : 1950) 152:5268-5274 Tatituri RV et al. (2013) Recognition of microbial and mammalian phospholipid antigens by NKT cells with diverse TCRs Proceedings of the National Academy of Sciences of the United States of America 110:1827-1832 doi:10.1073/pnas.1220601110 Teitell M, Holcombe H, Cheroutre H, Aldrich CJ, Stroynowski I, Forman J, Kronenberg M (1993) The alpha 3 domain of the Qa-2 molecule is defective for CD8 binding and cytotoxic T lymphocyte activation The Journal of experimental medicine 178:2139-2145 Tilloy F et al. (1999) An invariant T cell receptor alpha chain defines a novel TAP- independent major histocompatibility complex class Ib-restricted alpha/beta T cell subpopulation in mammals The Journal of experimental medicine 189:1907-1921 Tomasec P et al. (2000) Surface expression of HLA-E, an inhibitor of natural killer cells, enhanced by human cytomegalovirus gpUL40 Science 287:1031 Treiner E (2015) Mucosal-associated invariant T cells in inflammatory bowel diseases: bystanders, defenders, or offenders? Frontiers in immunology 6:27 doi:10.3389/fimmu.2015.00027 Treiner E et al. (2003) Selection of evolutionarily conserved mucosal-associated invariant T cells by MR1 Nature 422:164-169 doi:10.1038/nature01433 Treiner E, Liblau RS (2015) Mucosal-Associated Invariant T Cells in Multiple Sclerosis: The Jury is Still Out Frontiers in immunology 6:503 doi:10.3389/fimmu.2015.00503 Tsujimura K, Obata Y, Iwase S, Matsudaira Y, Ozeki S, Takahashi T (2000) The epitope detected by cytotoxic T lymphocytes against thymus leukemia (TL) antigen is TAP independent International immunology 12:1217-1225 Tsujimura K et al. (2003) Thymus leukemia antigen (TL)-specific cytotoxic T lymphocytes recognize the alpha1/alpha2 domain of TL free from antigenic peptides International immunology 15:1319-1326 Tsujimura K, Obata Y, Matsudaira Y, Ozeki S, Yoshikawa K, Saga S, Takahashi T (2001) The binding of thymus leukemia (TL) antigen tetramers to normal intestinal intraepithelial lymphocytes and thymocytes Journal of immunology (Baltimore, Md : 1950) 167:759-764 Tsujimura K, Takahashi T, Morita A, Hasegawa-Nishiwaki H, Iwase S, Obata Y (1996) Positive selection of gamma delta CTL by TL antigen expressed in the thymus The Journal of experimental medicine 184:2175-2184 Tupin E et al. (2008) NKT cells prevent chronic joint inflammation after infection with Borrelia burgdorferi Proceedings of the National Academy of Sciences of the United States of America 105:19863-19868 doi:10.1073/pnas.0810519105 Tvinnereim A, Wizel B (2007) CD8+ T cell protective immunity against Chlamydia 243 pneumoniae includes an H2-M3-restricted response that is largely CD4+ T cell- independent Journal of immunology (Baltimore, Md : 1950) 179:3947-3957 Tyznik AJ, Verma S, Wang Q, Kronenberg M, Benedict CA (2014) Distinct requirements for activation of NKT and NK cells during viral infection Journal of immunology (Baltimore, Md : 1950) 192:3676-3685 doi:10.4049/jimmunol.1300837 Ugrinovic S, Brooks CG, Robson J, Blacklaws BA, Hormaeche CE, Robinson JH (2005) H2-M3 major histocompatibility complex class Ib-restricted CD8 T cells induced by Salmonella enterica serovar Typhimurium infection recognize proteins released by Salmonella serovar Typhimurium Infect Immun 73:8002-8008 doi:10.1128/IAI.73.12.8002-8008.2005 Ulbrecht M, Martinozzi S, Grzeschik M, Hengel H, Ellwart JW, Pla M, Weiss EH (2000) Cutting edge: the human cytomegalovirus UL40 gene product contains a ligand for HLA-E and prevents NK cell-mediated lysis Journal of immunology (Baltimore, Md : 1950) 164:5019-5022 Ulrichs T, Moody DB, Grant E, Kaufmann SH, Porcelli SA (2003) T-cell responses to CD1-presented lipid antigens in humans with Mycobacterium tuberculosis infection Infect Immun 71:3076-3087 Urdahl KB, Liggitt D, Bevan MJ (2003) CD8+ T cells accumulate in the lungs of Mycobacterium tuberculosis-infected Kb-/-Db-/- mice, but provide minimal protection Journal of immunology (Baltimore, Md : 1950) 170:1987-1994 Urdahl KB, Sun JC, Bevan MJ (2002) Positive selection of MHC class Ib-restricted CD8(+) T cells on hematopoietic cells Nature immunology 3:772-779 doi:10.1038/ni814 Ussher JE et al. (2014) CD161++ CD8+ T cells, including the MAIT cell subset, are specifically activated by IL-12+IL-18 in a TCR-independent manner European journal of immunology 44:195-203 doi:10.1002/eji.201343509 van Meijgaarden KE, Haks MC, Caccamo N, Dieli F, Ottenhoff TH, Joosten SA (2015) Human CD8+ T-cells recognizing peptides from Mycobacterium tuberculosis (Mtb) presented by HLA-E have an unorthodox Th2-like, multifunctional, Mtb inhibitory phenotype and represent a novel human T-cell subset PLoS pathogens 11:e1004671 doi:10.1371/journal.ppat.1004671 Van Rhijn I et al. (2013) A conserved human T cell population targets mycobacterial antigens presented by CD1b Nature immunology 14:706-713 doi:10.1038/ni.2630 Vance RE, Jamieson AM, Raulet DH (1999) Recognition of the class Ib molecule Qa- 1(b) by putative activating receptors CD94/NKG2C and CD94/NKG2E on mouse natural killer cells The Journal of experimental medicine 190:1801-1812 Vance RE, Kraft JR, Altman JD, Jensen PE, Raulet DH (1998) Mouse CD94/NKG2A is a natural killer cell receptor for the nonclassical major histocompatibility complex (MHC) class I molecule Qa-1(b) The Journal of experimental medicine 188:1841- 1848 Wang CR, Castano AR, Peterson PA, Slaughter C, Lindahl KF, Deisenhofer J (1995) Nonclassical binding of formylated peptide in crystal structure of the MHC class Ib molecule H2-M3 Cell 82:655-664 Wang CR, Loveland BE, Lindahl KF (1991) H-2M3 encodes the MHC class I molecule 244 presenting the maternally transmitted antigen of the mouse Cell 66:335-345 Wang EC et al. (2002) UL40-mediated NK evasion during productive infection with human cytomegalovirus Proceedings of the National Academy of Sciences of the United States of America 99:7570-7575 doi:10.1073/pnas.112680099 Warner CM, Gollnick SO, Flaherty L, Goldbard SB (1987) Analysis of Qa-2 antigen expression by preimplantation mouse embryos: possible relationship to the preimplantation-embryo-development (Ped) gene product Biol Reprod 36:611- 616 Weber DA et al. (2002) Peptide-independent folding and CD8 alpha alpha binding by the nonclassical class I molecule, thymic leukemia antigen Journal of immunology (Baltimore, Md : 1950) 169:5708-5714 Wesley JD, Tessmer MS, Chaukos D, Brossay L (2008) NK cell-like behavior of Valpha14i NK T cells during MCMV infection PLoS pathogens 4:e1000106 doi:10.1371/journal.ppat.1000106 Wieland Brown LC et al. (2013) Production of alpha-galactosylceramide by a prominent member of the human gut microbiota PLoS Biol 11:e1001610 doi:10.1371/journal.pbio.1001610 Williams MA, Bevan MJ (2005) Cutting edge: a single MHC class Ia is sufficient for CD8 memory T cell differentiation Journal of immunology (Baltimore, Md : 1950) 175:2066-2069 Wu M, van Kaer L, Itohara S, Tonegawa S (1991) Highly restricted expression of the thymus leukemia antigens on intestinal epithelial cells The Journal of experimental medicine 174:213-218 Xu H, Chun T, Choi HJ, Wang B, Wang CR (2006) Impaired response to Listeria in H2- M3-deficient mice reveals a nonredundant role of MHC class Ib-specific T cells in host defense The Journal of experimental medicine 203:449-459 doi:10.1084/jem.20051866 Yeager M, Kumar S, Hughes AL (1997) Sequence convergence in the peptide-binding region of primate and rodent MHC class Ib molecules Mol Biol Evol 14:1035- 1041 Yokoyama K, Stockert E, Old LJ, Nathenson SG (1982) Structural evidence that the small subunit found associated with the TL antigen is beta 2-microglobulin Immunogenetics 15:543-549 Zajonc DM et al. (2005) Molecular mechanism of lipopeptide presentation by CD1a Immunity 22:209-219 doi:10.1016/j.immuni.2004.12.009 Zeng L et al. (2012) A structural basis for antigen presentation by the MHC class Ib molecule, Qa-1b Journal of immunology (Baltimore, Md : 1950) 188:302-310 doi:10.4049/jimmunol.1102379 Zhao J, Siddiqui S, Shang S, Bian Y, Bagchi S, He Y, Wang CR (2015) Mycolic acid- specific T cells protect against Mycobacterium tuberculosis infection in a humanized transgenic mouse model Elife 4 doi:10.7554/eLife.08525 Zhou XY et al. (1998) HFE gene knockout produces mouse model of hereditary hemochromatosis Proceedings of the National Academy of Sciences of the United States of America 95:2492-2497 245 APPENDIX III: NFIL3 EXPRESSION DISTINGUISHES TRNK AND CNK-LIKE CELLS IN THE MOUSE SUBMANDIBULAR GLANDS Originally published in The Journal of Immunology, September 15, 2016 Volume 197, Number 6, Pages 2485 – 2491 http://www.jimmunol.org/content/197/6/2485.long Copyright © 2016 The American Association of Immunologists, Inc. 246 NFIL3 expression distinguishes trNK and cNK-like cells in the mouse submandibular glands Timothy K. Erick,1 Courtney K. Anderson,1 Emma C. Reilly,1 Jack R. Wands,2 and Laurent Brossasy1 1 Department of Molecular Microbiology and Immunology, Division of Biology and Medicine, Brown University, Providence, Rhode Island, 02912, USA 2 Liver Research Center, Rhode Island Hospital and the Department of Medicine, Warren Alpert Medical School at Brown University, Providence, Rhode Island, 02912, USA This work was supported by National Institutes of Health Research Grants AI46709 and AI122217 (to LB), 1F31DE024360 (to TKE) and 1F31AI124556 (to CKA). 247 The Journal of Immunology NFIL3 Expression Distinguishes Tissue-Resident NK Cells and Conventional NK-like Cells in the Mouse Submandibular Glands Timothy K. Erick,* Courtney K. Anderson,* Emma C. Reilly,* Jack R. Wands,† and Laurent Brossay* The submandibular salivary gland (SMG), a major site of persistent infection for many viruses, contains a large NK cell population. Using NFIL3-deficient mice, PLZF reporter/fate mapping mice, and mixed bone marrow chimeras, we identified two distinct pop- ulations of NK cells in the SMG. Although phenotypically unique, the main population relies on NFIL3, but not PLZF, for devel- opment and, therefore, is developmentally similar to the conventional NK cell subset. In contrast, we found that approximately one quarter of the SMG NK cells develop independently of NFIL3. Interestingly, NFIL3-independent SMG tissue-resident NK (trNK) cells are developmentally distinct from liver trNK cells. We also demonstrated that the SMG NK cell hyporesponsive phenotype Downloaded from http://www.jimmunol.org/ by guest on March 14, 2018 during murine CMV infection is tissue specific and not cell intrinsic. In contrast, NFIL3-independent SMG trNK cells are intrin- sically hyporesponsive. Altogether, our data show that the SMG tissue environment shapes a unique repertoire of NK-like cells with distinct phenotypes. The Journal of Immunology, 2016, 197: 2485–2491. C onventional NK (cNK) cells are derived from the common response during encounters with the invading trophoblast cells of the lymphoid progenitor in the bone marrow (1). From there, placenta, despite possessing the full complement of activating re- they develop into committed NK cell precursors that de- ceptors and cytotoxic machinery (9–11). Thymic NK cells represent velop into immature NK (iNK) cells upon acquisition of NK1.1 another population that develops from unique precursors; they are expression. iNK cells progress into mature NK cells with a CD122+ Ly49lowCD11blow and CD127+CD69high, in contrast to cNK cells NK1.1+NKp46+DX5+ phenotype. In addition to cNK cells, several (4, 5). A unique population of trNK cells also was discovered re- distinct populations of tissue-resident NK (trNK) cells have been cently in the kidneys (6). The current understanding is that cNK identified, with unique developmental pathways and phenotypic cells, together with liver and skin trNKs (ILC1s), uterine NK cells, attributes (2–7). The liver contains a population of cNK cells, as thymic NK cells, and kidney trNK cells, account for multiple distinct well as a population of trNK cells (phenotypically similar to group NK cell lineages (3, 7). 1 innate lymphoid cells [ILC1s]) that maintains a CD49a+DX52 NFIL3 (also called E4BP4) is a basic leucine-zipper transcription TRAIL+ phenotype and develops from a liver-specific precursor factor that is linked to a number of immune processes and is crucial pool (3, 8). The skin also harbors a trNK/ILC1 subset, and there is for the early development of cNK cells (12–14). In contrast, the evidence to indicate that skin and liver trNK cells arise from the different trNK cell subsets have unique developmental require- same developmental lineage (3). Uterine NK cells are another ments. Although NFIL3 deficiency results in ablation of cNK cells unique population with a distinct phenotype from cNK and liver/skin in the periphery, its activity is mostly dispensable for the devel- trNK cells. Uterine NK cells do not produce an effector or cytotoxic opment of trNK cells in the liver (15), uterus, and skin (3), despite contrasting evidence that NFIL3 is necessary for the development *Department of Molecular Microbiology and Immunology, Division of Biology and of all innate lymphoid cell (ILC) lineages (16–19). T-bet and Medicine, Brown University, Providence, RI 02912; and †Liver Research Center, Eomes are also necessary, albeit at different levels, for the de- Rhode Island Hospital and the Department of Medicine, Warren Alpert Medical velopment of mature cNK cells (20), and there is evidence that School at Brown University, Providence, RI 02912 these two transcription factors are regulated by NFIL3 (14). ORCIDs: 0000-0003-1731-624X (J.R.W.); 0000-0002-7497-8488 (L.B.). However, liver and skin trNK cells develop independently of Received for publication June 23, 2016. Accepted for publication July 17, 2016. Eomes, and uterine trNK cells do not require T-bet (3, 21). This work was supported by National Institutes of Health Research Grants AI46709 The requirements of these transcription factors for the devel- and AI122217 (to L.B.), 1F31DE024360 (to T.K.E.), and 1F31AI124556 (to C.K.A.). opment of NK cells in the submandibular salivary gland (SMG) T.K.E. conceived, performed, and analyzed the experiments and wrote the manu- script; C.K.A. and E.C.R. conceived, performed, and analyzed the experiments; J.R.W. have not been clearly defined. In this study, using NFIL3-deficient contributed reagents and analysis tools; and L.B. conceived and analyzed the experi- mice, PLZF reporter/fate mapping mice, and mixed bone marrow ments and wrote the manuscript. chimeras, we show that the murine SMG contains two distinct Address correspondence and reprint requests to Dr. Laurent Brossay, Department of populations of NK cells: a main cNK-like cell subset that relies on Molecular Microbiology and Immunology, Division of Biology and Medicine, Box G-B618, Brown University, Providence, RI 02912. E-mail address: Laurent_Brossay@brown. NFIL3 for development and a smaller trNK cell subset that is edu NFIL3 independent. Our findings also demonstrate that SMG trNK The online version of this article contains supplemental material. cells represent another distinct ILC lineage with a unique devel- Abbreviations used in this article: AhR, aryl hydrocarbon receptor; B6, C57BL/6; opmental pathway. Importantly, using the murine CMV (MCMV) ILC, innate lymphoid cell; ILC1, group 1 innate lymphoid cell; iNK, immature NK; model of infection, we also show that the hyporesponsive phenotype LSK, Lin2Sca-1+cKit+; MCMV, murine CMV; 1% PBS-serum, PBS supplemented of NFIL3-dependent SMG NK cells is due to tissue environmental with 1% FBS; SMG, submandibular salivary gland; trNK, tissue-resident NK. factors, whereas NFIL3-independent SMG NK cells are intrinsically Copyright ! 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 poor effector cells. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601099 248 2486 SUBMANDIBULAR GLAND NK CELL DEVELOPMENT AND FUNCTIONS Flow cytometric analysis, Abs, and reagents Lymphocyte samples were incubated in 1% PBS-serum with the blocking mAb 2.4G2 and stained with specific mAbs for 20 min at 4˚C. For intra- cellular cytokine staining, cells were stained with extracellular mAbs, fixed with Cytofix/Cytoperm (BD Bioscience) for 20 min, and stained with in- tracellular mAbs in 13 Perm/Wash (BD Biosciences) for 20 min. For intranuclear transcription factor staining, cells were stained with intra- cellular Abs using the Foxp3 transcription factor staining reagents (BD Bioscience). Events were collected on a FACSAria (BD), and the data were analyzed using FlowJo (TreeStar). FITC-DX5, PE-Ly49H, PE–IFN-g, PE- E4BP4, PE-TCRb, PE-CD27, PE-NK1.1, PECy5-DX5, PECy7-NKp46, PECy7–T-bet, PECy7–Sca-1, PerCPCy5.5-CD127, PerCPCy5.5-NK1.1, PerCP–eFluor 710–NKG2A/C/E, allophycocyanin-CD3, allophycocyanin- Ly49H, allophycocyanin–IFN-g, allophycocyanin–TNF-a, allophycocyanin- TRAIL, allophycocyanin-CD45.1, allophycocyanin–eFluor 780–CD45.2, allophycocyanin–eFluor 780–CD117, eFluor 450–CD11b, eFluor 450–IFN-g, eFluor 450–CD3, and eFluor 450–Eomes were purchased from eBioscience (San Diego, CA). PE-CD49a, allophycocyanin-CD49a, Pacific Blue–Lineage, BV421-CD127, BV605-CD3, BV605-NK1.1, BV785-CD3, and BV785-NK1.1 Downloaded from http://www.jimmunol.org/ by guest on March 14, 2018 FIGURE 1. SMG CD32 NK1.1+ cells are significantly reduced in NFIL32/2 mice. (A) Representative staining of spleen, liver, and SMG NK cells in NFIL3+/+ and NFIL32/2 mice. (B) Frequency of SMG NK cells in NFIL3+/+ (n = 23), NFIL3+/2 (n = 23), and NFIL32/2 (n = 22) mice. (C) Absolute SMG NK cell number in NFIL3+/+ (n = 14), NFIL3+/2 (n = 14), and NFIL32/2 (n = 13) mice. Data are mean 6 SEM and are pooled from nine experiments. ****p , 0.0001, ***p = 0.0001–0.001, **p = 0.001–0.01. Materials and Methods Mice C57BL/6, B6.SJL, AhR2/2, and Rag22/2IL-2Rg 2/2 mice were purchased from Taconic Biosciences (Germantown, NY). T-bet2/2, R26R-EFYP, and PLZFGFPcre mice were purchased from The Jackson Laboratory (Bar Harbor, ME). R26R-EFYP mice were bred with PLZFGFPcre mice to produce PLZFGFPcre+/2ROSA26-floxstop-YFP mice. NFIL32/2 mice were a generous gift from Dr. Hugh J.M. Brady (13) and were bred in-house. All mice were maintained in pathogen-free facilities at Brown University. Both sexes were included, and no differences were observed. Infection and treatment protocols MCMV infections were carried out as previously described (2). Isolation of murine lymphocytes Mice were sacrificed with isoflurane, and cardiac puncture was performed prior to organ removal. Spleens were processed on the spleen01.01 program on a GentleMACS dissociator (Miltenyi Biotec), filtered through nylon mesh, and layered on Lympholyte-M (Cedarlane Laboratories). Lympho- cytes were harvested from the gradient interface and washed once in PBS supplemented with 1% FBS (1% PBS-serum). Livers were perfused with 1% PBS-serum before removal, processed in 1% PBS-serum on the E.01 program on the GentleMACS, and filtered through nylon mesh. The samples were washed three times with 1% PBS-serum, resuspended in 40% Percoll, and layered on 70% Percoll. Lymphocytes were harvested from the gradient interface and washed once with 1% PBS-serum. SMGs were processed manually to remove lymph nodes, processed in collagenase IV (Sigma-Aldrich) on the heart01.01 program on the GentleMACS, in- cubated at room temperature or 37˚C for 10 min, filtered through nylon FIGURE 2. SMG NK cells express E4BP4 protein, and their number is mesh, and washed once with 1% PBS-serum before being layered on a reduced in aged mice. (A) Representative intracellular E4BP4 staining in Lympholyte-M gradient. Lymphocytes were harvested from the gradient the NK cells of the spleen, liver, and SMG of NFIL3+/+ and NFIL32/2 interface and washed once in 1% PBS-serum. We report that Ly49 markers mice. To detect E4BP4 by intracellular staining, activation of NK cells is and TRAIL are sensitive to collagenase IV, leading to false negatives in required, and mice had been infected with MCMV for 38 h. Data are some studies. SMGs can be processed without collagenase to ascertain expression of these markers, but the number of lymphocytes recovered is representative of two experiments. (B) Frequency of NK cells in the SMG very low. To circumvent this issue, we screened a variety of enzymes and of NFIL3+/+ mice and NFIL32/2 mice at different ages. Data are mean 6 identified Liberase-DL (Sigma-Aldrich), which does not affect these SEM and are pooled from two experiments. (C) Representative staining of markers. Whenever the expression of these markers was assessed, colla- DX5 and CD49a expression on SMG NK cells from NFIL3+/+ mice and genase IV was replaced with Liberase-DL. NFIL32/2 mice at different ages. ****p , 0.0001, **p = 0.001–0.01. 249 The Journal of Immunology 2487 Statistical analysis All statistical analyses were performed with Prism Version 7.0 (GraphPad Software). Unpaired two-tailed Student t tests were used to compare cell populations from different mice. Paired two-tailed Student t tests were used for experiments involving adoptive transfer or chimeric mice. Results NFIL3 deficiency significantly reduces the frequency and number of SMG NK cells Although cNK cells depend on NFIL3 for development, trNK cells in the liver, skin, kidneys, and uterus develop mostly independently of NFIL3 (3, 10). It also was reported recently that salivary gland NK cells develop entirely independently of NFIL3 activity (22, 23). In contrast to these findings, we found a significant reduction in the frequency (Fig. 1A, 1B) and number (Fig. 1C) of SMG NK cells in NFIL32/2 mice. Intracellular staining with an anti-NFIL3 Ab also shows that a large subset of SMG NK cells express NFIL3 (Fig. 2A). Salivary gland structural development continues for several weeks after birth and is completed when the mice are 10–12 Downloaded from http://www.jimmunol.org/ by guest on March 14, 2018 wk old (24, 25). One possible explanation for the discrepancy with previous findings is that NFIL3-dependent NK cells initially seed the SMG and are later replenished by NFIL3-independent trNK cells. However, we found that NFIL32/2 mice have a significant decrease in SMG NK cell frequency compared with NFIL3+/+ mice with similar phenotype, regardless of their age (Fig. 2B, 2C). To unequivocally determine the origin of these two subsets, we generated PLZF reporter/fate mapping mice by crossing PLZFGFPcre+/2 reporter mice with mice carrying the ROSA26- FIGURE 3. PLZF reporter/fate-mapping mice demonstrate that SMG floxstop-YFP fate-mapping allele, as described recently (26). CD32NK1.1+ cells are primarily cNK cells. (A) YFP expression by indi- Bendelac and colleagues (26) reported that most ILCs, including cated cell types in the spleen of PLZFGFPcre+/2ROSA26-floxstop-YFP ILC1, but not cNK cells, are YFP+ in these mice. It should be bone marrow chimeras. (B) YFP expression by cNK and trNK cells from noted that, in the resulting mice (PLZFGFPcre+/2ROSA26-floxstop- PLZFGFPcre+/2ROSA26-floxstop-YFP chimeras. (C) YFP expression by YFP), ∼30% of the cells become YFP+ before hematopoiesis (26) CD32 NK1.1+ cells in the spleen, liver, and SMG of PLZFGFPcre+/2 (data not shown). To circumvent this issue, we generated chimeric ROSA26-floxstop-YFP chimeras. (D) Representative staining of DX5 and mice reconstituted with sorted YFP2 LSK bone marrow precursors CD49a expression on YFP+ and YFP2 SMG NK cells from PLZFGFPcre+/2 (26) (Supplemental Fig. 1A). In agreement with previous findings ROSA26-floxstop-YFP chimeras. Data are representative of three experi- (26), iNKT cells express YFP in the chimeric mice (Supplemental ments. Four chimeric mice were pooled per experiment. Fig. 1B), whereas B cells and conventional T cells are mostly un- labeled (Fig. 3A). In the liver, ∼60% of trNK cells were YFP+, were purchased from BioLegend (San Diego, CA). FITC-Ly49C/I was purchased from BD Pharmingen. FITC-DX5, PE-NK1.1, anti-CD5 magnetic whereas only ∼20% of the cNK cells were labeled (Fig. 3B), which beads, and anti-CD19 magnetic beads were purchased from Miltenyi Biotec. is in agreement with previous studies (26). Importantly, in the SMG To detect E4BP4 by intracellular staining, activation of NK cells is required. of these mice, ∼90% of the NK cells were YFP2, indicating that they originated from the cNK lineage (Fig. 3C). Interestingly, the Generation of PLZFGFPcre+/2ROSA26-floxstop-YFP bone marrow chimeras B6.SJL recipient mice (CD45.1+) were lethally irradiated with 1050 rad and placed on antibiotic treatment for 2 wk. One day postirradiation, donor bone marrow cells were harvested under sterile conditions from PLZFGFPcre+/2ROSA26-floxstop-YFP mice (CD45.2+/CD45.1+), pooled, and stained for Lineage, Sca-1, and cKit. YFP2 Lin2Sca-1+cKit+ (LSK) cells were sorted and injected i.v. into recipients at ∼10,000 cells/mouse. The recipients were allowed to reconstitute for $8 wk. Generation of mixed bone marrow chimeras Recipient mice were lethally irradiated with 1050 rad and placed on antibiotic treatment for 2 wk. One day postirradiation, recipient mice were injected with a 1:1 mixture of sorted LSK cells or DX5 and CD5–depleted cells from B6. SJL and NFIL32/2 bone marrow. The recipients were allowed to reconsti- tute for 8 wk. Adoptive transfer of NK cells FIGURE 4. SMG NK cells originate preponderantly from NFIL3+ bone marrow. (A) Percentage of spleen, liver, and SMG NK cells derived from NK cells were sorted under sterile conditions from the spleen of C57BL/6 donor B6.SJL and NFIL32/2 bone marrow. (B) Percentage of spleen, liver, (B6) (CD45.2+) mice, the SMG of B6.SJL (CD45.1+) congenic mice, or the SMG of NFIL32/2 (CD45.2+) mice. Donor NK cells were injected into and SMG non-NK cells derived from donor B6.SJL and NFIL32/2 bone recipient Rag22/2IL-2Rg 2/2 mice. Recipient mice were allowed to recon- marrow. Data are mean 6 SEM and are pooled from two experiments. stitute for 7 d before being infected i.p. with 5 3 104 PFU MCMV. Recipient Four chimeric mice were analyzed individually per experiment. ****p , mice were sacrificed for experiments 38 h postinfection. 0.0001. 250 2488 SUBMANDIBULAR GLAND NK CELL DEVELOPMENT AND FUNCTIONS marrow (Fig. 4B). Altogether, these data demonstrate that the SMG harbors at least two populations of NK cells: a prepon- derant NFIL3-dependent population that is developmentally similar to the cNK lineage and an NFIL3-independent tissue- resident population. SMG trNK cells are phenotypically and developmentally different from liver trNK cells We next examined whether SMG trNK cells were developmentally related to liver trNK cells (3). In the liver, cNK cells develop under the coordinated influence of the transcription factors NFIL3, T-bet, and Eomes, whereas the trNK population develops independently of NFIL3 and Eomes but requires T-bet (3). Moreover, there is a clear distinction between Eomes+ cNK and Eomes2 trNK cells, with NFIL3 deficiency causing a bias toward liver Eomes2 trNK cells (Fig. 5A) (15). In contrast, in the SMG of wild-type mice, a large proportion of the NK cells are Eomes+T-bet+, and their relative frequency in NFIL3 2 /2 animals remains mostly unchanged (Fig. 5A). Liver NK cells are also clearly divided between DX5+ CD49a2 cNK cells and DX52CD49a+ trNK cells, whereas the Downloaded from http://www.jimmunol.org/ by guest on March 14, 2018 majority of SMG NK cells are CD49a+, regardless of DX5 ex- pression (Fig. 5B). In addition, NFIL3-independent Eomes2 liver trNK cells are mostly DX52, whereas .50% of the NK cells in the SMGs of wild-type and NFIL32/2 mice are DX5+ (Fig. 5C). With regard to their effector functions, it was shown that liver Eomes2DX52 NFIL3-independent trNK cells express TRAIL (3, 20, 21, 27). Similarly, we found that ∼30% of SMG NK cells FIGURE 5. SMG trNK cells have a unique phenotype compared with liver trNK cells. (A) Representative staining of T-bet and Eomes expression on spleen, liver, and SMG NK cells from wild-type control (C57BL/6 or NFIL3+/+ littermate controls) and NFIL32/2 mice. Data are representative of four experiments. Two or three mice were pooled in each experiment. (B) Representative staining of DX5 and CD49a expression on spleen, liver, and SMG NK cells from wild-type control (C57BL/6 or NFIL3+/+ littermate controls) and NFIL32/2 mice. Data are representative of four experiments. Two or three mice were pooled in each experiment. (C) Representative staining of DX5 and Eomes expression in spleen, liver, and SMG NK cells from wild-type control (C57BL/6 or NFIL3+/+ littermate controls) and NFIL32/2 mice. Data are representative of four experiments. Two or three mice were pooled in each experiment. Spleens from NFIL32/2 mice were enriched for NK cells using anti-CD5 and anti-CD19 magnetic beads. remaining YFP + NK cells had lower DX5 expression than the FIGURE 6. SMG trNK cells are less mature compared with cNK-like YFP2 SMG NK cells, consistent with a possible NFIL3 indepen- cells. (A) Representative staining of TRAIL expression on NK cells from the spleen, liver, and SMG of wild-type control (C57BL/6 or NFIL3+/+ dency (Fig. 3D). To exclude potential extrinsic factors, which could littermate controls) and NFIL32/2 mice. Spleens from NFIL32/2 mice explain the difference between this study and the one by Cortez et al. were enriched for NK cells using anti-CD5 and anti-CD19 magnetic beads. (22), we also generated B6.SJL/NFIL32/2 mixed bone marrow chi- Data are representative of three experiments. Two or three mice were meras (Supplemental Fig. 1C). We found that ∼90% of SMG NK pooled in each experiment. (B) Representative staining of CD127 and cells were derived from B6.SJL donor bone marrow in all organs NK1.1 on NKp46+ lymphocytes in the SMG and lamina propria of C57BL/6 tested, including the SMG (Fig. 4A). In contrast, non-NK lymphocytes and AhR2/2 mice. Data are representative of three experiments. Three mice were derived equally from B6.SJL and NFIL32/2 donor bone were pooled in each experiment. 251 The Journal of Immunology 2489 constitutively express TRAIL, independently of NFIL3 expression (Fig. 6A). However, in contrast to the liver, TRAIL expression does not mark a specific subset of cells (i.e., DX52Eomes2), because the SMG TRAIL+ NK cells are mostly DX5+Eomes+. In NFIL32/2 animals, the DX5+Eomes+TRAIL+ SMG NK cell population is retained (data not shown). Altogether, these data demonstrate that the NFIL3-independent population of NK cells in the SMG is distinct from liver trNK cells with regard to cell surface phenotype and developmentally. The SMG does not contain group 3 ILCs Having identified a novel population of NFIL3-independent NK cells in the SMG, we sought to determine whether group 3 ILCs were also present in this organ. Group 3 ILCs develop under the influence of RORgt and aryl hydrocarbon receptor (AhR) (28). Therefore, we examined whether the absence of AhR affects the development of SMG lymphocytes. We did not find NKp46+ CD127+NK1.12 lymphocytes in the SMG from B6 or AhR2/2 mice (Fig. 6B). However, we found, as previously reported (28), that these cells are reduced in the lamina propria of AhR2/2 mice Downloaded from http://www.jimmunol.org/ by guest on March 14, 2018 (Fig. 6B). These results are consistent with our previous findings FIGURE 7. SMG cNK-like cell hyporesponsiveness, but not SMG trNK showing that no significant number of RORgt+ cells were found in cell hyporesponsiveness, can be reversed by tissue environment. (A) NK this organ using RORgt reporter mice (2). cell frequency from the two different donors. (B) Frequency of donor IFN-g+ B6 splenic-derived NK cells and IFN-g+ B6.SJL SMG-derived NK cells in SMG cNK-like cell hyporesponsiveness is dependent on the the spleen and liver of recipient Rag22/2IL-2Rg2/2 mice, 38 h after MCMV tissue environment, whereas SMG trNK cells are intrinsically infection. (C) Frequency of donor IFN-g+ SMG NK cells from B6.SJL and hyporesponsive NFIL32/2 mice in the spleen and liver of recipient Rag22/2IL-2Rg 2/2 We (2) and others investigators (22) showed that SMG NK cells mice, 38 h after MCMV infection. C57BL/6 + B6.SJL data are pooled from seven experiments. NFIL32/2 data are pooled from five experiments. Two are hyporesponsive during MCMV infection. Having identified recipient Rag22/2IL-2Rg 2/2 mice were pooled in each experiment. Data two subsets of NK cells in this organ, we sought to revisit these are mean 6 SEM. **p = 0.001–0.01. findings and examine whether this phenotype was reversible. To determine whether SMG NK cells were capable of producing an effector response in new tissue environments, NK cells were location), the main population of SMG NK cells expresses Eomes sorted from the SMGs of B6.SJL mice (CD45.1+) and the spleens and T-bet and requires NFIL3 for development, indicating that they of C57BL/6 mice (CD45.2+). The sorted NK cells were mixed in a are developmentally similar to cNK cells. The remaining NK cells 1:1 ratio and adoptively transferred into Rag22/2IL-2Rg2/2 mice. in this organ develop independently of NFIL3; therefore, they can The adoptively transferred NK cells were allowed to reconstitute be classified as trNK cells, yet they are phenotypically different for 7 d before the mice were infected i.p. with MCMV. We found from the recently described resident NK cells in other organs. Al- that the frequency of the two donor populations was unchanged though our results are in agreement with recent reports from and roughly at a 1:1 ratio (Fig. 7A). Importantly, at 38 h postin- Colonna’s group (22, 23) with regard to the phenotype and the fection, the magnitude of the IFN-g response from the spleen and functions of the SMG NK cells, they differ with regard to NFIL3 SMG-derived NK cells was comparable (Fig. 7B). This result dependence. The differences between these two studies might be indicates that the hyporesponsive phenotype seen in SMG NK explained by variations in extrinsic parameters, such as microbiota cells in situ is not cell intrinsic but is caused by properties of the or housing-dependent inflammation. In fact, the identification of an SMG microenvironment. To assess the effector capacity of SMG MCMV-driven population of peripheral NK cells in NFIL32/2 mice NFIL3-independent trNK cells, SMG trNK cells were sorted from supports this possibility (30). However, we found only residual NFIL32/2 mice and adoptively transferred into Rag22/2IL-2Rg2/2 seeding of NFIL32/2-derived NK cells in the salivary glands in mice. We found that NFIL32/2 SMG NK cells produced signifi- mixed bone marrow chimeras (Fig. 4), ruling out host-derived ex- cantly less IFN-g than did B6.SJL SMG NK cells under the same trinsic factors independent of microbiota. In addition, we detected conditions (Fig. 7C). Therefore, in contrast with NFIL3-dependent NFIL3 protein in SMG NK cells (Fig. 2A) from wild-type animals, SMG NK cells, NFIL3-independent trNK cells appear not to respond and ∼90% of CD32NK1.1+NKP46+ cells never express PLZF optimally to MCMV infection, regardless of the tissue environment. during their development (Fig. 3C). Altogether, these data advocate for the presence of cNK-like cells in this organ. In support of this Discussion conclusion, a recent report showed that the transcription factor Although NK cells have been studied and characterized for decades, Runx3 similarly affects splenic cNK and SMG NK cells (31). our understanding of their developmental pathways is still incom- Although NFIL3-independent SMG trNK cells share similarity plete. cNK cells were discovered decades ago, but the last 10 y have with other trNK cells, they have unique phenotypic and effector seen the emergence of several new classes of trNK cells, each with characteristics. In contrast to liver and skin trNK cells, but similarly unique properties and developmental pathways. Moreover, NK cells to uterine trNK cells, SMG trNK cells from NFIL3-deficient animals as a whole make up one subset of ILCs, a diverse group of im- express DX5 and Eomes. Several transcription factors are known to mune lymphocytes that may represent the innate analog of play critical roles during ILC development. A committed a4b7+ T cells (29). In this article, we show that the SMG in naive C57BL/6 PLZF+ precursor to all helper-like ILCs (excluding cNK and LTi) mice contains at least two distinct populations of NK cells. Al- was identified by Bendelac and colleagues (26), whereas the Die- though phenotypically unique (most likely as a result of their tissue fenbach group (32) identified the common helper-like innate lymphoid 252 2490 SUBMANDIBULAR GLAND NK CELL DEVELOPMENT AND FUNCTIONS precursor as a4b7+ID2high. The development of this common ILC References precursor is dependent on NFIL3 , which directly regulates Id2 to 1. Vosshenrich, C. A., and J. P. Di Santo. 2013. Developmental programming of promote the development of the common helper-like innate lymphoid natural killer and innate lymphoid cells. Curr. Opin. Immunol. 25: 130–138. 2. Tessmer, M. S., E. C. Reilly, and L. Brossay. 2011. Salivary gland NK cells are precursor from the common lymphoid progenitor (19). Under this phenotypically and functionally unique. PLoS Pathog. 7: e1001254. paradigm, the NFIL3-independent NK cells of the SMG would rep- 3. Sojka, D. K., B. Plougastel-Douglas, L. Yang, M. A. Pak-Wittel, M. N. Artyomov, Y. Ivanova, C. Zhong, J. M. Chase, P. B. Rothman, J. Yu, et al. 2014. Tissue- resent yet another ILC subset, independent from helper-like ILC1s. In resident natural killer (NK) cells are cell lineages distinct from thymic and con- addition to cNK-like cells and NFIL3-independent trNK cells, other ventional splenic NK cells. eLife 3: e01659. small NK1.1+ subsets are found in the SMG. This includes a subset of 4. Vosshenrich, C. A., M. E. Garcı´a-Ojeda, S. I. Samson-Ville´ger, V. Pasqualetto, L. Enault, O. Richard-Le Goff, E. Corcuff, D. Guy-Grand, B. Rocha, NK cells that is strictly dependent on T-bet and similar to liver trNK A. Cumano, et al. 2006. A thymic pathway of mouse natural killer cell devel- cells (Supplemental Fig. 1D, see gate DX52CD49a+). In addition, a opment characterized by expression of GATA-3 and CD127. Nat. Immunol. 7: subset of T cells not detected in B6 mice can be observed in the SMG 1217–1224. 5. Vargas, C. L., J. Poursine-Laurent, L. Yang, and W. M. Yokoyama. 2011. De- of NFIL3-deficient animals (Supplemental Fig. 1E). Although the velopment of thymic NK cells from double negative 1 thymocyte precursors. characterization of these CD3+NK1.1+NKp46+ T cells is beyond the Blood 118: 3570–3578. scope of this article, our preliminary data indicate that these T cells are 6. Victorino, F., D. K. Sojka, K. S. Brodsky, E. N. McNamee, J. C. Masterson, D. Homann, W. M. Yokoyama, H. K. Eltzschig, and E. T. Clambey. 2015. not semi-invariant iNKT cells (data not shown). Tissue-resident NK cells mediate ischemic kidney injury and are not depleted by We also showed previously that SMG NK cells are hyporesponsive anti-asialo-GM1 antibody. J. Immunol. 195: 4973–4985. 7. Erick, T. K., and L. Brossay. 2016. Phenotype and functions of conventional and to MCMV infection, both in situ and during in vitro cytokine- non-conventional NK cells. Curr. Opin. Immunol. 38: 67–74. stimulation assays (2). However, we show in this study that when 8. Peng, H., X. Jiang, Y. Chen, D. K. Sojka, H. Wei, X. Gao, R. Sun, W. M. Yokoyama, wild-type SMG NK cells are isolated from their native environment and Z. Tian. 2013. Liver-resident NK cells confer adaptive immunity in skin-contact inflammation. J. Clin. Invest. 123: 1444–1456. and allowed to reconstitute in peripheral tissues, they regain the Downloaded from http://www.jimmunol.org/ by guest on March 14, 2018 9. Kopcow, H. D., D. S. Allan, X. Chen, B. Rybalov, M. M. Andzelm, B. Ge, and ability to produce an effector response to MCMV. cNK cell effector J. L. Strominger. 2005. Human decidual NK cells form immature activating plasticity was reported in other contexts (33, 34). The ability of the synapses and are not cytotoxic. Proc. Natl. Acad. Sci. USA 102: 15563–15568. 10. Doisne, J. M., E. Balmas, S. Boulenouar, L. M. Gaynor, J. Kieckbusch, SMG NK cells (Fig. 7) and splenic NK cells (33, 34) to regain ef- L. Gardner, D. A. Hawkes, C. F. Barbara, A. M. Sharkey, H. J. M. Brady, et al. fector functions reinforces our finding that the majority of NK cells 2015. Composition, development, and function of uterine innate lymphoid cells. J. Immunol. 195: 3937–3945. (∼75%, Fig. 1) in the B6 SMG are developmentally and functionally 11. Vacca, P., E. Montaldo, D. Croxatto, F. Moretta, A. Bertaina, C. Vitale, similar to cNK cells. These findings also indicate that environmental F. Locatelli, M. C. Mingari, and L. Moretta. 2016. NK cells and other innate factors in the SMG influence NK cell effector potential. A role for lymphoid cells in hematopoietic stem cell transplantation. Front. Immunol. 7: 188. 12. Kamizono, S., G. S. Duncan, M. G. Seidel, A. Morimoto, K. Hamada, TGF-b, which is 100-fold more abundant in the SMG than in the G. Grosveld, K. Akashi, E. F. Lind, J. P. Haight, P. S. Ohashi, et al. 2009. Nfil3/ spleen, was demonstrated recently in the salivary glands (23). The E4bp4 is required for the development and maturation of NK cells in vivo. phenotypic change induced by TGF-b appears to be reversible. In- J. Exp. Med. 206: 2977–2986. 13. Gascoyne, D. M., E. Long, H. Veiga-Fernandes, J. de Boer, O. Williams, deed, addition of TGF-b to splenic NK cells induces them to dif- B. Seddon, M. Coles, D. Kioussis, and H. J. Brady. 2009. The basic leucine ferentiate into tissue resident–like NK cells, whereas blocking TGF-b zipper transcription factor E4BP4 is essential for natural killer cell development. Nat. Immunol. 10: 1118–1124. signaling (23) or relocation of SMG NK cells into low TGF-b en- 14. Male, V., I. Nisoli, T. Kostrzewski, D. S. Allan, J. R. Carlyle, G. M. Lord, vironments (Fig. 7) reinstates their effector functions. Cortez et al. A. Wack, and H. J. Brady. 2014. The transcription factor E4bp4/Nfil3 controls (23) proposed that TGF-b drives the progressive differentiation commitment to the NK lineage and directly regulates Eomes and Id2 expression. J. Exp. Med. 211: 635–642. of CD49a2 NFIL3-dependent SMG ILCs into CD49a+ NFIL3- 15. Crotta, S., A. Gkioka, V. Male, J. H. Duarte, S. Davidson, I. Nisoli, H. J. Brady, independent mature SMG ILCs. Although our data and their data and A. Wack. 2014. The transcription factor E4BP4 is not required for extra- support a linear differentiation model for SMG NK cells, it is unclear medullary pathways of NK cell development. J. Immunol. 192: 2677–2688. 16. Yu, X., Y. Wang, M. Deng, Y. Li, K. A. Ruhn, C. C. Zhang, and L. V. Hooper. how NFIL3-dependent SMG NK cells become independent in this 2014. The basic leucine zipper transcription factor NFIL3 directs the develop- model. Instead, we propose that this model is better explained by the ment of a common innate lymphoid cell precursor. eLife 3: e04406. 17. Seillet, C., L. C. Rankin, J. R. Groom, L. A. Mielke, J. Tellier, M. Chopin, existence of two distinct populations in this organ. N. D. Huntington, G. T. Belz, and S. Carotta. 2014. Nfil3 is required for the NK cells also were shown to limit salivary gland inflammation development of all innate lymphoid cell subsets. J. Exp. Med. 211: 1733–1740. and tissue damage during MCMV infection (35), indicating that 18. Geiger, T. L., M. C. Abt, G. Gasteiger, M. A. Firth, M. H. O’Connor, C. D. Geary, T. E. O’Sullivan, M. R. van den Brink, E. G. Pamer, A. M. Hanash, they play an immunoregulatory role. Recent studies have begun to and J. C. Sun. 2014. Nfil3 is crucial for development of innate lymphoid cells address whether NK cell regulation occurs via a viral load de- and host protection against intestinal pathogens. J. Exp. Med. 211: 1723–1731. crease or is mediated by a more complex mechanism. In support 19. Xu, W., R. G. Domingues, D. Fonseca-Pereira, M. Ferreira, H. Ribeiro, S. Lopez-Lastra, Y. Motomura, L. Moreira-Santos, F. Bihl, V. Braud, et al. 2015. of the second possibility, a recent report demonstrated that NFIL3 orchestrates the emergence of common helper innate lymphoid cell TRAIL+ NK cells in the SMG specifically eliminate CD4+ T cells, precursors. Cell Rep. 10: 2043–2054. which are critical for the clearance of active MCMV from the 20. Daussy, C., F. Faure, K. Mayol, S. Viel, G. Gasteiger, E. Charrier, J. Bienvenu, T. Henry, E. Debien, U. A. Hasan, et al. 2014. T-bet and Eomes instruct the salivary glands (27). The investigators argue that NK cell–mediated development of two distinct natural killer cell lineages in the liver and in the T cell killing would prolong MCMV infection but also reduce in- bone marrow. J. Exp. Med. 211: 563–577. 21. Seillet, C., N. D. Huntington, P. Gangatirkar, E. Axelsson, M. Minnich, flammatory damage to the delicate SMG tissues, allowing the virus H. J. Brady, M. Busslinger, M. J. Smyth, G. T. Belz, and S. Carotta. 2014. to be cleared slowly without causing irreversible damage to the host. Differential requirement for Nfil3 during NK cell development. J. Immunol. 192: Our data add to these findings and show that NK cells are rendered 2667–2676. 22. Cortez, V. S., A. Fuchs, M. Cella, S. Gilfillan, and M. Colonna. 2014. Cutting hyporesponsive by the salivary gland environment, presumably edge: salivary gland NK cells develop independently of Nfil3 in steady-state. benefiting the host. J. Immunol. 192: 4487–4491. 23. Cortez, V. S., L. Cervantes-Barragan, M. L. Robinette, J. K. Bando, Y. Wang, T. L. Geiger, S. Gilfillan, A. Fuchs, E. Vivier, J. C. Sun, et al. 2016. Transforming growth factor-b signaling guides the differentiation of innate lymphoid cells in Acknowledgments salivary glands. Immunity 44: 1127–1139. We thank Kevin Carlson for cell sorting, Ce´line Fuge`re for tail vein injec- 24. Gattone II, V. H., D. A. Sherman, D. A. Hinton, F. W. Niu, R. T. Topham, and tions, and Dr. Hugh J.M. Brady for providing NFIL3 2/2 mice. R. M. Klein. 1992. Epidermal growth factor in the neonatal mouse salivary gland and kidney. Biol. Neonate 61: 54–67. 25. Redman, R. S. 2008. On approaches to the functional restoration of salivary glands damaged by radiation therapy for head and neck cancer, with a review of Disclosures related aspects of salivary gland morphology and development. Biotech. Histo- The authors have no financial conflicts of interest. chem. 83: 103–130. 253 The Journal of Immunology 2491 26. Constantinides, M. G., B. D. McDonald, P. A. Verhoef, and A. Bendelac. 2014. 31. Ebihara, T., C. Song, S. H. Ryu, B. Plougastel-Douglas, L. Yang, D. Levanon, A committed precursor to innate lymphoid cells. Nature 508: 397–401. Y. Groner, M. D. Bern, T. S. Stappenbeck, M. Colonna, et al. 2015. Runx3 specifies 27. Schuster, I. S., M. E. Wikstrom, G. Brizard, J. D. Coudert, M. J. Estcourt, lineage commitment of innate lymphoid cells. Nat. Immunol. 16: 1124–1133. M. Manzur, L. A. O’Reilly, M. J. Smyth, J. A. Trapani, G. R. Hill, et al. 2014. 32. Klose, C. S., M. Flach, L. Mo¨hle, L. Rogell, T. Hoyler, K. Ebert, C. Fabiunke, TRAIL+ NK cells control CD4+ T cell responses during chronic viral infection D. Pfeifer, V. Sexl, D. Fonseca-Pereira, et al. 2014. Differentiation of type 1 ILCs to limit autoimmunity. Immunity 41: 646–656. from a common progenitor to all helper-like innate lymphoid cell lineages. Cell 28. Kiss, E. A., C. Vonarbourg, S. Kopfmann, E. Hobeika, D. Finke, C. Esser, and 157: 340–356. A. Diefenbach. 2011. Natural aryl hydrocarbon receptor ligands control organ- 33. Joncker, N. T., N. Shifrin, F. Delebecque, and D. H. Raulet. 2010. Mature natural ogenesis of intestinal lymphoid follicles. Science 334: 1561–1565. killer cells reset their responsiveness when exposed to an altered MHC envi- 29. Eberl, G., M. Colonna, J. P. Di Santo, and A. N. McKenzie. 2015. Innate lym- ronment. J. Exp. Med. 207: 2065–2072. phoid cells. Innate lymphoid cells: a new paradigm in immunology. Science 348: 34. Elliott, J. M., J. A. Wahle, and W. M. Yokoyama. 2010. MHC class I-deficient aaa6566. natural killer cells acquire a licensed phenotype after transfer into an MHC class 30. Firth, M. A., S. Madera, A. M. Beaulieu, G. Gasteiger, E. F. Castillo, I-sufficient environment. J. Exp. Med. 207: 2073–2079. K. S. Schluns, M. Kubo, P. B. Rothman, E. Vivier, and J. C. Sun. 2013. Nfil3- 35. Carroll, V. A., A. Lundgren, H. Wei, S. Sainz, K. S. Tung, and M. G. Brown. independent lineage maintenance and antiviral response of natural killer cells. 2012. Natural killer cells regulate murine cytomegalovirus-induced sialadenitis J. Exp. Med. 210: 2981–2990. and salivary gland disease. J. Virol. 86: 2132–2142. Downloaded from http://www.jimmunol.org/ by guest on March 14, 2018 254 Sup. Fig. 1 A B Liver iNKT Normalized to mode Stop PLZF GFP.cre Codon EYFP PLZFGFPcre+/- ROSA26-YFP CD45.1+ CD45.2+ loxP loxP eYFP Sorted eYFP-Lin- PLZFGFPcre+/-ROSA26-YFP+/- D C57BL/6 T-bet -/- Sca-1+ckit+ bone CD45.1+CD45.2+ marrow 1050 Rad Spleen B6.SJL CD45.1+ Analysis of CD45.1+CD45.2+ cells: GFP-YFP- = Never PLZF+ GFP-YFP+ = PLZF+ in fate history GFP+YFP+ = Currently PLZF+ DX5 Liver SMG C Donors B6.SJL NFIL3-/- CD45.1+ CD45.2+ CD49a 1:1 E NFIL3+/+- NFIL3-/- 1050 Rad CD45.1+ B6.SJL CD3 Analysis NK1.1 Supplementary Figure 1. (A) Diagram representing the generation of PLZFGFPcre+/-ROSA26-floxstop-YFP chimeras. (B) YFP expression by liver iNKT cells from PLZFGFPcre+/-ROSA26-floxstop-YFP chimeras. (C) Diagram representing the generation of B6.SJL/NFIL3-/- mixed bone marrow chimeras. (D) Representative staining of DX5 and CD49a on NK cells in the spleen, liver, and SMG of C57BL/6 and T-bet-/- mice. Spleens from T-bet-/- mice were enriched for NK cells using anti-CD5 and anti-CD19 magnetic beads. Data are representative of two experiments. Three mice were pooled in each experiment. (E) A CD3+NK1.1+NKP46+ T cell subset is revealed in the NFIL3-/- SMG. 255