The Pennsylvania State University. The Graduate School. College of Agricultural Sciences

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1 The Pennsylvania State University The Graduate School College of Agricultural Sciences ANALYSIS OF T AND NKT CELL ACTIVATION AND FUNCTION IN RESPONSE TO SEB IN VIVO A Thesis in Pathobiology by Melanie Jean Ragin Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2005

2 ii The thesis of Melanie Ragin has been reviewed and approved* by the following: Avery August Associate Professor of Immunology Thesis Adviser Chair of Committee Pamela Correll Associate Professor of Immunology Biao He Assistant Professor of Veterinary Science Andrea Mastro Professsor of Microbiology and Cellular Biology Andy Henderson Associate Professor of Veterinary Science Nandini Vasudevan Assistant Professor of Biology C. Channa Reddy Distinguished Professor of Veterinary Science Head of the Department of Veterinary Science *Signatures are on file at the Graduate School

3 iii Abstract Exposure to Staphylococcal Enterotoxin B (SEB), a bacterial superantigen, from Staphylococcus aureus (S. aureus), is characterized by its ability to specifically expand Vβ8 + T cell populations, induce massive cytokine release (IL-2, IFN-γ, and TNFα), followed by rapid deletion and anergy of these populations. This results in illnesses such as food poisoning and lethal toxic shock in humans. SEB responses have been shown to be mediated primarily by T cells. The Tec family kinase has been shown to be involved in T cell activation in vitro. NKT cells, a T cell subset, are known to be rapid responders to infection, causing rapid release of cytokines such as IFN-γ, IL-4 and IL-2. Therefore, using SEB as a model antigen we aimed to study the role of the Tec family kinase ITK in activation and function of T and NKT cells in vivo. We examined proliferative responses to SEB, showing that both in vitro and in vivo ITK null T cells have reduced proliferative responses. We show that, in response to SEB, ITK deficient mice showed increased expression of the early activation marker CD69, although lower than that of WT mice. Since ITK regulates downstream signals we analyzed the role of ITK in regulating JNK pathway activation using phosporylated-c-jun as a measure of JNK pathway activation; showing that ITK mediates and is necessary for JNK pathway activation. Since NKT cells are known to be important immunomodulatory cells by their immediate responses through rapid cytokine release, we analyzed the regulatory role ITK has on NKT cells, using the potent NKT cell stimulator α- Galactosylceramide (α-galcer). We analyzed proliferative responses and cytokine production of these cells in ITK null mice, showing NKT cells in ITK null mice exhibit reduced proliferative responses in vitro and secrete reduced levels of IL-2 in vivo.

4 iv However, showing less of an effect of ITK on NKT cell secretion of IL-4, IFN-γ, and TNF-α in vivo compared to WT. Based on the ability of NKT cells to respond to antigen quickly and their ability to inhibit responses to disease we analyzed NKT cells responses to SEB, using NKT cell null mice. We analyzed the ability of T cells to expand in these NKT cell null mice, and found no differences compared to WT mice, suggesting that the Vβ8 + T cell population is expanded by SEB in these mice. When we analyzed cytokine production in response to SEB we found that Jα18 and CD1d null mice, thus NKT cells, do not seem to be necessary for IL-2 production, but are necessary for IL-4 secretion whereas Jα18, not CD1d seems to be required for the production of IFN-γ; CD1d, however, is more important for the production of TNF-α, suggesting differential regulation of cytokines in response to SEB. Together these data suggest a role for ITK in regulating downstream T cell signal transduction in response to SEB, as well as regulation of NKT cell proliferation, but not development, and function, evidence that ITK may serve as an important target for T cell mediated diseases. Further, our studies suggest that NKT cells, in response to SEB, may differentially regulate disease, through their cytokine secretion patterns.

5 v Table of Contents List of Figures.ix List of Tables...x List of Abbreviations..xi Acknowledgements..xiv Chapter 1: Literature Review Introduction Staphylococcal Enterotoxin B SEB in disease manifestation Natural Killer T cells Role of NKT cells in disease development TcR signal transduction The Tec kinase ITK Tec Kinases and T Cell Differentiation and Cytokine Production..19 Chapter 2: A role for the Tec family kinase ITK in regulating SEB induced Interleukin-2 production in vivo via the JNK pathway Abstract Background Materials and Methods Mice In vivo expansion assays...24

6 vi Intracellular staining In vitro proliferation In vitro cytokine analysis In vivo cytokine analysis Results ITK deficient T cells proliferate less efficiently than WT T cells in response to varying concentrations of SEB in vitro ITK null T cells secrete less IL-2 in response to SEB in vitro Reduced expansion of Vβ8 + CD4 + population in ITK null animals in response to SEB exposure in vivo Defective SEB induced IL-2 secretion in vivo in ITK null mice Defective JNK pathway activation by SEB in ITK null T cells Similar toxicity of SEB on WT and ITK null mice Discussion 41 Chapter 3: Regulation of NKT cell function by the Tec Family Kinase Abstract Background Materials and Methods Mice..46

7 vii In vivo staining In vitro proliferation In vivo cytokine analysis Results ITK is not required for NK or NKT cell development ITK regulates NKT cell proliferation in vitro Differential cytokine production by ITK null NKT cells in vivo in response to α-galcer Discussion 53 Chapter 4: Differential SEB-induced cytokine regulation by NKT cells in vivo Abstract Background Materials and Methods Mice Analysis T cell expansion in vivo in response to SEB Analysis of in vivo cytokines Analysis of in vivo toxicity Results Similar expansion of Vβ8 + CD4 + population in NKT null and WT

8 viii animals in response to SEB exposure in vivo Differential cytokine regulation by Jα18 and CD1d null mice in vivo in response to SEB Role of NKT cells in SEB induced toxicity Discussion 64 Chapter 5: Discussion and Conclusions SEB and T cell function in vivo Regulation of NKT cells by ITK NKT cell responses to SEB induced disease...70 Chapter 6: Miscellaneous Experiments Introduction Results WT and ITK deficient livers appear unaffected by SEB-induced toxicity Lack of NKT cells may protect CD1d-deficient mice from liver damage NKT cells are involved in IL-2 production in response to SEB in vivo ITK regulates AHR in response to intranasal SEB exposure...76 References..80

9 ix List of Figures Figure 1.1 Figure 1.2 Figure 1.3 SEB-MHC-TcR Crystal structures.3 Structure of the T cell receptor..12 Models of class I peptide-mhc (pmhci)-tcr-cd8 (a) and pmhcii- TCR-CD4 (b) ternary complex structures..13 Figure 1.4 Figure 1.5 Figure 2.1 Figure 2.2 Figure 2.3 Figure 2.4 In vitro T cell activation signal transduction pathways.14 Structure of ITK.15 ITK regulates proliferation in response to SEB.28 ITK regulates IL-2 secretion in vitro in response to SEB..29 Reduced T cell expansion in response to SEB exposure in vivo...32 ITK null mice secrete reduced IL-2 in response to SEB stimulation in vivo 34 Figure 2.5 SEB induces activation of the JNK pathway specifically in responding T cells in vivo 36 Figure 2.6 SEB induces activation of the JNK pathway specifically in responding T cells in vivo as early as 15 minutes 38 Figure 2.7 ITK is required for SEB mediated activation of the JNK pathway in responding T cells in vivo..39 Figure 2.8 Figure 3.1 Figure 3.2 Figure 3.3 ITK null cells are activated by SEB...40 ITK does not regulate NKT cell development...48 ITK regulates in vitro proliferation of NKT cells..49 ITK regulates NKT cell IL-2 production in vivo...49

10 x Figure 3.4 Figure 4.1 ITK does not appear to regulate NKT cell cytokine production in vivo 52 Similar expansion of Vβ8 + CD4 + populations in NKT null and WT animals in response to SEB exposure in vivo..60 Figure 4.2 Differential cytokine regulation by NKT null mice...62 Figure 4.3 NKT cells negatively regulate production of TNF-α in response to SEB 63 Figure 6.1 WT and ITK deficient livers appear unaffected by SEB-induced-toxicity 73 Figure 6.2 Lack of NKT cells may protect CD1d-deficient mice from liver damage 74 Figure 6.3 Figure 6.4 NKT cells are involved in IL-2 production in response to SEB in vivo 76 ITK regulates AHR in response to intranasal SEB exposure...77 Figure 6.5 ITK does not appear to regulate responses in the lung to intranasal SEB 78

11 xi List of Tables Table 2.1 Table 3.1 Similar Toxicity of SEB in WT and ITK null mice...41 ITK is not required for NKT cell development as shown by Total numbers of NKT cells in the Spleen, Liver, and Bone Marrow...49 Table 3.2 ITK is not required for NKT cell development shown as NKT cells as a percent of total T cells in the Spleen, Liver, and Bone Marrow 49 Table 4.1 NKT cells do not contribute a significant protective effect against SEB induced disease..64

12 xii Abbreviations ADAP α-galcer AHR AICD APC Adhesion and degranulation-promoting adaptor protein alpha-galactosylceramide airway hyperresponsiveness Activation-Induced Cell Death Antigen Presenting Cell AP-1 Activator protein 1 CD CsA CTLA-4 DAG D-GalN DIG DN DRM EAE ERK Cluster of Differentiation Cyclosporin A cytotoxic T lymphocyte antigen-4 Diaclyglycerol D-Galactosamine detergent-insoluble glycolipid-enriched membranes Double negative detergent-resistant membranes Experimental Autoimmune Encephalomyelitis Extracellular signal Regulated Kinase GATA3 GATA-binding protein 3 GEM GM-CSF Gpi Grb2 IL-2 glycosphingolipid-enriched membranes Granulocyte Macrophage-Colony Stimulating Factor glycosylphosphatidyinositol growth-factor-receptor-bound protein Interleukin-2

13 xiii igb3 ITK JNK Lck LCM MAPK NCK NFAT NK PBS PH PI3k PIP2 PKC PLCγ1 PMA PTEN Rlk SAg SEB SH SHIP SLP-76 isoglobotrihexosylceramide Interleukin-2 inducible T cell Kinase c-jun N-terminal kinase Lstra cell kinase Lymphocytic Choriomeningitis Mitogen Activated Protein Kinase noncatalytic region of tyrosine kinase Nuclear Factor of Activated T cells Natural Killer cells Phosphate Buffered Saline Pleckstrin homology domain, Phosphatidylinsitol-3-kinase Phosphatidylinositol-4,5-bisphosphate Protein kinase C Phospholipase C gamma Phorbol Myristic Acid phosphatase and tensin homologue Resting lymphocyte kinase Superantigen Staphylococcal Enterotoxin B Src Homology SH2-domain containing inositol-5-phosphatase SH2 domain-containing phoshoprotein of 76kDa

14 xiv Syk TcR TH Th1 Th2 VS Zap-70 Spleen tyrosine kinase T-cell Receptor Tec Homology domain T-helper 1 cells T-helper 2 cells Vesicular Stomatitis Zeta-chain Associated protein-70

15 xv Acknowledgements First, I would like to dedicate this thesis to my mother, because without her friendship, guidance and support I never would have made it, not only, in this program, but I would not have grown to become the woman I am today if it had not been for her. I am also eternally grateful to my dad for also providing support and direction when I needed it. I would also like to thank, my mentor and friend, Dr. Avery August, for standing by me and giving me the room to grow and find myself through this process, when others may have given up on me, it truly means a lot. I would like to give my thanks to past and present lab members that have assisted me with information, guidance, and conversation. I would also like to acknowledge the cooperation and assistance of neighboring labs: Dr. Andy Henderson, Dr. Pamela Correll, Dr. Biao He and Dr. Bob Paulson. I will also the treasure the relationships I have formed while traveling through this journey, called graduate school, where I have seen many of my friends and peers turn into men and women and become mothers and fathers. I have learned many life lessons and gained a new perspective on myself and the world, because of you. Last, but not least I wish everyone peace, fulfillment, and contentment in your next phase and future endeavors.

16 1 Chapter 1 Literature Review 1.0 Introduction Immunis meaning exempt in Latin, is the basis for the English word immunity, meaning the state of protection from infectious disease. Immunity is also the basis for the study of Immunology, the study of immune responses to various pathogenic organisms and dysregulations, thereof. Using the superantigen, SEB, as a model we aim to gain a better understanding of the regulation of immune system as a whole but more specifically, physiological T cell immune responses. 1.1 Staphylococcal Enterotoxin B Staphylococcal Enterotoxin B (SEB), a toxin and superantigen, from gramnegative bacterium Staphylococcus aureus is best known for its superantigenic activity on T cells (1-3). T cell superantigenic activity is defined by the ability of a substance to bind specific Vβ region of the TcR and residues in the α chain of the class II MHC molecule; it induces activation of all T cells that express TcR with a particular Vβ domain (1, 2). Superantigens, such as SEB are also known for their ability to form a bridge between the TcR and MHC II without being processed, unlike conventional peptides which need to be processed and presented as short peptides (Figure1.1) (2, 3). In order for a T cell to recognize conventional antigen it must be processed into smaller peptides that form complexes with MHC I or MHC II proteins. How a conventional antigen is presented in the context of MHC I or II depends on the way in which an

17 2 antigen enters a cell. If the antigen is exogenous, i.e. produced outside of the cell, it is degraded and the peptide is presented in the context of MHC II, since expression of MHC II is limited to antigen presenting cells (APCs) (macrophages, dendritic cells, and B cells) (4, 5). However, if antigen is endogenous, i.e. produced within the host cell, it is degraded into peptide fragments and binds MHC I proteins (4, 5). The ability of SEB to induce T cell activation through forming a bridge between the TCR and the MHC II allows for minute concentrations of SEB to activate T cells because of their strong binding avidity to T cell antigen receptors and class II molecules of the MHC (4, 5). Superantigens, specifically SEB, are characterized by their ability to activate up to 5-20% of Vβ Τ cells, in the case of SEB activation of Vβ3, 7, 8 positive T cells, compared to.001% of T cell by conventional antigens (1, 2, 6). Additionally, exposure to SEB results in large amounts of cytokines being released including IL-2, IFN-γ, TNF-α and systemic shock (1, 7). 2-3 days after undergoing clonal expansion and massive cytokine production in response to SEB, the responding T cells undergo clonal deletion followed by induction of anergy in the remaining cells. These anergic cells are characterized by their inability to secrete IL-2 and to proliferate after stimulation with antigen in vitro and in vivo (7). Superantigens (Sags) have also been reported to be able to break tolerance (the state of becoming unresponsive to a specific antigen) through repeated exposure. This has led to speculations that they are sometimes involved in causing autoimmune diseases such as multiple sclerosis and rheumatoid arthritis (1, 2). There is still a lot to be understood about superantigens and why they exist; it is speculated that superantigens give microbes, such as, S. aureus, the ability to colonize through promoting local

18 3 inflammation, thereby increasing the blood and nutrient supply, disturbing a normal immune response, potentially prolonging survival (2). Staphylococcal enterotoxins, toxic shock syndrome toxin-1 (TSST-1) and certain other bacterial products and viral products are commonly referred to as superantigens because of their profound effects on the immune system (1, 3). Figure 1.1 Crystal structures of SEB-MHC-TcR interactions. A) Model of the structure of SEB (blue) and the interaction with MHCα (green) and MHCβ (light blue) with peptide (purple) present. B) Model of interactions between TcRα (brown) - TcRβ (red) - MHCα (green) and MHCβ (light blue) with peptide (purple) present in the absence of SEB (blue). C) Model of SEB (blue) forming a bridge between the TcRα (brown)-tcrβ (red) and MHCα (green)-mhcβ (light blue) complex with peptide (purple) present. Curr Opin Immun :36-44

19 4 1.2 SEB in Disease manifestation Primates, including humans, are the most sensitive to SEB because the binding affinity of their MHC class II molecules is greater than those of mice (6, 8). Food poisoning and Toxic shock syndrome are two most commonly known diseases to be caused by superantigens produced by S. aureus (1, 3, 9). Staphylococcal enterotoxins are among the most common causes of food poisoning in humans (1, 2, 10). This colonization of food by S. aureus is associated with a form of gastroenteritis, inflammation of the mucous membrane of the stomach and intestine that is diagnosed clinically as emesis (vomiting), with or without diarrhea thought to be associated with histamine and leukotriene release from mast cells (10). In contrast, it has been suggested that the enterotoxic effects of bacterial superantigens are directly related to their superantigenic activities, i.e. dependent on T cell stimulation and massive cytokine release (2, 10). Toxic shock syndrome is an acute, life-threatening intoxication characterized by high fever, hypotension, rash multiorgan dysfunction and cutaneous desquamation that is caused by S. aureus produced superantigens, such as TSST-1. The interaction of these pyrogenic toxins with the TcR and MHC activates the T cells for secretion of TNF-β, IL-2 and IFNγ in TSS patients (2, 10). S. aureus produced superantigens, such as SEB and TSST-1, are also potent potential biological warfare agents not only because of their ability to cross mucosal surfaces, but due to the fact that their potent emetic effects could possibly disable an army of men exposed in their food or aerosolized, causing emesis, diarrhea and other gastrointestinal dysfunction (10).

20 5 1.3 Natural Killer T cells Effective immunity, against such diseases, involves regulated cytokine production by lymphocytes (11, 12). Conventional T cells are an important source of cytokines during infection, but they must clonally expand, differentiate into effector cells and migrate to sites of infection over the course of several days to perform their function (12). To make up for the time it takes before antigen-specific cytokine production during the activation and expansion of adaptive immunity, several lymphocyte populations, including Natural Killer T cells, surround the sites of pathogen entry where they are able to secrete cytokines within minutes to hours of infection (13). NKT cells represent a unique subset of T cells that co-express an invariant T cell receptor (TcR) and NK cell-related surface markers, including NK1.1 (NKRP1C) and Ly-49 comprised primarily of two subsets: CD4 + or CD4 - CD8 - (14, 15). They are usually defined as αβtcr + cells that express the surface marker NK1.1 (16, 17). The majority of NKT cells are restricted to the MHC-I-related molecule CD1d, preferentially using an invariant TcRα chain consisting of the Vα14-Jα18 and also have a bias in TcRβ gene usage as is used by 50% of these cells (12, 14). NKT cells recognize synthetic glycolipid antigens such as the marine sponge derived α-galactosylceramide (α-galcer) presented by CD1d, as well as other endogenous ligands such as isoglobotrihexosylceramide (igb3) and glycosylphosphatidyinositol (GPi) (12, 18). CD1d-restricted NKT cells that preferentially use the invariant Vα24-Jα18 TCR gene segment with similar phenotypic characteristics have been described in humans (11, 19). NKT cells are most frequently found in the liver and bone marrow (20-40% of T cell population), but are also detected

21 6 in the thymus and spleen (1.5%) and are rare in the lymph nodes (12, 19). TcR Vα14/Jα18-deficient mice lack only Vα14 NKT cells, whereas other conventional cells of the immune system, such as T cells, B cells, and NK cells, remain intact (12, 14, 19). Analysis of CD1d knockout animals indicate that CD1d expression is required for the development of NKT cells and plays a critical role for CD1d-dependent T cells in various disease conditions (12, 20). Other types of NKT cells include those that also recognize CD1d, but have more diverse TCRs (21). These cells appear to be less numerous in mice and are either CD4 + or CD4/CD8 double negative (DN), the role these particular NKT cells play in immune regulation is still unclear (21). In addition, there are those NKT cells which are CD1d independent, have diverse TCR s and can be CD8 +, CD4 +, or DN. These NK1.1 + cells are most prevalent in the spleen and bone marrow compared with the thymus and liver, the function of these NKT cells has yet to be identified (21). The last identifiable type of NKT cells are those that are defined by their expression of CD49b (α2/β1-integrin) which is recognized by the DX5 antibody (21). There is very little overlap of this population and the most well recognized Vα14i T cells. These cells may play a more substantial role in antigen-specific suppression of immune responses, such as autoimmune diabetes (21). NKT (Vα14i T cells) cells were originally identified for their ability to rapidly produce IL-4 and IFN-γ the most well characterized cytokines. Other cytokines, such as IL-2, TNF, IL-5, IL-13, and Granulocyte Macrophage-Colony Stimulating Factor (GM- CSF) are also produced, upon stimulation using anti-cd3 antibodies (14). NKT cells, like T cells arise from thymocyte progenitor cells (14, 22). Currently, it is unclear how early

22 7 prior to TcR expression, progenitor cells become committed to the NKT lineage since they are identified by their TcR specificity (12, 22). Following TcR expression, CD1d restricted cells can be identified even before NK1.1 expression based on the ability to bind α-galcer/cd1d tetramers (12, 23). Based on expression of the cell surface marker DX5, CD1d reactive NKT cells have been divided in 4 stages of development (14, 15, 24). The first two stages are immature, with the most immature stages lacking both DX5 and NK1.1. These cells are highly CD4 + as are cells in the next stage, DX5 + /NK1.1 - (14, 15, 24). Mature NKT cells are divided almost equally into DX5 + /NK1.1 + and DX5 - /NK1.1 + cells (14, 15, 24). In contrast to conventional T cells, which develop in the fetus and are present at birth, NKT cells are not found until approximate 5 days post-partum in mice (14, 15, 24). In addition to the invariant TcR, most NKT cells express NK1.1 and DX5, and mature NKT cells express Ly49 and a memory phenotype CD44 hi /CD69 + /CD49b low, and produce large amounts of IL-4 and IFN-γ upon activation (14, 15, 24). It is unclear how the secretion of various cytokines by NKT cells results in a regulated immune response, however there are several possibilities: one possibility is that there are subsets of Vα14i NKT and Vα24i NKT cells, based on anatomic location and/or cells surface phenotype and that the activation of one subset or the other could have a selective influence dependent on the cytokine being produced (12). The other possibility is that the quality of the TCR signal influences the cytokine profile produced, thus the nature of the immune response (12). The third possibility is that cytokine production by the Vα14i NKT cells may be determined by the integration of signals from different types of receptors, and this may influence the pattern of cytokines produced. A

23 8 fourth possibility is that the context in which antigen is presented has an influence on the pattern of cytokines produced (12). NKT cells are a very complex and important cell population that require further investigation. 1.4 Role of NKT cells in disease development NKT cells have been implicated in the regulation of several different autoimmune diseases in mice and humans, including type 1 diabetes, experimental autoimmune encephalomyelitis (a mouse model for multiple sclerosis), and atherosclerosis (14, 19). It has been shown that the NKT cell ligand α-galcer can inhibit disease in diabetes-prone non-obese diabetic mice (NOD)(14, 19). Diabetes is the most notable disease associated with NKT cells (11, 14). The NOD mouse strain, used to study type 1 diabetes, is used because of the spontaneous development of the disease with many of the features common to that seen in humans (25). The possibility that NKT cells may be involved in diabetes was suggested when Hammond, et al, found that NOD mice are specifically deficient in NKT cells compared to other strains (25, 26). Based on studies which repeatedly injected α-galcer into these mice, a response was observed in which the number of NKT cells increased accompanied by increased insulin levels, suggesting that NKT cells may be regulating this disease. Overcoming defective activation of NKT cells and polarizing the autoimmune response in NOD mice to a less pathogenic Th2 profile may be effective treatments in this disease (19, 25, 27). Recent studies have focused on the interaction between NKT cells and antigen presenting cells (APC) and it is likely that α-galcer activated NKT cells

24 9 influence helper T cell differentiation through their interactions on APC s, which is influenced by the cytokine microenvironment and costimulation (14, 19, 25). However, NKT cells do not always play a protective role in immunity; rather they sometimes play a detrimental role, as in the cases of airway hypersensitivity (AHR) and atherosclerosis where these diseases are exacerbated by the activation of NKT cells (12, 14, 19). It has been shown in mice that lack NKT cells that AHR and inflammation is virtually nonexistent (12). Additionally, in the case of atherosclerosis, NKT cells seem to enhance the size of atherosclerotic plaques in mice that are genetically determined to develop atherosclerosis or in apolipoprotein-e deficient mice fed high fat diets (12). This is suggested to be caused by the increase secretion proinflammatory cytokines by NKT cells (14). The discovery that the deficiency of NKT cells in NOD mice was directly related to their susceptibility to diabetes led to the suggestion that these cells could represent a target for potential new treatment strategies in Type 1 diabetes, particularly when human diabetics were found to have lower numbers of these cells than that of their non-diabetic siblings (11, 25). A deficiency in function and number of NKT cells may also be involved in the increased susceptibility of SJL mice to EAE, thus the decrease of NKT cells observed in multiple sclerosis patients is of interest (19, 25). However, before α- GalCer is considered for human use, several studies have shown that α-galcer can be toxic, causing liver damage, in older mice, presumably due to their higher NKT cell numbers, sufficient enough to cause death (21, 25). It is possible that α-galcer may not be toxic in humans as they have a lower proportion of NKT cells (21, 25).

25 10 This evidence suggests that NKT cells play a role in controlling human autoimmune diseases, however it is more difficult to demonstrate a causal relationship in humans (12, 25). On the other hand, the ability to specifically target and manipulate these cells in mouse models of autoimmune disease, using natural and synthetic ligands such as α-galcer, increases the possibility that NKT cell manipulation represents a feasible approach to immunotherapeutic intervention for autoimmune disease in human (12, 14, 19). 1.5 TcR signal transduction T cell activation is initiated by the interaction of the T cell Receptor (TcR)-CD3 complex with an antigenic peptide bound to MHC II molecules on the surface of an antigen presenting cell (28-30). APC's are distinguished by their ability to express MHC II and deliver a costimulatory signal. These cells include dendritic cells, macrophages and B cells, which are classified as professional APC's (31). Dendritic cells however are the most effective APC because they constitutively express high levels of MHC II and costimulatory ligand, including B7-1/B7-2 and CD80/CD86 activity (31). The TcR, is a multicomponent structure expressed on the cell surface of T cells includes a heterodimer αβ or γδ chains which detect antigen presented by APC's in the context of appropriate major histocompatibility complex (MHC) proteins (32). The αβ or γδ chains have very small cytoplasmic tails and are unable to generate signals upon antigen binding. Instead, they associate with transmembrane proteins CD3γ, CD3δ and CD3ε and a CD3 zeta chain homodimer (TCRζ) (Figure 1.2) (32-34). The CD3 and TCRζ components of the receptor are responsible for transmitting the signal into the cell via a structurally

26 11 conserved amino acid motif present in their cytoplasmic domains, known as an immunoreceptor tyrosine-based activation motif (ITAM) (35). Signaling by the TCR is also facilitated by the CD4 and CD8 coreceptors, which interact with MHC molecules expressed on APC's during antigen presentation (Figure 1.3) (33). Successful stimulation of the TcR leads to the activation of a number of signaling pathways that involve generation of second messengers, increased transcriptional activity and production of new proteins that mediate effector functions of activated T cells (36). Upon receptor stimulation, the first biochemical event is phosphorylation of the tyrosine residues present within ITAMs (34). The Src-family kinases Lck and Fyn have been implicated in this phosphorylation. Lck interacts with the cytoplasmic tail of the CD4 and CD8 coreceptors (36). These receptors bind to MHC molecules on APC's during antigen presentation, bringing Lck in proximity to the TcR, facilitating ITAM phosphorylation. Phosphorylated ITAMs form docking sites for the Src homology (SH) 2 domains of the cytosolic tyrosine kinase ZAP-70 (30, 37-39). Upon recruitment to the TCR, ZAP-70 is phosphorylated, most likely by Lck, and activated. A substrate for ZAP-70 activity is the adapter molecule LAT (linker for activation of T cells). Proteins that directly bind phospho-lat include phospholipase Cγ1 (PLCγ1), phosphatidylinositol-3-kinase (PI3K) and the adapter molecules Grb2 and Gads (30, 37-39). The Tec-family tyrosine kinases ITK Emt and Txk Rlk are also involved in PLCγ1 regulation following TcR stimulation. Another adapter molecule that participates in this signaling process through its interaction with Gads is SLP-76, which is also critical in linking early to late TCR signaling events (30, 37-39). As a result of these signaling events intracellular Ca 2+ is mobilized and stimulation of the Ras MAP (mitogen activated protein) kinase pathway occurs downstream (30, 37-39).

27 12 The action of enzymes, adapter molecules and second messengers along with costimulatory pathways regulated by CD28, leads to increased activity of transcription factors like nuclear factor (NF)-AT, AP-1 and NFκ-B and expression of proteins such as CD69, CD25 and cytokines such as IL-2, IL-4, IFN-γ (30, 37-39). Figure 1.4 present an overview of pathways activated during T cell activation. Figure 1.2 T-cell receptor (TCR) structure. The TCR protein complex is composed of a αβ or γδ dimer associated with two CD3ε, one each of CD3γ and CD3δ, and a TCRζ homodimer. The CD3 and TCRζ components of the receptor are responsible for transmitting the signal into the cell interior via a structurally conserved amino acid motif present in their cytoplasmic domains, known as immunoreceptor tyrosine-based activation motif (ITAM).

28 Figure 1.3 Models of class I peptide-mhc (pmhc I) TCR CD8 (a) and pmhc II TCR CD4 (b) ternary complex structures. At the interface of T-cell APC contact these three partners form a V -shape complex structure based on the pmhc II CD4 complex crystal structure and short stalk region in CD8. In the models, peptides are colored in orange, class I heavy chain and class II α chain in green, class I β2m and class II β chain in yellow, CD8αα in red and blue, CD4 in blue, TCR α chain in purple and TCR β chain in cyan. 13

29 Figure 1.4 Overview of In vitro T cell activation signal transduction pathways. TcRmediated signal results in the activation of various signal transduction pathways which lead to the formation of several products, including transcription factors NF-κB, NF-AT, and activated MAPK along with the production of various cytokines. In one pathway ZAP-70 activates PLCγ 1 by tyrosine phosphorylation. Activated PLCγ 1 hydrolyzes PIP2, a membrane phospholipid, to produce the second messenger DAG and IP 3. DAG, together with Ca 2+ released by the action of IP 3, collaboratively activate the PKC, which phosphorylates Iκ-B, causing the release of the transcription factor NF-κB. Ca 2+, acting with calmodulin, activates calcineurin, a phosphatase which removes a phosphate from cytoplasmic NF-AT, which then translocates to the nucleus where it acts as a potent transcription factor for a number of genes. In another pathway, ZAP-70 initiates a sequence of events that activates the Ras pathway. Ras initiates a cascade of phosphorylations, known as a MAP kinase cascade, which culminates in the activation of MAPK, which translocates in the nucleus and activates a transcription factor that upregulates transcription of the gene encoding Fos. Fos joins with Jun to form the transcription factor known as AP-1. The engagement of the costimulatory molecule CD28 by B7 also induces the activation of signal transduction pathways, one of which is the MAPK cascade initiated by the small G protein Rac. The end result of this pathway is the phosphorylation of Jun kinase (Jnk). Activated Jnk then translocates to the nucleus where it activates Jun by phosphorylation. Activated Jun joins Fos in AP-1. Together these signaling pathways increase the expression of several genes and induce the expression of others including the gene that encodes the T cell growth promoting cytokine IL-2. 14

30 The TEC Family kinase ITK ITK, a member of the Tec family of kinases which includes ITK, Txk/RLK, BTK, ETK and Tec, and is primarily expressed in T cells, NK cells and mast cells (Figure 1.5) (28-30, 37-41). Figure 1.5. Structure of ITK. Pleckstrin Homology (PH) domain is involved in protein:phospholipid interactions; Tec Homology (TH) domain consists of a proline rich (PR) region and a zinc binding domain; Src Homology domains (SH3/2/1). The SH3 domain binds to proline rich regions and can engage the PR region in an intramolecular fashion. The SH2 domain binds to phosphorylated tyrosine residues. The SH1 (kinase domain) has tyrosine-kinase catalytic activity. Tec kinases are characterized by a common domain organization, consisting of a Phospahtidyinositol-3,4,5-Triphosphate(PtdIns(3,4,5)P3)-binding Pleckstrin homology (PH) domain, followed by a Tec homology (TH) domain, then a Src homology (SH1/2/3) domains (Fig. 1.5) (28-30, 37-41). The Tec kinases are the only tyrosine kinases that have pleckstrin-homology domains, which inducibly recruit Tec-family members to the plasma membrane by binding the phosphatidylinositol-3-kinase (PI3K) product PtdIns(3,4,5)P3, thereby promoting their activation (28-30, 37-40). Activation of Tec-family kinases involves several steps: first, recruitment to the plasma membrane through interactions between their pleckstrin homology domains and the products of PI3K and/or other

31 16 proteins; phosphorylation by Src family kinases; interactions with other proteins that bring the Tec-family kinases into antigen receptor signaling complexes (28-30, 37-40). Membrane localization and activation of the PH domain-containing Tec kinases are regulated by the balance of PI3K and the lipid phosphatases PTEN (phosphatase and tensin homologue) and SHIP (SH2-domain containing inositol-5-phosphatase), which catalyze the breakdown of PtdIns (3,4,5)P3 (28-30, 37-40). Following membrane localization the activation of Tec family kinases requires phosphorylation by the Src kinase, Lck (28-30, 37-40). This activating phosphorylation event is also dependent on zeta-associated protein of 70kDa (ZAP-70) and linker for activation of T cells (LAT) (28-30, 37-40). Although the activation of ITK by TcR requires ZAP-70 and LAT, ITK is not a substrate of ZAP-70, but does bind LAT in vitro (28-30, 37-40). ITK has also been shown to bind SH2 domain-containing phosphoprotein of 76kDa (SLP-76) which binds LAT via interaction with Gads in a ZAP-70-dependent manner (28-30, 37-40). Once ITK has been activated and is bound to LAT via SLP-76, it is capable of phosphorylating phospholipase C-γ (PLC-γ) and/or interacting with the adaptors GRB2 (growth-factorreceptor-bound protein 2), NCK (non-catalytic region of tyrosine kinase) and ADAP (adhesion- and degranulation-promoting adaptor protein), and the guanine nucleotideexchange factor VAV1 (28-30, 37-40). This TCR-signaling complex is crucial for downstream effector functions, including mobilization of Ca 2+, activation of mitogenactivated protein kinases (MAPKs) and downstream transcription factors, and regulation of the actin cytoskeleton. This phosphorylation leads to the activation of PLC-γ, which then hydrolyzes PIP 2 into second messengers inositol (3, 4, 5)-triphosphate (IP 3 ) and diacylglycerol (DAG) (28-30, 37-40). The production of IP 3 results in calcium

32 17 mobilization while DAG activates protein kinase C (PKC) and Ras-GRP, thereby leading to activation of Ras/Raf/mitogen activated protein kinase (MAPK) pathways (28-30, 37-40). Subsequent to Ca 2+ mobilization, the nuclear factor for activated T cells (NFAT) transcription factors are dephosphorylated, translocate to the nucleus and activate a number of genes, including those that encode cytokines (28-30, 37-40). The activation of PKC and the Ras/MAPK pathways affect a number of serine/threonine kinases including Erk1/2, p38, and c-jun-n-terminal kinase (JNK) (28-30, 37-40). These pathways result in the activation of additional transcription factors, such as NF-κB and c-jun, which regulates genes involved in cytokine signaling, survival, and differentiation (28-30, 37-40). Thus following the activation of PLC-γ by the Tec kinases, multiple processes important for T cell development, activation, and effector function are affected (28-30, 37-40). Together, these signaling intermediates are crucial for the production of cytokines and the expression of activation markers by T cells (28-30, 37-40). Accordingly, T cells from mice that are deficient in Tec kinases show defective phosphorylation of PLC-γ, production of Ins(1,4,5)P3, influx of Ca 2+ and activation of NFATc1 (nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1) and NFATc (28-30, 37-40). Impaired activation of the MAPKs ERK (extracellular-signal-regulated kinase) and JNK (JUN amino-terminal kinase) and of the transcription factor AP1 (Activation Protein-1) are also observed in these T cells (28-30, 37-40). Mice which lack ITK exhibit the following phenotype: impaired CD4 + T-cell development (28-30, 37-40) and reduced T cell differentiation, preferentially affecting T H 2 cell differentiation, however mildly affecting Th1 responses (42-44). TCR-CD3 signaling events in vitro, including tyrosine phosphorylation and activation of PLCγ1,

33 18 calcium mobilization, reduced IL-2 production and T cell proliferation are also decreased (28-30, 37-40). These mice also have weakened immune responses to infection, such as Toxoplasma gondii that require T H 2 responses, but not to Leishmania infection that requires T H 1 responses and reduced Activation-Induced Cell Death (AICD), substantiating the importance of ITK in adaptive immunity (28-30, 37-40, 42). It has been suggested that other Tec kinases may play a compensatory role in the absence of ITK, such as the Tec kinase Rlk (Resting lymphocyte kinase; also known as TXK) (28-30, 37-41). Rlk is an atypical Tec kinase lacks a pleckstrin homology domain, having instead a palmitoylated string of cysteine residues, which leads to the constitutive membrane association of Rlk, independent of PI3K activity unlike ITK (28-30, 37-41). Although ITK and Rlk are found in T cells they are expressed at different levels and by different subpopulations with ITK being the predominant Tec kinase being expressed in naïve mouse T cells with Rlk expressing 3-10 fold lower levels (28-30, 37-41). Upon T cell activation ITK expression is increased in Th2 cells, whereas Rlk expression is increased in Th1 cells (28-30, 37-41). Mice deficient in Rlk show moderate defects in T cell function, whereas they are more severe in ITK deficient mice (28-30, 37-41). Mice lacking both Rlk and ITK show more severe defects in naïve T cell function suggesting redundancy between these two kinases. However, unlike the case with ITK null mice, ITK -/- Rlk -/- mice have been shown to have effective Th2 responses, suggesting more complex regulation of T cell differentiation by Tec family kinases (39, 41). Although biochemical and cellular defects are important in the appropriate T cell signals being transduced there may be other defects or cell types that are regulated by ITK or that regulate ITK. Along with the defects stated previously, ITK null mice have

34 19 been suggested to have abnormal development of NKT cells, which are responsible for immediate IFN-γ and IL-4 production after infection (28-30, 37-40). In addition, ITK also binds and is activated by the co-stimulatory molecule CD28, which is necessary for Th2 differentiation and may be is also another important signaling pathway that regulates the positive and negative effects of ITK (28-30, 37-40). Using SEB as a model antigen and mice lacking ITK or NKT cells the work in this thesis tests the hypothesis that ITK regulates T and NKT cell responses to SEB in vivo and that NKT cells play a role in SEB-mediated cytokine responses Tec Kinases and T Cell Differentiation and Cytokine Production There are two main classes of T cells: cytotoxic T cells and T helper cells. Cytotoxic T cells kill infected cells, whereas helper T cells help activated macrophages, B cells and cytotoxic T cells (31). Upon recognition of an antigen-mhc complex a primary response is initiated leading to the secretion of cytokines and proliferation or differentiation into memory or effector T cell populations (31). Effector CD4 + T cells carry out specialized functions and form two main populations: Th1, which secrete IL-2, IFN-γ, and TNF-β and Th2 which secrete IL-4, IL- 5, IL-6, IL-10, and IL-13 (31). The Th1 subset is responsible for cell-mediated functions, such as delayed type-hypersensitivity and the activation of cytotoxic T cells, defending an animal against intracellular pathogens (31). The Th2 subset functions effectively as a helper for B cell activation and defends animals against extracellular pathogens (31). The decision of naïve helper T cells to differentiate into Th1 or Th2 effector cells influences the type of adaptive immune response that will be mounted against the

35 20 pathogen (31, 41). The specific cytokines present during helper T cell activation determine the type of effector cell produced (31, 41). Microbes at a site of infection stimulate dendritic cells to produce specific types of cytokines (31, 41). The dendritic cell then migrates to a peripheral lymphoid organ, such as the spleen, lymph nodes or thymus, and activates naïve T helper cells to differentiate into either Th1 or Th2 effector cells, depending on the cytokines the dendritic cells produce (31, 41). If IL-12 is present Th1 development is initiated. If IL-4 is present, then Th2 cells develop (31, 41). Once Th1 or Th2 cells develop they inhibit the differentiation of the other type of T helper cell (IFN-γ produced by Th1 cells inhibits the development of Th2 cells, while IL-4 produced by Th2 cells inhibit the development of Th1 cells) (31, 41). Two transcription factors important in determining Th cell differentiation are T- bet and GATA3 (31, 41). T-bet directs IFN-γ expression by CD4 + T cells and is required for differentiation into Th1 cells (31, 41). GATA3 is the master regulator of differentiation into Th2, as it is required to remodel chromatin at the locus that encodes IL-4, IL-5, and IL-13 (31, 41). Expression of these transcription factors is regulated by both cytokines and signals from the TCR (31, 41). The role of Tec kinases in T cell differentiation is poorly understood, however it is clear that there is a role as shown in studies where over-expression of the Tec kinase Rlk increases IFN-γ production, shifting T cells towards Th1 development and the fact that Th2 populations express much less Rlk than Th2 populations (31, 41). In the case of ITK, which directs T cells towards Th2 differentiation, for which there is strong evidence, the exact mechanisms are poorly defined (31, 41).

36 21 Chapter 2 A role for the Tec family kinase ITK in regulating SEB induced Interleukin-2 production in vivo via the JNK pathway 2.1 Abstract Exposure to Staphylococcal Enterotoxin B (SEB), a bacterial superantigen secreted by the Gram-positive bacteria Staphyloccocus aureus results in the expansion, eventual clonal deletion and anergy of Vβ8 + T cells, as well as massive cytokine release, including Interleukin-2 (IL-2). This IL-2 is rapidly secreted following exposure to SEB and may contribute to the symptoms seen following exposure to this bacterial toxin. The Tec family kinase ITK has been shown to be important for the production of IL-2 by T cells stimulated in vitro and may represent a good target for blocking the production of this cytokine in vivo. In order to determine if ITK represents such a target, mice lacking ITK were analyzed for their response to SEB exposure. It was found that T cells from mice lacking ITK exhibited significantly reduced proliferative responses to SEB exposure in vitro, as well as in vivo. Examination of IL-2 production revealed that ITK null mice produced reduced levels of this cytokine in vitro, and more dramatically, in vivo. In vivo analysis of JNK pathway activation, previously shown to be critical for regulating IL-2 production, revealed that this pathway was specifically activated in SEB reactive Vβ8 + (but not non-reactive Vβ6 + ) T cells from WT

37 22 mice, but not in Vβ8 + T cells from ITK null mice. However, toxicity analysis indicated that both WT and ITK null animals were similarly affected by SEB exposure. These data show that ITK is required for IL-2 production induced by SEB in vivo, and may regulate signals leading to IL-2 production, in part by regulating activation of the JNK pathway. The data also suggest that perturbing T cell activation pathways leading to IL-2 does not necessarily lead to improved responses to SEB toxicity. 2.2 Background Superantigens (SAGs) are microbial toxins of bacterial and viral origin with the ability to activate 5-20% of the T cell population, causing T cell activation, cytokine release and systemic shock (2, 45). Most superantigens share the ability to simultaneously bind the class II major histocompatibility complex molecules and the variable region of the T cell receptor β-chain, without the need to be processed by antigen presenting cells (2, 45). Thus SEB can interact directly with MHC class II molecules on APCs and activate T cells bearing the proper TcR Vβ chains. The result of this interaction is largescale stimulation of any T cell that expresses the proper TCR Vβ chain. A number of studies have shown that when mice are challenged with a superantigen such as Staphylococcal Enterotoxin B (SEB), toxicity results from massive induction of cytokines derived from T-helper-type-1 (Th1) type cells such as IL-2, IFN-γ, TNF-α and TNF-β (2, 8). This cytokine production is accompanied by expansion of the numbers of SEB reactive T cells, followed by cell death and the induction of functional anergy (46-48).

38 23 In order for a T cell to be activated antigen presenting cells (APC) must present antigen, either an antigenic peptide or superantigen. In vitro, this interaction with the TcR has been shown to lead to the activation of a number of tyrosine kinases, including the Src family kinase Lck, the Syk family kinase Zap-70 and the Tec family kinase ITK (for review see (30, 49-51)). This then results in the activation of a number of signaling pathways including members of the MAPK family of kinases, ERK, JNK and p38, followed by transcription factor activation (51). In vivo, activation of these T cells lead to the induction of cytokine secretion within hours of SEB exposure (2, 8). ITK is expressed primarily in T cells, NK cells and mast cells (28, 52, 53). In T cells, it is rapidly activated following TcR crosslinking in vitro (54, 55). Mice lacking ITK exhibit reduced IL-2 production in vitro, and reduced T cell differentiation in vitro and in vivo, with Th2 cell differentiation preferentially affected (38, 55-58). However, these animals are not entirely immunocompromised, with residual responses against LCM, Vaccinia and VS viruses (59). We have tested whether ITK null mice are susceptible to SEB induced IL-2 secretion. We show here that mice lacking ITK have much reduced IL-2 production and T cell expansion in response to SEB in vitro and in vivo. We also show that SEB induced the activation of the JNK MAPK pathway in responding T cells in vivo, and that ITK null T cells were defective in the activation of this pathway in vivo. However, toxicity analysis indicated that both WT and ITK null animals were similarly affected by SEB exposure. Our data suggest that ITK is required for full IL-2 secretion following SEB exposure, and that this may be due to the regulation of the JNK pathway by ITK in vivo. However, reducing T cell signals do not necessarily lead to better physiological responses to SEB exposure.