LINEAGE SPECIFIC ROLE FOR ADHESION AND DEGRANULATION PROMOTING ADAPTOR PROTEIN (ADAP) IN iNKT CELL DEVELOPMENT AND FUNCTIONS By Trevor Glenn Gohl A THESIS Submitted to Michigan State University In partial fulfillment of the requirements for the degree of Physiology – Master of Science 2018 ABSTRACT LINEAGE SPECIFIC ROLE FOR ADHESION AND DEGRANULATION PROMOTING ADAPTOR PROTEIN (ADAP) IN iNKT CELL DEVELOPMENT AND FUNCTIONS By Trevor Glenn Gohl Invariant natural killer T (iNKT) cells comprise a sub-lineage of T lymphocytes that express an "invariant" T cell receptor (TCR) with specificity for glycolipid antigens when presented in complex with the MHC class I molecule CD1d. Following TCR engagement, iNKT cells rapidly secrete cytokines and transactivate the anti-tumor functions of dendritic cells (DC), natural killer (NK), T and B cells as well as directly kill tumor cells. Although TCR-CD1d interactions are required for iNKT cell functions, the signaling mechanisms that co-operate with the TCR to promote maximal iNKT cell responses remain poorly understood. Collective studies from our laboratory and others have established that the adaptor protein SAP and its signaling partner, Fyn is critical for iNKT cell development and functions. We investigated the role of ADAP (adhesion and degranulation promoting adaptor protein), a Fyn-binding protein in iNKT cell development and functions. Our studies demonstrate that ADAP is critical for homeostatic maintenance of these cells in peripheral organs. Furthermore, we observe that iNKT cytokine production, and bystander immune cell activation are remarkably reduced in Adap−⁄− mice. These findings are not restricted to the murine system, as ADAP-silenced human iNKT cells also have significantly impaired cytokine release. Importantly, we demonstrate that ADAP is essential for TCR-induced iNKT cell cytotoxicity against leukemia targets in vitro and in vivo. Collectively, our data suggest that ADAP plays a unique linage-specific role in iNKT cells. These studies will have an impact by establishing new paradigms for iNKT cell signaling and offering insights into how iNKT cells can be best activated to enhance host immunity and treat cancer. I dedicate this thesis to my Mother, Father, Sister & Grandparents; and of course, my dog, Milo. iii ACKNOWLEDGEMENTS I write here to express my deepest gratitude to those who have helped me along the way, for the successes I have had are largely due to them. I first must thank my mentor, Dr. Rupali Das, as she has taken me under her wing and gone beyond her call of duty to teach me how to question, conduct, and present among the realm of research. Her willingness to help me in all areas, inside and outside of research, has helped improve my scientific literacy, self-discipline, and communication. Her support more than anything else has exponentially excelled any improvements thus far. Most of all, she has instilled some of her passion for science into me, a gift which will last a lifetime. Thanks must also be delivered to my committee members. Particularly, Dr. Hariharan Subramanian has been a second mentor to me. Among the shared lab space, he was consistently assisting me in various ways, such as direct protocol teachings or scientific discussions. He also provided extremely valuable critiques on work I was presenting, and was incessantly encouraging and supportive. Continuing, Dr. Kathy Gallo has my gratitude and respect, for providing outside feedback on my work and progress. Her reviews and comments allowed me to think about my work from different perspectives, and improve on it. Additionally, she was a large help in conveying to me what was required as a Master’s student. Other faculty members in the department to whom I owe my gratitude include Dr. Erica Wehrwein. She has helped me in ways of showing strong enthusiasm and effective communication when teaching. Her altruistic nature has expressed to me the importance of iv voicing your opinion and defending what is correct; both valuable traits in a scientific or other community. Her encouragement was a backbone to my success. Dr. Louis King, director of the Flow Core Facility has my most gratituditous thanks for closely working with me for long hours during my FACS sorting and analysis, a large component of my research. Among lab personnel, a magnitude of thanks goes to Dr. Hyun Hee Lee, “Hanny”, for her experimental assistance, and guidance in the lab. Over the two years we have worked together, she provided valuable advice from her past experiences, and was encouraging in time of need. Her long hours of help to assist in my success deserves my all of my gratitude. More so, Dr. Ananth Kammala, Tanwir Hashem, and Ryan Mack have greatly contributed to the lab work and enjoyable nature of the lab, which was very beneficial to my progress. Many other students have helped with my larger experiments in the lab, thus expediting my accomplishments, whose names are listed for recognition: Sami Abdelaziz, Ryan Griffin, Kellie Mullaney, Manthan Patel, Christopher Occhuito and others. Last but certainly not the least, I must thank my family and close friends, who have given me unconditional love and support along the way. Both my mother and father have raised me in such a way that allowed for me to succeed as I have. The invaluable lessons they teach, and the backing they have given to me will be something I will be eternally grateful for. My sister, as a best friend knows me unlike any other, and thus was always a voice of understanding and comfort. Lastly, I must thank Frances Greathouse and her family, for providing me balance in my life. These people are my roots and without them, I could not have grown. v PREFACE The primary motivator, and basis of my research stems from clinical stories and experiences I have witnessed, and brought me to the ongoing search for a “cure” for cancer. Dr. Rupali Das has enlightened me on the emerging potential of immunotherapy, and its successes and deficits. I performed these studies with hopes of one day contributing to advancement and discovery of more powerful, and directed medicine. vi TABLE OF CONTENTS LIST OF TABLES..........................................................................................................................ix LIST OF FIGURES.........................................................................................................................x KEY TO ABBREVIATIONS.........................................................................................................xi OVERALL INTRODUCTION........................................................................................................1 iNKT classifications.............................................................................................................2 Invariant NKT cells in disease states...................................................................................4 Signaling mechanisms of iNKT cells..................................................................................6 ADAP regulates lymphocyte development and functions...................................................7 CHAPTER 1 ADAP REGULATES HOMEOSTATIC MAINTENANCE OF iNKT CELLS BUT NOT ITS THYMIC DEVELOPMENT OR MATURATION..............................................12 Introduction........................................................................................................................13 Developmental stages of iNKT cells in the thymus...............................................13 Transcriptional regulation of iNKT cells...............................................................14 SLAM-SAP-Fyn axis in iNKT cell ontogeny........................................................15 Materials and Methods.......................................................................................................17 Mice.......................................................................................................................17 Antibodies and Flow Cytometry............................................................................17 Isolation of purified populations of invariant NKT (iNKT) cells..........................17 Human iNKT cell expansion.................................................................................18 Western Blotting....................................................................................................18 Quantitative real-time PCR....................................................................................19 Statistics.................................................................................................................19 Results................................................................................................................................20 ADAP is dispensable for the thymic development of iNKT cells but is essential for their maintenance in the spleen and liver.............................................20 ADAP is not required for iNKT cell maturation or subset distribution.................24 Altered CXCR6 expression of thymic and splenic iNKT cells of Adap-/- mice...29 Expression of integrin CD11a and CD11b is normal in iNKT cells of Adap-/- mice...............................................................................................33 Discussion..........................................................................................................................36 CHAPTER 2 ADAP REGULATES INVARIANT NATURAL KILLER T CELL ACTIVATION AND IMMUNOMODULATORY FUNCTION.................................................42 Introduction........................................................................................................................43 Materials and Methods.......................................................................................................47 Mice.......................................................................................................................47 Reagents.................................................................................................................47 Flow cytometry and Western Blotting...................................................................47 vii Transient gene silencing in human iNKT cells......................................................47 In vivo cytokine production...................................................................................48 In vitro cytokine assay...........................................................................................48 Statistics.................................................................................................................49 Results................................................................................................................................50 ADAP is required for TCR-induced iNKT cell cytokine production....................50 ADAP regulates iNKT cell cytokine release.........................................................53 ADAP is required for human iNKT cell cytokine release.....................................56 ADAP is required for TCR-induced iNKT cell-mediated bystander cell activation....................................................................................................59 Discussion..........................................................................................................................62 CHAPTER 3 ADAP IS INDISPENSABLE FOR INVARIANT NATURAL KILLER T CELL ANTI-TUMOR RESPONSES............................................................................................66 Introduction........................................................................................................................67 Materials and Methods.......................................................................................................70 Mice.......................................................................................................................70 Reagents and Antibodies........................................................................................70 In vitro Cytotoxicity assay.....................................................................................70 Human iNKT cell expansion, purification and gene silencing..............................71 In Vivo tumor model..............................................................................................71 In vivo and ex vivo BLI..........................................................................................71 Histologic analysis.................................................................................................72 Statistics.................................................................................................................72 Results................................................................................................................................73 ADAP is required for optimal TCR-induced iNKT cell cytotoxicity in vitro.......73 ADAP is required for iNKT cell-mediated control of tumor growth in vivo.........77 Discussion..........................................................................................................................84 OVERALL DISCUSSION............................................................................................................88 BIBLIOGRAPHY..........................................................................................................................93 viii LIST OF TABLES Table 1. Frequency of iNKT cell subsets in various organs..........................................................23 Table 2. Absolute cell number of iNKT cell subsets in various organs........................................23 Table 3. Frequency and Absolute cell number of CXCR6+ iNKT cells........................................32 Table 4. Total flux from ex vivo organ BLI...................................................................................83 Table 5. Frequency of EL4 and iNKT cells in various organs ex vivo..........................................83 Table 6. Absolute cell number of EL4 and iNKT cells in various organs ex vivo.........................83 ix LIST OF FIGURES Figure 1. Schematic view of iNKT cell versus conventional ab T cell development.....................9 Figure 2. Molecular domains of ADAP and their binding partners...............................................11 Adap-/- mice....................................................................................................................21 Figure 3. iNKT cell incidence and absolute numbers are reduced in the spleen and liver of Figure 4. ADAP is neither required for the early nor late stages of iNKT cell Figure 5. ADAP is not required for subset distribution of iNKT cells..........................................27 maturation.......................................................................................................................25 Figure 6. Reduced expression of CXCR6 in iNKT cells of Adap-/- mice.....................................30 Figure 7. Expression of CD11a and CD11b is not altered in Adap-/- iNKT cells.........................34 Figure 8. Cellular interactions between iNKT cells and other immune cells...............................46 Figure 9. ADAP is required for adequate cytokine production and release in iNKT cells............51 pulsed EL4 cells in vitro...............................................................................................54 Figure 10. ADAP-deficient iNKT cells produce less cytokines in response to antigen- Figure 11. Silencing ADAP gene expression in human iNKT cells results in reduced Figure 12. ADAP is required for iNKT-cell mediated transactivation of immune cells...............60 cytokine production......................................................................................................57 Figure 13. Anti-tumor mechanisms of iNKT cells........................................................................69 Adap-/- iNKT cells........................................................................................................74 Figure 14. Basal expression of death receptors and lytic molecules are not altered in Figure 15. Adap-/- iNKT cells exhibit defective killing of antigen-pulsed target cells in vitro....76 EL4 cells......................................................................................................................78 Figure 16. Adap-deficient iNKT cells exhibit defective in vivo control of antigen-pulsed x KEY TO ABBREVIATIONS 51Cr ADAP Ag Chromium-51 Adhesion and degranulation-promoting adaptor protein Antigen ANOVA Analysis of variance AP-1 APC CD CPM Activator protein 1 Antigen presenting cell Cluster of differentiation Counts per minute CRACC CD2-like receptor-activating cytotoxic cells CXCL CXCR DC DN DP E:T EBV ELISA EVH1 FACS FSC FYB Chemokine (C-X-C motif) ligand Chemokine (C-X-C motif) receptor Dendritic cell Double negative Double positive Effector to target ratio Epstein-Barr Virus Enzyme-linked immunosorbent assay Drosophila enabled/vasodilator-stimulated phosphoprotein homology 1 Fluorescence activated cell sorting Forward scatter Fyn-binding protein xi GAPDH Glyceraldehyde 3-phosphate dehydrogenase H&E IACUC Hematoxylin and eosin Institutional Animal Care and Use Committee IFN-g Interferon gamma IGB3 IL Isoglobotriosylceramide Interleukin iNKT Invariant natural killer T-cell LAT LFA Liv Luc Mac-1 MACS MFI MHC MTOC NFkB NK NLS NSG PBMC PD-1 PH Linker for activation of T-cells Lymphocyte function-associated antigen Liver Luciferase Macrophage-1 antigen Magnetic-activated cell sorting Mean fluorescence intensity Major histocompatibility complex Microtubule-organizing center Nuclear factor kappa-light-chain-enhancer of activated B cells Natural killer cell Nuclear localization sequence Non-obese diabetic (NOD) scid gamma Peripheral blood mononuclear cell Programmed cell death protein 1 Pleckstrin homology domain xii PLZF Promyelocytic leukemia zinc finger protein PRAM-1 Promyelocytic leukemia retinoic acid receptor-alpha regulated adaptor molecule-1 RORgt RAR-related orphan receptor gamma SAP SD SEM SLAM-associated protein Standard deviation Standard error of the mean siRNA Short-interfering ribonucleic acid SKAP55 Src kinase-associated phosphoprotein SKAP55R Src kinase-associated phosphoprotein-related protein SLAM Signaling lymphocytoic activation molecule SLAP-130 SH2 domain-containing Slp-76-associated phosphoprotein of 130 kDa SLP-76 SMAC Spl SSC TAM TBI Tcon TCR TEC Tet ThPOK Thy SH2 domain containing leukocyte protein of 76kDa Supramolecular activation complex Spleen Side Scatter Tumor-associated macrophage Total body imaging Conventional T-cell T-cell receptor Thymic epithelial cells Tetramer Th inducing POZ-Kruppel factor Thymus xiii TME TRAIL Tumor microenvironment Tumor necrosis factor (TNF)-related apoptosis-inducing ligand ZAP-70 Zeta-chain-associated protein kinase 70 aGC Alpha-galactosylceramide xiv OVERALL INTRODUCTION 1 Natural Killer T (NKT) cells comprise a unique subset of T lymphocytes that differ in both their development and functional requirements, as compared to conventional T-cells (Tcons) [1]. As both NKT and T-cells develop in the thymus, they are by definition T-cells, however they differ in their selection process. It is well studied that Tcons undergo a selection process by peptide antigen presentation by major histocompatibility complex (MHC), of class I, or II on the surface of epithelial cells in the thymus. In contrast, NKT cells develop by selection of an endogenous glycolipid antigen presented by CD4+CD8+ thymocytes, termed double-positive (DP) thymocytes [1, 2], as shown in Figure 1. Presentation of the glycolipid is by a monomorphic MHC class I-like molecule, termed CD1d [1]. This engagement is a positive selection process for NKT cells, as only T-cell progenitors expressing a highly invariant T-cell receptor (TCR), will engage with the CD1d and commit to becoming an invariant NKT (iNKT) cell [1]. In addition, iNKT cells express NK-cell surface markers, such as NK1.1, and possess an innate-like memory [3]. Much like NK cells, iNKT cells contain lytic molecules for direct cytolysis [4]. Unique to iNKT cells, they contain preformed vesicles for Th1 and Th2 cytokines, which upon activation, are secreted rapidly and in a large amounts [5, 6]. iNKT classifications Mature iNKT cells can be further divided into sub-types, based on their co-receptor expression, and cytokine release profile. As a result of their T-cell lineage, mature iNKT cells can either be CD4+ or CD4−CD8− (double negative, DN) [5, 6], that can vary in their functional phenotypes. Another classification of NKT cells, is dependent on their cytokine release profile, Th1, Th2, or Th17 [7]. At large, release of Th1 cytokines, including IFN-g and IL-12, are commonly considered inflammatory inducing cytokines, as they recruit and assist in activation of other 2 effector immune cells. Th2 cytokines, on the other hand are commonly referred to as anti- inflammatory, and include cytokines such as IL-4, IL-5, and IL-13 that usually help to suppress an immune response. However these cytokines can sometimes be detrimental to the host in certain conditions such as in asthma and allergic diseases [8]. Lastly, the pro-inflammatory Th17 group of cytokines, such as IL-17, IL-21, and IL-22 [9, 10], are currently under investigation to ascertain their roles in modulating iNKT cell functions [8]. Thus, iNKT cells are designated as NKT-1, NKT-2, or NKT-17, corresponding to their Th1, Th2, or Th17 cytokine release profiles, respectively. While iNKT cells can release various amounts of cytokines in different settings, the type of release can be skewed which is largely dependent on the glycolipid antigen presented and the co-stimulatory molecules at play. Invariant NKT cells, as described, contain a highly restricted TCR repertoire, consisting of Va14 that usually pairs with Vb8.2, 2, or 7 in mice and Va24 and Vb11 in humans [11, 12]. This invariant TCR is the phenotype of Type-I or invariant NKT (iNKT) cells. These iNKT cells are dependent on the transcription factor promyelocytic zinc finger (PLZF) in order to develop into their fully functional, TCR-restricted phenotype [13, 14]. The TCR of iNKT cells are exclusively reactive to glycolipid antigens, which is commonly modeled by use of a-galactosylceramide (aGC) when presented by CD1d on an antigen- presenting cell (APC) [15]. The use of this reagent has excelled the study of iNKT cells, as CD1d tetramers loaded with aGC has allowed for this population to be more accurately analyzed by flow cytometry [15]. These iNKT cells are commonly found in highest incidence in the liver lymphocytes of both mice and humans, at about 30% and 1% respectively, however much less studied in humans [16, 17]. They are also found in about 1% of thymic and splenic lymphocytes in mice, and about .01% in humans [18, 19]. Additionally, iNKT cells can be found in circulating peripheral blood mononuclear cells (PBMCs), at around 1% in both mice and 3 humans, however this number can vary greatly on different factors[19]. Another subtype of NKT cells classified as Type-II NKT cells express more variable TCR ab chain combinations [20]. While they are still CD1d restricted, they are activated by sulfatide form of a glycolipid [20]. Additionally, they can lack expression of NK1.1, however are still classified as a NKT cell due to their CD1d dependence for activation. As iNKT cells release both Th1 and Th2 cytokines, they can act as a protector against certain infectious diseases, while promoting others such as asthmatic allergy. Type-II NKT cells appear to play dual roles as well, however seem to play beneficial roles in regulation of autoimmune diseases, but have suppressive roles in in host immunity against cancer, which is usually opposing to the protective role mediated by type I or iNKT cells. Invariant NKT cells in disease states Invariant NKT cells can either confer protection or mediate promotion of different disease states. Certain studies have shown iNKT cells to play a role in the clearance of some infectious diseases, whether it be bacterial, parasitic, or viral, although these studies are not fully confirmed [21-23]. However, studies in different murine models, that lack of iNKT cells demonstrate greater incidence and proliferation of the infection, indicating that iNKT cells play some key role in the protection from infection, most likely by indirect mechanisms. Continuing, iNKT cells have been shown to play a protective role against autoimmune induced diseases, such as type-1 diabetes, multiple sclerosis and rheumatoid arthritis [24-27]. In each case, primarily via cytokine release, certain subtypes of iNKT cells are present in greater incidence at the autoimmune target sites resulted in slower progression of the disease. However, due to the dual cytokine profile of iNKT cells, studies have also shown that release of Th1 cytokines can recruit 4 effector cells that amplify the disease progression. This leaves more room for iNKT cells to be studied and manipulated to prevent progression of pathological diseases. In certain asthmatic conditions modeled in mice, secretion of Th2 cytokines by iNKT exacerbated the airway hyper- reactivity, suggesting a detrimental role for these cells in the amplification of asthmatic conditions [28]. Studies have further shown that manipulation of iNKT cells to skew their cytokine profile to a Th1 release, have deterred the incidence of asthma [29]. More so, previous studies have shown iNKT cells have direct cytotoxic effects against tumor targets, as well as indirect activation of other cytolytic effectors. Activation of iNKT cells by aGC presentation has been shown to be sufficient in recruiting cytolytic effectors such as NK cells and CD8+ T- cells, however this response was short-lived as iNKT cells soon became unresponsive or anergic [30, 31]. Anergic iNKT cells are in a state such that they are functionally inactivated, but still present [31]. To prevent this state, incorporation of DCs pulsed with aGC resulted in a more sustained attack on the tumor, most likely due to the co-stimulation from the DC, such as CD40- CD40L interactions [30]. At large, shown in both murine and human cancers, increased iNKT cell incidence results in a slower progression of various tumor types [32, 33]. In contrast, iNKT cell-deficient mice (Cd1d−⁄− or Ja18−⁄−) are highly susceptible to spontaneous, carcinogen- induced or adoptively transferred tumors and reconstitution of the iNKT cell compartment in these animals results in prevention of slower tumor formation. These studies coupled with data from retrospective clinical studies highlight the importance of iNKT cells in tumor control [34, 35]. While this is true, iNKT cells can play a paradoxical role in cancer, as it can both promote and suppress the immune responses for recruitment or lack there of cytotoxic effectors [36]. The suppression of tumor cells occurs through a two-pronged attack, direct and indirect cytotoxicity. Tumors expressing CD1d can present a glycolipid and be directly lysed by iNKT cells through 5 release of perforin and Granzyme-B, or by Fas Ligand or TRAIL interactions [37]. Additionally, the recruitment of CD8+ T and NK cells by Th1 cytokine release amplifies the cytotoxic response [38]. More so, iNKT cells also possess the ability to kill tumor associated macrophages (TAMs), which function to keep the tumor microenvironment in an immunosuppressive state, hiding it from immuneosurveillance [39]. On the contrary, release of Th2 cytokines by Type-II NKT cells, or other subtypes of NKT cells, can also result in immunosuppression and thus deterred tumor clearance [40]. Taken together, iNKT cells play a prominent role in the protection, detection, and clearance of cancerous cells. Further characterization of the mechanism(s) of how iNKT cells function, or can be altered to function, in different tumor types can be critical in understanding how to eliminate cancers using novel immunotherapeutic approaches. Signaling mechanisms of iNKT cells Although it is well recognized that iNKT cells play critical role in host immunity against several pathogens or malignant cells, little is known regarding the signals that promote iNKT functions. Previous studies from our laboratory demonstrated for the first time that the adaptor molecule, SAP (Signaling Lymphocytic Activation Molecule [SLAM]-associated protein) is essential for not only iNKT cell linage development but also for TCR-induced iNKT cell lysis of multiple cellular targets in vitro and in vivo, where mice reconstituted with SAP-null iNKT cells exhibit extremely poor control of T-cell lymphoma growth. TCR-induced iNKT cell lysis was dependent upon Fyn, which often functions in conjunction with SAP to promote cell activation. Importantly, siRNA-mediated silencing of SAP or FYN in human iNKT cells significantly impaired TCR-induced target cell lysis. Collectively, these findings highlight the central role for 6 SAP-associated signals in iNKT cell cytotoxicity. Thus the goal of the current study was to identify and characterize the downstream signaling molecules that regulate iNKT cell development and functions. To that end, we investigated the role of the adaptor protein ADAP (Adhesion and Degranulation-Promoting Adapter Protein) in iNKT cell development and functions. The results obtained from these studies are significant as they increase our understanding of iNKT cell biology and provide insights into how iNKT cell functional responses can be further enhanced to improve host immunity against various diseases, specifically for the treatment of hematopoietic cancers such as leukemia and lymphoma. ADAP regulates lymphocyte development and functions In this study, we specifically chose to investigate the role of ADAP in iNKT cell development and function, as it is well established that upon TCR stimulation, ADAP binds to Fyn to modulate T cell functional responses. It was originally cloned as a phosphoprotein that inducibly binds SLP-76 and Fyn upon TCR stimulation [41, 42]. Accordingly, ADAP was formerly known as Fyb (Fyn binding protein) or SLAP 130 (Slp76-associated adaptor protein of 130kD) [43]. ADAP contains a large central proline-rich domain, an enabled vasodilator-stimulated phosphoprotein homology 1-binding domain [42], three putatively phosphorylated tyrosine residues, and a C-terminal SH3-like domain [42]). Two isoforms, 120 and 130 kDa, differing by a single, differentially spliced exon of 138 bp, have thus far been identified [42, 44], as shown in Figure 2. Early studies overexpressing ADAP in the Jurkat human T cell line implicated ADAP in TCR-induced IL-2 production [42]. ADAP has also been shown to be phosphorylated downstream of β1 integrin ligation in Jurkat T cells [45, 46]. Studies in ADAP-deficient mice demonstrate that ADAP is required for optimal passage through multiple thymic developmental 7 checkpoints and that ADAP may modulate TCR-dependent adhesive functions that are critical for thymocyte development and selection. Upon TCR activation, ADAP-deficient peripheral T cells have intact proximal signaling but inefficiently up-regulate early activation markers CD25 and CD69, fail to produce IL-2, and proliferate poorly in response to TCR stimulation [47]. ADAP-deficient T cells also demonstrate impaired inside-out activation of integrins, with decreased clustering of LFA-1 and impaired adhesion to ICAM-1 upon TCR ligation [48]. Employing ADAP-deficient mice (Adap−⁄−) and gene-silencing approaches in human iNKT cells, we specifically examined the requirement of ADAP in iNKT cell development and maturation as well as it role in direct and indirect immune-surveillance against cancer cells. 8 Thymus Conventional ab T cells CD3-CD4-CD8- DN Invariant NKT cells CD3-CD4-CD8- DN Selecting cell Precursor cell Precursor cell Selecting cell Precursor cell Selecting cell MHC I TCR Peptide DP TEC DP TCR MHC II Peptide TEC CD8 CD4 DP iTCR CD1d Glycolipid Antigen Immature NKT DP Mature NKT NKT Mature Periphery Spleen Lymph Nodes CD8 CD4 NKT Immature Naive phenotype Memory phenotype Spleen Liver Adapted from Das, et al., Immunol Rev., 2010 Figure 1. Schematic view of iNKT cell versus conventional ab T cell development. NKT cell precursors diverge from conventional ab T cells at the CD4+CD8+ double-positive (DP) stage. The invariant TCR a-chain (iTCR) on iNKT cell precursors recognizes glycolipid antigens presented by the CD1d molecule expressed at the surface of “selecting” DP thymocytes. In contrast, CD4+ and CD8+ T cells express diverse TCRs that recognize peptide antigens presented by MHC class II or class I molecules, respectively, expressed by thymic cortical epithelial cells (TEC). Mature thymic emigrants of the conventional ab T cell lineage have a naïve phenotype, as opposed to iNKT cells, which acquire a memory/effector phenotype during thymic development. Immature NKT cells can acquire the NK1.1 marker after emigration to 9 Figure 1. (cont’d) peripheral tissues or within the thymus. While a subset of NK1.1+ iNKT cells remain in the thymus as “long term-residents”, some also migrate to the periphery. 10 ADAP-130 PPP NLS Pro SLP-76 FYN Y595DDV Y625DGI Y651DDV SH3 EVH1- binding site 616-624 46aa NLS hSH3 Ena and VASP ADAP-120 PPP NLS Pro SH3 NLS hSH3 Figure 2. Molecular domains of ADAP and their binding partners. Adapted from Rudd, et al., Trends Cell Biol., 2008 ADAP and its major molecular domains as shown, within its two major isoforms, 130 kD (top) and 120 kD (bottom). ADAP interaction sites with SLP-76 and Fyn at tyrosine sites 595 & 651 and 625, respectively. EVH1 shown to bind to 616-624 sites. Abbreviations: PPP, proline-rich domain; NLS, nuclear localization sites; EVH1, Ena (enabled)-VASP (vasodilator-stimulated phosphor-protein) homology-1; PH, Pleckstrin homology domain; SH3, Src homology 3 domain. 11 ADAP REGULATES HOMEOSTATIC MAINTENANCE OF iNKT CELLS BUT NOT ITS THYMIC DEVELOPMENT OR MATURATION CHAPTER 1 12 Introduction Invariant natural killer T (iNKT) cells comprise a rare lymphocyte sub-lineage with phenotypic and functional properties similar to T and NK cells. Much like T-cells which develop in the thymus, development of iNKT cells also begin from a lymphoid progenitor [49]. During ontogenesis, iNKT cells diverge from the conventional T cells at the stage when T-cells express both CD4 and CD8 co-receptors, termed as double positive (DP) cells [49]. The T-cell receptor (TCR), in general, can express highly variable alpha (a) and beta (b) chain combinations following V(D)J recombination [49]. However, in the case of iNKT cells, the TCR consists of highly invariant Va14-Ja18 chains that often pair with Vb 8.2, 7, or 2 in mice and Va24-Ja18 and Vb11 in humans [49]. While conventional T cells are selected by peptide antigens in complex with major histocompatibility complex (MHC) class I or II molecules present on the surface of thymic epithelial cells, iNKT cells develop following selection by self-glycolipid antigens in complex with the MHC class I–like molecule CD1d, when presented by DP thymocytes [15]. Although still unclear, it is thought the endogenous glycolipid is or likely to be similar to iGb3 (isoglobotrisylceramide) [49]. Developmental stages of iNKT cells in the thymus After positive selection, when a cell is committed into becoming an iNKT cell, it enters a naïve stage, also termed stage 0, when phenotypically it is CD24hi, CD44lo, and NK1.1neg [1, 50] as shown in Figure 1. As this cell continues to develop into stages 1 through 3, it undergoes further changes in the surface expression of CD24, CD44 and NK1.1 as: expressing CD24lo, CD44lo, and NK1.1neg (stage 1), CD24lo, CD44hi, and NK1.1neg (stage 2) and CD24lo, CD44hi, and NK1.1pos (stage 3) [50]. In addition, CD4 is expressed exclusively from stages 0-3, where at the 13 end of stage 3, CD4 will remain expressed, or be down regulated, resulting in mature, CD4+ or CD4−CD8− (double negative; DN) iNKT cells [1]. At this stage, iNKT cells can remain in the thymus, or migrate to secondary lymphoid organs such as the spleen, and lymph nodes [51] or the liver and lungs. Migration to these organs is determined by a balance of chemokine receptor CXCR6 expression on iNKT cells, and CXCL16 (ligand for CXCR6) on resident organs, and expression is determined by the niche of the immune milieu in each of these organs [51, 52]. Transcriptional regulation of iNKT cells While several transcription factors that regulate iNKT cell development and maturation are shared with T cells, there are some unique transcription factors that exclusively regulate the developmental pathway of iNKT cells. In particular, mice deficient in Rag-1 or Rag-2 genes lack iNKT cells, as theses genes are required for V(D)J recombination into the invariant TCR [53]. In addition, any genetic deletion of the proper TCRa/b chains of iNKT cells or any of the TCR signaling molecules, such as Lck, ZAP-70, LAT, and SLP-76, results in diminished iNKT cells [54-57]. Along the stages of development for iNKT cells, the first major genetic requirement is expression of transcription factors RORgt and AP-1, required for expression of CD4 and CD8 before stage 0 [58, 59]. The transition from stage 0 to stage 1 requires a key regulator of iNKT cells called promyelocytic zinc finger protein (PLZF), unique to iNKT cells in the thymus [14, 60]. Mice deficient in this transcription factor result in decreased iNKT cell incidence in the thymus in mice [14]. Moreover, iNKT cells deficient in PLZF result in decreased functional capacity, including failure to secrete Thi and Th2 cytokines upon activation, emphasizing its importance to iNKT cells [14]. The transition from stage 1 to 2 and beyond to full maturation is required by multiple transcription factors, including Egr2, NF-kB, c-Myc, 14 GATA-3, and ThPOK being required for NK1.1 expression as well as iNKT functionality and migration [61-65]. One of the final maturation factors required is the T-Box transcription factor (T-bet) whose expression increases from stage 1-3, resulting in functionally mature, iNKT cells [66, 67]. SLAM-SAP-Fyn axis in iNKT cell ontogeny While transcriptional regulation is quite well defined, the signaling mechanisms they induce and regulate in iNKT cell development are yet to be fully understood. In particular, an additional required signaling event must occur during the DP selection process while TCR is engaged with CD1d. A family of receptors, known as SLAM (signaling lymphocytic activation molecule) receptors mostly act as self-ligands for each other, inducing activation of many required transcription factors downstream [68]. The family consists of varied expression of SLAM, Ly108, Ly-9, CD84, and CRACC of which its ligand is the same homotypic molecule, and 2B4, which uniquely binds to CD48, a heterotypic molecule [68]. While it is well characterized that SLAM-150 and Ly108 have known to be important for iNKT development, expression of other SLAM family receptors on iNKT cells, their intracellular signaling pathways are yet to be fully understood. However, a molecule downstream of all of these SLAM receptors, termed SAP (SLAM associated protein) is required in expression and functions of its downstream components [68]. This adaptor protein is required for an appropriate interaction with target molecules, and immunological synapse formation [68]. More so in the thymus, SAP is an indispensable molecule for iNKT development, as mice deficient in SAP lack any iNKT cells whatsoever [68]. After a SLAM receptor is bound, SAP recruits another protein, termed Fyn, which is a tyrosine kinase that is required for the downstream signaling events to occur [68]. 15 Thus, in addition to TCR ligation in the thymus, the SLAM-SAP-Fyn axis is required as well for proper iNKT development [68]. Earlier studies claimed that the adapter protein ADAP (a well-known Fyn-binding protein) is dispensable for conventional T cell development [43], however, data from more recent studies demonstrate that ADAP is required for optimal development of TCRab cells in the thymus, although it is dispensable for splenic B cell [69] and NK cell development [70]. Interestingly, it is now known that ADAP not only regulates intraepithelial innate T cells, CD8aa homeostasis in the small intestinal epithelium, it might also play a role in the development of non-T hematopoietic lineages [69]. In this study, we used a flow cytometry-based approach to determine the role of ADAP in iNKT cell development, maturation, homeostatic maintenance as well as subset distribution. We observed that while ADAP is dispensable for the development of mature iNKT cells in the thymus, it is required for the migration and homeostatic maintenance of these cells in the spleen as well as the liver. Our studies indicate that the decreased number of iNKT cells in the periphery could be partly due to the reduced expression of CXCR6 in thymic iNKT cells. We also demonstrate that the decrease in iNKT cells in the spleen and livers of Adap−⁄− mice is independent of CD11a (a chain of LFA-1) and CD11b ((a chain of Mac-1) expression. 16 Materials and Methods Mice C57BL/6 (B6), and Va14-Ja18 transgenic mice Tg(Cd4-TcraDN32D3)1Aben (Va14), were purchased from Jackson Laboratories (Bar Harbor, ME). Adap−/− mice on B6 background were kindly provided by Dr. Gary Koretzky (Weill Cornell University, New York, NY). All animal studies were approved by the Campus Animal Resources facilities of Michigan State University (MSU) and used under IACUC standards. Antibodies and Flow Cytometry The antibodies used for immunofluorescence staining included CD4, CD8, NK1.1, CD44, CD3, and CD56, CD11a, CD11b (BD Biosciences, San Jose, CA); TCRb, CD24, CXCR6, and fluorochrome attached isotype matched IgG1 and IgG2b (BioLegend, San Diego, CA); Va14 and Vb11 (Beckman Coulter, Brea, CA). Fluorochrome conjugated CD1d-tetramer (CD1d-Tet) loaded with glycolipid antigen (PBS57), or unloaded controls were provided by the NIH Tetramer Core Facility (Emory University, Atlanta, GA). All flow data were collected on a LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, OR). Isolation of purified populations of invariant NKT (iNKT) cells Single-cell suspensions were prepared from the liver, spleen and thymus. Hepatic mononuclear cells were first isolated using density centrifugation with Percoll (GE, Piscataway, NJ) and then stained with NK1.1 and TCRb antibodies and sorted for NK1.1+TCRb+ cells using BD Influx (BD Biosciences) to obtain pure populations of iNKT cells. For spleens and thymi, iNKT cells 17 were enriched by depletion of B220+ B and CD8+ T cells using anti-B220 and anti-CD8 magnetic beads, respectively, (Miltenyi Biotec; Auburn, CA) per manufacturer’s instructions. Subsequently, iNKT cells were flow-sorted from these enriched lymphocyte fractions as described above. Cells obtained using this method were routinely >97% NK1.1+ and TCRb+ and were also largely iNKT cells (>92%), when stained using PBS57-loaded CD1d tetramers. Human iNKT cell expansion Human blood samples (de-identified) were obtained from Gulf Coast Regional Blood Center (Houston, TX). Human peripheral blood mononuclear cells (PBMCs) were isolated with Ficoll- Paque (GE Healthcare) and subsequently cultured in complete AIM-V media (Gibco, Waltham, MA) with 10% fetal bovine serum (FBS), alpha-galactosylceramide, (aGC) (Enzo Lifesciences, Farmingdale, NY) at 500 ng/mL and recombinant human (rh) IL-2 (Peprotech, Rocky Hill, NJ) at 50U/mL. On the fourth day, rhIL-15 (Peprotech) and rhIL-2 were added at 10ng/mL and 10U/mL respectively to the culture. After four more days, human iNKT cells were purified by staining with FITC-conjugated anti-Va24 antibody, followed by anti-FITC magnetic beads, per manufacturer’s instructions (Miltenyi Biotech). Post-sort FACS analysis revealed >95% PBS57- CD1d tetramer reactive cells. Western Blotting Cells were lysed in RIPA lysis buffer (Thermo Scientific; Rockland, IL) with complete protease inhibitors (Roche; Basel, Switzerland) as per manufacturer’s instructions. Protein concentration was determined by Bradford assay (Biorad; Hercules, CA); 30µg of total protein was resolved by 8% SDS-PAGE and transferred to nitrocellulose membrane (GE Healthcare). Blots were probed 18 with the following antibodies: anti-ADAP and anti-GAPDH-HRP (Cell Signaling Technologies, Danvers, MA). Bound antibodies were detected using the appropriate anti-IgG coupled to HRP (Biorad, Hercules, CA) and ECL reagents (GE Healthcare). Quantitative real-time PCR Total RNA was extracted from purified iNKT cells from thymi and livers of Adap−⁄− and B6 mice using TRIzol solution (Invitrogen). RNA (1 µg) was reverse transcribed into cDNA using SuperScript III in a 20 µl reaction volume according to manufacturer's instructions (Applied Biosystems; Foster City, CA). Real-time quantitative PCR was performed using Quant Studio™ 3 system (Applied Biosystems), validated Taqman ADAP and GAPDH primers and Fast Advanced Master Mix according to manufacturers' instructions. Relative gene expression data (fold change) between samples was accomplished using the ΔΔCt method and GAPDH was used as the internal reference control [68] . Statistics Student’s t-test and One-way ANOVA with Tukey’s post-hoc test were used as indicated in the figure legends. Significance is shown as *(p<0.05), **(p<0.01), or ns for non-significant values. All statistical data was determined using GraphPad Prism (GraphPad, San Diego, CA). 19 Results ADAP is dispensable for the thymic development of iNKT cells but is essential for their maintenance in the spleen and liver Given that SAP and Fyn are both required for proper development of iNKT cells [71], and that ADAP is a known binding partner of Fyn [72], we sought to determine the role of ADAP in iNKT cell development, using Adap−⁄− mice and C57BL/6 (B6) as wild type controls. The burst of iNKT cell development occurs during the first few weeks of post-natal life [73], so we allowed mice to mature until 8 weeks of age. We then investigated the distribution and cell numbers of iNKT cells in the thymus, spleen, and livers of age and sex-matched animals from each genotype by flow cytometry analysis. The percentage iNKT cell populations were identified as TCRb+CD1d-tetramer+ (Figure 3A). While the incidence of iNKT cells in the thymus of B6 was comparable to those from Adap−⁄− mice (Table 1), these frequencies were significantly reduced in the peripheral organs (Fig. 3A and B) of Adap−⁄− mice. Consistent with previous studies and as depicted in Figure 3B, the incidence of iNKT cells in the spleen and liver of B6 mice were (0.72%±0.12%) and (16.6%±2.0%) respectively. In contrast, this was two- and over three-fold reduced in spleen (0.35%±0.06%) and liver (5.1%±0.4%), respectively of Adap−⁄− mice. Similarly, the total number of thymic iNKT cells in B6 mice was indistinguishable from those of Adap-/- mice (Table 2). However, iNKT cells in spleen and liver of Adap−⁄− mice were consistently and significantly (for livers) reduced as compared to their wild-type counterparts (Figure 3C and Table 2). These results show that ADAP is not required for iNKT development within the thymus, but becomes required as the iNKT cells migrate and maintain themselves in the peripheral organs. 20 A Thy Spl Liv 0.5 0.7 t e t - d 1 D C 1.1 0.2 18.0 B6 5.0 Adap-/- TCRb B6 Adap-/- C Frequencies ** iNKT Numbers * Thy 0.0 ADAP T B6 T Spl ADAP S B6 S Liv ADAP L B6 L Figure 3. iNKT cell incidence and absolute numbers are reduced in the spleen and liver of Adap−⁄− mice. (A) Populations of iNKT cells were identified by FACS analysis of PBS57-CD1d tetramer+ TCRβ+ cells in the thymus (Thy), spleen (Spl), and liver (Liv) of B6 and Adap−⁄−mice. Representative density plots are from 5 separate experiments. (B) Average frequency (percent of total live cells) and (C) absolute number of PBS57-CD1d tetramer+ TCRβ+ iNKT cells from the organs of B6 and Adap−⁄− mice were pooled. Data is presented as the mean of each group ± 21 B 20 15 10 5 2 s l l T K N e C T K N i t n e c r e P i % 1 * Thy 0 ADAP T B6 T Spl ADAP S B6 S Liv ADAP L B6 L 0.8 0.6 0.4 0.2 ) 6 0 1 x ( r e 6 ^ b 0 1 m x r e b u m N u N T K N e C T K N l l i i Figure 3. (cont’d) standard error of the mean (SEM) of at least 10 mice per organ, per genotype. Significance was determined by unpaired t-test between B6 and Adap−⁄− mice for each organ. (*p<0.05, **p<0.01). 22 Table 1. Frequency of iNKT cell subsets in various organs Thy Spl Liv Frequency B6 Adap−⁄− B6 Adap−⁄− B6 Adap−⁄− iNKTs CD44hiNK1.1hi CD4+ DN *p<0.05 5.1±0.4* 0.61±0.09 0.69±0.13 0.72±0.13 0.35±0.06* 16.6±2.0 84.4±2.72 79.3±8.28 76.28±6.7 68.98±4.12 95.7±1.2 43.72±5.55 42.45±6.0 75.12±4.05 67.23±4.41 75.1±1.87 72.7±1.41 27.5±2.54 23.0±1.91 25.3±1.42 38.13±2.4 37.0±4.05 20.0±2.71 83.9±6.3 Adap−⁄− 0.46±0.07 0.58±0.1 0.55±0.08 0.42±0.08 0.27±0.04 0.2±0.04 0.25±0.04 0.18±0.03 B6 B6 0.5±0.1 Adap−⁄− Adap−⁄− 0.15±0.03* 0.49±0.14 0.31±0.11 0.4±0.16 0.25±0.11 0.59±0.16 0.18±0.02* 0.32±0.13 0.22±0.09 0.47±0.12 0.15±0.01* 0.12±0.06 0.12±0.06 0.13±0.03 0.05±0.00 Table 2. Absolute cell number of iNKT cell subsets in various organs Absolute Cell Number (x106) Thy Spl B6 Liv iNKTs CD44hiNK1.1hi CD4+ DN *p<0.05 23 ADAP is not required for iNKT cell maturation or subset distribution To precisely and quantitatively determine the stages of iNKT cell development affected by the absence of ADAP, we analyzed various maturation markers expressed during defined stages of development and migration from the thymus [50] by flow cytometry. Accordingly, TCRb+CD1d-tetramer+ iNKT cells from thymus, spleen, and liver of B6 and Adap−⁄− mice were analyzed for their surface expression of CD24, CD44, and NK1.1 (Figure 4). No significant differences in either frequency (Figure 4A-C) or absolute number (Figure 4D) of iNKT cells in stage 0 (CD24hiCD44−), stage 1 (CD24loCD44−), stage 2 (CD24−CD44hi) or stage 3 (CD44hiNK1.1+) were observed in Adap−⁄− mice compared to wild-type controls, in any of the organs. Based on the CD4 expression, two iNKT cell subsets can be distinguished: CD4+ and CD4−CD8− (double negative [DN]). We analysed whether lack of ADAP could affect the development and/or tissue distribution of one particular iNKT cell subset. Comparison of the frequency and absolute number of iNKT cells in B6 and Adap−⁄− mice revealed a significant reduction in both CD4+ and DN iNKT cells in the livers of Adap−⁄− mice (Figure 5C, E). However, there was no skewing toward one particular subset in the thymus or peripheral organs (Figure 5A-E, Table 2). Taken together, our data suggests that ADAP is not required for iNKT cell maturation or subset development. Importantly, although iNKT cell numbers are diminished in the spleen and livers of Adap−⁄− mice, they are fully mature. 24 A Thy 2.5 1.2 0.3 Spl 2.8 0.1 Liv 2.0 4 2 D C 1.9 0.8 94.4 0.6 1.1 0.6 95.8 3.6 0.1 0.1 97.8 2.3 0.9 97.8 1.5 94.2 0.2 97.5 B Thy 0.6 95.9 0.2 CD44 Spl 96.4 0.4 Liv 98.4 4 4 D C 0.8 0.1 2.7 98.8 0.1 0.7 3.3 91.9 0.0 1.7 1.1 98.1 B6 Adap-/- B6 Adap-/- 0.4 0.8 0.1 7.2 0.0 0.1 NK1.1 B6 Adap-/- D ) 6 0 1 x ( ) 6 s 0 T 1 x K ( r N e b i m + 1 u N . 1 l l K e C N + 4 4 D C NumbersCD44+NK1.1+ * 0.8 0.6 0.4 0.2 0.0 Spl Thy Spl ADAP Spl B6 Liv B6 Thy B6 Liv ADAP Thy ADAP Liv C CD44+NK1.1+ i s T K N e c i n + e 1 d . c 1 n K I t N e s b + u 4 S 4 D C % 100 90 80 70 60 Figure 4. ADAP is neither required for the early nor late stages of iNKT cell maturation. Spl Thy Spl ADAP Spl B6 Liv B6 Thy B6 Liv ADAP Thy ADAP Liv (A, B) Representative density plots for CD24 and CD44 (A); CD44 and NK1.1 (B) expression in 25 Figure 4. (cont’d) iNKT cells within the thymus (Thy), spleen (Spl), and liver (Liv) of B6 and Adap−⁄− mice. (C-D) Pooled data from 3 experiments showing percent (C) and absolute number (D) of CD44hi NK1.1+ iNKT cells (n=6 for each condition) in the various organs. Data is presented as mean ± SEM. Statistical analysis was performed by unpaired t-test between B6 and Adap−⁄− mice for each organ (*p<0.05). 26 A Thy 0.4 8.7 2.0 Spl 1.5 0.3 Liv 1.4 8 D C 41.4 0.1 49.5 7.0 17.8 1.2 78.7 1.4 21.6 0.5 76.6 1.2 B6 Adap-/- 39.7 53.3 23.5 73.9 28.6 69.7 CD4 B6 Adap-/- CD4+ B 100 i e T c n K e d N c i n + I 4 t e D s b C u S % 80 60 40 20 Spl Thy Spl ADAP Spl B6 Liv B6 Thy B6 Liv ADAP Thy ADAP Liv DN D 50 40 30 20 i e c T n e K d N c n i I N t e s D b u % S C ) 6 ) 0 6 0 1 1 x x ( ( r s e b T m K u N N i l l e + C 4 D C E ) ) 6 6 0 0 1 1 x x ( r ( e b s m T u K N N e i C N D l l NumbersCD44+NK1.1+ * 0.8 0.6 0.4 0.2 0.0 Spl Thy Spl ADAP Spl B6 Liv B6 Thy B6 Liv ADAP Thy ADAP Liv NumbersDN 0.4 0.3 0.2 0.1 * 10 Spl Thy Spl ADAP Spl B6 Liv B6 Thy B6 Liv ADAP Thy ADAP Liv Figure 5. ADAP is not required for subset distribution of iNKT cells. (A) Representative FACS plots of CD4 and CD8 (gated on iNKT cells) in B6 and Adap−⁄− mice within the thymus 27 0.0 Spl Thy Spl ADAP Spl B6 Liv B6 Thy B6 Liv ADAP Thy ADAP Liv Figure 5. (cont’d) (Thy), spleen (Spl), and liver (Liv). (B-E) Pooled data from 3 experiments show percent (B, D) and absolute cell numbers (C, E) of CD4+ (B, C) and DN (D, E) iNKT cells. Data is shown as mean ± SEM, with n=6 for each condition. Statistical significance was determined by unpaired t- test between B6 and Adap−⁄− mice for each organ. (*p<0.05). 28 Altered CXCR6 expression on thymic and splenic iNKT cells of Adap−/− mice CXCR6, a chemokine receptor, which is expressed on iNKT cells, controls the selective accumulation of these cells in the liver, in part by transmitting survival signals [51]. To investigate a potential role for CXCR6 in the reduced frequency of iNKT cells in the spleen and liver of Adap−/− mice, we examined the expression of this chemokine receptor on these cells in B6 and Adap−/− mice. As shown in Figure 6A, the percentage of CXCR6 expressing iNKT cells were significantly decreased in thymus and spleen of Adap−/− mice when compared to B6 animals (Table 3). Interestingly, while there was no difference in the incidence of CXCR6+ hepatic iNKT cells (Figure 6B), the absolute numbers of CXCR6+iNKT cells were significantly reduced in the livers of Adap−/− mice (Figure 6C). Taken together, our data indicate that one of the possibilities for the reduced numbers of iNKT cells in the livers of Adap−/− mice could be due to the reduced expression of this chemokine receptor on thymic iNKT cells in these mice. However, the Adap−/− iNKT cells that do home to the liver, express CXCR6. 29 Spl 2365 87.8 1623 70.2 Liv 3905 97.9 B6 3862 94.9 Adap-/- CXCR6 B6 Adap-/- CXCR6 Expression ** C l l ) 6 0 1 x ( ) r 6 e 0 b 1 m x u ( N s T e K C N T i K + 6 N R + C 6 X R C C X C i 1.0 0.8 0.6 0.4 0.2 0.0 1245 77.4 680 63.0 CXCR6 Expression ** A Thy B 100 ) i % T ( K T N K N + i 6 + R 6 C R X C C X C % 80 ** 60 Spl Thy Spl ADAP Spl B6 Liv B6 Thy B6 Liv ADAP Thy ADAP Liv Spl Thy Spl ADAP Spl B6 Liv B6 Thy B6 Liv ADAP Thy ADAP Liv Figure 6. Reduced expression of CXCR6 in iNKT cells of Adap−⁄− mice. (A) Representative histograms depicting CXCR6 expression in iNKT cells from the thymus (Thy), spleen (Spl), and liver (Liv) of B6 and Adap−⁄− mice. Numbers on gate shows percentage greater than isotype controls, per organ. MFI of representative histograms are shown in the upper right corner of each plot. Percentages (B) and absolute numbers (C) of CXCR6+ iNKT cells from each organ 30 Figure 6. (cont’d) and genotype were pooled from 2 experiments (n= 4-9 mice per genotype). Data is presented by mean ± SEM. Significance was determined by unpaired t-test between B6 and Adap−⁄− mice for each organ (**p<0.01). 31 Table 3. Frequency and Absolute cell number of CXCR6+ iNKT cells Thy Spl Liv B6 Adap−⁄− B6 Adap−⁄− B6 Adap−⁄− Frequency 77.1±1.17 64.1±1.4* 88.3±0.43 76.2±4.3* 97.9±0.06 92.72±2.06 Absolute Cell Number (x106) *p<0.05 0.38±0.07 0.26±0.08 0.14±0.04 0.09±0.02 0.81±0.12 0.2±0.02* 32 Expression of integrin CD11a and CD11b is normal in iNKT cells of Adap-/- mice Previous studies have demonstrated clearly that iNKT cells are retained locally within the liver or spleen through constitutive interactions of integrins such as LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) with their natural ligand–intercellular adhesion molecule 1 (ICAM-1) [74, 75]. However, as shown in Figure 7A-C, we observed no difference in either the frequency or the absolute number of CD11a+ or CD11b+ iNKT cells in the Adap-/-mice when compared to those in B6 mice, suggesting that decreased iNKT cells in the peripheral organs of Adap-/- mice is not likely due to integrin expression. 33 Isotype CD11a 98.1 Isotype CD11b 19.2 B6 94.2 21.8 Adap-/- CD11a CD11b B6 Adap-/- C ) 6 0 1 x ( r e b m u N l l e C 80 70 60 30 20 10 0 A B n o i s s e r p x E % 120 100 80 40 30 20 10 0 CD11a CD11b CD11a CD11b Figure 7. Expression of CD11a and CD11b is not altered in Adap-/- iNKT cells. (A) Representative histograms of CD11a (left) and CD11b (right) expression in iNKT cells from the spleens of B6 and Adap-/- mice. Numbers within histograms depict percent positive expression for CD11a or CD11b. Positive gate was determined by incidence greater than isotype controls, per organ. Percentage (B) and total numbers (C) of iNKT cells positive for CD11a and CD11b 34 Figure 7. (cont’d) surface expression is shown. Data is presented as mean ± SEM from 1 of 2 experiments with n= 3-9 mice tested per genotype. 35 Discussion Adapter molecules mediate protein-protein interactions in signal transduction cascades. These signaling cascades translate information from cell surface receptors into cellular responses. It is known that the adaptor ADAP plays a role in integrin-mediated signaling pathways that regulate thymocyte development as well as adhesion and functional responses in T lymphocytes. However, little is known about the role of ADAP in iNKT cell development or functions. In the current study, we examined the role of ADAP in the development of iNKT cells using a genetically engineered mouse lacking this molecule. Here, we provide evidence that although the development or maturation of iNKT cells is not affected by the lack of ADAP, their numbers in the liver and spleens are significantly reduced in Adap−⁄− mice. Thus our findings reveal a unique linage-specific role of ADAP in iNKT cell homeostatic maintenance. Similar to conventional T cells, development of iNKT cells occurs in the thymus. These cells are absent in nude mice [1, 76], fail to develop in thymectomized mice [77, 78], and are first detected in the thymus in the perinatal period [49, 50, 79, 80]. Both conventional αβ T cells and iNKT cells proceed through the same early triple negative (CD3−CD4−CD8−) stage and have almost identical signaling requirements for the development of their respective precursors [81]. Rather, it is during the double positive (CD4+CD8+) stage that Vα14 to Jα18 rearrangement and invariant TCR expression occurs, which dictate iNKT lineage commitment [2, 58, 82, 83]. iNKT cell development at the DP stage is highly dependent on signals generated by engagement of the SLAM family (SLAMf) of surface receptors, which are expressed on developing iNKT cell precursors as well as cortical DP thymocytes. The SLAM receptors include SLAM, 2B4, CD84, Ly9, Ly108, and CRACC (CD2-like receptor activating cytotoxic cells). With the exception of 36 2B4, which interacts with CD48 as its ligand, these receptors all function in a homotypic manner and bind to like receptors with high affinity [83]. SLAM receptor signaling is mediated SAP, which binds via its phosphotyrosine-binding pocket to one or more conserved tyrosine-based motifs present within the SLAM receptor cytoplasmic tails [84]. Using a distinct region of its SH2 domain centered at arginine 78, SAP also binds to the Src family tyrosine kinase Fyn, leading to an increase in kinase activity and generation of a SLAM receptor-SAP-Fyn-mediated tyrosine phosphorylation signal [85, 86]. Evidence for the crucial role of SLAM-SAP signaling in iNKT cell development came from studies wherein patients with the immunodeficiency X- linked lymphoproliferative disease (XLP), who harbor inactivating mutations in the gene encoding SAP, and Sap−/− mice, lack iNKT cells [71, 87, 88]. Interestingly, similar results are seen in mice lacking Fyn or expressing a mutant version of SAP that cannot bind to Fyn (SapR78A) [55, 89]. It is currently unclear as to how SLAMf receptor-SAP signals coordinate with those emanating from the TCR to foster proper iNKT cell development. ADAP binds to both SH2‐containing leukocyte phosphoprotein of 76 kDa (SLP‐76) and the Src‐ family kinase Fyn [42, 90]. As both these molecules control iNKT cell development, we postulated that ADAP is a likely downstream candidate of the SAP-Fyn axis and/or is required for TCR –induced signals during development. ADAP is clearly required for efficient maturation of thymocytes undergoing TCR‐dependent positive and negative selection [90]. ADAP protein and mRNA are upregulated during the DP‐to‐SP transition, a phase of thymocyte maturation driven by signals emanating from the TCR [91]. In addition, ADAP is specifically increased in TCRβhigh and CD69high DP subsets, suggesting that the TCR signals known to enhance expression of these surface proteins may also be driving augmentation of ADAP protein levels. 37 These observations are intriguing because they suggest that protein levels of ADAP and of its binding partner SLP‐76 are regulated largely in parallel. SLP‐76 is a hematopoietic adaptor protein that is absolutely required for pre‐TCR and TCR‐dependent signaling leading to thymocyte maturation [92]. The lack of a requirement for ADAP in the development of iNKT cells despite the fact that they clearly express ADAP protein suggests the presence of an ADAP‐ independent signaling cascade. These observations are similar to those seen in other non- conventional T lymphocytes, such as the CD8aa T cells, the most abundant among intestinal intraepithelial lymphocytes (IEL). The marked reduction in contribution by ADAP‐deficient cells to the IEL CD8aa T cell compartment in mixed chimeras suggests a defect either in homeostasis or in migration/homing of these cells. Indeed, our results indicate that the reduced iNKT cell numbers in the peripheral organs of Adap−⁄− mice may be in part due defective migration/homing of these cells to the liver and spleen. Following positive selection, NKT cells progress down a pathway of maturation that is marked by the sequential acquisition of specific patterns of surface marker expression: CD24, CD44 and NK1.1. The most immature iNKT cells are defined as CD24hiCD44loNK1.1− (Stage 0), followed by CD24loCD44loNK1.1− (Stage 1), CD24loCD44hiNK1.1− (Stage 2), and finally CD24loCD44hiNK1.1 + (Stage 3) [93]. In the absence of ADAP, we neither observed a defect in transition of the immature iNKT cells to the mature stages nor did we observe preferential skewing to a particular subset development. The most significant changes observed in our study were the alterations in peripheral iNKT compartment in Adap−/− mice. ADAP deficiency specifically impacted cells within the liver and spleen but not the thymus. Adap−/− iNKT cells exhibit a peripheral homeostatic defect most likely due to altered tissue-specific migration and/or 38 retention. The migration and retention of iNKT cells within peripheral tissues is mediated by a complex interplay between integrins (LFA-1 [94] and Mac-1 [95] and chemokines (CXCL16 [96]). We observed no reduction in the levels of CD11a or CD11b integrin expression on Adap−/− iNKT cells derived from all tissues. However, we did observe significant reduction in the frequency of CXCR6+ cells in the thymus of Adap−/− mice. Although CXCR6 is not required for thymic iNKT cell development, it plays a vital role in the accumulation of iNKT cells in the liver via interaction with its ligand, CXCL16. The redistribution of iNKT cells to bone marrow of Cxcr6−/− mice suggests a role for CXCR6 in iNKT cell homing or retention in the liver and lungs. Thus the reduced frequency of CXCR6 on thymic iNKT cells of Adap−/− mice explains in part, the reduced number of iNKT cells in the spleens and livers. Furthermore, the reduced frequency of CXCR6 on splenic iNKT cells of Adap−/− mice suggests that although thymic iNKT cells migrate to the spleen, reduced CXCR6 expression fail to retain them in the spleen. Interestingly, we observed that the incidence of CXCR6 on liver iNKT cells were indistinguishable between B6 and Adap−/− mice. Of note, the reduced numbers of iNKT cells in the liver and/or spleen did not coincide with increased accumulation of these cells in the thymus or peripheral lymph nodes. Taken together, we believe that thymic iNKT cells that express CXCR6 home to the spleen and liver. However either due to lack of CXCL16 expression in the respective tissues or due to their inability to maintain surface expression of CXCR6 in the absence of ADAP, these cells fail to either proliferate or survive rather than migrating to the lymph nodes. Our studies thus favor a role for ADAP in proliferation and/or survival of iNKT cells, in part via CXCR6 expression. Consistent with this notion, Geissmann et al. [51] reported that Cxcr6−/− iNKT cells exhibited enhanced apoptosis in culture. However, other studies [96] 39 exhibited no differences in the apoptosis (annexin V staining) of cultured Cxcr6+/+ and Cxcr6−/− iNKT cells [96]. In addition, anti-CXCL16 treatment does decrease survival of resident liver NKT cells, as cell numbers remained the same as in isotype-treated mice [96]. In addition to chemokines and integrins, peripheral iNKT homeostasis is dependent on a steady provision of co-stimulatory signals. While thymic iNKT cell development depends on CD28-B7 interactions [97], peripheral survival relies on co-stimulatory signals delivered through ICOS/ICOS-L interaction [98]. Whether ADAP is involved in CD28-mediated signaling or regulates ICOS signaling has not yet been established. Although T cells and NK cells share a common lymphoid progenitor in the bone marrow, their molecular requirements for development are distinct. While T cell maturation requires pre-TCR- and TCR-mediated signal transduction in addition to signals mediated by select cytokines [e.g. IL-7 [99]], NK cells primarily rely on cytokine or growth factor signaling components for their development [100]. Normal NK development and distribution in Adap−⁄− mice suggest that ADAP does not regulate signaling by cytokines that utilize the common gamma chain [IL-2 and IL-15 [101]]. Given that ADAP signals downstream of ITAM-associated molecules [e.g. TCR [102] and the FcεRI [103]] rather than of cytokine receptors, it is not surprising that ADAP is indispensable for NK cell development. Taken together, our findings define a pivotal role for ADAP in peripheral iNKT homeostasis: ADAP is important for iNKT migration to the peripheral tissues and is involved in promoting cellular retention within the tissues, probably in part by regulating chemokine receptor CXCR6 expression. While ADAP is required for the homeostasis of iNKT cells, the mechanism(s) 40 involved are yet to be defined. Studies are currently underway to identify whether the decreased peripheral iNKT cell numbers in Adap−/− mice are likely due a marked reduction in homeostatic proliferation or higher susceptible to apoptosis or both. 41 ADAP REGULATES INVARIANT NATURAL KILLER T CELL ACTIVATION AND IMMUNOMODULATORY FUNCTIONS CHAPTER 2 42 Introduction Invariant natural killer T (iNKT) cells are innate-like lymphocytes as they exhibit characteristics of both innate and adaptive immune cells. However, unlike conventional T cells, iNKT cells bear a semi-invariant TCR that recognizes different lipid antigens presented by monomorphic CD1d molecules. Soon after the TCR-antigen-CD1d engagement, iNKT cells rapidly secrete copious amounts of cytokines, mainly IFN-g and IL-4, a characteristic reminiscent of innate immune responses [104-107]. Besides the CD1d-dependent mode, iNKT cells are also activated by cytokines, which is common when the lipid antigen is weakly immunogenic [108]. In a physiological context, iNKT cells become activated by microbial or self-lipid antigens bound to CD1d molecules. For example, isoglobotriosylceramide (iGB3), a neutral glycosphingolipid, has been identified as a weak self-lipid antigen for human and murine iNKT cells [21, 109, 110], although its role as the only positive-selecting self-lipid in the thymus remains controversial [21, 109, 110]. Lysophospholipids and glycosphingolipids have been shown to be self-lipid antigens in different contexts [21, 111, 112]. Self-lipid antigens are weakly immunogenic and iNKT cell activation in this case is often largely driven by IL-12 and IL-18. Although CD1d-activated iNKT cells can undergo further activation via cytokines secreted from matured DCs, cytokines IL-12 and IL-18 are alone sufficient to activate iNKT cells [113, 114]. Unlike peptide-MHC restricted conventional T cells, which emerge from the thymus “naïve” iNKT cells leave the thymus fully matured and able to perform their effector functions without priming [115-118]. Depending on how the iNKT cells are activated, this can include both regulatory cytokines (e.g., IL-4, IL-10) [115-118] and/or pro-inflammatory cytokines (e.g., IL-2, IL-17, TNF-a, and/or IFN-g) [115-118]. Since iNKT cells respond rapidly and without the need 43 for priming, they are some of the first cells within an immune response to be activated and therefore act as a “bridge” between the innate and adaptive immune systems. As shown in Figure 8, iNKT cell activation via TCR engagement or IL-12 or both causes iNKT cells to upregulate IL-12 receptor and CD40L, while also inducing maturation and production of IL-12 in dendritic cells (DCs). This IL-12 release then in turn greatly increases IFN-g production by iNKT cells, leading to a positive feedback loop for Th1 immunity [115-118]. Additionally, this maturation of DCs leads to trans-activation of NK cells and increased MHC class I and II antigen presentation to T and B cells, allowing for both innate and adaptive immune responses to be established [115- 118]. Since iNKT cells are uniquely placed at the interface between innate and adaptive immunity, they have a tremendous influence in shaping immune responses. Although the frequency of iNKT cells in humans ranges from 0.01 to 0.1% in peripheral blood (lower than in mice), this frequency is still orders of magnitude higher than that of naïve peptide-specific T cells [119, 120]. Cytokine stimulation and cognate interaction between iNKT cells and dendritic cells (DCs), B cells, neutrophils, and macrophages often polarizes these cells toward a pro- inflammatory phenotype [121-123]. In general, CD4+ iNKT cells are able to express more Th2- related cytokines like IL-4, although they can express as much Th1 cytokines at the same time [115-119]. Human DN iNKT cells are biased toward a Th1-related phenotype, and preferentially make IFN-g. However, CD4 + iNKT cells can make IFN-g and DN iNKT cells can make IL-4, at least partially depending on the stimuli given [115-119]. Mouse iNKT cells are less clearly biased. Relatively Th1-like iNKT cells tend to be enriched within the spleen and liver, while Th2-like iNKT cells are associated with the lungs and intestine [115-119]. There are also Th17- 44 like iNKT cells that express cytokines like IL-17 and are enriched within the lungs, intestine, lymph nodes, and skin [124]. A recently described subset, adipose iNKT cells tend to make anti- inflammatory cytokines like IL-10 and unlike other mature iNKT cells, lack PLZF [125, 126]. Because iNKT cells can rapidly produce IFN-γ, IL-4, or both, they have been found to play a role in various diseases by establishing a Th1- or Th2-based immune response. However, the mechanisms that control iNKT cell functions are poorly understood. In light of the shared functional properties of T, NK and iNKT cells, and the requirement for ADAP in efficient but specific functions of T [43, 127] and NK cells [70, 128], we hypothesized that ADAP would also play a role in iNKT cell activation and immunomodulatory functions. ADAP is a positive regulator of T cell activation and is required for both T cell proliferation and IL-2 production. The role of ADAP in NK cell cytokine production seems controversial. While earlier studies showed that ADAP is dispensable for NK cell cytokine secretion [70], more recent studies demonstrate that signaling by Fyn-ADAP via the Carma1-Bcl10-MAP3K7 signalosome exclusively regulates inflammatory cytokine production in NK cells [128]. In this study, using B6 and Adap−⁄− mice as well as purified iNKT cells from these animals, we demonstrate that ADAP is required for both iNKT cell cytokine production as well as its release. We also demonstrate that in the absence of ADAP, murine iNKT cells fail to modulate activation of T, B and NK cells as to promote maturation of dendritic cells (DC). Importantly, this observation is not limited to murine iNKT cells, as gene silencing of ADAP in human iNKT cells also results in significant reduction in cytokine levels in culture supernatants. 45 Adapted from Bendelac, et al., Annu. Rev. Immunol., 2007 Figure 8. Cellular interactions between iNKT cells and other immune cells Invariant NKT cells are activated by TCR engagement with a glycolipid presented by CD1d on dendritic cells (DC), B cells, or macrophages (MF). Additional co-stimulation between CD40L on iNKT cells and DCs or DC release of IL-12 amplifies the activation of iNKT cells. iNKT cell release of IFN-g recruits and activates NK cells and CD8 killer T-cells. Release of IL-4 and IL- 13 helps recruit CD4 helper T-cells. Migration of iNKT cells to the liver is by interaction between CXCR6 on iNKT cells and CXCL16 on resident liver sinusoids. 46 Materials and Methods Mice Mice were used as described in Chapter 1. Reagents EL4 T lymphoblastic cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA). Recombinant human (rh) IL-2 was from Peprotech (Rocky Hill, NJ) and rhIL-15 from Sigma Aldrich (St. Louis, MO). KRN7000 (aGC) and its analog, OCH were purchased from Enzo Life Sciences (Farmingdale, NY). Antibody list includes anti: NK1.1, CD69, CD3, CD56, IL-4, IFN-g, CD11b, CD11c, and B220 (BD Biosciences, San Jose, CA); TCRb, (BioLegend, San Diego, CA); Va14 and Vb11 (Beckman Coulter, Brea, CA). CD1d-tetramer loaded with glycolipid antigen PBS57, was provided by the NIH Tetramer Core Facility (Emory University, Atlanta, GA). Murine and human iNKT cells were obtained as in Chapter 1. Flow cytometry and Western Blotting Immunofluorescence staining, cell sorting and flow cytometry as well as SDS-PAGE and western blotting were performed as described in the “Materials and Methods” of Chapter 1. Transient gene silencing in human iNKT cells Human iNKT cells were subjected to a transient gene knockdown as per protocol provided in the Amaxa Human T-cell Nucleofector Kit (Lonza, Basel, Switzerland). Briefly, 2x106 human iNKT cells were resuspended in 100µL nucleofector solution, and transfected with 300 pmol of control (Invitrogen) or ADAP-specific (SMARTpool: ON-TARGETplus FYB siRNA; Dharmacon, 47 Lafayette, CO) siRNA oligonucleotides by using Amaxa nucleofection protocol O-17. Cells were immediately transferred to complete RPMI media supplemented with 10% FBS in the presence of 50U/mL rhIL-2, for 48 hours at 37ºC before use. Knockdown efficiency was verified by western blotting using human specific antibodies against ADAP and GAPDH (loading control). In vivo cytokine production For in vivo stimulation, mice were injected i.p. with 4 µg of PBS57 in 300 µl of sterile PBS. After 2-4 h, serum was collected from mice by retro-orbital bleeds and analyzed for cytokines using specific sandwich enzyme-linked immunosorbent assay (ELISA; BD OptEIA™). All samples were run in duplicate. Splenocytes and liver lymphocytes were isolated and stained to detect cell surface markers or intracellular cytokines, as indicated in the respective section. In vitro cytokine assay Purified iNKT cells (80x103 cells in 100 µL) were co-cultured with EL4 cells (80x103 cells in 100 µL) that were either left untreated or loaded with PBS44 (100ng/mL), in a total volume of 200 µL. After 24 hours, culture supernatant was harvested and analyzed for cytokine release by ELISA (BD OptEIA™). Optical density was measured at 450 nm on a FlexStation3 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA) and converted into sample concentration through SoftMax Pro Software (Molecular Devices). 48 Statistics Statistical analysis was performed as described in the “Materials and Methods” section of Chapter 1. 49 Results ADAP is required for TCR-induced iNKT cell cytokine production A primary role of iNKT cells is to modulate the activation and recruitment of other immune cells, which is accomplished by release of both Th1 and Th2 cytokines [129]. Injection of aGC into B6 mice leads to iNKT cell activation [130], resulting in the rapid production of both Th1- and Th2-type cytokines. To investigate whether deletion of Adap influences iNKT cell cytokine responses, B6 and Adap−⁄− mice were injected i.p. with PBS44, an analogue of aGC. After 2 hours, serum was collected and analyzed for the presence of cytokines. Both IFN-g and IL-4 were markedly reduced in the serum from Adap−⁄− mice, whereas robust levels of both the cytokines were found in B6 mice (Figure 9A-C). To investigate whether the reduced serum cytokine levels were due to the lower number of cytokine producing iNKT cells in Adap−⁄− mice, defect(s) in their ability to produce cytokine, or a combination of both factors, splenocytes and liver lymphocytes were harvested and evaluated for iNKT cell cytokine production by intracellular staining. Using this approach, we observed that both B6 and Adap−⁄− mice produced detectable levels of IFN-g and IL-4 directly ex vivo (Figure 9D-G). However, the frequency of IFN-g or IL-4-producing iNKT cells in the spleen and liver of Adap−⁄− mice were significantly reduced as compared to those from B6 controls (Figure 9H, I). These data indicate that in the absence of ADAP, the reduced cytokine levels in the serum could be either due to lower number of IFN-g or IL-4-producing iNKT cells in Adap−⁄− mice or their impaired ability to release cytokines following challenge with glycolipid antigen, or both. 50 A l / m g p D B6 Adap−⁄− No Ag B ** B6 Adap−⁄− + Ag 4000 IL-4 3000 ** C 2000 1000 0 500 IFN-g 400 300 200 100 0 n o i t c u d e R % 100 90 80 70 60 50 IFN-g IL-4 B6 Adap−⁄− E B6 Adap−⁄− 64.2 44.3 Spl 71.2 51.8 Spl 88.8 68.6 Liv 70.8 53.2 Liv No Ag + Ag IFN-g F 6000 IFN-g ** I F M 4000 2000 ns 0 Spl IFN-g ** H T K N i + g - N F I 100 80 60 40 20 0 % IL-4 ns G 6000 IL-4 I F M 4000 2000 * Spl Liv IL-4 ** * Liv ** I T K N i + 4 - L I % 0 100 80 60 40 20 0 B6 Adap−⁄− B6 Adap−⁄− No Ag + Ag B6 Adap−⁄− B6 Adap−⁄− No Ag + Ag Spl Liv Spl Liv Figure 9. ADAP is required for adequate cytokine production and release in iNKT cells. Age and sex matched B6 and Adap−⁄− mice were injected intraperitoneally (i.p) with 4µg of 51 Figure 9. (cont’d) PBS44 or left untreated. After 2 hours, serum was collected and IFN-g (A) and IL-4 (B) levels were measured by ELISA. Data represent the mean ± SEM from 9 independent experiments. (C) Mean percent reduction ± SEM in the serum levels of IFN-g and IL-4 in PBS44–injected Adap−⁄− mice as compared to similarly treated B6 mice. Data are averaged from 9 independent experiments. (D-G) After 2 hours, the percentages of splenic and hepatic iNKT cells producing IFN-g (D) and IL-4 (E) directly ex vivo were analyzed by intracellular cytokine staining and flow cytometry. Representative histograms are shown in D, E as indicated. Bar graphs show MFI (F, G) and % positive iNKT cells (H, I) for IFN-g (F, H) and IL-4 (G, I) in the spleen and liver. Data in F-I represent the mean ± SEM and are pooled from 4 independent experiments. Significance was determined by unpaired t-test. *p<0.05,**p<0.01. MFI: mean fluorescence intensity 52 ADAP regulates iNKT cell cytokine release In order to determine if the cytokine release defect in Adap−⁄− mice was due to an intrinsic iNKT cell defect, or because of a decreased numbers of these cells in the peripheral organs, equal numbers of purified B6 and Adap−⁄− iNKT cells were cultured with antigen-pulsed or unpulsed EL4 cells. After 18 hours, iNKT cell cytokine production was assessed. B6 iNKT cells secreted detectable levels of IFN-g and IL-4 when cultured with lipid-antigen loaded but not unloaded EL4 cells. Compared to B6, Adap−⁄− iNKT cells produced significantly less cytokines when cultured with antigen-loaded cells establishing that ADAP regulates both Th1 and Th2 cytokine release by iNKT cells (Figure 10). These findings also help explain the reduced in vivo cytokine responses of PBS44-injected Adap−⁄−mice. 53 B6 Adap-/- No Ag B6 Adap-/- + Ag A l / m g p B l / m g p C n o i t c u d e R % 800 600 400 200 0 500 400 300 200 100 0 100 80 60 40 20 0 IFN-g * IL-4 * IFN-g IL-4 Figure 10. ADAP-deficient iNKT cells produce less cytokines in response to antigen-pulsed EL4 cells in vitro. iNKT cells from B6 or Adap−/− mice were co-cultured with either PBS44- 54 Figure 10. (cont’d) loaded or unloaded EL4 cells. iNKT and target cells were mixed at a 1:1 ratio in a total volume of 200µL and incubated overnight at 37°C. After 24 hours, culture supernatants were harvested and analyzed for IFN-g (A) and IL-4 (B) levels by ELISA. (C) Percent reduction in cytokine (IFN-g and IL-4) release by Adap−⁄− iNKT cells as compared to those from B6 mice. Data in A-C represent the mean ± SEM from 3-6 experiments. Statistical significance was determined by unpaired two-tail t-test. * p < 0.05. 55 ADAP is required for human iNKT cell cytokine release To examine whether ADAP is also required for human iNKT cell cytokine production, we expanded and purified iNKT cells from the blood of normal donors [68] and examined their ability to produce Th1 and Th2 cytokines. Gene expression of ADAP in human iNKT cells was significantly lower than in conventional T cells; however both the lymphocyte lineages expressed comparable protein levels of ADAP (Figure 11A, B). Interestingly, both primary T and iNKT cells express varying levels of both the isoform ADAP (Figure 11B). Human iNKT cells exhibit robust cytokine responses against PBS44-loaded but not unloaded EL4 cells [68]. Expanded iNKT cells from 1 donor was independently transfected with ADAP-specific siRNA or left untransfected (WT) and used for in vitro cytokine assay. ADAP-silenced cells expressed almost no ADAP protein (Figure 11C) and similar to murine iNKT cells, exhibited a significant reduction in cytokine release (Figure 11D, E). Together, these studies indicate that ADAP is a critical mediator of human iNKT cell cytokine response. 56 A Tcon iNKT n n o o i i s s s s e e r r p p x x e E e e n n e e g G P P A A D D A A * 100 90 80 70 60 20 10 0 Tcons iNKTs 1 2 3 4 5 6 C ADAP GAPDH kDa 130 120 37 WT KD kDa 130 37 WT hNKT No Ag WT hNKT ADAP KD hNKT + Ag IFN-g E IL-4 40 30 20 10 0 B ADAP GAPDH D l / m g p 200 150 100 50 0 Figure 11. Silencing ADAP gene expression in human iNKT cells results in reduced cytokine production. (A) Gene expression of ADAP in human conventional T-cells (Tcons) and 57 Figure 11. (cont’d) human iNKT cells expanded from PBMCs from human donors. (B) Representative western blotting of ADAP (top) and GAPDH (bottom) in human T (lanes 1-3) and iNKT (lanes 4-6) cells from three separate human PBMC samples. (C) Human iNKT cells were transfected with ADAP- specific siRNA (KD) or left untransfected (WT). After 48 hours, knockdown of ADAP expression was assessed by immunoblotting. (D, E) Untransfected or ADAP-silenced human iNKT cells were co-cultured with either PBS44-loaded or unloaded EL4 target cells. iNKT and target cells were mixed in a 1:1 ratio in a total volume of 200µL and incubated overnight at 37°C. After 24 hours, culture supernatants were harvested and analyzed for IFN-g (D) and IL-4 (E) levels by ELISA. Data is from 1 donor and is represented as mean ± SD. 58 ADAP is required for TCR-induced iNKT cell-mediated bystander cell activation. The secretion of cytokines by iNKT cells contributes to the in vivo activation of other immune cell lineages [131]. Therefore, we next investigated whether Adap−/− mice showed abnormalities in iNKT cell cytokine–dependent events. Animals were injected with PBS44 and 4 hours later, lymphocytes harvested and examined for up-regulation of the CD69 activation marker on splenic dendritic cells (DC), T, B, and NK cells and production of IFN-g by splenic and liver NK cells. In B6 mice, PBS44 induced robust up-regulation of CD69 on T, B, NK and DCs (Figure 12A), stimulated the production of IFN-γ by ∼20-30% of splenic and liver NK cells (Figure 12C-E), as well as exhibited robust serum cytokine responses. However, most of these antigen-induced responses were markedly reduced in Adap−/− mice, indicating that ADAP is required iNKT cell- mediated immunomodulatory functions. 59 A T 28.4 No Ag + Ag B 69.1 NK 35.9 DC B B6 Adap−⁄− 56.8 B6 80 ** 9.8 17.4 18.1 29.6 Adap−⁄− 60 40 + 9 6 D C % 20 ** ** ** CD69 0 T B NK DC C 1 . 1 K N Spl B6 3.7 Adap−⁄− 1.5 D 1 . 1 K N E Liv B6 10.3 Adap−⁄− 6.0 TCRb TCRb 34.3 3.3 26.2 7.3 s l l e C K N + g - N F I IFN-g B6 Adap−⁄− + Ag IL-4 * H IFN-g B6 Adap−⁄− No Ag IFN-g * G 3500 3000 2500 2000 1500 1000 500 0 F l / m g p 3500 3000 2500 2000 1500 1000 500 0 % n o i t c u d e R % 100 90 80 70 60 50 No Ag B6 Adap−⁄− B6 Adap−⁄− + Ag ** * Spl Liv 30 20 10 0 IFN-g IL-4 Figure 12. ADAP is required for iNKT-cell mediated transactivation of immune cells. Age and sex-matched B6 and Adap−⁄− mice were injected i.p with 4µg of PBS44 (Ag) or left untreated 60 Figure 12. (cont’d) (No Ag). After 4 hours, splenocytes were analyzed for the percentage of TCRb+ (T), B220+ (B), NK1.1+ (NK), and CD11c+ (DC) cells expressing CD69. Representative histograms are shown in (A). (B) Bar graph showing % positive T, B, and NK cells and DC expressing CD69 following activation, is pooled from 2 independent experiments. Splenocytes (C) and intrahepatic lymphocytes (D) were stained with NK1.1 and TCRβ antibodies and the percentage of NK1.1+TCRβ− cells (as gated in the upper density plots) producing IFN-g directly ex vivo was determined by intracellular staining and flow cytometry. Data are representative of 2 experiments with 5-9 mice of each genotype analyzed per condition. (E) Bar graph showing % IFN-g+ NK cells in the spleen and liver are pooled from 2 independent experiments. Serum was collected and IFN-g (F) and IL-4 (H) levels were measured by ELISA. Data represent the mean ± SEM from 2 independent experiments. (H) Mean percent reduction ± SEM in the serum levels of IFN-g and IL-4 in PBS44-injected Adap−⁄− mice as compared to similarly treated B6 mice. Data are averaged from 2 independent experiments. Significance was determined by unpaired t-test. *p<0.05, **p<0.01. 61 Discussion As one of the hallmark functions of iNKT cells is the large magnitude of cytokines released upon activation, understanding the intercellular signaling requirements becomes crucial. iNKT cells possess mechanisms of both conventional T-cells and NK cells, and in regards to ADAP, it is essential for each. Previous studies have shown ADAP to be required for normal levels of IL-2 release from CD4+ T-cells[43], and IFN-g from NK cells [128]. Consistent with the role of ADAP in T & NK cells, our studies have shown that iNKT cells are critically dependent on ADAP to both produce and release cytokines upon activation as shown in Figure 9. However, our studies reveal that there was a great defect in cytokine release than cytokine production by Adap−⁄− iNKT cells, in both major Th1 and Th2 cytokines, IFN-g and IL-4, respectively. This leads us to believe that there is a greater requirement for ADAP in the functional cytokine release from iNKT cells than there is for production of cytokines. We have previously shown a decreased number of iNKT cells in the spleen and liver of Adap−⁄− mice (Chapter 1, Figure 3), thus leading to the possibility that this may be the cause of the decreased serum cytokine levels we observed. To test this possibility, we performed an in vitro cytokine release assay, as to control for the number of iNKT cells. As our findings showed significant reductions of IFN-g and IL-4 on a per cell basis, this aided to our mode of thought that ADAP is a critical requirement for iNKT cell cytokine release. Our findings in mice are recapitulated in human iNKT cells. Using equal numbers of wild type (untransfected) and ADAP gene-silenced human iNKT cells, we observed a similar dependence on ADAP for cytokine (IFN-g and IL-4) release following activation. 62 Previous studies have characterized the requirements of vesicular release of cytokines from both NK cells and T-cells. In particular, NK cells and T-cells alike are dependent on several Rab family proteins such as Rab11, GTPase required for proper vesicular trafficking and exocytosis of cytokine loaded vesicles [132]. Additionally, several classes of syntaxins, such as syntaxin 11, may be required for the docking and release of such cytokine vesicles of T-cells and NK cells for vesicular release as well [133]. Additional data from our lab shows the gene expression as determined by real-time PCR, of Rab11 GTPase is relatively decreased in Adap−⁄− iNKT cells as compared to B6, where as the syntaxin levels are unchanged. While gene expression may or may not be changed, the functionality of these proteins may be dependent on ADAP. As is important with what is termed “inside-out” signaling, the requirement of ADAP for integrin LFA-1 reorganization and proper immune synapse formation may be required for integrin dependent signaling for vesicle trafficking and release [48]. As it is yet to be studied the importance of these vesicular release proteins in iNKT cells, it is possible ADAP may be influencing these; future studies are warranted in these areas for expression of these proteins. Invariant NKT cells are known to have pre-formed mRNA for IFN-g and IL-4 to assist in the rapid translation and production upon activation. This mechanism is separate of the preformed cytokines, ready for immediate release. Thus, due to the fact we observe some release, albeit much less, of IFNg and IL4 post in vivo iNKT cell activation, it is possible ADAP is playing influence into the gene expression of these pre-formed mRNAs. These processes are thought to be regulated by a combination of several transcription factors, such as PLZF and NFkB, and ThPOK [14, 62, 64]. While also yet to be fully understood, it remains possible ADAP is regulating the amount of mRNA encoding for IFN-g and IL-4 by influencing these transcription 63 factors, thus dampening the release of each. Of note, it has been shown ThPOK expression in iNKT cells is required for development CD4+ iNKT cells as well secretion of both IFN-g and IL- 4 [64, 134]. Due to our observations of decreased CD4 iNKT cells in Adap−⁄− mice (Chapter 1 Figure 4), and decreased cytokine secretion, it is consistent with our data that ADAP is possibly influencing expression of ThPOK. More so, an upstream transcriptional regulator of ThPOK, called GATA-3, is found to act downstream of TCR signaling. Additionally, it has been shown that a lack of this transcription factor resulted in apoptosis in iNKT cells, as seem by a decreased survival in the liver [135]. As ADAP acts downstream of TCR as well, it is possible the decreased homeostasis or proliferation seen in iNKT cells in Adap−⁄− mice, is because of influence on GATA-3, however future studies in these areas are warranted. The premier role of cytokine release by iNKT cells is to recruit and assist in the activation of other bystander immune cells. Consistent with our hypothesis, decreased cytokine release from iNKT cells resulted in the decreased activation of other immune cells, as shown by CD69 expression. As ADAP is expressed in T-cells [136], NK-cells [70], and dendritic cells (DCs) [137], is possible the complete lack of ADAP in these mice is influencing the activation of these cells by cytokine dependent mechanisms. However, as we observed a significant decrease in B- cell activation, this can more accurately explain the inherent defect in iNKT transactivation ability, as B-cells do not express ADAP [138]. More so, as iNKT cells can signal for differentiation of DCs into a mature state through cytokine dependent mechanisms, iNKT cell cytokine release is further required for immune regulation [139]. Additionally, NK cells depend on ADAP for only the release of Th1 cytokines, we can confer the decreased serum release of IFN-g in Adap−⁄− mice was dependent on both NK cells and iNKT cells. More so, the defect in 64 secretion of IL-4 in the serum must be from iNKT cells alone, as no other immune cells will produce Th2 cytokines within 4 hours of aGC administration. Taken together, we find ADAP to be essential in the release and to a lesser magnitude production of both Th1 and Th2 cytokines by iNKT cells. As this is the primary method of bridging the adaptive and innate immune systems, a lack of this function emphasizes importance of ADAP in iNKT cell immune functions. We have shown the requirement of ADAP in the transactivation of bystander immune cells, by iNKT cell activation alone, highlighting a critical role for this molecule in regulation of host immunity in several disease states. 65 CHAPTER 3 ADAP IS INDISPENSABLE FOR INVARIANT NATURAL KILLER T CELL ANTI-TUMOR RESPONSES 66 Introduction Previous studies have shown that iNKT cells aid in protection from tumors, through multiple mechanisms. Heterozygous mutations in the well-studied tumor suppressor p53, results in increased tumor incidence, the susceptibility to which increases in the absence of iNKT cells [140]. Similarly, carcinogen-induced tumor incidence was much greater in mice lacking iNKT cells (Cd1d−⁄−) as compared to mice with normal iNKT cells [141]. On the contrary, reincorporation of wild-type iNKT cells into iNKT deficient mice halted growth of the carcinogen-induced tumors [141]. In addition, activation of endogenous, wild-type iNKT cells by injection of aGC alone decreased the growth of spontaneously-induced [142] or carcinogen- induced tumors [143]dependent on release of IFN-g. In human models, cytotoxic iNKT cells were able to enhance the cytotoxic T-cell lysis of Epstein-Barr Virus-positive (EBV+) human tumors by means of IFN-g release [144]. Taken together, iNKT cells appear to play a vital role in the control of tumor burden in both mice and humans. iNKT cells are an enticing cell type for means of tumor control, as they possess multiple ways of inducing anti-tumor effects. In particular, iNKTs main mode of killing involves the recruitment and activation of other cytolytic immune cells, such as natural killer cells (NK cells) and CD8+ T-cells (cytotoxic T-cells), by means of release of IFNg and expression of the co-stimulatory molecule CD40 [145, 146]. These mechanisms result in dendritic cell (DC) activation, a professional antigen presenting cell for iNKT cells, which assist in the responses of both cytotoxic and helper T-cells [141], by IL-12 release [141]. These methods of killing are termed indirect iNKT tumor control (Figure 13A). However, iNKT cells also possess the mechanisms of direct tumor lysis (Figure 13B). In particular, upon iNKT cell activation by TCR interaction 67 with a glycolipid presented by CD1d, iNKTs will upregulate and release cytolytic molecules such as perforin and Granzyme B [147, 148]. In addition, they upregulate and localize the surface death receptors Fas ligand and TRAIL, of which induce apoptosis in the target cell when bound [149]. Although it is known that the iNKT cytolytic functional responses are highly dependent on CD1d-glycolipid antigen presentation interaction with a functional TCR, the signaling mechanisms that regulate these cytotoxic responses are not completely understood. However, studies have shown both the protein SAP and Fyn, which interact with each other downstream of SLAM family receptors upon iNKT cell activation, are essential in the cytotoxic functional response of iNKT cell [68]. In this study, we examined the role of ADAP (adhesion and degranulating-promoting adaptor protein) in iNKT cell cytotoxicity, as it is a known binding partner of Fyn. These studies are important as better characterization of the intracellular signaling for optimal activation of iNKT cells will lend greater understanding of the use of iNKT cells in tumor control and prevention. To understand the role of ADAP in iNKT cytotoxic functions, we sort-purified iNKT cells from B6 and Adap−⁄− mice and tested their ability to target EL4 T lymphoma cells both in vitro and in vivo. Our results demonstrate that ADAP is required for murine iNKT cell cytotoxicity in vitro as well as iNKT cell-mediated control of tumor growth in vivo. 68 A IL-12 Activation DC αGC NKT CD8 T IL-12 IFN-γ CD40 stimulation NK Tumor Tumor cell Death B NKT Direct Cytotoxicity Fas L Fas Glycolipid Ag Perforin Direct Cytotoxicity Tumor Tumor cell Death Figure 13. Anti-tumor mechanisms of iNKT cells. (A) Indirect mechanism of iNKT cell cytotoxicity. The cross talk between iNKT cells and antigen presenting cells (such as DCs), presenting a tumor-derived glycolipid, leads to the activation of iNKT cells, IFN-g release and CD40 stimulation. This iNKT cell-derived IFN-g induces DC production of IL-12, which further augments IFN-g production by iNKT and NK cells, and serves to stimulate CD8+ T cell- and NK cell-dependent killing of tumor cells. (B) iNKT cells recognize glycolipid antigens presented by CD1d on tumor cells and mount direct cytotoxicity via perforin/granzyme exocytosis or Fas-Fas ligand (Fas L) interactions. 69 Materials and Methods Mice Mice were used as described in Chapter 1, with the addition of NOD.CgPrkdcscid Il2rgtm1Wjl/SzJ (NSG) mice, which were purchased from Jackson Laboratories (Bar Harbor, ME), and bred at MSU animal facility. All Animal studies were approved by the Institutional Animal Care and Use Committee (IACUC), MSU. Reagents and Antibodies Reagents and antibodies used are as described in Chapters 1 & 2, with the addition of Granzyme-B and Thy1.2 (BioLegend, San Diego, CA); Perforin, Fas L, and TRAIL (BD Bioscience, San Diego, CA). Luciferase-expressing EL4 (EL4-Luc) was purchased from Caliper Life Sciences (Hopkinton, MA). EL4-Luc cells were thoroughly tested and certified to be pathogen-free prior to injection into NSG mice. In vitro Cytotoxicity assay Invariant NKT cell cytotoxicity was evaluated by 51Cr-release assay. EL4 cells were used as target cells (T, 1x 106) were labeled with 100µCi 51Cr (Na2CrO4; Perkin Elmer; Waltham, MA) for 1-2 h at 37°C and then washed extensively. 51Cr-labeled targets were either loaded with PBS44 (100ng/mL) or left untreated; then co-cultured in triplicate with effector cells (E) at varying E:T ratios. After 16 h, supernatants were sampled (experimental counts per minute [CPM]) and applied to LumaPlates (Perkin Elmer). Cultures of target cells alone were assayed in a similar fashion to determine spontaneous release (Spont CPM). Total CPM was assessed by 70 complete lysis of target cells by using 0.1% IGEPAL (Sigma Aldrich) in water. Percent specific lysis was calculated using the following formula: % Specific lysis = 100 x [Experimental CPM – Spont CPM]/[Total CPM – Spont CPM] Human iNKT cell expansion, purification and gene silencing As described in Chapter 1 and 2. In Vivo tumor model Age-matched NSG mice received purified iNKT (4x105) cells from either B6 or Adap−⁄− animals via tail vein injection. Animals in control group received no iNKT cells. Three days later (Day 0), mice in all the groups were challenged with PBS44-loaded EL4-Luc cells (1x105). Tumor dissemination was visualized by bioluminescence imaging (BLI) at different time points post tumor cell injection. Two weeks later, mice were sacrificed; livers and spleens were removed and weighed. Tumor clearance was evaluated using ex vivo BLI, flow cytometry and histology. Mice were euthanized during the study if they developed hind-limb paralysis. In vivo and ex vivo BLI For in vivo BLI, mice were given an i.p injection of D-luciferin (Caliper Life Sciences; 150 mg/kg body weight) and then anesthetized with isoflurane gas using a Xenogen XGI Gas Anesthesia System. Ten minutes after injection of the substrate, mice were imaged in groups using the IVIS Imaging system (Xenogen; Baltimore, MD). To image specific tissues for tumor involvement, mice were injected with an additional dose of luciferin (50 mg/kg body weight). Five minutes later, animals were killed; spleens and liver were harvested and imaged. Tissue 71 processing and imaging were conducted within a period of 3 minutes to minimize signal decay. For both in vivo and ex vivo BLI, data were collected until either the mid-range of the linear scale or the maximal exposure settings were achieved (f stop 1, large binning and 120 s). Imaging data were analyzed and quantified with Living Image v3.2 Software (Xenogen). Histologic analysis Representative samples of liver were obtained from all the animals in each cohort, washed in PBS and fixed in 10% formalin. Samples were then embedded in paraffin, cut into 5-µm-thick sections and stained with hematoxylin and eosin (H&E). Digital images of sections were obtained using a Nikon Eclipse 50i (Nikon, Minato, Tokyo, Japan) and INFINITY-3, using INFINITY ANALYZE 6.5.4 software (Lumenera Corporation, Ottawa, Ontario, Canada). Statistics Student’s t-test and one-way or two-way ANOVA with Tukey’s post-hoc test were used as indicated. Significance shown as *(p<0.05), **(p<0.01), or ns for non-significant values. All statistical data was determined using GraphPad Prism (GraphPad, San Diego, CA). 72 Results ADAP is required for optimal TCR-induced iNKT cell cytotoxicity in vitro. The SAP and Fyn axis is required for robust murine and human iNKT cell cytotoxicity and this requirement is conversed among other cytotoxic effectors, including T as well as NK cells. However, ADAP-deficient NK cells exhibit no cytotoxic defects, and the role of ADAP in CD8+ T cells remains undefined. To address whether ADAP regulates iNKT cell cytotoxicity, we evaluated the expression of death promoting proteins in B6 and Adap−⁄− iNKTs and observed that they expressed comparable levels of perforin, granzyme B, FasL and TRAIL-R (Figure 14). Next, we assessed the killing ability of iNKT cells sort-purified from the livers of B6 and Adap−⁄− mice. iNKT cells were placed in culture with 51Cr-labeled EL4 T-lymphoma cells that had been loaded with OCH (weak antigen, Figure 15A) or PBS44 (strong agonist, Figure 15B) or left unloaded. Regardless of the antigen used, B6 iNKT cells exhibited potent killing of OCH or PBS44 loaded but not unloaded EL4 cells (Figure 15A, B). In contrast, Adap−⁄− iNKT cells exhibited diminished killing, that was 46 ± 9% (OCH) and 52 ± 10% (PBS44) less than the activity of B6 cells (E:T ratio 20:1) (Figure 15C). Together, these studies reveal that ADAP is required for optimal in vitro TCR-induced iNKT cell cytotoxic responses. 73 35.7 21.2 30.2 5.7 B6 35.4 21.5 25.5 4.8 Adap-/- Perforin Granzyme B FasL Isotype Sample TRAIL-R A B T K N i e v i t i s o P % C 300 200 100 0 I F M Perforin 50 40 30 20 10 0 B6 Granzyme B 25 20 15 10 5 0 150 100 50 0 TRAIL-R Adap-/- FasL 40 30 20 10 0 300 200 100 0 8 6 4 2 0 150 100 50 0 Figure 14. Basal expression of death receptors and lytic molecules are not altered in Adap−⁄− iNKT cells. (A) Splenocytes from B6 and Adap−⁄− mice (gated on CD1d-tetramer+TCRb+iNKT cells) were analyzed for surface expression of FasL (CD95L) and TRAIL receptor-R2. Cells were also fixed, permeabilized and stained for intracellular levels of perforin and granzyme B. Representative flow cytometric data from 1 of 2 independent experiments is shown. (B) Percent 74 Figure 14. (cont’d) % iNKT cells and (C) MFI for the lytic molecules and death receptors are shown as indicated. Data is presented as mean ± SD from 1 of 2 independent experiments (n=5-7 per genotype). 75 A 10.0 s i s y L c i f i c e p S % 7.5 5.0 2.5 0.0 B s i s y L c i f i c e p S % 25 20 15 10 5 0 ** * B6, OCH B6 +OCH ADAP KO, OCH Adap-/- B6, No Ag B6 No Ag ADAP KO, No Ag Adap-/- 1.25 2.5 5 10 20 Effector:Target B6 +Ag B6 +Ag ADAP KO + Ag Adap-/- B6 No Ag B6 No Ag ADAP KO No Ag ** Adap-/- ** ** * 1.25 2.5 5 10 20 Effector:Target C n o i t c u d e R % 80 60 40 20 0 5 2.5 Effector:Target 10 20 Figure 15. Adap−⁄− iNKT cells exhibit defective killing of antigen-pulsed target cells in vitro. EL4 target cells were pulsed with 100 ng/ml of antigen: OCH (A) or PBS44 (B) or left untreated. Cytolysis of OCH-loaded (A) or PBS44 (B) or –unloaded EL4 cells by B6 or Adap−⁄− iNKT cells. (C) Mean percent reduction ± SEM in cytolysis of PBS44-loaded EL4 cells by Adap−⁄− iNKT cells compared to B6 iNKT cells. Data in A is from 1 representative experiment and data in B, C are averaged from 5 independent experiments. Statistical significance was determined by two-way ANOVA test. * p < 0.05, ** p < 0.01 76 ADAP is required for iNKT cell-mediated control of tumor growth in vivo. Having observed that ADAP was required for optimal in vitro anti-tumor cytotoxic responses, we next sought to address whether it was also required for iNKT cell-mediated tumor clearance in vivo. For these studies, immunodeficient NOD-SCID IL-2Rg–/–(NSG) mice were reconstituted or not with purified B6 or Adap−⁄− iNKT cells and 3 days later challenged with PBS44-loaded luciferase-expressing EL4 cells (EL4-LUC). Tumor dissemination was monitored by bioluminescence imaging (BLI) and tumor cell burden and iNKT reconstitution by flow cytometric and histological analyses 3 weeks after tumor cell injection. Compared to mice that received no iNKT cells, animals receiving B6 iNKT cells exhibited a significantly lower tumor burden as determined by lower live animal radiance (Figure 16A, B), and reduced radiance (Figure 16C, D) and weights (Figure 16E) of tumor-bearing organs. Similarly, flow cytometric analyses of B6 iNKT cell-reconstituted mice demonstrated a lower number of tumor cells in the liver and spleen (Figure 16F-I) and histological analyses showed very few if any tumor cell aggregates (Figure 16L). Consistent with their in vitro cytolytic defects, Adap−⁄− iNKT cells exhibited poorer control of EL4 cell growth in vivo compared to B6 iNKT cells (Figure 16A-I, L). Of note, there was no significant difference in the number of B6 or Adap−⁄− iNKT cells recovered from the organs of tumor-bearing mice (Figure 16J, K). Thus, the suboptimal in vivo clearance of tumor is not due to the inability of Adap−⁄− iNKTs to traffic to the tumor site, but instead due to their impaired cytotoxic capacity. 77 / ] s p [ x u F l l a t o T 1.5×1010 1.0×1010 5.0×109 0.0 AP30+AP35 4 14 Days 18 20 1.5 1.0 0.5 No iNKT No NKT B6 NKT B6 ADAP KO NKT Adap−⁄− * 14 4 20 Days Post Injection 18 A C D No iNKT B6 Adap−⁄− B D4 D14 D18 D20 Spleen Liver Lung Bone ) 0 1 0 1 x ( ] s / p [ x u F l l a t o T No iNKT B6 Adap−⁄− Spleen Liver 10 8 6 4 2 8 6 4 2 Lung ) 8 0 1 x ( ] s p [ x u F l / l a t o T ) 8 0 1 x ( ] s p [ x u F l / l a t o T No iNKT B6 iNKT Adap−⁄− iNKT Bone 8 6 4 2 4 3 2 1 Figure 16. Adap-deficient iNKT cells exhibit defective in vivo control of antigen-pulsed EL4 cells. NSG mice were reconstituted or not (No iNKT cells) with 4x105 iNKT cells (B6 or Adap−⁄−). Three days later, mice were challenged with 1x105 EL4-Luc cells. (A) Whole body 78 Figure 16. (cont’d) distribution of EL4-Luc cells over the first 3 weeks using in vivo BLI. (B) Quantification of BLI images at serial time points. Data points are average radiance emitted from 4 mice per group. (C) Total radiance of spleen, liver, lung and bones from a representative mouse from each group is shown. Organ (D) ex vivo BLI, (E) weights and (F) total cell counts are shown (spleens and livers). (G) Gating strategy for EL4 and iNKT cell analysis from the liver is shown. (H-K) Percent (H, J) and absolute number (I, K) of EL4 (H, I) and iNKT cells (J, K) was determined in the spleens and livers of mice by flow cytometry. (L) Tumor burden in the liver was assessed histologically. Black arrows indicate tumor aggregates. Data are pooled from 2 experiments with 3-4 mice in each cohort and error bars represent SEM. Statistical significance was determined by two-way ANOVA. * p < 0.05, ** p < 0.01 79 Figure 16. (cont’d) 400 300 200 100 E ) g m i ( t h g e W n a g r O F ) 6 ) 0 6 1 0 ( 1 x s ( t n s u t n o u C o C l l l l e C a t o T l e C G C S S 0 80 60 40 20 0 No NKT 87.7 40.3 FSC No iNKT B6 iNKT Adap−⁄− iNKT Liv Liver Liv Spl Spleen Spl 2000 1800 1600 1400 30 20 10 ) 6 0 1 x ( s t n u o C l l e C ADAP NKT B6 NKT 0 No NKT ADAP NKT B6 NKT 28.1 66.0 99.2 C S S TCRb CD1d-tet b R C T 65.2 CD1d-tet 18.4 Thy1.2 80 Figure 16. (cont’d) H 100 Spl 100 Liv No iNKT B6 iNKT Adap−⁄− iNKT 4 L E % ) 6 0 1 x ( s l l e C 4 L E T K N i % I J K ) 3 0 1 x ( s l l e C T K N i 95 90 85 80 15 10 5 0 40 30 20 10 0 20 15 10 5 0 Spl Spl Spl 95 90 85 80 15 10 5 0 60 40 20 0 600 400 200 0 81 Liv Liv * No iNKT B6 iNKT Adap−⁄− iNKT Liv Figure 16. (cont’d) L No iNKT B6 iNKT Adap−⁄− iNKT 82 Table 4. Total flux from ex vivo organ BLI Total Flux [p/s](x108) Spleen Liver Lung Bone No iNKT B6 iNKT Adap-/- iNKT 8.19±1.26 4.91±1.11 5.15±0.96 2.62±1.06 1.0±0.43 1.11±0.47 0.95±0.35 0.03±0.01 5.53±2.46 2.26±0.56 3.08±1.09 0.32±0.12 Table 5. Frequency of EL4 and iNKT cells in various organs ex vivo No iNKT B6 iNKT Frequency Spl Liv Spl Liv Adap−⁄− iNKT Spl Liv EL4 iNKT * p < 0.05 96.1±0.58 93.5±0.1 2.14±0.18 5.94±1.51 88.81±2.1 23.7±6.9 84.5±3.5 45.3±7.86 90.3±2.3 91.15±1.3 8.35±2.2 19.0±4.58* Table 6. Absolute cell number of EL4 and iNKT cells in various organs ex vivo Absolute Cell No iNKT B6 iNKT Adap−⁄− iNKT Liv Spl Number Spl Liv Spl Liv EL4 (x106) 12.8 ±2.4 8.01±2.3 iNKT (x103) 2.6±0.78 65.4±28.9 4.0±1.0 2.95±0.88 2.6±1.19 5.1±1.23 12.95±5.0 346.3±132.6 6.8±3.34 118.4±36. 1 83 Discussion Lymphocyte signaling cascades responsible for anti-tumor cytotoxicity and inflammatory cytokine production must be tightly regulated in order to control an immune response. Disruption of these cascades can cause immune suppression as seen in a tumor microenvironment, and loss of signaling integrity can lead to autoimmunity and other forms of host-tissue damage. Therefore, understanding the distinct signaling events that exclusively control specific effector functions of “killer” lymphocytes (T and NK cells) is critical for understanding disease progression and formulating successful immunotherapy. Elucidation of divergent signaling pathways involved in receptor-mediated activation has provided insights into the independent regulation of cytotoxicity and cytokine production in lymphocytes. Specifically, the Fyn signaling axis represents a branch point for killer cell effector functions and provides a model for how cytotoxicity and cytokine production are differentially regulated. While the Fyn–PI(3)K pathway controls multiple functions, including cytotoxicity, cell development, and cytokine production, the Fyn–ADAP pathway preferentially regulates cytokine production in NK and T cells. In this study, we provide evidence that unlike in T or NK cells, ADAP regulates both cytokine as well as cytotoxic responses of iNKT cells. The primed nature of iNKT cells in addition to their multiple mechanisms of cytolysis make them prime candidates for the immune surveillance of human tumors. While this is true, the intracellular signaling requirements of these cells are less well known. In regards to ADAP, we found it to be required for direct cytolysis by murine iNKT cells. This is consistent with our previous studies regarding SAP and Fyn, as a lack of either protein significantly reduces target cell lysis of iNKT cells [68]. As ADAP binds to Fyn, this extends 84 our previous understanding of the signaling pathways for iNKT cell cytolysis. Although SAP and ADAP differentially regulate iNKT cell development, their requirement for iNKT cell cytolytic activity might be similar or overlapping. Both SAP and ADAP are required for stable conjugate formation between T cell and the antigen-presenting cell [150]. SAP has been shown to be required in the formation of a functional lytic synapse, which is requires actin polymerization and movement of the microtubule organization center at the site of contact [68]. Interestingly, ADAP links TCR signaling to reorganization of the actin cytoskeleton. As seen in T-cells upon activation, ADAP engages with Evl of the Ena/VASP family of proteins, WASP (Wiskott- Aldrich syndrome protein), and the Arp2/3 complex, inducing actin rearrangement, which may play a role in TCR clustering and signal strength [151, 152]. In our studies, we observed an inherent defect in cytotoxicity by Adap−⁄− iNKT cells, which was independent of TCR signal strength. Consistent with this notion, Adap−⁄− iNKT cells failed to mount a robust cytotoxic response both in the presence of a weak or strong agonist like OCH (truncated version of aGC) and aGC respectively. ADAP is unique in that it has binding partners of two separate pathways; ADAP binds to Fyn downstream of SLAM family receptors [68, 86] and to phosphorylated Slp76 downstream of the TCR [153]. As Fyn is required for iNKT cell cytotoxicity upon TCR engagement, and Slp76 is in NK cells [154], it leads us to believe ADAP expression may be a converging point of each pathway. Previous studies reveal that in vitro and in vivo lysis of EL4 T cell lymphoma relies on iNKT cell expression of perforin where as Fas/FasL pathway is required for iNKT cell-mediated control of CD1d-transfected A20 B-lymphoma cells. However, A20-CD1d cells express significantly higher levels of CD1d and Fas compared to EL4s [155, 156], and thus they may induce more 85 FasL upregulation on iNKT cells and/or be more susceptible to FasL-mediated apoptosis. Loss of perforin expression does not fully abolish EL4 lysis in vitro, while loss of both perforin and FasL does. Therefore perforin and FasL both contribute to iNKT cell cytotoxicity. Taken together, results from these studies imply that the mechanism of iNKT cell-mediated tumor control may be modulated by several variables, including the levels of cytotoxic proteins and ligands expressed by the iNKT cells, the apoptosis-inducing receptors borne by the targets, and other external factors that influence iNKT:target cell interactions. We observed comparable levels of both cytotoxic molecules (perforin and granzyme) and death-promoting receptors (FasL and TRAIL) on B6 and Adap−⁄− iNKT cells, suggesting that the cytolytic defect exhibited by and Adap−⁄− is independent of the levels of cytotoxic proteins. Our most clinically relevant finding was that ADAP is required for the tumor protective properties of iNKT cells in vivo. We demonstrate that ADAP expression in iNKT cells is required for direct EL4 tumor control. This was apparent by increased total body as well as organ specific BLI in animals reconstituted with Adap−⁄− iNKT cells. While monitoring expansion of the tumor by live animal imaging, allowed us to monitor growth over time at a macroscale, FACS analysis allowed us to quantitate EL4 growth in the spleen and liver. Histological assessment of liver also showed a greater visualized infiltration (black arrow) of EL4 cells (dark purple) near the blood vessels of the organ. As the EL4 cell line is a T-cell lymphoma, it does not commonly form solid tumors, but will greatly accumulate around highly vascularized areas, especially in a T-cell deficient niche as in the NSG liver. Consistent with our hypothesis, we observed a decrease in the number of reconstituted Adap−⁄−iNKT cells in both the spleen and liver as compared to B6. This leads us to further believe that ADAP may be required for the 86 peripheral maintenance or proliferation of iNKT cells, as described in Chapter 1. Taken all together, we conclude ADAP is required in iNKT cells for direct cytolysis and tumor immuneosurveillance in vivo. Understanding iNKT cell signaling becomes of vital importance when trying to modulate or adapt them for tumor regulation. Comparative analysis of certain tumor types, in particular breast cancer, have found correlatively lower metastasis rates with CD1d expressing tumors [157], indicating an tumor control role of iNKT cells. More so than just direct tumor lysis, iNKT cells can inhibit tumor growth by other mechanisms. Particularly, iNKT cells can kill both myeloid-derived suppressor cells (MDSCs) [158] and tumor associated macrophages (TAMs) [39], both of which can skew the tumor microenvironment to an immunosuppressive state, and promote tumor growth. Future studies are warranted as to the role ADAP plays in these functions, however our findings lead us to believe it to be important in each. More so, as it has been studied in cytotoxic CD8+ T-cells, deficiencies in ADAP resulted in a decrease in PD-1 expression, and thus enhanced the anti-tumor response by the T-cells [159]. Future studies understanding how ADAP can be influencing iNKT cell tumor protection will become vital in clinical immunotherapies. In conclusion, we found ADAP in iNKT to be required in the direct cytotoxicity to tumor targets, as seen in our in vitro models. Given that ADAP is dispensable for NK cell cytotoxicity and has no known role in T cell anti-tumor response, our studies highlight a lineage –specific role for ADAP in iNKT cell cytotoxicity. 87 OVERALL DISCUSSION 88 The application of iNKT cells have been implicated in clinical settings for protection for various cancer types. It has been observed that greater incidences of iNKT cells have correlated with decreased rates of tumor growth and metastasis in various solid tumors such as colon [32], lung [33], head and neck[160], and neuroblastomas [161]. Additionally, as thought be a protective mechanism of the tumor by deterring immuneosurveillance, iNKT cells are shown to be greatly reduced in circulation independent of tumor type or load [162]. It is common for tumors to influence the tumor microenvironment (TME) to an immunosuppressive state, the ability of iNKT cells to secrete Th1 cytokines assists in the recruitment and activation of other immune cells for attack against a tumor. Additionally, iNKT cells can directly kill CD1d positive tumors or tumor-associated macrophages (TAM), which assist in the immunosuppressive TME [39]. Due to the nature of Th1 and Th2 cytokine release by iNKT cells, understanding how to influence these responses by regulating the signaling proteins downstream of TCR activation proves crucial in future cancer immunotherapies. In particular to our study, we found ADAP to required for both the release of cytokines, and cytotoxic functions of iNKT cells, thus highlighting its importance in the intracellular signaling requirements of iNKT cells. While we observe these functional defects, it is yet to be fully determined the mechanisms of which ADAP is regulating. However, it is likely that ADAP is regulating the functional immune synapse in iNKT cells, as was previously shown in T-cells. T- cell conjugation with an antigen presenting cell was shown to be dependent on ADAP co- localization with integrin LFA-1 and supramolecular activation cluster (SMAC) [163]. While these ADAP dependent mechanisms could be regulating iNKT cell functions, it is likely multi- faceted, as ADAP functions downstream of multiple signaling pathways. In particular, ADAP 89 binds to Slp-76 downstream of TCR [151], and to Fyn downstream of SLAM-R[68], and possibly cytokine receptor signaling. This highlights ADAP as a possible convergence point, of multiple pathways, thus better characterizing its role in iNKT cell function. ADAP serves as a binding protein, and other binding interactions downstream may be the cause of its functional dependencies. In particular, a protein termed SKAP55 (src kinase-associated phosphoprotein of 55-kD), known to constitutively bind to ADAP in T-cells, is specifically required for LFA-1 clustering [164, 165]. More so, ADAP is required to prevent degradation of SKAP55 [166]. Preliminary studies in our lab have found similar findings in iNKT cells; diminished SKAP55 expression in Adap−⁄−mice. While not yet fully understood, this data leads us to believe that SKAP55 may be a functional regulatory protein in iNKT cells a well. A protein with nearly identical structure to SKAP55, called SKAP55R, is thought to play similar roles as SKAP55 [165]. Preliminary studies from our lab reveal that SKAP55R have greater basal expression than SKAP55 in iNKT cells, and more so SKAP55R expression increases upon polyclonal TCR activation, while SKAP55 levels are unchanged. Whether SKAP55 and/or SKAP55R preferentially bind to ADAP following stimulation to regulate iNKT cell functions remain to be determined. A structural homologue of ADAP that is thought to play a role in integrin-mediated signaling cascade for integrin-dependent is termed PRAM (PML-retinoic acid receptor alpha regulated adaptor molecule 1) [167, 168]. PRAM shares multiple similar binding partners with ADAP, including Slp-76, Lyn (structurally similar to Fyn) and SKAP55R (structurally similar to SKAP55)[167, 168]. While similar in many ways to ADAP, PRAM differs in its expression within cell types, as PRAM is most commonly expressed in myeloid immune cell lineages such 90 as neutrophils, whereas ADAP is more commonly expressed in lymphoid cells. However, is interesting to note that while iNKT cells express PRAM, basal expression of ADAP is 4-5 fold higher in Pram−⁄− mice. Furthermore, our preliminary analysis demonstrates that Pram−⁄− iNKT cells to have greater antigen-induced functional response, as seen by increased direct specific lysis and cytokine release in vitro. Given that both ADAP and PRAM can bind to Slp-76 as well as SKAP55 and/or SKAP55R, it is likely that ADAP and PRAM compete for binding with either of these molecules and thereby regulate the functions of each other. Alternatively, it is also possible that ADAP and PRAM function co-operatively to promote iNKT cells functions. Studies are currently underway to gain mechanistic insights into how ADAP regulates iNKT cell homeostatic maintenance, cytokine production and release as well as has the ability to mount robust target cell lysis both in vitro and in vivo. Cancer immunotherapy is aimed at harnessing the power and specificity of the immune system in the treatment of specific malignancies. Based on their combined ability to kill tumor cells and promote the anti-tumor functions of NK and CD8+ T cells, iNKT cells represent an excellent candidate for incorporation into novel immunotherapeutic trials for cancer. Indeed, several clinical trials demonstrate that iNKT cell therapies are well tolerated, transiently activate the human immune system, and promote objective yet partial tumor responses. Although encouraging, there is need for improvement. At present, the challenge lies in better understanding how iNKT cells recognize and respond to tumors and developing more effective methods to promote their anti-tumor functions. By further defining the role of ADAP in murine and human iNKT cell functions, these studies will provide new and important insights into the signals controlling iNKT interaction with, and responses to, normal, malignant and/or infected cells. 91 These studies will have an impact by establishing new paradigms for iNKT cell signaling and offering insights into how iNKTs can be best activated to enhance host immunity and treat cancer. 92 BIBLIOGRAPHY 93 BIBLIOGRAPHY Pellicci, D.G., et al., A natural killer T (NKT) cell developmental pathway iInvolving a thymus-dependent NK1.1(-)CD4(+) CD1d-dependent precursor stage. J Exp Med, 2002. 195(7): p. 835-44. 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. McNab, F.W., et al., The influence of CD1d in postselection NKT cell maturation and homeostasis. J Immunol, 2005. 175(6): p. 3762-8. Li, Y., et al., CD4+ natural killer T cells potently augment aortic root atherosclerosis by perforin- and granzyme B-dependent cytotoxicity. Circ Res, 2015. 116(2): p. 245-54. Ballas, Z.K. and W. Rasmussen, NK1.1+ thymocytes. Adult murine CD4-, CD8- thymocytes contain an NK1.1+, CD3+, CD5hi, CD44hi, TCR-V beta 8+ subset. J Immunol, 1990. 145(4): p. 1039-45. Yoshimoto, T. and W.E. Paul, CD4pos, NK1.1pos T cells promptly produce interleukin 4 in response to in vivo challenge with anti-CD3. J Exp Med, 1994. 179(4): p. 1285-95. Mars, L.T., et al., Invariant NKT cells inhibit development of the Th17 lineage. Proc Natl Acad Sci U S A, 2009. 106(15): p. 6238-43. 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. Park, H., et al., A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat Immunol, 2005. 6(11): p. 1133-41. Harrington, L.E., et al., Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol, 2005. 6(11): p. 1123-32. Godfrey, D.I., S. Stankovic, and A.G. Baxter, Raising the NKT cell family. Nat Immunol, 2010. 11(3): p. 197-206. Imai, K., et al., Sequence and expression of transcripts of the T-cell antigen receptor alpha-chain gene in a functional, antigen-specific suppressor-T-cell hybridoma. Proc Natl Acad Sci U S A, 1986. 83(22): p. 8708-12. Savage, A.K., et al., The transcription factor PLZF directs the effector program of the NKT cell lineage. Immunity, 2008. 29(3): p. 391-403. 94 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 18. 20. 23. 25. 26. 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. 19. Marsh, R.A., et al., Patients with X-linked lymphoproliferative disease due to BIRC4 mutation have normal invariant natural killer T-cell populations. Clin Immunol, 2009. 132(1): p. 116-23. 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. 21. Mattner, J., et al., Exogenous and endogenous glycolipid antigens activate NKT cells during microbial infections. Nature, 2005. 434(7032): p. 525-9. 22. Mallevaey, T., et al., Activation of invariant NKT cells by the helminth parasite schistosoma mansoni. J Immunol, 2006. 176(4): p. 2476-85. Ilyinskii, P.O., et al., CD1d mediates T-cell-dependent resistance to secondary infection with encephalomyocarditis virus (EMCV) in vitro and immune response to EMCV infection in vivo. J Virol, 2006. 80(14): p. 7146-58. 24. Wilson, S.B., et al., Extreme Th1 bias of invariant V alpha 24J alpha Q T cells in type 1 diabetes. Nature, 1998. 391(6663): p. 177-181. 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. Kawano, T., et al., CD1d-restricted and TCR-mediated activation of valpha14 NKT cells by glycosylceramides. Science, 1997. 278(5343): p. 1626-9. Benlagha, K., et al., In vivo identification of glycolipid antigen-specific T cells using fluorescent CD1d tetramers. J Exp Med, 2000. 191(11): p. 1895-903. 17. Matsuda, J.L., et al., Tracking the response of natural killer T cells to a glycolipid antigen using CD1d tetramers. J Exp Med, 2000. 192(5): p. 741-54. Kukreja, A., et al., Multiple immuno-regulatory defects in type-1 diabetes. Journal of Clinical Investigation, 2002. 109(1): p. 131-140. Van Kaer, L., L. Wu, and V.V. Parekh, Natural killer T cells in multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Immunology, 2015. 146(1): p. 1-10. 95 Kojo, S., et al., Dysfunction of T cell receptor AV24AJ18+,BV11+double-negative regulatory natural killer T cells in autoimmune diseases. Arthritis and Rheumatism, 2001. 44(5): p. 1127-1138. 28. 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. Terabe, M., et al., A nonclassical non-Valpha14Jalpha18 CD1d-restricted (type II) NKT cell is sufficient for down-regulation of tumor immunosurveillance. J Exp Med, 2005. 202(12): p. 1627-33. 37. Wingender, G., et al., Antigen-specific cytotoxicity by invariant NKT cells in vivo is CD95/CD178-dependent and is correlated with antigenic potency. J Immunol, 2010. 185(5): p. 2721-9. 38. Metelitsa, L.S., et al., Human NKT cells mediate antitumor cytotoxicity directly by recognizing target cell CD1d with bound ligand or indirectly by producing IL-2 to activate NK cells. J Immunol, 2001. 167(6): p. 3114-22. Song, L., et al., Valpha24-invariant NKT cells mediate antitumor activity via killing of tumor-associated macrophages. J Clin Invest, 2009. 119(6): p. 1524-36. 96 Knothe, S., et al., The NKT cell ligand alphagalactosylceramide suppresses allergic airway inflammation by induction of a Th1 response. Vaccine, 2011. 29(25): p. 4249-55. Nagaraj, S., et al., Dendritic cells pulsed with alpha-galactosylceramide induce anti- tumor immunity against pancreatic cancer in vivo. Int Immunol, 2006. 18(8): p. 1279-83. Parekh, V.V., et al., Glycolipid antigen induces long-term natural killer T cell anergy in mice. J Clin Invest, 2005. 115(9): p. 2572-83. Tachibana, T., et al., Increased intratumor Valpha24-positive natural killer T cells: a prognostic factor for primary colorectal carcinomas. Clin Cancer Res, 2005. 11(20): p. 7322-7. 33. Motohashi, S., et al., Anti-tumor immune responses induced by iNKT cell-based immunotherapy for lung cancer and head and neck cancer. Clin Immunol, 2011. 140(2): p. 167-76. Chang, D.H., et al., Inflammation-associated lysophospholipids as ligands for CD1d- restricted T cells in human cancer. Blood, 2008. 112(4): p. 1308-16. Dhodapkar, M.V., et al., A reversible defect in natural killer T cell function characterizes the progression of premalignant to malignant multiple myeloma. J Exp Med, 2003. 197(12): p. 1667-76. 27. 29. 30. 31. 32. 34. 35. 36. 39. 40. Terabe, M. and J.A. Berzofsky, The immunoregulatory role of type I and type II NKT cells in cancer and other diseases. Cancer Immunol Immunother, 2014. 63(3): p. 199- 213. 41. Myung, P.S., et al., Differential requirement for SLP-76 domains in T cell development and function. Immunity, 2001. 15(6): p. 1011-26. da Silva, A.J., et al., Cloning of a novel T-cell protein FYB that binds FYN and SH2- domain-containing leukocyte protein 76 and modulates interleukin 2 production. Proc Natl Acad Sci U S A, 1997. 94(14): p. 7493-8. Griffiths, E.K., et al., Positive regulation of T cell activation and integrin adhesion by the adapter Fyb/Slap. Science, 2001. 293(5538): p. 2260-3. da Silva, A.J., et al., Biochemical analysis of p120/130: a protein-tyrosine kinase substrate restricted to T and myeloid cells. J Immunol, 1997. 158(5): p. 2007-16. Huang, Y., et al., Deficiency of ADAP/Fyb/SLAP-130 destabilizes SKAP55 in Jurkat T cells. J Biol Chem, 2005. 280(25): p. 23576-83. 42. 43. 44. 45. 46. 49. 50. 51. Hunter, A.J., et al., Cutting edge: a novel function for the SLAP-130/FYB adapter protein in beta 1 integrin signaling and T lymphocyte migration. J Immunol, 2000. 164(3): p. 1143-7. 47. Mueller, K.L., et al., Adhesion and degranulation-promoting adapter protein (ADAP) positively regulates T cell sensitivity to antigen and T cell survival. J Immunol, 2007. 179(6): p. 3559-69. 48. Wang, H., et al., SLP-76-ADAP adaptor module regulates LFA-1 mediated costimulation and T cell motility. Proc Natl Acad Sci U S A, 2009. 106(30): p. 12436-41. Benlagha, K., et al., Characterization of the early stages of thymic NKT cell development. J Exp Med, 2005. 202(4): p. 485-92. Benlagha, K., et al., A thymic precursor to the NK T cell lineage. Science, 2002. 296(5567): p. 553-5. Geissmann, F., et al., Intravascular immune surveillance by CXCR6+ NKT cells patrolling liver sinusoids. PLoS Biol, 2005. 3(4): p. e113. 52. Wei, D.G., et al., Expansion and long-range differentiation of the NKT cell lineage in mice expressing CD1d exclusively on cortical thymocytes. J Exp Med, 2005. 202(2): p. 239-48. 53. Marrella, V., et al., A hypomorphic R229Q Rag2 mouse mutant recapitulates human Omenn syndrome. J Clin Invest, 2007. 117(5): p. 1260-9. 97 Arase, H., et al., Developmental arrest of NK1.1+ T cell antigen receptor (TCR)- alpha/beta+ T cells and expansion of NK1.1+ TCR-gamma/delta+ T cell development in CD3 zeta-deficient mice. J Exp Med, 1995. 182(3): p. 891-5. Gadue, P., N. Morton, and P.L. Stein, The Src family tyrosine kinase Fyn regulates natural killer T cell development. J Exp Med, 1999. 190(8): p. 1189-96. Zhang, W., et al., Essential role of LAT in T cell development. Immunity, 1999. 10(3): p. 323-32. Iwabuchi, K., et al., Defective development of NK1.1+ T-cell antigen receptor alphabeta+ cells in zeta-associated protein 70 null mice with an accumulation of NK1.1+ CD3- NK-like cells in the thymus. Blood, 2001. 97(6): p. 1765-75. 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. Rincon, M. and R.A. Flavell, Regulation of AP-1 and NFAT transcription factors during thymic selection of T cells. Mol Cell Biol, 1996. 16(3): p. 1074-84. Flavell, S.W., et al., Activity-dependent regulation of MEF2 transcription factors suppresses excitatory synapse number. Science, 2006. 311(5763): p. 1008-12. Karin, M. and Y. Ben-Neriah, Phosphorylation meets ubiquitination: the control of NF- [kappa]B activity. Annu Rev Immunol, 2000. 18: p. 621-63. Sivakumar, V., et al., Differential requirement for Rel/nuclear factor kappa B family members in natural killer T cell development. J Exp Med, 2003. 197(12): p. 1613-21. 63. Macian, F., NFAT proteins: key regulators of T-cell development and function. Nat Rev Immunol, 2005. 5(6): p. 472-84. 64. Wang, L., et al., Distinct functions for the transcription factors GATA-3 and ThPOK during intrathymic differentiation of CD4(+) T cells. Nat Immunol, 2008. 9(10): p. 1122- 30. 54. 55. 56. 57. 58. 59. 60. 61. 62. 65. Lazarevic, V., et al., The gene encoding early growth response 2, a target of the transcription factor NFAT, is required for the development and maturation of natural killer T cells. Nat Immunol, 2009. 10(3): p. 306-13. 66. Matsuda, J.L., et al., Temporal dissection of T-bet functions. J Immunol, 2007. 178(6): p. 3457-65. 98 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. Townsend, M.J., et al., T-bet regulates the terminal maturation and homeostasis of NK and Valpha14i NKT cells. Immunity, 2004. 20(4): p. 477-94. Das, R., et al., The adaptor molecule SAP plays essential roles during invariant NKT cell cytotoxicity and lytic synapse formation. Blood, 2013. 121(17): p. 3386-95. Dluzniewska, J., et al., Immature hematopoietic cells display selective requirements for adhesion- and degranulation-promoting adaptor protein in development and homeostatsis. Eur J Immunol, 2007. 37(11): p. 3208-19. Fostel, L.V., et al., ADAP is dispensable for NK cell development and function. Int Immunol, 2006. 18(8): p. 1305-14. Nichols, K.E., et al., Regulation of NKT cell development by SAP, the protein defective in XLP. Nat Med, 2005. 11(3): p. 340-5. Schraven, B., A. Marie-Cardine, and G. Koretzky, Molecular analysis of the fyn- complex: cloning of SKAP55 and SLAP-130, two novel adaptor proteins which associate with fyn and may participate in the regulation of T cell receptor-mediated signaling. Immunol Lett, 1997. 57(1-3): p. 165-9. Bendelac, A., et al., A subset of CD4+ thymocytes selected by MHC class I molecules. Science, 1994. 263(5154): p. 1774-8. Thomas, S.Y., et al., PLZF induces an intravascular surveillance program mediated by long-lived LFA-1-ICAM-1 interactions. J Exp Med, 2011. 208(6): p. 1179-88. Han, Y., et al., Pathogen-expanded CD11b+ invariant NKT cells feedback inhibit T cell proliferation via membrane-bound TGF-beta1. J Autoimmun, 2015. 58: p. 21-35. Levitsky, H.I., P.T. Golumbek, and D.M. Pardoll, The fate of CD4-8- T cell receptor- alpha beta+ thymocytes. J Immunol, 1991. 146(4): p. 1113-7. Coles, M.C. and D.H. Raulet, NK1.1+ T cells in the liver arise in the thymus and are selected by interactions with class I molecules on CD4+CD8+ cells. J Immunol, 2000. 164(5): p. 2412-8. Hammond, K., et al., Three day neonatal thymectomy selectively depletes NK1.1+ T cells. Int Immunol, 1998. 10(10): p. 1491-9. Gadue, P. and P.L. Stein, NK T cell precursors exhibit differential cytokine regulation and require Itk for efficient maturation. J Immunol, 2002. 169(5): p. 2397-406. 80. Makino, Y., et al., Development of Valpha4+ NK T cells in the early stages of embryogenesis. Proc Natl Acad Sci U S A, 1996. 93(13): p. 6516-20. 99 Pear, W.S., L. Tu, and P.L. Stein, Lineage choices in the developing thymus: choosing the T and NKT pathways. Curr Opin Immunol, 2004. 16(2): p. 167-73. Dao, T., et al., Development of CD1d-restricted NKT cells in the mouse thymus. Eur J Immunol, 2004. 34(12): p. 3542-52. 83. Ma, C.S., K.E. Nichols, and S.G. Tangye, Regulation of cellular and humoral immune responses by the SLAM and SAP families of molecules. Annu Rev Immunol, 2007. 25: p. 337-79. Starr, T.K., S.C. Jameson, and K.A. Hogquist, Positive and negative selection of T cells. Annu Rev Immunol, 2003. 21: p. 139-76. Clements, J.L., et al., Requirement for the leukocyte-specific adapter protein SLP-76 for normal T cell development. Science, 1998. 281(5375): p. 416-9. 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. Ohteki, T., et al., Cutting edge: LFA-1 is required for liver NK1.1+TCR alpha beta+ cell development: evidence that liver NK1.1+TCR alpha beta+ cells originate from multiple pathways. J Immunol, 1999. 162(7): p. 3753-6. 100 Veillette, A., Immune regulation by SLAM family receptors and SAP-related adaptors. Nat Rev Immunol, 2006. 6(1): p. 56-66. Latour, S., et al., Binding of SAP SH2 domain to FynT SH3 domain reveals a novel mechanism of receptor signalling in immune regulation. Nat Cell Biol, 2003. 5(2): p. 149-54. Chan, B., et al., SAP couples Fyn to SLAM immune receptors. Nat Cell Biol, 2003. 5(2): p. 155-60. 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. Chung, B., et al., Signaling lymphocytic activation molecule-associated protein controls NKT cell functions. J Immunol, 2005. 174(6): p. 3153-7. Nunez-Cruz, S., et al., Differential requirement for the SAP-Fyn interaction during NK T cell development and function. J Immunol, 2008. 181(4): p. 2311-20. 90. Wu, J.N., et al., Adhesion- and degranulation-promoting adapter protein is required for efficient thymocyte development and selection. J Immunol, 2006. 176(11): p. 6681-9. 81. 82. 84. 85. 86. 87. 88. 89. 91. 92. 93. 94. Emoto, M., et al., Critical role of leukocyte function-associated antigen-1 in liver accumulation of CD4+NKT cells. J Immunol, 1999. 162(9): p. 5094-8. Germanov, E., et al., Critical role for the chemokine receptor CXCR6 in homeostasis and activation of CD1d-restricted NKT cells. J Immunol, 2008. 181(1): p. 81-91. 97. Williams, J.A., et al., Regulation of thymic NKT cell development by the B7-CD28 costimulatory pathway. J Immunol, 2008. 181(2): p. 907-17. Akbari, O., et al., ICOS/ICOSL interaction is required for CD4+ invariant NKT cell function and homeostatic survival. J Immunol, 2008. 180(8): p. 5448-56. Berg, L.J. and J. Kang, Molecular determinants of TCR expression and selection. Curr Opin Immunol, 2001. 13(2): p. 232-41. 100. Vosshenrich, C.A., S.I. Samson-Villeger, and J.P. Di Santo, Distinguishing features of developing natural killer cells. Curr Opin Immunol, 2005. 17(2): p. 151-8. 101. Kovanen, P.E. and W.J. Leonard, Cytokines and immunodeficiency diseases: critical roles of the gamma(c)-dependent cytokines interleukins 2, 4, 7, 9, 15, and 21, and their signaling pathways. Immunol Rev, 2004. 202: p. 67-83. 102. Griffiths, E.K. and J.M. Penninger, Communication between the TCR and integrins: role of the molecular adapter ADAP/Fyb/Slap. Curr Opin Immunol, 2002. 14(3): p. 317-22. 103. Geng, L., et al., Adaptor FYB (Fyn-binding protein) regulates integrin-mediated adhesion and mediator release: differential involvement of the FYB SH3 domain. Proc Natl Acad Sci U S A, 2001. 98(20): p. 11527-32. 104. 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. 105. Wesley, J.D., et al., NK cell-like behavior of Valpha14i NK T cells during MCMV infection. PLoS Pathog, 2008. 4(7): p. e1000106. 106. 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. 107. 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. 108. Zeissig, S., et al., Hepatitis B virus-induced lipid alterations contribute to natural killer T cell-dependent protective immunity. Nat Med, 2012. 18(7): p. 1060-8. 95. 96. 98. 99. 101 109. Zhou, D., et al., Lysosomal glycosphingolipid recognition by NKT cells. Science, 2004. 306(5702): p. 1786-9. 110. Porubsky, S., et al., Globosides but not isoglobosides can impact the development of invariant NKT cells and their interaction with dendritic cells. J Immunol, 2012. 189(6): p. 3007-17. 111. 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. 112. Gumperz, J.E., et al., Murine CD1d-restricted T cell recognition of cellular lipids. Immunity, 2000. 12(2): p. 211-21. 113. 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. 114. 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. 115. Salio, M., et al., Biology of CD1- and MR1-restricted T cells. Annu Rev Immunol, 2014. 32: p. 323-66. 102 116. Constantinides, M.G. and A. Bendelac, Transcriptional regulation of the NKT cell lineage. Curr Opin Immunol, 2013. 25(2): p. 161-7. 117. Rossjohn, J., et al., Recognition of CD1d-restricted antigens by natural killer T cells. Nat Rev Immunol, 2012. 12(12): p. 845-57. 118. Brennan, P.J., M. Brigl, and M.B. Brenner, Invariant natural killer T cells: an innate activation scheme linked to diverse effector functions. Nat Rev Immunol, 2013. 13(2): p. 101-17. 119. Berzins, S.P., M.J. Smyth, and A.G. Baxter, Presumed guilty: natural killer T cell defects and human disease. Nat Rev Immunol, 2011. 11(2): p. 131-42. Jenkins, M.K. and J.J. Moon, The role of naive T cell precursor frequency and recruitment in dictating immune response magnitude. J Immunol, 2012. 188(9): p. 4135- 40. 121. Liu, T.Y., et al., Distinct subsets of human invariant NKT cells differentially regulate T helper responses via dendritic cells. Eur J Immunol, 2008. 38(4): p. 1012-23. 122. Oleinika, K., et al., CD1d-dependent immune suppression mediated by regulatory B cells through modulations of iNKT cells. Nat Commun, 2018. 9(1): p. 684. 120. 123. Zhang, H., et al., M2-specific reduction of CD1d switches NKT cell-mediated immune responses and triggers metaflammation in adipose tissue. Cell Mol Immunol, 2017. 124. Monteiro, M. and L. Graca, iNKT cells: innate lymphocytes with a diverse response. Crit Rev Immunol, 2014. 34(1): p. 81-90. 125. Lynch, L., et al., Invariant NKT cells and CD1d(+) cells amass in human omentum and are depleted in patients with cancer and obesity. Eur J Immunol, 2009. 39(7): p. 1893- 901. 126. Exley, M.A., et al., Interplay between the immune system and adipose tissue in obesity. J Endocrinol, 2014. 223(2): p. R41-8. 127. Peterson, E.J., et al., Coupling of the TCR to integrin activation by Slap-130/Fyb. Science, 2001. 293(5538): p. 2263-5. 128. Rajasekaran, K., et al., Signaling by Fyn-ADAP via the Carma1-Bcl-10-MAP3K7 signalosome exclusively regulates inflammatory cytokine production in NK cells. Nat Immunol, 2013. 14(11): p. 1127-36. 129. Tomura, M., et al., A novel function of Valpha14+CD4+NKT cells: stimulation of IL-12 production by antigen-presenting cells in the innate immune system. J Immunol, 1999. 163(1): p. 93-101. 130. Uldrich, A.P., et al., NKT cell stimulation with glycolipid antigen in vivo: costimulation- dependent expansion, Bim-dependent contraction, and hyporesponsiveness to further antigenic challenge. J Immunol, 2005. 175(5): p. 3092-3101. 131. Carnaud, C., et al., Cutting edge: Cross-talk between cells of the innate immune system: NKT cells rapidly activate NK cells. Journal of immunology, 1999. 163(9): p. 4647-50. 132. Reefman, E., et al., Cytokine secretion is distinct from secretion of cytotoxic granules in NK cells. J Immunol, 2010. 184(9): p. 4852-62. 133. D'Orlando, O., et al., Syntaxin 11 is required for NK and CD8(+) T-cell cytotoxicity and neutrophil degranulation. Eur J Immunol, 2013. 43(1): p. 194-208. 134. Engel, I., et al., Co-receptor choice by V alpha14i NKT cells is driven by Th-POK expression rather than avoidance of CD8-mediated negative selection. J Exp Med, 2010. 207(5): p. 1015-29. 135. Kim, P.J., et al., GATA-3 regulates the development and function of invariant NKT cells. J Immunol, 2006. 177(10): p. 6650-9. 103 136. Musci, M.A., et al., Molecular cloning of SLAP-130, an SLP-76-associated substrate of the T cell antigen receptor-stimulated protein tyrosine kinases. J Biol Chem, 1997. 272(18): p. 11674-7. 137. Togni, M., et al., The adapter protein ADAP is required for selected dendritic cell functions. Cell Commun Signal, 2012. 10(1): p. 14. 138. Kliche, S., et al., The ADAP/SKAP55 signaling module regulates T-cell receptor- mediated integrin activation through plasma membrane targeting of Rap1. Mol Cell Biol, 2006. 26(19): p. 7130-44. 139. Hegde, S., et al., NKT cells direct monocytes into a DC differentiation pathway. Journal of Leukocyte Biology, 2007. 81(5): p. 1224-1235. 140. Swann, J.B., et al., Type I natural killer T cells suppress tumors caused by p53 loss in 104 mice. Blood, 2009. 113(25): p. 6382-5. 141. Smyth, M.J., et al., Differential tumor surveillance by natural killer (NK) and NKT cells. J Exp Med, 2000. 191(4): p. 661-8. 142. Smyth, M.J., et al., Sequential production of interferon-gamma by NK1.1(+) T cells and natural killer cells is essential for the antimetastatic effect of alpha-galactosylceramide. Blood, 2002. 99(4): p. 1259-66. 143. Crowe, N.Y., M.J. Smyth, and D.I. Godfrey, A critical role for natural killer T cells in immunosurveillance of methylcholanthrene-induced sarcomas. J Exp Med, 2002. 196(1): p. 119-27. 144. Yuling, H., et al., EBV-induced human CD8+ NKT cells suppress tumorigenesis by EBV- associated malignancies. Cancer Res, 2009. 69(20): p. 7935-44. 145. Fujii, S., et al., Activation of natural killer T cells by alpha-galactosylceramide rapidly induces the full maturation of dendritic cells in vivo and thereby acts as an adjuvant for combined CD4 and CD8 T cell immunity to a coadministered protein. J Exp Med, 2003. 198(2): p. 267-79. 146. Hayakawa, Y., et al., Differential regulation of Th1 and Th2 functions of NKT cells by CD28 and CD40 costimulatory pathways. J Immunol, 2001. 166(10): p. 6012-8. 147. Dao, T., W.Z. Mehal, and I.N. Crispe, IL-18 augments perforin-dependent cytotoxicity of liver NK-T cells. J Immunol, 1998. 161(5): p. 2217-22. 148. Matsuda, J.L., et al., T-bet concomitantly controls migration, survival, and effector functions during the development of Valpha14i NKT cells. Blood, 2006. 107(7): p. 2797- 805. 149. Nieda, M., et al., TRAIL expression by activated human CD4(+)V alpha 24NKT cells induces in vitro and in vivo apoptosis of human acute myeloid leukemia cells. Blood, 2001. 97(7): p. 2067-74. 150. Lettau, M., et al., The adapter proteins ADAP and Nck cooperate in T cell adhesion. Mol Immunol, 2014. 60(1): p. 72-9. 151. Krause, M., et al., Fyn-binding protein (Fyb)/SLP-76-associated protein (SLAP), Ena/vasodilator-stimulated phosphoprotein (VASP) proteins and the Arp2/3 complex link T cell receptor (TCR) signaling to the actin cytoskeleton. J Cell Biol, 2000. 149(1): p. 181-94. 152. Coppolino, M.G., et al., Evidence for a molecular complex consisting of Fyb/SLAP, SLP- 76, Nck, VASP and WASP that links the actin cytoskeleton to Fcgamma receptor signalling during phagocytosis. J Cell Sci, 2001. 114(Pt 23): p. 4307-18. 153. Danzer, C., et al., A mutation within the SH2 domain of slp-76 regulates the tissue distribution and cytokine production of iNKT cells in mice. Eur J Immunol, 2016. 46(9): p. 2121-36. 154. Hidano, S., et al., Distinct regulatory functions of SLP-76 and MIST in NK cell cytotoxicity and IFN-gamma production. Int Immunol, 2008. 20(3): p. 345-52. 155. Bassiri, H., et al., iNKT cell cytotoxic responses control T-lymphoma growth in vitro and in vivo. Cancer Immunol Res, 2014. 2(1): p. 59-69. 156. Cohen, N.R., et al., Shared and distinct transcriptional programs underlie the hybrid nature of iNKT cells. Nat Immunol, 2013. 14(1): p. 90-9. 157. Hix, L.M., et al., CD1d-expressing breast cancer cells modulate NKT cell-mediated antitumor immunity in a murine model of breast cancer metastasis. PLoS One, 2011. 6(6): p. e20702. 158. De Santo, C., et al., Invariant NKT cells reduce the immunosuppressive activity of influenza A virus-induced myeloid-derived suppressor cells in mice and humans. J Clin Invest, 2008. 118(12): p. 4036-48. 159. Li, C., et al., ADAP and SKAP55 deficiency suppresses PD-1 expression in CD8+ cytotoxic T lymphocytes for enhanced anti-tumor immunotherapy. EMBO Mol Med, 2015. 7(6): p. 754-69. 160. Schneiders, F.L., et al., Circulating invariant natural killer T-cell numbers predict outcome in head and neck squamous cell carcinoma: updated analysis with 10-year follow-up. J Clin Oncol, 2012. 30(5): p. 567-70. 105 161. Metelitsa, L.S., et al., Natural killer T cells infiltrate neuroblastomas expressing the chemokine CCL2. J Exp Med, 2004. 199(9): p. 1213-21. 162. Molling, J.W., et al., Peripheral blood IFN-gamma-secreting Valpha24+Vbeta11+ NKT cell numbers are decreased in cancer patients independent of tumor type or tumor load. Int J Cancer, 2005. 116(1): p. 87-93. 163. Wang, H., et al., ADAP-SLP-76 binding differentially regulates supramolecular activation cluster (SMAC) formation relative to T cell-APC conjugation. J Exp Med, 2004. 200(8): p. 1063-74. Jo, E.K., H. Wang, and C.E. Rudd, An essential role for SKAP-55 in LFA-1 clustering on T cells that cannot be substituted by SKAP-55R. J Exp Med, 2005. 201(11): p. 1733-9. 165. Liu, J., et al., FYB (FYN binding protein) serves as a binding partner for lymphoid protein and FYN kinase substrate SKAP55 and a SKAP55-related protein in T cells. Proc Natl Acad Sci U S A, 1998. 95(15): p. 8779-84. 166. Wang, H. and C.E. Rudd, SKAP-55, SKAP-55-related and ADAP adaptors modulate integrin-mediated immune-cell adhesion. Trends Cell Biol, 2008. 18(10): p. 486-93. 167. Moog-Lutz, C., et al., PRAM-1 is a novel adaptor protein regulated by retinoic acid (RA) and promyelocytic leukemia (PML)-RA receptor alpha in acute promyelocytic leukemia cells. J Biol Chem, 2001. 276(25): p. 22375-81. 168. Clemens, R.A., et al., PRAM-1 is required for optimal integrin-dependent neutrophil function. Mol Cell Biol, 2004. 24(24): p. 10923-32. 164. 106