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Characterization of the role of p59Fyn in T cell development and anergy Utting, Oliver B. 2001

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CHARACTERIZATION OF THE R O L E OF p59Fyn IN T C E L L DEVELOPMENT AND ANERGY by Oliver B. Utting B.Sc, University of Calgary, 1995 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF T H E REQUIREMENT FOR T H E DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Microbiology and Immunology)  We accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH COLUMBIA March 2001 © Oliver B. Utting, 2001  UBC  3/26/01 12:52 PM  Special Collections - Thesis Authorisation Form  In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced d e g r e e a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the head o f my d e p a r t m e n t o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s for f i n a n c i a l gain s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n .  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r , Canada  http://www.library.ubc.ca/spcoll/thesauth.html  Columbia  Page 1 of 1  Abstract  Signaling via the T cell receptor (TCR) is essential for the development of thymocytes and the mounting of peripheral T cell responses. Studies of proximal T C R signaling have demonstrated that two kinases, Lck and Fyn, are involved in the initial T C R phosphorylation events that eventually lead to T cell activation. Studies of these two kinases generally point to Lck having a major role in TCR signaling and to Fyn playing a secondary, perhaps redundant role. In this thesis I have investigated the hypothesis that the role Fyn plays in T cell signaling is not redundant, and that Fyn is important for both thymocyte selection and peripheral T cell responses. Using transgenic mice that express TCRs with different affinities for their ligands, I have found that Fyn plays an important role in thymocyte selection and that Fyn is required for T cell responses to low affinity ligands. T cell anergy is an important form of T cell tolerance. Fyn has often been implicated in T cell anergy, but the requirement for Fyn in this phenomenon is still unclear. I have characterized a model for in vivo induced T cell anergy that involves a mature CD4CD8" a P T C R population of peripheral T cells. Using this model system, I found that deletion +  of Fyn led to a partial recovery from the proliferation defect observed in anergic cells. These anergic cells also have an enhanced ability to survive in vitro. Enhanced survival of anergic cells was dependent on signaling through elevated expression of IL-2RJ3 in a Fyn-dependent manner. It appears that Fyn plays unique roles in the induction of T cell anergy and the survival of anergic T cells. Biochemical characterization of anergic T cells revealed signaling defects associated with incomplete phosphorylation of TCR-^, a failure to phosphorylate ZAP-70 and L A T , and a failure to mobilize intracellular calcium. Signaling via the Ras pathway is completely intact in these cells, and they have a lowered triggering threshold for activation and hyperproliferate i f exogenous IL-2 is added. These unique properties of this population of anergic T cells indicate that these cells may be re-activated by bystander immune responses.  ii  Table of Contents Abstract  "  Table of Contents  iii  List of Figures  y  List of Tables  ii i  List of Abbreviations  x  x  Acknowledgements  xiii  Chapter 1 Introduction  1  1.0.  The role of T cells in the immune response  1  1.1.  The T cell receptor  3  1.2.  T cell signaling  3  1.2.1.  T C R proximal signaling  6  1.2.2.  Src Kinases  7  1.2.3.  ZAP-70 Kinase  8  1.2.4.  L A T and SLP-76 Adaptor Proteins  9  1.2.5.  PLC-yl  10  1.2.6.  Ras  10  1.2.7.  Lipid Rafts  11  1.3.  T cell development and selection  12  1.4.  Anergy  16  1.5.  Fyn Src Kinase  19  1.5.1.  Cloning of Fyn  19  1.5.2.  Fyn knockout mice  20  1.5.3.  Fyn kinase activity  21  1.5.4.  Fyn and T cell anergy  21  1.6.  Thesis Goals.....  22  1.7.  Approach  23  1.8.  Summary of Thesis  23  1.9.  Publications arising from work in this thesis  24  iii  Chapter 2 Materials and Methods 2.0.  26  Mice  26  2.0.1.  H-2 2CMice  26  2.0.2.  H-2  2C Mice  26  2.0.3.  H-2 H - Y Mice  26  2.0.4.  Fyn"'" Mice  26  2.0.5.  Other Mice  26  2.1.  b  b/d  b  Reagents  27  2.1.1.  Antibodies against TCR components  27  2.1.2.  Antibodies against cell surface markers  27  2.1.3.  Antibodies against signaling molecules  27  2.1.4.  Secondary antibodies/reagents  28  2.1.5.  Cytokines  28  2.2.  Methods  28  2.2.1.  Isolation of lymphocytes  28  2.2.2.  Purification of CD8 cells  29  2.2.3.  Purification of Double negative cells  29  2.2.4.  Proliferation assays  30  2.2.5.  Flow cytometric analysis of lymphocytes  31  2.2.6.  Whole-cell lysate and immunoprecipitation studies  33  2.2.7.  RT-PCR  34  +  Chapter 3 Fyn plays a role in T cell development and in an optimal T cell proliferation response  38  3.0  Introduction  38  3.1  Results  39  3.1.1.  Phenotypic analysis of thymocytes and lymph node cells from 2C/2C Fyn- - and H - Y / H - Y F y n mice 7  3.1.2.  A  39  Deletion of Fyn leads to a less mature population of T cells entering periphery  42  iv  3.1.3.  CD8 SP thymocytes and lymph node cells with the Fyn" mutation are /_  hyporesponsive to low affinity antigenic ligands 3.1.4.  46  CD8 SP thymocytes and lymph node cells with the Fyn " mutation are _/  hyporesponsive to low affinity antigenic ligands 3.2  46  Discussion  52  Chapter 4 Characterization of an anergic DN T cell population  58  4.0  Introduction  58  4.1  Results  60  4.1.1.  Anergic H-2  b/d  2C D N cells proliferate in response to a low affinity ligand  and exogenous IL-2  60  4.1.2.  Impaired tyrosine phosphorylation of L A T in anergic D N cells  63  4.1.3.  ZAP-70 phosphorylation is reduced in H-2  67  4.1.4.  Anergic D N cells have impaired mobilization of intracellular calcium... 69  4.1.5.  Normal phosphorylation of SLP-76 and ERK1/2 activation in TCR-  b/d  2C D N cells  stimulated anergic D N cells 4.2  72  Discussion  76  Chapter 5 Fyn promotes the survival of anergic DN T cells but negatively regulates their proliferation response to antigenic stimulation  80  5.0  Introduction  80  5.1  Results  81  5.1.1.  The deletion of Fyn from anergic D N cells leads to partial recovery of their proliferation defect  81  5.1.2.  H-2  b/d  Fyn" " D N cells do not have improved IL-2 production  81  5.1.3.  H-2  b/d  Fyn"'" D N cells display enhanced upregulation of the high affinity  7  IL-2 receptor 5.1.4.  82  Elevated Expression of IL-2R(3 on H - 2 D N cells and their enhanced b/d  survival in culture 5.1.5.  IL-15 or IL-2 interacts with IL-2R(3 to promote the survival of H-2 cells  86 b/d  DN 89  5.2  Discussion  93  Chapter 6 Final discussion and future studies  98  6.0  Fyn and low affinity interactions  98  6.1  Fyn and the upregulation of IL-2 receptors  100  6.2  Signaling in anergic D N cells  101  6.3  Double negative cells  102  References  104  VI  List of Figures Figure 1.1. Schematic representation of the T cell receptor complex  4  Figure 1.2 Schematic depicting a simplified view of TCR signaling T C R signaling leading to IL-2 gene transcription  5  Figure 3.1. Increased CD8 expression and numbers of DP thymocytes in the 2C Fyn"'" but not H - Y Fyn"'" mice  40  Figure 3.2. Increased CD8 and H - Y T C R expression by thymocytes from male H-2 H - Y b  TCR transgenic mice with the Fyn"'" mutation  44  Figure 3.3. CD8 SP Fyn"'" thymocytes maintained elevated HSA expression levels  45  Figure 3.4. Down-regulation of HSA by CD8 SP peripheral T cells from Fyn"'" mice.... 47 Figure 3.5. Fyn"'" thymocytes expressing the H - Y T C R are hyporesponsive to CD3 stimulation  48  Figure 3.6. H - Y Fyn"'" thymocytes and lymph node cells are hyporesponsive to anti-male stimulation  50  Figure 3.7. 2C Fyn'" thymocytes and lymph node cells exhibit normal proliferative responses to stimulation by BDF1 spleen cells  51  Figure 3.8. 2C Fyn"'" thymocytes are hyporesponsive to stimulation by the high affinity L /p2Ca ligand at low ligand density  53  d  Figure 3.9. 2C Fyn"'" thymocytes and lymph node cells are hyporesponsive to a low affinity ligand  54  Figure 4.1. Exogenous IL-2 restores the proliferation response of Ag-stimulated H-2 D N cells Figure 4.2. D N cells from H-2  b/d  2C 61  b/d  2C mice are activated by the low-affinity p2Ca/K  ligand  b  62  Figure 4.3. Induction of CD25 and CD69 by low-affinity ligands in anergic D N cells... 64 Figure 4.4. L A T phosphorylation is defective in TCR-stimulated anergic D N cells  65  Figure 4.5. Hypophosphorylation of ZAP-70 in TCR-stimulated anergic D N cells  68  vii  Figure 4.6. Phosphorylation and expression levels of TCR^ in D N cells from H-2 2C and b  H-2  b/d  2C mice  70  Figure 4.7. Fyn, but not Lck, is expressed at higher levels in D N cells from H-2  b/d  2C  mice  71  Figure 4.8. Less efficient mobilization of intracellular calcium in TCR-stimulated H-2 2C D N cells  b/d  73  Figure 4.9. Normal phosphorylation of SLP-76 in H-2 Figure 4.10. Normal activation of ERK1/2 in H-2  b/d  b/d  2C D N cells  74  2C D N cells  75  Figure 5.1. Deletion of Fyn allows enhanced proliferation of anergic H - 2 D N cells.... 83 b/d  Figure 5.2. Defective IL-2 production by H-2  b/d  D N cells is not rescued by deletion of  Fyn  84  Figure 5.3. Efficient upregulation of CD25 on antigen-stimulated H-2  b/d  Fyn D N cells. /_  85 Figure 5.4. Enhanced responsiveness to IL-2 accounts for the reversal of the proliferation defect in H-2  b/d  Fyn" D N cells  87  A  Figure 5.5. Immediately ex-vivo H-2  b/d  D N have elevated IL-2RP expression  Figure 5.6. Enhanced survival of cultured H-2  b/d  88  D N cells is dependent on Fyn  Figure 5.7. IL-2 and IL-15 enhance the survival of cultured D N cells  90 91  Figure 5.8. Blastogenic response of H - 2 D N cells to IL-15 impaired by Fyn mutation. b/d  /_  92 Figure 5.9. Enhanced survival of H - 2 D N cells is blocked by anti-IL-2R(3 antibody.... 94 b/d  viii  List of Tables Table 2.1 Antibody concentrations and dilutions for Western Blotting and Immunprecipitations 35 Table 3 1 Effect of Fyn on the proportions of thymocyte subsets in TCR transgenic mice. 43  ix  List of Abbreviations  2C TCR  Transgenic T C R specific for the p2Ca peptide presented by K and L class I b  d  MHC Ab  Antibody  Ag  Antigen  AP-1  Activator protein 1  APC  Antigen presenting cell  APL  Altered peptide ligand  CD  Cluster of differentiation  CDR  Complementarity-determining region  ConA  Conconavalin A  DAG  Diacylglycerol  DMSO  Dimethyl Sulfoxide  DN  Double negative  DP  Double positive  ERK  Extra-cellular signal regulated protein kinase  F,  Filial 1  Fab  Fragment of antibody with antigen binding site  FACS  Fluorescence activated cell sorter  FITC  Fluorescein Isothiocyanate  Fluo-3-AM  Fluo-3-acetoxymethyl ester  GAP  GTPase activating proteins  GEF  Guanine nucleotide exchange factors  Grb2  Growth factor receptor bound 2  H-2  Histocompatibility-2  HPRT  Hypoxanthine phosphoribosyltransferase  x  HRP  Horseradish peroxidase  HSA  Heat stable antigen  H - Y TCR  Transgenic TCR specific for a male peptide presented by D class I M H C b  Ig  Immunoglobulin  IL  Interleukin  IP  Inositol 1,4,5-trisphosphate  3  IT A M  Immunoreceptor tyro sine-based activation motif  JNK  c-Jun N-terminal kinase  LAT  Linker for activation of T cells  LB  Luria-Bertani  mAb  Monoclonal antibody  MAPK  Mitogen activated protein kinase  MHC  Major histocompatibility complex  NFAT  Nuclear factor of activated T cells  p2Ca  The peptide LSPFPFDL (single letter amino acid code)  pg PIP  Phycoerythrin Phosphatidylinositol 4,5-bisphosphate  2  PKC  Protein kinase C  PTK  Protein tyrosine kinase  PTPase  Protein tyrosine phosphatase  pTa  Pre-Ta  rIL-2  Recombinant IL-2  SEA  Staphylococcal enterotoxin A  SH  Src homology  SOS  Son of sevenless  SP  Single positive  TAP  Transporters associated with antigen processing  xi  T cell receptor Thymidine deoxyribose  Acknowledgements  I would like to thank a number of people for their help over the course of my graduate work. Specifically, I would like to give special thanks to my supervisor Dr. Hung-Sia Teh for his guidance and patience over the years; to Soo-Jeet Teh for several key observations that I was able to follow up on and also for an incredible amount of help over the years— without which much of this work would not have been possible; to Dr. John Priatel for helpful discussions, for making the initial observations regarding IL-2R0 expression that I was able to follow up on, and for his assistance with RT-PCR; to Simon Ip and Edward K i m for their work as technicians in the lab; and to all past and present members of the Teh lab. I would also like to thank my supervisory committee (Drs. Mike Gold, Linda Matsuuchi and Pauline Johnson) for their advice and help. I would also like to thank the following individuals for key reagents: Dr. Eisen for the 1B2 hybridoma, Dr. Loh for H 2 2C breeders, Dr. Perlmutter for Fyn" mice, Dr. Koretzky for anti-SLP-76 antibody, Dr. b  A  Cresswell for the T2-L and K cell lines, and Dr. Locksely for the pPQRS competitors. d  b  xiii  Chapter 1 Introduction  1.0.  The role of T cells in the immune response The immune response is broadly divided into an innate response and an adaptive  response. The innate immune response provides the first line of defense against many pathogens and also plays a role in initiating the adaptive immune response. Lymphocytes make up an important arm of the adaptive immune response, with B cells being critical to humoral immunity and T cells playing a role in both humoral and cell mediated immunity. To help mount these varied immune responses there are two types of T cell, those that express the CD4 coreceptor and those that express the CD8 coreceptor (for more details on background information in this section please see (1)). T cells recognize antigenic peptides that are presented by major histocompatibility molecules (MHC). These peptides are derived by the processing of protein molecules from distinct cellular compartments. Peptides derived from cytosolic proteins are processed by a protease complex called the proteasome. Peptides derived from cytosolic proteins are loaded onto M H C I molecules by the transporter associated with antigen processing (TAP) 1 and 2 molecules. This loading is done while the M H C molecules are in the lumen of the endoplasmic reticulum, and once loaded M H C I molecules are transported via the Golgi complex to the surface of the cell. CD8 T cells recognize M H C I and the expression of the M H C I molecules on most +  tissue types complements the role of CD8 T cells in their surveillance of the body's +  tissues. Immunological protection offered by CD8 T cells is mainly against viruses that +  have gained entry into cells. Peptides derived from viral proteins are usually presented by M H C I molecules. Recognition of these peptide/MHC complexes on antigen presenting cells (APC) by T cells can initiate the differentiation of CD8 T cells into killer cells, and +  can elicit the killer response of mature cytotoxic CD8 cells. Cytotoxic CD8 cells kill +  +  their targets by triggering apoptosis. This triggering is mediated either by lytic granules that contain perforin and granzymes, or by the expression of the ligand for Fas (FasL).  1  Both of these methods can initiate the target cell's own apoptotic pathway, and lead to the rapid destruction of the virally infected cell. The peptides presented by M H C II molecules are derived from proteins present in vesicles. These proteins may be derived from pathogens that replicate in intracellular vesicles or from pathogens that have recently been phagocytosed by phagocytes. Phagocytic vesicles are also known as endosomes. These endosomes become increasingly acidic as they move to the interior of the cell. Within these vesicles are proteases that are activated at low pH. It is these proteases that degrade the proteins found in endosomes and create the peptides to be presented by M H C II molecules. CD4 T cells +  recognize M H C II molecules presenting peptides derived from this extracellular processing pathway. CD4 T cells responding to M H C II molecules can differentiate into T 1 or T 2 cells, +  H  H  depending on the avidity of the TCR/MHC-peptide interaction and the cytokine environment. For example, IL-12 promotes the generation of a T 1 response, whereas ILH  4 promotes a T 2 response. T 1 cells offer defense largely against pathogens that infect H  H  macrophages. Within these phagocytic cells, pathogens can replicate in intracellular vesicles. T 1 CD4 recognize M H C class II/peptide ligands on macrophages and deliver +  H  activating signals to macrophages. These activating signals include CD40 ligand and cytokines such as IFN-y, GM-CSF and TNF-a. Activated macrophages are able to eliminate intracellular pathogens much more efficiently that non-activated ones. T 2 cells are largely involved in facilitating the humoral response by providing help H  to B cells. A B cell interacts with an antigen via its antigen receptor, takes up this antigen and processes it via the extracellular processing pathway, and presents peptide fragments of the protein to T cells via M H C II molecules. T 2 CD4 T cells then provide the B cell +  H  with the necessary costimulation (CD40/CD40 ligand interactions) and cytokines (IL-4, IL-5 and IL-6) required for differentiation and antibody production. These antibodies are critical to the body's defense against extracellular pathogens. The T cell functions discussed above require specific recognition of M H C molecules and the peptides they present. T cells recognize these peptide/MHC complexes using their T cell receptor.  2  1.1.  The T cell receptor The T cell receptor (TCR) expressed on thymocytes and mature T cells is formed by  an a and a p chain, linked by a disulfide bond (1). The structure of each of these chains consists of two immunoglobulin-like domains, along with a highly variable aminoterminal region. These chains are largely extracellular, with only a short two to seven amino acid tail stretching into the cytoplasm. The signaling function of the TCR is mediated not by the a and (3 chains, but by the components of the non-covalently associated CD3 complex. The CD3 complex consists of invariant accessory chains, CD3y, CD35, CD3e and the TCR-C, chains. The TCR/CD3 complex is organized as follows: TCRocp, CD3e5, CD37E and T C R ^ and is depicted in Figure 1.1. Within the cytoplasmic tails of the CD3 molecules are protein motifs critical to the signaling function of the TCR. These motifs are known as immunoreceptor-tyrosine based activation motifs, or ITAMs. The I T A M motif consists of tyrosine residues flanked by specific sequences (YXXL/I-(X) _ -YXXL/I (2) (single letter amino acid code amino 6  8  acid, with X being any amino acid). Each of the CD3 chains contains one I T A M , while the TCR-^ chains each contain three, bringing the total for each CD3 complex to ten. The ITAMs are critical to signaling from the TCR, with their modification by the addition of a phosphate to the tyrosine residues by kinases being essential to TCR signaling.  1.2.  T cell signaling A T cell must be able to "know" when its TCR has interacted with an appropriate  ligand. Signals are sent from the TCR to the nucleus of the cell via a complex pathway of signaling events. I will briefly introduce this pathway before giving details on the molecules relevant to this thesis (references are given below, in the relevant sections). Key molecules are also depicted in Figure 1.2. Upon interaction of the TCR with an appropriate ligand, the ITAMs of the CD3 chains are phosphorylated by protein tyrosine kinases of the Src family. These phosphorylation events allow for the recruitment of another protein tyrosine kinase, ZAP-70. Once recruited, ZAP-70 kinase activity is activated and ZAP-70 can phosphorylate L A T . L A T is a transmembrane protein that  3  T cell membrane  T T or + = Charge on Transmembrane Domain j = Disulphide Bond ITAM  = Ig like domain  Figure 1.1. Schematic representation of the T cell receptor complex  As described in the text, the TCR complex consists of the TCR itself, and the associated CD3 signaling molecules.  4  Figure 1.2 Schematic depicting a simplified view of TCR signaling leading to IL-2 gene transcription. Please see text for details  5  recruits numerous other proteins to the cell membrane. Two of the recruited proteins are PLC-yl and Grb2. PLC-yl hydrolyzes a membrane phospholipid to generate second messengers. One of these second messengers stimulates the release of intracellular calcium, which in turn allows for the activation of calcineurin and the eventual activation of the N F A T transcription factor. The recruitment of Grb2 allows for the recruitment of SOS to the membrane. SOS is an exchange factor for Ras, and recruiting SOS allows for the TCR to activate the Ras pathway. The activation of Ras signaling ultimately leads to the activation of transcription.  1.2.1.  TCR proximal signaling  As mentioned above (section 1.1), the cytoplasmic tails of the TCRap chains are only 1  two to seven amino acids long and can not transduce TCR signals on their own. The associated CD3 complex, with its extended cytoplasmic tails, allows for the communication of the TCR with the interior of the cell (2). Modification of the tyrosine residues within the IT A M motifs in the TCR/CD3 chains by phosphorylation is the earliest known indicator of TCR triggering. Phosphorylation of the TCR-^ chains leads to the generation of different molecular weight phospho-isoforms depending on the number of ITAMs that have been phosphorylated. These isoforms include the partially phosphorylated p21, and the fully phosphorylated p23. The importance of the phosphorylation of individual tyrosine residues has recently been examined by sitedirected mutagenesis of individual or of combinations of tyrosines. The p21 form was shown to phosphorylated on the four C-terminal tyrosines (two tyrosines per ITAM) (referred to as tyrosines 3-6), and the p23 form requires phosphorylation of all six tyrosines in the three ITAMs of each ^-chain (3). Incomplete phosphorylation of the TCR-^ chains has recently been shown to have an inhibitory effect on TCR signaling (4).These researchers transfected CD8/^ hybrids (either wildtype or various I T A M tyrosine to phenylalanine mutants) into non-immortalized T cells and found that partially phosphorylated ITAMs (constructs with mutated ITAMs) had an inhibitory effect on the T cell proliferation response. These results suggested that partial phosphorylation of the ^-chain may play a regulatory role.  6  TCR transgenic mice that lack functional ^-chains have been produced (5, 6), though the outcome of these experiments differ. In the former, H - Y TCR transgenic mice (see below for details) showed a dependence on ^-chains for positive and negative selection. In the latter, P14 transgenic mice lacking functional ^-chains were analyzed and were found to not require ^-chains for development. In addition, the development of peripheral T cells in these mice allowed for more detailed analysis of T cell functions such as proliferation and cytokine production (IL-2 and IFN-y were studied). These functions were found to be largely normal, although IFN-y production was reduced. These results suggest that CD3-y5e chains can also mediate most TCR signals in the place of ^-chains, though the data from the H - Y mice indicate that this is not true for TCRs of all affinities. The initial addition of phosphate residues to the ITAMs is mediated by kinases from the Src family. 1.2.2.  Src Kinases  Src kinase family members are non-receptor protein tyrosine kinases. Src was initially discovered based on its ability to act as the dominant oncogenes of transforming retroviruses (7). Initial work demonstrated that \-src (as well as other virus associated oncogenes) had acquired the ability to transform based on mutations that allowed for deregulation of kinase activity. The Src kinase family now counts among its members Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk (reviewed in (8)). The Src kinases all share common characteristics such as domains, referred to as Src homology domains, or SH domains. While SHI refers to the kinase domain, SH2 domains are involved in phosphotyrosine interactions. SH2 domains are protein modules of-100 amino acids that recognize phosphotyrosine residue containing peptides in the context of 3-6 carboxyterminal amino acids (9-11). Most SH2 domains require phosphorylation of their peptide ligand for high affinity binding, but SH2 domains vary in their ability to recognize residues carboxy-terminal to the phosphorylated tyrosine. This allows for SH2 domains on different proteins to have distinct affinities for phosphorylated tyrosines based on the surrounding motifs (12). SH3 domains are similar to SH2 domains in that they recognize residues in the context of their carboxy-terminal amino acid sequences, but SH3 domains function in the recognition of poly-proline motifs. Both SH2 and SH3 domains are  7  critical to TCR signaling in terms of the assembly of multi-molecular signaling complexes (reviewed in (13)), such as those assembled around the adaptor protein L A T (see below). Of critical importance to TCR signaling are the Src family kinases Lck and Fyn. Both of these kinases have been shown to be able to phosphorylate ITAMs on the TCR/CD3 complex. Transfection experiments in COS-18 cells demonstrated that co-expression of either Lck or Fyn with ZAP-70 results in an increase in the phosphorylation of the ITAMs of a CD8-^ chimera (14). It is these phosphorylation events that are critical to the initiation of TCR signaling. Lck, by virtue of its association with the CD4 and CD8 coreceptors (15), is believed to come into close proximity with the TCR chains during the initial T C R / M H C interaction. The importance of Lck to T cells was underscored by the production of mice lacking Lck in which T cell development was severely hampered and very few T cells were found in the periphery (16). Besides phosphorylating ITAMs, Lck can also phosphorylate and activate ZAP-70 (17). This was demonstrated by overexpression studies in which ZAP-70 activity required co-expression with Lck. Although Lck can perform many of the signaling functions required by the TCR, it has become clear that another kinase also plays a role in proximal T C R signaling. This second Src kinase intimately involved in TCR signaling is Fyn. As this kinase is a major focus of this thesis Fyn will be discussed in detail below (see 1.5). 1.2.3.  ZAP-70 Kinase  Once a chain in the TCR/CD3 complex has been doubly phosphorylated on adjacent I T A M tyrosines by Lck or Fyn, ZAP-70 can interact with the TCR chains by virtue of its tandem SH2 domains (14, 18). This recruitment is essential to ZAP-70 activation . Agents that can block ZAP-70 recruitment prevent ZAP-70 activation (19). ZAP-70 signaling is critical to T cells, since humans and mice that lack ZAP-70 expression have severely compromised immune systems (20, 21). Once ZAP-70 has bound the ITAMs, it can be phosphorylated on tyrosine 493 by Lck or Fyn, allowing for full ZAP-70 tyrosine kinase activity (17). ZAP-70 has several phosphorylation sites other than tyrosine 493 (reviewed in (13)). These include sites of negative regulation such as tyrosine 292, a  8  residue that can be bound by the negative regulator c-Cbl (22, 23). Activated ZAP-70 can phosphorylate the adapter molecules L A T (24) and SLP-76 (25).  1.2.4.  L A T and SLP-76 Adaptor Proteins  L A T (linker for activation of T cells) is a palmitoylated transmembrane protein expressed by (26) T cells, mast cells, N K cells and megakaryocytes (notably, L A T is not expressed in B cells) (24). L A T is phosphorylated by ZAP-70 and upon its phosphorylation can interact with P L C - y l , Grb2, Gads and Grap via their SH2 domains (24). L A T can also interact with Vav, c-Cbl and SLP-76 indirectly, perhaps by virtue of these molecule's interactions with Grb2 (reviewed in (27)). That L A T plays a crucial role in T cell development is demonstrated by data from LAT" ~ mice (28) showing T cell development to be blocked at the double negative stage (CD4"CD8"CD25 CD44") in these +  mice. In Jurkat cells with very low expression of L A T , the phosphorylation of P L C - y l , SLP-76 and Vav are reduced upon TCR stimulation, and activation of M A P K and the flux of intracellular calcium are defective (29). Ultimately, the upregulation of the CD69 (an activation marker) and the activation of the transcription factors AP-1 and N F A T are disrupted in these cells (29). Recent data from altered peptide ligand stimulation of T cell clones reveals a failure to phosphorylate L A T in these cells, but a maintained ability to phosphorylate SLP-76. This data suggests that L A T phosphorylation is not essential for SLP-76 activity (30) (see chapter 4 for more discussion). SLP-76, a cytoplasmic adapter protein, is also deemed essential for T cell development, with SLP-76 mice showing a T cell developmental arrest at the double A  negative stage (CD4"CD8"CD25 CD44) (31, 32). Jurkat cells lacking SLP-76 expression +  have a defect in Ras activation, in the upregulation of CD69 and in N F A T activity at the IL-2 promoter upon TCR ligation (33). Exactly how SLP-76 is integrated into TCR signaling remains to be elucidated, though it has recently been demonstrated that L A T may serve to recruit SLP-76 to the membrane compartments known as lipid rafts (see below) (34). This report suggests that once L A T is phosphorylated, the adapter molecule Gads is able to bring SLP-76 to L A T , and therefore into close proximity with the membrane and to the membrane microdomains.  9  1.2.5.  PLC-yl  One of the key signaling molecules that interacts with L A T is P L C - y l . This interaction requires the phosphorylation of L A T (most likely on tyrosine 132 of L A T ) and is mediated by PLC-yl's N-terminal SH2 domain (35). The interaction of P L C - y l with L A T has been shown to be crucial for the phosphorylation and activation of PLC-yl (29, 36). ZAP-70 is believed to be a candidate for the kinase that phosphorylates and activates P L C - y l , as mutation of ZAP-70 at tyrosine 319 prevents the activation of PLCyl in Jurkat cells (37). Once activated, PLC-yl hydrolyzes the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP ) to generate the products inositol 1,4,52  trisphosphate (IP ) and diacylglycerol (DAG). IP stimulates the release of intracellular 3  3  calcium stores, which in turn leads to the activation of calcineurin (reviewed in (38)). Calcineurin dephosphorylates the cytoplasmic pNFAT, allowing this transcription factor to enter the nucleus. Activation specific T cell genes, including IL-2, IL-3, IL-4, G M CSF and TNFoc have been found to have N F A T transcriptional regulatory regions within their promoters (reviewed in (39)). D A G stimulates the activation of protein kinase C (PKC)(40). P K C has been shown to be important in the activation of numerous transcription factors, including AP-1 and N F A T (reviewed in (41)). The functional nuclear N F A T transcription factor actually consists of N F A T (which is found in resting T cells), and a nuclear factor that is induced upon T cell stimulation. This nuclear factor is the AP-1 complex, which is composed of dimers of the Fos and Jun proteins (42).  1.2.6. Ras Ras is a membrane bound guanine nucleotide binding protein that is an important regulator of T cell activation. Ras is regulated by guanine nucleotide exchange factors (GEFs) which promote the transition of Ras to an active GTP-bound state, and by GTPase activating proteins (GAPs) which stimulate Ras's GTPase activity, resulting in hydrolysis of the bound GTP. Activated Ras recruits the serine/threonine kinase Raf-1 to the cell membrane where it is activated. Activated Raf-1, in turn, can activate M E K ,  10  which activates Erkl and Erk2 (extra-cellular signal regulated protein kinases, also known as mitogen activated protein kinases, or MAPKs). The Ras pathway acts on several important transcription factors including Elk-1, the serum response factor, AP-1 and members of the N F A T family (reviewed in (43). L A T provides a scaffolding for the assembly of Ras-activating proteins at the cell membrane. Within the protein sequence of L A T are several Grb2 binding motifs. Grb2 is able to recruit the Ras exchange factor SOS via interactions between Grb2's amino terminal SH3 domain and SOS (reviewed in (44)). SLP-76 is also believed to be involved in the activation of Ras, but the exact mechanism is still unclear (45). Recently a second Ras exchange factor as been found to be important for the activation of Ras in T cells. RasGRP (Ras guanyl-releasing protein) was described in 1998 (46). Along with the catalytic domain, the sequence of RasGRP revealed a D A G binding domain. Using mutants that lacked the D A G binding domain, these researchers demonstrated that RasGRP is recruited to the membrane by virtue of its interaction with D A G . RasGRP"'" mice revealed a deficiency in single positive thymocytes, and double positive thymocytes showed an inability to activate Erk in response to TCR stimulation (47). The relationship between Grb-2/SOS activation of Ras and RasGRP activation of Ras will be discussed below.  1.2.7.  Lipid Rafts  While the above discussion of TCR signaling focuses on some of the molecules involved in the signaling process, structural aspects of the cell membrane also play a role in T cell signaling. The plasma membrane contains defined lipid microenvironments that are enriched for sphingolipids and cholesterol. These regions will be referred to here as lipid rafts, but they are also known as detergent insoluble membranes, glycosphingolipid enriched domains, and detergent insoluble glycolipid-rich membranes. The saturated hydrocarbon chains of the sphingolipids in lipid rafts create an area distinct from the remaining membrane composed of unsaturated phospholipids. Certain proteins have differing affinities for the lipid rafts. L A T is constitutively associated with rafts, and this association requires L A T palmitoylation (26). By mutating two membrane proximal cysteines in L A T , researchers demonstrated not only that L A T palmitoylation is essential to its targeting to lipid rafts, but that without this targeting the 11  phosphorylation of L A T upon TCR ligation was lost. Using sucrose gradients to isolate lipid rafts, Xavier et al. demonstrated that the TCR-^ chain targets to rafts upon T C R ligation (48). In addition to the relocalization of TCR-^, these researchers also found that the Src kinases Lck and Fyn were constitutively associated with the rafts, while ZAP-70 was recruited upon TCR ligation. While disruption of lipid rafts (for example by cholesterol depletion (48)) attenuates TCR signaling, it is still not clear how exactly lipid rafts contribute to receptor signaling. Several theories have been proposed and these are either based on signaling being mediated by single rafts or by the aggregation of rafts (for review see (49)). In the single raft theory, either molecules within a raft are aggregated by receptor ligation, or receptor aggregation leads to the recruitment (or exclusion) of signaling molecules to the raft. The raft aggregation theory proposes that receptor ligation leads to the aggregation of several rafts and the mixing of varied proteins from the different rafts. This mixing of different raft contents would lead to the activation of signaling events.  1.3.  T cell development and selection Functional T cells are derived from hematopoietic stem cells that reside in the bone  marrow. Pro-T cells are derived from these hematopoietic stem cells, and these pro-T cells migrate to the thymus, a primary lymphoid organ located in the thorax just above the heart. The thymic stroma triggers the differentiation and proliferation of T cell precursors into mature T cells. This maturation process takes the T cell precursors through different stages of development characterized by distinctive patterns of surface marker expression, and culminating in the development of T cells expressing TCRoc(3 and either CD4 or CD8. One of the earliest points along the developmental pathway of a thymocyte is the expression of the CD44 adhesion molecule. At this point the thymocyte resides in the outer regions of the thymus, known as the thymic cortex. Thymocytes at this stage of development are known as double negative thymocytes for their lack of both the CD4 and CD8 coreceptors, though they also do not express the TCRa|3 chains. Thymocytes go through a CD44 CD25 stage during which the rearrangement of the +  +  TCRfJ chain occurs (CD25 is a member of the high affinity IL-2 receptor complex and is also referred to as IL-2Roc). Once successfully rearranged, the TCR(3 chain pairs with the  12  pre-Toc (pTa) chain, as well as TCR-^ and the CD3 complex. This pairing allows for the expression of the pre-TCR on the surface of the CD44~CD25 thymocyte (reviewed in +  (50)). Expression of the pre-TCR complex on the thymocyte surface leads to abrogation of the TCRJ3 chain gene rearrangement, a phenomenon termed allelic exclusion. Signaling through the pre-TCR drives the differentiation of the thymocyte to the CD44" CD25"stage. At this point the thymocyte also begins to express both the CD4 and CD8 coreceptors and is known as a double positive thymocyte (DP). DP thymocytes rearrange their T C R a chain genes, and a successfully rearranged a chain pairs with the (3 chain to form the TCR. It is this population of immature DP TCRocp expressing cells that are subject to thymic selection (51, 52). The terms positive and negative selection describe the processes by which a developing thymocyte is stringently tested for the M H C restriction and the self reactivity of its TCR. These processes are necessary because of the random generation of TCR specificity by the rearrangement of the V , D and J gene segments that make up the complementarity-determining regions (CDRs) of the amino-terminal region of the TCR a and (3 chains (1). Positive selection was discovered in classical bone marrow chimera mice experiments. In these experiments, bone marrow from mice heterozygous at the M H C locus was used to rescue lethally irradiated mice that were homozygous at the M H C locus. It was found that the responses of T cells from such chimeric mice are limited by the M H C of the host, suggesting the thymocytes learn to recognized M H C during development and that they are not preprogrammed (reviewed in (51)). A large body of work on T cell selection has been done with TCR transgenic mice. As an example I will discuss the H - Y TCR, a transgenic system used in some of the work described in this thesis. This TCR transgenic mouse was produced using the rearranged TCRaP chains from a CD4"CD8 cytolytic T cell clone specific for the male minor +  histocompatibility antigen (H-Y) presented in the context of M H C I (H-2D ) (53). These b  mice revealed the deletion of autoreactive thymocytes when the transgenic TCR was expressed in male mice, and the positive selection of CD4"CD8 thymocytes in female +  mice. As well, in female mice expressing the TCR transgene it was discovered that the positive selection of the H - Y receptor proceeded only in H-2 mice but not in H-2 or H b  k  2 mice (54). The M H C mismatched mice there was a higher incidence of CD4 CD8" T d  +  cells expressing the transgenic TCR P chain but an endogenous TCR a chain. From these observations it was concluded that the selection of the transgenic receptor required the  13  expression of the appropriate M H C haplotype. While these experiments and others clarified the importance of the T C R / M H C interaction to thymocyte selection, the signals involved in positive and negative selection were not clear. Models have been proposed to explain how the signaling in positive and negative selection works. In the qualitative model (55, 56), positive and negative selection are mediated by peptides that differ from one another qualitatively. The positively selecting peptide is an antagonist of mature T cell proliferation, and the negatively selecting peptide is an agonist of mature T cell function. The antagonist peptide is believed to engage the TCR without inducing a conformational change critical to TCR signaling, whereas agonist peptides induce the necessary conformational changes for TCR signaling (55, 56). Evidence for this model comes from fetal thymic organ cultures (FTOC) in which thymic lobes from fetal mice, consisting largely of double negative T cells, are cultured under conditions that allow the addition of peptides. Using FTOC and a transgenic TCR, Hogquist et al. (57) found that antagonist peptides were able to induce positive selection, whereas peptides defined as agonists induced negative selection. Later, using the same system, Hogquist et al. found that even low concentrations of agonist peptides could not mediate positive selection (58). However, in this report, low concentrations of a weaker agonist were capable of inducing some positive selection, though it was demonstrated that the positively selected cells were not functional when challenged with antigenic peptide in a proliferation assay. The quantitative model of selection (59, 60) argues that it is the avidity of the selecting ligand that is important. The avidity of the T C R / M H C interaction takes into account the number of T C R / M H C interactions and their collective affinities. The T C R / M H C affinity is typically expressed as a K value, determined as the concentration d  of ligand required to achieve half maximal receptor saturation. This model predicts that negative selection is induced by T C R / M H C interactions of high avidity, whereas positive selection is induced by relatively weak interactions. In the quantitative model, signals from multiple T C R / M H C interactions are integrated to form a signaling gradient that will ultimately dictate the outcome of the T C R / M H C interaction. In FTOC systems, the addition of a broad range on peptide concentrations allows for either the positive or negative selection of thymocytes, depending on the concentration used. Higher concentrations of peptide mediate negative selection, whereas low concentrations induce positive selection (61, 62). Despite differences in observations between the studies on qualitative or quantitative models, the data are consistent with the quantitative model for thymic positive and negative selection (51).  14  While the quantitative model of selection suggests that the avidity of the TCR interaction is critical to thymocyte selection other factors influencing thymocyte/ligand interactions are also important. Experiments done with the 2C TCR transgenic mice have provided insight into the importance of CD8 expression and selection (please see section 1.7 from details on the 2C TCR transgenic mice). With a two fold increase in the surface expression levels of CD8 on thymocytes, the positively selecting H-2 background was b  converted to a negatively selecting one (63). This observation demonstrates that enhancing the ability of the thymocyte to recognize M H C can lead to conversion of a positive selecting ligand to a negative one and demonstrates that changes in coreceptor expression levels are important to thymocyte selection. Recent work aimed at understanding the signaling involved in thymocyte selection has suggested how the signals of positive and negative selection might differ. Mice heterozygous for the adaptor protein Grb2 have revealed differences in the activation thresholds of Erk, JNK and p38 kinases (64). In these mice, the negative selection of H - Y TCR transgenic thymocytes in male mice was hampered by the Grb2 insufficiency, while positive selection in female mice was normal. As well, evidence was provided demonstrating the decreased activation of Ras in Grb2 " thymocytes. This data, in +/  conjunction with the observed decrease in JNK and p38 activation but normal Erk activation, suggests that these various M A P K family members have different thresholds of activation. These different thresholds of activation may be critical to the thymocyte distinguishing the signals for positive and negative selection, with Erk signaling being required for positive selection, and with signals mediated by Erk, JNK and p38 being important to negative selection. It has recently been demonstrated that RasGrp is essential for positive selection of thymocytes, and in RasGrp " thymocytes there is a marked defect in Erk activation (47). 7  The combination of the observations from Grb2 " mice and RasGrp"'" suggest a possible +/  model for the consequences of TCR engagement during thymocyte selection (discussed in (65)). This model proposes that the signals for positive and negative selection require sustained versus transient Erk activation, respectively. The strong but transient signals of negative selection would be mediated by Grb2-SOS dependent activation of Erk, JNK and p38. The weak but sustained stimulation of positive selection would be mediated by RasGRP.  15  1.4.  Anergy The interaction of the TCR with peptide/MHC complexes and the signals these  interactions produce are not sufficient for the generation of an immune response. Brescher and Cohn (66) theorized that the activation of lymphocytes must require two signals. Without this requirement for lymphocyte activation, they argued that autoimmune responses would occur when a single self-reactive lymphocyte was produced. The interaction of CD28 with its ligands CD80 or CD86 (B7.1 and B7.2 respectively) can provide the necessary costimulation for full T cell activation (67). The expression of B7.1 and B7.2 is restricted to professional antigen presenting cells and activated APCs and T cells (reviewed in (68)). Thus the restriction of the expression of costimulatory molecules helps insure the maintenance of self tolerance. The ligation of the TCR in conjunction with costimulation allows for the efficient activation of ' transcription factors required for proliferation and differentiation of naive T cells into effector cells. While the ligation of the TCR in conjunction with costimulation leads to the effector responses, TCR ligation on its own results in the induction of a non-responsive, or anergic, state. In 1987 Jenkins and Schwartz (69) observed that exposure of T cell clones or T cells to antigen presenting cells that have been chemically fixed with l-ethyl-3-(3dimethylaminopropyl) carbodiimide (ECDI) results in an unresponsive T cell state that is characterized by the inability of the T cells to proliferate. The ECDI fixing of the APCs was believed to have inactivated the ability of the APCs to provide the necessary accessory signals to fully activate T cells. At the time it was not known what the ECDI sensitive molecule was, but it was clear that TCR occupancy in the absence of accessory signals resulted in the induction of an unresponsive state. Later that year, Jenkins et al. (70) found that exposure of T cells to ECDI treated APCs results in the failure of T cells to produce IL-2. In the same study, it was found that a critical parameter to the induction of the unresponsive state is a partial flux in intracellular calcium, and that removal of calcium from the growth media prevents the induction of the unresponsive state. In 1992 it was discovered that CD28 expressed on T cells (murine CD4 ) could be +  crosslinked with agonist antibodies to provide the co-stimulatory signal required for optimal T cell proliferation (67). Blocking the CD28 signal using anti-CD28 Fab (the  16  fragment of an antibody with the antigen binding site) blocked co-stimulation provided by APCs. As well, by providing the CD28 signal during stimulations with ECDI fixed APCs the induction of anergy is blocked. From these results it was concluded that CD28 signaling is important to the response of T cells, and that TCR ligation in the absence of co-stimulation results in the induction of a non-responsive state in the T cells, a state characterized by a failure to produce IL-2. With the above experiments done in vitro, it was important to demonstrate that this non-responsive state could also be induced in vivo. This was first demonstrated by Rammensee et al. in 1989 (71). T cells expressing the V(36 TCR gene interact with the M l s - l antigen. Using anti-V(36 mAb these researchers were able to demonstrate in vivo a  induced T cell non-responsiveness in mice injected with M l s - l expressing cells. a  Although it was expected that the V(36 expressing ( M l s - l specific) cells would be a  deleted, it was found that V(36 expressing T cells persist in these mice and they do not proliferate to M l s - l when stimulated in vitro. However, they are capable of upregulating a  IL-2 receptors and increasing their cell size in response to antigen. These observations demonstrate that the unresponsive state is not strictly restricted to in vitro systems. In 1992 Kang et al. (72) discovered that the activation factor AP-1 was affected in anergic T cell clones. Anergizing the CD4 clone A.E7 with Conconavalin A (ConA) +  stimulation, these researchers used electrophoretic mobility shift assays to determine the D N A binding of AP-1, N F - K B and N F A T in anergic cells. They found that AP-1 is the only transcription factor afflicted in their anergic cells. It was later found that the failure to activate AP-1 may be due to a block in the Ras pathway. These investigations were done by Fields et al. (73) in murine T 1 clones anergized by anti-TCR mAb stimulation. H  While it was clear that Ras in the anergic cells does not form the active GTP-bound form it was not clear why this was the case. It was later discovered that the failure to activate Ras may be due to antagonism by activated Rapl (74). Here, researchers anergized human T cell clones with fibroblasts that did not express the costimulatory molecule B71. A series of biochemical studies demonstrated that Fyn constitutively associates with cCbl in anergic cells and that this c-Cbl is constitutively phosphorylated. Phosphorylated c-Cbl was found to be associated with CrkL. CrkL is an adapter protein that constitutively associates with C3G, an exchange factor for the Ras family member Rapl.  17  Thus c-Cbl phosphorylation was believed to result in the recruitment of the Rapl activator C3G to the membrane. Transfection studies with constitutively active Rapl demonstrated a 94% decrease in IL-2 transcription by Jurkat cells. It was hypothesized then that the failure of anergic cells to activate Ras is due to inhibition of Ras activation by Rapl, and that preferential Rapl activation leads to a failure to transcribe the IL-2 gene. In other models where human T cell clones are anergized by high concentrations of soluble peptide, an impaired intracellular calcium response has been credited with the reduced IL-2 production, by means of impaired binding of the N F A T transcription factor to distal response elements within the IL-2 enhancer (75). In this human T cell model, AP-1 is hardly observed to be affected (76). While the above mentioned alterations in AP-1 or N F A T function provide a distal biochemical basis for the anergic T cell's inability to produce IL-2, signaling defects more proximal to the TCR also exist in anergic cells and may be causal to the reduced activation of these transcription factors. Anergic cells exhibit increased expression of Fyn, along with increased Fyn protein tyrosine kinase (PTK) activity (77). Fyn has been reported to be constitutively associated with c-Cbl in anergic cells (74) and this association has been shown to lead to increased activity of Rapl, as discussed above. More recently, c-Cbl has been shown to be a negative regulator of ZAP-70 and c-Cbl"'" cells show increased ZAP-70 P T K activity (22, 78). c-Cbl binds to ZAP-70 and this binding is dependent on phosphorylation of tyrosine 292 on ZAP-70 (79). Anergic T cells have been reported to be unable to activate ZAP-70 upon TCR ligation (80) but it is unclear whether this is due to negative regulation of ZAP-70 by c-Cbl in anergic T cells. T cells stimulated with specific peptides altered by single amino acid substitutions at TCR contact sites (altered peptide ligands, or APL) can be induced into a state of anergy. Functionally, T cells stimulated with A P L fail to proliferate, but can exhibit other hallmarks of stimulation. For example, in early studies with APLs it was demonstrated that a T 2 clone stimulated with an A P L could produce IL-4 and provide help to B cells, H  but failed to proliferate (81). Significantly, examinations of the phosphotyrosine profiles of these T cells stimulated with APLs reveals differences in the phosphorylation patterns  18  compared to T cells stimulated with full agonist peptides. These differences include a failure to phosphorylate ZAP-70, significant reduction in CD3e phosphorylation, and greatly reduced induction of the p23 form of the TCR-^ chain. While the above anergy models are all based on different cell types and anergy induction protocols, they all seem to be based on a similar definition of anergy. Anergy in these models is typically defined as a cellular state in which a lymphocyte is alive but fails to display certain functional responses when optimally stimulated through both its antigen receptor and any other receptors that are normally required for full activation (82). It is typically characterized by a defect in IL-2 production and T cell proliferation (82). Typically, IL-2 gene transcription is decreased by about 8 fold, and IL-2 secretion decreased by ~ 20 fold (72). The anergy model used in this thesis also fits this definition. The cells defined as anergic in our system fail to proliferate or produced detectable IL-2 when stimulated with their antigenic ligand (83). The specifics of this model will be discussed in Chapter 4.  1.5.  Fyn Src Kinase  1.5.1.  Cloning of Fyn  In 1986, a new member of the Src kinase family was cloned using probes from v-fgr (84) or v-yes (85) to screen cDNA libraries. Although the new clones (then referred to as slk for src-like kinase and syn for src/yes-related novel proto-oncogene) had high sequence homology to src, the amino-terminal region of 82 amino acids showed no significant homology to Src (84, 85) and were thus determined to be a distinct protein. By 1988, this new Src kinase had been designated p59' "' (Fyn) (86). 5  Elucidation of the role of Fyn in lymphocytes began with the discovery that Fyn kinase activity associates with TCR-^ (87). The N-terminal 10 amino acid residues of Fyn were found to be crucial for this association (88). Fyn kinase activity was later shown to increase 2-4 fold upon CD3 ligation in human T cells (resting peripheral T cells, a CD4 T cell clone, and a leukemic T cell line) (89). Studies with mice genetically engineered to express high levels of Fyn also provided insights into the role of Fyn in T  19  cell functions. In these mice, Fyn was placed under the control of the Lck proximal promoter (90). Thymocyte numbers and thymus size were not markedly different in these mice. Thymocyte subset representation, along with peripheral T cell numbers were also not significantly altered. T cells displayed abnormally vigorous activation responses, including proliferation to P M A and CD3 stimulation, production of IL-2, and intracellular calcium flux in response to either CD3 stimulation or ConA. The phosphorylation of several TCR proximal substrates was also enhanced (including proteins with the following mobilities: 130 kDa, 100 kDa, 71 kDa, 65 kDa, 60 kDa, and 56 kDa).  1.5.2.  Fyn knockout mice  In 1992, the Fyn gene was knocked out by two separate groups (91, 92). Despite the physical association between Fyn and the TCR-^ chain, however, initial work with Fyn~'~ mice showed that thymocytes in these mice appear to undergo normal development (91, 92). Thymocytes derived from these mice revealed no differences in CD4/CD8 F A C S staining profiles compared with control mice, and thymocyte yields were unaffected. Several lines of evidence do, however, indicate that Fyn plays a role in thymocyte development. Studies in Lck'Fyn"'" mice demonstrate that T cell development was completely halted by the concurrent disruption of the lck and fyn genes (93, 94), with thymocytes halted at the CD44"  /l0W  CD25 D N stage. As mice lacking a functional lck +  gene (16) or overexpressing a catalytically inactive form of Lck (95) display a substantial but not absolute reduction in single positive (SP) thymocyte numbers, these results suggest that Fyn is at least able to perform some of the signaling functions required for T cell development. Studies on SP thymocytes from Fyn"'" mice showed that although T cell development was apparently normal, SP thymocytes from Fyn"'" mice demonstrated functional defects in TCR (91, 92) and Thyl (96) mediated responses. Proliferation responses, calcium ion fluxes, and IL-2 production were significantly depressed in Fyn"'" thymocytes. However there was some discrepancy in studies of peripheral T cell responses by these two groups. Stein et al. (91) found that proliferation was largely normal but IL-2 secretion and calcium flux were decreased. In contrast, Appleby et al. (92) found that the proliferative response was decreased, as was calcium flux. Both groups found decreases in tyrosine  20  phosphorylated substrates in peripheral Fyn " T cells. Because of the importance of _/  understanding T cell development, it is of interest to further elucidate the role played by Fyn in this process.  1.5.3.  Fyn kinase activity  Fyn kinase activity is largely regulated by phosphorylation of a few critical residues. Autophosphorylation within the kinase domain on tyrosine 417 potentiates kinase activity, whereas phosphorylation within the C-terminal domain on the negative regulatory tyrosine 528 leads to inhibition of kinase activity (97). Phosphorylation within the C-terminal domain allows for intramolecular interactions between the phosphotyrosine and Fyn's own SH2 domain, causing folding of the protein and the inhibition of the catalytic activity (98, 99). Csk is the kinase that phosphorylates this residue in Fyn (97). The CD45 transmembrane protein tyrosine phosphatase (PTPase) removes the inhibitory phosphate from this residue (100). The balance between phosphorylation and dephosphorylation is critical to the activation status of Fyn (8). It has been demonstrated that Fyn phosphorylates tyrosine residues within immunoreceptor tyrosine-based activation motifs on the TCR/CD3 chains upon TCR stimulation (101). As mentioned above, Fyn has also been shown to phosphorylate Z A P 70 leading to activation of this kinase (101). As well, Fyn has been shown to associate with phosphatidylinositol-3' kinase (PI3K) (102), with c-Cbl (103) and with Pyk2 (104).  1.5.4.  Fyn and T cell anergy  Fyn has been implicated in T cell anergy by a number of studies. In the T 1 clone H  A.E7, anergy induced by anti-CD3 stimulation leads to two to four fold increases in Fyn protein expression as observed by Western blot (105). A dramatic decrease in Lck protein expression (varying between 40% and 80% depending on the experiment) was observed in the same anergic clone. These observed variations in protein expression were consistent whether the cells were lysed with SDS or NP-40, suggesting the observed differences could not be accounted for by a redistribution of the proteins. Fyn kinase activity (as determined by autophosphorylation) was also seen to increase, whereas Lck  21  kinase activity decreased. In another report, the T 1 clone pGLlO was anergized with a H  similar protocol but provided slightly different results (106). In this study, the protein expression levels of Fyn and Lck were normal in anergic clones, but there is a four to five fold increase in the kinase activity associated with Fyn. Lck kinase activity is normal. In this study the anergic cells also fail to flux intracellular calcium in response to TCR stimulation. The anergic clones have elevated basal levels of intracellular calcium, and this is associated with elevated basal levels of IP . Finally, in these anergic cells PLC-yl 3  has elevated basal levels of phosphorylation. The association of Fyn with the TCR-^ chains has also been shown to be important in anergy induction in alloantigen specific human T cells (107), with Fyn phosphorylating the TCR-^ chains but not the y, 8 or e chains. In the same human T cells Fyn has been shown to constitutively associate with c-Cbl (74). The significance of this finding was discussed above. With Jurkat cells lacking Lck but expressing Fyn it has recently been demonstrated that preferential TCR signaling via Fyn results in reduced ZAP-70 activation, reduced phosphorylation of L A T , impaired IL-2 production and cell growth , although SLP 76 phosphorylation and CD69 induction are normal (108). These data provide support for the idea that Fyn activity may be important for T cell anergy. However more work is required to further clarify Fyn's role in this process.  1.6.  Thesis Goals  Hypothesis: Fyn plays unique roles in T cell development during the selection of TCRs with various affinities, in TCR signaling from specific TCRs, and in T cell anergy. Goals: 1)  To determine the role played by Fyn in the selection of thymocytes expressing TCRs with high or low affinities for their selecting ligands.  2)  To determine whether Fyn plays an essential role in signaling from TCRs with lower affinity for their peptide ligand.  3)  To develop and characterize a model of in vivo induced T cell anergy.  22  To determine the role Fyn plays in anergic T cells.  4)  1.7.  Approach TCR transgenic mice were used extensively in this thesis. Here I will describe the two  TCR transgenic systems used in this thesis, the H - Y TCR and the 2C TCR. As mentioned above, the H - Y TCR was produced using the rearranged TCR ap chains from a CD4"CD8 cytolitic T cell clone specific for the male minor +  histocompatibility antigen (H-Y) presented in the context of M H C I (H-2D ) (53). Female b  mice of the H-2 genetic background, which lack the ligand (H-Y antigen) for the H - Y b  TCR, offer an opportunity to study positive selection (54). In contrast, male mice of the H-2 background are used to study negative selection as thymocytes expressing the b  transgenic T C R are deleted in response to the male antigen (53). The 2C transgenic mice were originally produced using the rearranged TCR a and p genes from an alloantigen specific T cell clone (109). CD4"CD8 T cells from H-2 mice +  b  expressing the transgenic T C R were found to have the same specificity as the original clone. T cells expressing the 2C T C R are very strongly selected in the H-2 genetic b  background by the K M H C molecule(l 10). The peptide ligand for the 2C TCR is derived b  from the mitochondrial protein 2-oxoglutarate dehydrogenase and is presented by L  d  M H C class I molecules (111, 112). This peptide is referred to as p2Ca and has the amino acid sequence LSPFPFDL (single letter amino acid code). The 2C TCR can also respond to the p2Ca peptide presented in the context of K , but with a much lower affinity than b  the 2C response to p2Ca/L . The affinities of these interactions are ~3 x 10 M" and ~2 x d  3  1  10 M" respectively (113). 6  1.8.  1  Summary of Thesis The results in this thesis can be divided into three parts. In the first section I will  discuss investigations into the role played by Fyn in the development of T cells. Fyn was found to provide an important signaling function during T cell development. I discovered that Fyn plays a different role depending on the affinity of the interaction between the TCR and the selecting ligand. Deletion of Fyn allowed for the survival of cells expressing TCRs with higher affinity for their selecting ligand which otherwise would have been deleted during negative selection. Deletion of Fyn also slowed the downregulation of  23  developmental markers, suggesting that Fyn plays a role in signaling during the development of all T cells. I also discovered that mature T cells involved in low affinity TCR/ligand interactions required Fyn for optimal proliferation responses. In the second section, I will outline work done on a model of anergic T cells. This model is based on a population of mature peripheral CD4"CD8"TCRafT (double negative or DN) T cells that have been chronically exposed to their antigenic ligand. I will discuss the unique characteristics of these cells, such as their ability to hyperproliferate upon the addition of exogenous IL-2 and their ability to rapidly upregulate activation markers. I also found that these cells have TCR signaling defects at the level of ZAP-70 phosphorylation and L A T phosphorylation, but that they maintain the ability to effectively signal through the Ras pathway. Interestingly, they also have elevated levels of Fyn protein expression. In the final results chapter I will discuss experiments designed to determine the role of Fyn in anergic T cells. The peripheral double negative cells that lack expression of the Fyn protein were studied in order to understand the role played by Fyn in in vivo induced T cell anergy. I found that deletion of Fyn from anergic double negative cells allowed for a partial recovery from the proliferation defect. I also found that the anergic double negative cells had an enhanced survival ability in culture, and that deletion of Fyn reduced this survival advantage. This was found to correlate with an inability of the Fyn"'" anergic cells to maintain the expression of IL-2R(3. 1.9.  Publications arising from work in this thesis  T cells expressing receptors of different affinity for antigen ligands reveal a unique role for p59*" in T cell development and optimal stimulation of T cells by antigen. Utting, O., Teh, S-J., Teh, H-S. 1998. The Journal of Immunology 160:5410-9. A population of In vivo anergized T cells with a lower activation threshold for the induction of CD25 exhibit differential requirements in mobilization of intracellular calcium and mitogen-activated protein kinase activation. Utting, O., Teh, S-J., Teh, H-S. 2000. The Journal of Immunology 164:2881-9.  24  Fyn promotes the survival of anergic CD4CD8 oc(3 T C R cells but negatively regulates +  their proliferative response to antigen stimulation. Utting, O., Priatel, J.J., Teh, S-J. and Teh, H-S. 2001 The Journal ofImmunology 166:1540-6.  25  Chapter 2 Materials and Methods 2.0.  Mice  2.0.1.  H-2 2C Mice b  Breeders for the H-2 2C TCR-transgenic mice (109, 114) were kindly provided by b  Dr. Dennis Loh (then at the University of Washington, St. Louis, MO). The H-2 2C TCR b  mice have been backcrossed onto a C57BL/6 (H-2 ) background. The 2C TCR is specific b  for the naturally processed peptide, p2Ca (LSPFPFDL), presented by L M H C class I d  molecules (113). The p2Ca peptide is derived from the mitochondrial protein 2oxoglutarate dehydrogenase (112). 2.0.2.  H-2  bd  2C Mice  H-2 2C mice were filial 1 (F,) mice obtained by mating DBA/2 mice with H-2 2C b/d  b  TCR mice. 2.0.3.  H-2 H-Y Mice b  H-2 H - Y TCR transgenic mice were produced as previously described (115). The H b  Y TCR transgenic mice have been backcrossed onto a C57BL/6 background. 2.0.4.  Fyn' Mice  Fyn"'" mice, on a mixed 129 (H-2 ) and C57BL/6 background, were kindly provided b  by Dr. R. Perlmutter (then of the Howard Hughes Medical Institute, University of Washington, Seattle, WA) (92). TCR transgenic mice and Fyn"'" mice were mated to produce transgenic animals with the Fyn"'" mutation. 2.0.5.  Other Mice  C57BL/6 (B6), DBA/2 (H-2 ), and B10.BR (H-2 ) mice were obtained from The d  k  Jackson Laboratory (Bar Harbor, ME). BDF1 mice were F, mice obtained by mating B6  26  mice with DBA/2 mice. A l l animals were maintained in the animal facility at the University of British Columbia in the Department of Microbiology and Immunology.  2.1.  Reagents  2.1.1.  Antibodies against TCR components  T3.70 (anti-Voc3, specific for the a chain of the H - Y TCR) (116, 117); F23.1 (118) (anti-Vp8; specific for the p chain of the H - Y or 2C TCR); 1B2 (anti-2C TCR, antiidiotypic) (119) (the 1B2 hybridoma was a kind gift from Dr. Eisen (Cambridge, MA)).  2.1.2.  Antibodies against cell surface markers  Anti-CD4 (GK1.5), anti-CD8 (53.67), anti-CD3 (145-2C11), anti-CD25 (PC61) and anti-HSA (Ml/69) were obtained from American Type Culture Collection (Manassas, V A ) with clone number indicated in parenthesis. Antibodies were ( N H ) S 0 precipitated 4  2  4  (GK1.5, 53.67, PC61 and Ml/69) or purified using Protein A columns (145-2C11) and where either Fluorescein Isothiocyanate Isomer 1 (FITC) (Sigma F-7250) labeled or biotinylated, depending on the study. Anti-CD69 (catalogue no. 01502D) and anti-IL-2Rp (catalogue no. 55336) mAbs were obtained from PharMingen (San Diego, CA) For blocking experiments ,the anti-IL-2Rp antibody was first extensively dialyzed against phosphate buffered saline (PBS, 150 m M NaCl, 2.6 raM KC1, 1.86 m M N a H P 0 H 0, 8.39 m M N a H P 0 7H 0) to remove sodium azide. 2  2.1.3.  4  2  2  4  2  Antibodies against signaling molecules  Anti-Fyn polyclonal (catalogue no. sc-16), anti-ZAP-70 polyclonal (catalogue no. sc574), anti-ERKl/2 polyclonal (catalogue no. sc-94), and anti-phosphoERKl/2 mAb (catalogue no. sc-7383) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). 4G10 anti-phosphotyrosine mAb (catalogue no. 05-321) and anti-LAT polyclonal (catalogue no. 06-807) were purchased from Upstate Biotechnology (Lake Placid, N Y ) .  27  Sheep anti-SLP-76 polyclonal antibodies were a kind gift from Dr. Koretzky (University of Pennsylvania, Philadelphia) (120). The anti-Lck Ab 54.3B is a peptide-specific (Nterminal residues 3-147) rabbit antisera (121). The anti-TCR-^ mAb (G3), specific for the cytoplasmic domain of TCR-£ was produced in our laboratory (122).  2.1.4.  Secondary antibodies/reagents  Fluoresceinated (FITC) goat F(ab') anti-mouse Ig Abs (catalogue no. 1012-02), 2  streptavidin Phycoerythrin (PE) (catalogue no. 7100-09L) and goat-anti-mouse IgGhorseradish peroxidase (HRP) (catalogue no. 1030-05) were purchased from Southern Biotechnology Associates, Birmingham, A L . Streptavidin tricolor (catalogue no. CLCSA1006) was purchased from Cederlane Laboratories, Hornby, ON. Protein A-HRP (catalogue no. N A 9120) was purchased from Amersham Pharmacia Biotech (Baie d'Urfe, Quebec, Canada).  2.1.5.  Cytokines  Recombinant mouse IL-2 was purchased from PharMingen (catalogue no. 1921 IT). Recombinant human IL-15 (catalogue no. 247-IL) was purchased from R & D Systems Inc. (Minneapolis, MN).  2.2.  Methods  2.2.1.  Isolation of lymphocytes  Lymph nodes, thymi and spleens were harvested from mice sacrificed by cervical dislocation. Organs were processed in RPMI 1640 (catalogue no. 31800-071 GibcoBRL , Burlington, Ontario) media supplemented with 2% Fetal Bovine Serum (FBS) (catalogue no. F-2442 Sigma) (heat inactivated for 30 minutes at 56°C). The organs were chopped into small pieces using surgical scissors, then teased through a steel sieve to remove clumps. The cell suspensions were centrifuged in a clinical centrifuge for 5 minutes at setting 6. Pellets were suspended in RPMI and fat clumps were discarded. Suspensions  28  were centrifuged again and resuspended in I M D M media (I media) (Life Technologies, Burlington, Ontario, Canada) supplemented with 5 x 10" M f i - M E , 10% FBS, and 5  antibiotics (10 units/ml penicillin G and 10 units/ml streptomycin (catalogue no. 15140148 GibcoBRL)). For spleen preparations, an additional red blood cell (RBC) lysis step was included. This consisted of resuspending the cell pellet in R B C lysis buffer (155 | j M Ammonium Chloride, 10 u M Tris Base, pH 7.3) for 5 minutes, then resuspending the cells in I media.  2.2.2.  Purification of CD8 cells +  Cells were then incubated with biotinylated anti-CD8 mAb in I media at 10 cells/ml 7  for 20 minutes on ice (optimum concentration of antibody was determined empirically using FACS staining). After incubation, cells were washed three times in MiniMacs buffer (0.5% B S A , 2mM EDTA, in PBS) and resuspended at 10 cells/ml. 10 jLtl of 8  Streptavidin-conjugated MicroBeads (Miltenyi Biotec, Auburn, CA) were added per 10  7  cells, and the suspensions were incubated for 20 minutes on ice. At end of the incubation period, the volume of cell suspension was brought up to 500 jul with the MiniMacs buffer and the cells were run through a Miltenyi Biotec column while attached to a Miltenyi Biotec magnet. Columns were washed 2 x with 1 ml of MiniMacs buffer. To remove the CD8 cells that remained bound to the column, 2 x 1 ml of MiniMacs buffer was put +  through the column immediately after it was detached from the magnet. The purity of the positively selected C D 8 cells was determined by staining the selected cells with 53.58 +  anti-CD8(3 FITC Ab and anti-CD4 PE Ab. T cells purified in this manner were typically 99% CD4"CD8 . As MiniMacs beads are very small and biodegradable they do not +  interfere with cellular function.  2.2.3.  Purification of Double negative cells  Double negative cells were purified by incubation with GK1.5 and 53.67 (anti-CD4 and anti-CD8a) Ab on ice for 20 minutes in I media at 10 cells/ml. After washing, cells 7  were resuspended in PBS:I media (1:1) and 50 | i l of M-450 Sheep anti-mouse IgG Dyna  29  beads (Dynal, Oslo, Norway) were added per 10 cells. Cells were incubated at room 7  temperature for 40 minutes with Dyna beads before magnetic separation. Cells remaining in solution were CD4CD8". The purity of the CD4CD8" cell population was verified in the same manner as for CD4"CD8 cells. +  2.2.4.  Proliferation assays  2.2.4.1. Cell isolation Thymocytes and lymph node cells were harvested from transgenic mice as described above, and were used as responder cells in proliferation assays.  2.2.4.2. Antibody stimulated proliferation assays Anti-CD3e (2C11) mAb stimulations were done using purified Ab and 20 U/ml of exogenous IL-2 (when indicated). 2C11 was coated to tissue culture plates (Falcon Microtest™ plates, from Becton Bickinson) at the desired concentration (typically 1 or 10 |lg/ml) by incubating in PBS for lhr at 37°C, or overnight at 4°C. After incubation, plates were washed 3 times with cold PBS.  2.2.4.3. Splenocyte stimulated proliferation assays For use as stimulator cells in proliferation assays, splenocytes were isolated as described above, resuspended at 10 cells/ml in cold PBS, and irradiated with 2000 rad 7  using the Department of Chemistry Gamma Cell. After irradiation, splenocytes were centrifuged and resuspended in I media and added to 96 well plate cultures, typically at 5 x 10 cells/well. 5  2.2.4.4. Antigen presenting cell stimulated proliferation assays The peptide transporter-deficient cell lines T2-L and T2-K (123) were a kind gift d  b  from Peter Cresswell and were derived by transfecting the human (T x B) hybridoma T2  30  with L or K . Before use, these cells were treated with mitomycin-C (catalogue no. M d  b  0503 Sigma) at 50 |J,g/ml for lhr at 37°C at a maximum density of 5 x 10 cells/ml in 6  serum free I media. After treatment, T2 cells were washed 3 times with I media to remove all mitomycin-C.  2.2.4.5. Harvesting proliferation assays For proliferation assays, cells were cultured in triplicates in a volume of 0.20 ml, in either 96-well round-bottom or flat bottom plates, depending on the experiment. The incubation periods differed depending on the cell type studied, but were generally 72 hrs at 37°C (5% C0 ). For assessment of proliferation, 1 uCi of [ H] thymidine (TdR) was 3  2  added to each culture well for the last 6 hours of incubation. After completion of the incubation period cells were harvested onto Whatman Glass Microfibre Filters (catalogue no. 1820866) (Whatman Nuclepore Canada, Toronto, ON). Wells were washed 6-7 times with distilled water. After washing, samples were fixed with methanol, transferred to Mini Poly-Q Vials (Beckman Coulter, Mississauga, ON) to dry. Once dry, 2 ml of scintiallation cocktail was added per vial. [ H] TdR incorporation was assessed using a 3  Liquid Scintillation Counting System from Beckman (LS 6000TA). The standard deviation (SD) of the triplicate samples was calculated as the square root of the variance. The variance was calculated as ((x, - xbar) + (x - xbar) + (x - xbar) )/(n-l) where xbar 2  2  2  2  3  is equal to the average of x, through x . Standard error of the mean (SEM) (as used in 3  Table 3.1) was calculated as by dividing the SD by the square root of n (number of observations). Where error bars are not shown, the SD was less than 10% of the mean.  2.2.5.  Flow cytometric analysis of lymphocytes  2.2.5.1. Cell Preparation and standard FACS staining Single cell suspensions of thymocytes and lymph node cells were prepared as described above. For immediately ex vivo phenotypic analysis cells from different mice were not pooled (such as Figures 3.1-4). Cells (typically 1-5 x 10 ) were stained with s  mAb for 15 minutes on ice in 50 ul of FACS buffer (PBS with 2% FBS). Cells were  31  washed and then incubated with secondary Ab for 15 minutes on ice in 50 ul of F A C S buffer. Cells were then washed and resuspended in FACS buffer for analysis on a FACScan IV flow cytometer using LYSIS II software or CELLQuest software (Becton Dickinson, Mountain View, CA).  2.2.5.2. 7-A AD Staining 7-amino actinomycin D (7-AAD) was purchased from Calbiochem-Novabiochem (catalogue no. 129935) (La Jolla, CA). Purified cells were cultured for the indicated time, washed, and stained with 7-AAD. 7-AAD staining was done in FACS media with a final concentration of 10 u\g/ml 7-AAD. After 15 minutes incubation on ice, cells were washed and then fixed with 4% paraformaldehyde. After fixing cells were analyzed with the FACScan flow cytometer using CELLQuest software (Becton Dickinson). Samples were collected in triplicates and the percentage of live cells (cells not stained with 7-AAD) was calculated. Standard deviations were calculated as the square root of the variance. The variance was calculated as ((X[ - xbar) + (x - xbar) + (x - xbar) )/(n-l) where xbar is 2  2  2  2  3  equal to the average of x, through x . The z test was used to determine whether results 3  were statistically significant. This was calculated as follows: (x ;  x )/root((s /n )+(s /n )) where s, equals the standard deviation of set 1, and n, equals the 2  2  1  2  1  2  2  number of values in this set. The values were deemed statistically different with a z score of greater than 4.5, indicating 99.99% confidence.  2.2.5.3. CD69, CD25 and IL-2Rpflowcytometry Single-cell suspensions of lymph node cells were prepared. Purified cells (1 x 10 ) 5  were stimulated with 1 x 10 mitomycin C-treated T2-L or T2-K cells plus the indicated 5  d  b  concentration of the p2Ca peptide in a 96-well plate in a volume of 0.2 ml. No exogenous IL-2 was added. After the culture period indicated in the figure legend (typically either 18 hours or 40 hours), the cells were collected and stained with biotinylated anti-CD69 or anti-CD25 mAb followed by streptavidin-Tricolor and analyzed with the FACScan flow cytometer using CELLQuest software. A total of 15,000 events were analyzed for each sample. 32  2.2.5.4. Intracellular calcium Flow cytometry was used to measure intracellular calcium levels in cells loaded with the calcium-binding dye fluo-3-acetoxymethyl ester (fluo-3-AM) (Molecular Probes, Eugene, OR) using the Chronys software package (Becton Dickinson, Mountain View, CA) as described previously (124). Fluo-3-AM was prepared by dissolving F-127 (catalogue no. P2443 Sigma) in dimethyl sulfoxide (DMSO) at 37.5 mg/ml (requires heating to 40°-55°C for a few minutes). 43.8 ul of F-127/DMSO was added to 1 vial (50 ug) fluo-3-AM to give 1 m M stock. Cells were prepared by washing 2 x with PBS (no calcium or magnesium) and then resuspending at 10 cells/ml in prewarmed (37°C) PBS. 7  4 jil of 1 m M fluo-3-AM stock was added and the suspension was incubated at 37°C (5% C 0 ) for 30 minutes. Cells were washed 1 x with RPMI and resuspended at 2 x 10  6  2  cells/ml in I-media with 2% FBS (prewarmed to 37°C) and incubated for 10 minutes at 37°C (5% C 0 ) . Cells were then washed 3 x with PBS and finally resuspended in C a  2+  2  media (25 m M HEPES, 140 m M NaCl, 10 m M glucose, 1.8 m M CaCl , 1 m M M g C l , 3 2  2  m M KC1) at 5 xlO cells/ml. Samples were stored at room temperature in the dark until 5  10 minutes prior to run, at which time they were warmed to 37°C. Samples were analyzed and calcium signals were collected as FL-1 signals.  2.2.6.  Whole-cell lysate and immunoprecipitation studies  Cells were prepared and resuspended in I M D M supplemented with 0.25% FCS at 10  7  cells/ml. Cells were pre-warmed to 37°C for 10 minutes, stimulated with 10 ug/ml 2C11 for the time period indicated in the figure legends (typically 3 min, 5 min or 10 min), washed with ice-cold PBS, and then lysed in lysis buffer. Lysis buffer consisted of the following: TNE (10 m M Tris-Cl, 150 m M NaCl, 1 m M EDTA) pH 7.6, 10 ug/ml aprotinin, 10 (J.g/ml leupeptin, 1 m M sodium orthovanadate, 1 m M sodium molybdate, 2 m M phenylmethylsulfonyl fluoride (PMSF), and 1% Triton X-100 (catalogue no. T-8787 Sigma). For the immunoprecipitation of L A T , Brij 97 (catalogue no. M-0503 Sigma) was substituted for Triton X-100. For whole-cell lysate studies, cells were lysed on ice for 10  33  minutes. Lysates were clarified by centrifugation at 12800 x g for 10 minutes at 4°C. Supernatants from the samples were mixed with 3 x protein sample buffer (New England Biolabs)/dithiothreitol (40 mM) before boiling for 5 minutes. For immunoprecipitations studies, lysates were clarified by centrifugation at 12800 x g for 10 minutes at 4°C before immunoprecipitation. Lysates were incubated for 2 hours with the appropriate Ab and 20 ul of packed protein A-Sepharose CL-4B (Amersham Pharmacia Biotech, Baie d'Urfe, Quebec, Canada). Immune complexes were washed 3 x with lysis buffer before the protein A beads were resuspended in protein sample buffer/DTT and boiled for 5 minutes.  2.2.6.1. SDS-PAGE Immune complexes and whole cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, M A ) for 1.3 h at 105 V . After transferring, membranes were blocked, typically overnight with 5% B S A in Tris-buffered saline (10 m M Tris-HCl, pH 8, 150 m M NaCl) with 0.1% Tween 20 (catalogue no. BP337-500 Fisher Biotech). Western blotting was conducted with the indicated Abs and developed with the appropriate HRP-conjugated secondary Ab and the enhanced chemiluminescence (Amersham Pharmacia Biotech) detection system. Where indicated, blots were stripped for 40 minutes at 55°C in stripping solution: 62.5 m M Tris-HCl (pH 6.8), 2.0% SDS, and 100 m M p-mercaptoethanol. Spot densitometry quantification was done using the Alphalmager 1200 v4.03 software on an Alpha Imager 1200 (Alpha Innotech, San Leandro, CA). 2.2.7.  RT-PCR  2.2.7.1. RNA preparation R N A was prepared from 1 x 10 cells stimulated with 5 x 10 T2-L antigen 5  4  d  presenting cells (APCs) + 1 (iM p2Ca in 0.2 ml wells. 20 wells were pooled (2x10 cells) 6  and R N A was prepared using the Qiagen RNeasy kit (Valencia, CA)(catalogue no. 74104) according to the manufacturers' instructions.  34  Vendor/Catalogue #  Antibody  Western Blot  IP.  Cells per IP.  Specificity 145-2C11  A.T.C.C.  N/A  6^g  6x10  4G1.0  U.B.I. 05-321  0.6 fig/ml  N/A  N/A  Erk  Santa Cruz sc-94  0.5 ug/ml  N/A  N/A  Fyn  Santa Cruz sc-16  1 |Xg/ml  N/A  N/A  G3  Teh Lab  2 ug/ml  N/A  N/A  LAT  U.B.I. 06-807  1 ug/ml  4 |Llg  6x10  1:2500  N/A  N/A  Lck (54.3B)  6  6  pERk  Santa Cruz sc-7383  0.5 ug/ml  N/A  N/A  SLP-76  Gift from G. Koretzky  1:500  1.5 ul  6x10  6  ZAP-70  Santa Cruz sc-574  1 |0,g/ml  1  6x10  6  Table 2.1 Antibody concentrations and dilutions for Western Blotting and Immunprecipitations The vendor, concentration or dilution of antibody solution and number of cells required for immunoprecipitation is indicated.  35  2.2.7.2. RT-PCR R N A was quantified and reverse transcribed as follows: 10 (il l Strand Buffer, 5 JLLI s l  lOOmM DTT, 5 fil random primers (3 |J,g/ml) (#48190-011), 1 (il RNase Inbibitor (10 u/ul) (15518-012), 2.5 JLLI I O U M dNTPs, 2 [ig R N A , 1 ul reverse transcriptase (28025013), ddH 0 to 50 | l l total volume (all catalogue numbers are for GibcoBRL). The 2  reaction was carried out in a Perkin Elmer GeneAmp System 2400 using the following program: 65°C 5 minutes, 4°C 5 minutes, 20°C 15 minutes, 37°C 60 minutes, 95°C 5 minutes, 4°C. The reverse transcriptase was added to the reaction once it had cooled to below room temperature after the initial 65°C incubation.  2.2.7.3. Competitive PCR cDNAs were normalized for content of the hypoxanthine phosphoribosyltransferase (HPRT) gene using competitive PCR (125) and the Alphalmager. Using normalized samples, IL-2 message was quantified using the pPQRS competitors (125) (a generous gift from Dr. Locksley). The plasmid pPQRS was used for competitive PCR. Before use, pPQRS was first digested with Sfil and Notl restriction enzymes. The digest was resolved on a 0.8% agarose gel and the 5 kb fragment was purified using Qiaex II Agarose Gel Extraction kit according to manufacturers instructions. The purified fragment was quantified by comparing titrations of plasmid to known concentrations of a control plasmid. Once quantified, 10 fold dilutions of pPQRS fragment were prepared and used in subsequent competitive PCR reactions. For these experiments, titrations of pPQRS fragment were added to PCR reactions. IL-2 message was compared between samples by amount of pPQRS fragment required to reach the equivalence point of pPQRS and cDNA (equal intensity bands on agarose gel). The following primers were used: 5' IL-2 5 ' C C A C T T C A A G C T C T A C A G C G G A A G 3 ' 3' IL-2 5 ' G A G T C A A A T C C A G A A C A T G C C G C A 3 ' 5' HPRT 5' G T T G G A T A C A G G C C A G A C T T T G T T G 3 '  36  3' HPRT 5' G A G G G T A G G C T G G C C T A T A G G C T 3 ' The following program was used for these reactions: 94°C 3min, 35 x (94°C 0.5 minutes, 62°C 0.5 minutes, 72°C 0.6 minutes), 72°C 5 minutes, 4°C «>. PCR products were resolved using agorose gel electrophoresis.  37  Chapter 3 3.0  Introduction Signaling from the pre-TCR and the TCRocP chains is essential for the development  of a normal peripheral T cell compartment (reviewed in (126)). Mice engineered to lack the expression of various signaling molecules have provided much insight into the role of these molecules in the development of the T cell compartment. Interestingly, initial analyses of Fyn"'" mice revealed that these mice have a similar thymocyte subset distribution as their control littermates (91, 92). As discussed in Chapter 1, this observation was taken as evidence that Fyn does not play a major role in signaling during T cell development. More recent work on Lck/Fyn double knockout mice revealed that Fyn does play some role in T cell selection. The nature of the role played by Fyn in T cell selection and in signaling for peripheral T cells is still unclear. Previous analyses of Fyn"'" mice were done in non-TCR transgenic mice (91, 92). These mice expressed a broad range of TCR specificities, and therefore a broad range of TCR affinities. The initial hypothesis investigated in this thesis was that Fyn plays a role in signaling for the development of a discrete subset of thymocytes with defined TCR affinities. It was hypothesized that the role of Fyn during T cell selection may be discernable under extreme TCR affinity conditions: either very weak selection or very strong selection. The role of Fyn in peripheral T cell signaling may also be evident under defined affinity conditions. To address the question of whether Fyn is required for the development of T cells bearing specific subsets of TCRs, we mated mice with the Fyn"'" mutation with mice transgenic for TCRs with differing affinities for their antigen ligands. Using this approach, we hoped to identify conditions in Fyn"'" mice in which TCR signaling was not sustained by the activities of other Src kinases. The 2C TCR, whose ligand has been identified as the naturally processed peptide p2Ca (LSPFPFDL) presented by L M H C d  class I molecules (111, 112) was chosen as an example of a high affinity TCR. The affinity of the 2C TCR for the p2Ca/L ligand is - 2 x 10 M" (113). The 2C TCR is very d  6  1  strongly positively selected on the H-2 M H C background (110). b  38  We studied the H - Y TCR (53) as a representative of a low affinity T C R (127). The H Y TCR is positively selected by H-2D M H C class I molecules in H-2 female mice, and b  b  is negatively selected in H-2 male mice (53, 54). b  My results demonstrate that Fyn plays a role both in signaling during thymocyte development and in signaling during the response of thymic and peripheral T lymphocytes to stimulation by specific antigens. The function of Fyn is most evident in the response of T cells to low affinity ligands.  3.1  Results  3.1.1.  Phenotypic analysis of thymocytes and lymph node cells from 2C/2C Fyn"'" and H-Y/H-Y Fyn' mice  The CD4/CD8 phenotype of thymocytes from female H-2 H - Y T C R transgenic mice b  with or without the Fyn"'" mutation is shown in Figure 3.1. The Fyn"'" mutation had several effects on thymocyte development in female H-2 H - Y T C R transgenic mice. In Fyn"'" b  mice, the proportion of DP thymocytes was slightly increased. A corresponding reduction in the proportion of D N thymocytes compensated for this increase. The expression of the CD4 and CD8 co-receptor molecules was slightly down-regulated on DP thymocytes from Fyn"'" mice. Furthermore, the number of Fyn"'" DP thymocytes that expressed higher levels of the transgenic TCR(3 chain (detected by the F23.1 mAb) and the transgenic T C R a chain (detected by the T3.70 mAb) was also increased. Previous studies have shown that DP thymocytes that have been positively selected by thymic M H C ligands upregulate T C R expression levels (128, 129). If the lack of Fyn facilitated positive selection, this would have been accompanied by an increase in the production of CD8 SP thymocytes in Fyn"'" mice. However, this was not observed (Fig. 3.1). One explanation for this observation is that while Fyn is not required for the up-regulation of TCR levels on positively selected DP thymocytes, it may be required for the efficient differentiation of positively selected DP thymocytes into SP thymocytes. Thus, the lack of Fyn may result in the accumulation of DP thymocytes that expressed higher levels of the H - Y TCR. The 2C T C R is positively selected in H-2 mice and negatively selected in H-2 mice b  d  (114). Previous studies have suggested that the 2C TCR is very strongly positively selected in H-2 mice. Thus, the expression of a transgenic CD8 molecule led to the b  39  2C / Fyn-  2C  H-Y  H-Y / Fyn-  *cfi8' 23 48  6 9  58 8 19 14  10 46 21 22  13 12  H-Y 2C / Fyn- " / CD8 expression by all thymocytes ]  72 11  7  H-Y / Fyn-  8  Figure 3.1. Increased CD8 expression and numbers of DP thymocytes in the 2C Fyn'' but not H-Y Fyn' mice. Thymocytes from H-2 2C and female H-2 H - Y TCR transgenic mice with or b  b  without the Fyn"'" mutation were triply stained with anti-CD4 PE, anti-CD8 FITC, and another cell surface molecule as indicated. DP cells were gated as indicated by the R2 gate (indicated by the boxed area in the upper right quadrant of each dot plot). The histograms directly below each dot plot indicate the expression of H S A and transgenic TCR expression (Tricolor) for this cell population. The F23.1 and 1B2 mAb detected the TCRp and Id of the 2C TCR, respectively. The TCR a and p chains of the H - Y T C R were detected by the T3.70 and F23.1 mAb, respectively. The percentages of thymocytes in each quadrant of the dot plots are indicated below each figure. The CD8 expression by all thymocytes from the indicated mouse line is indicated in the histograms at the bottom of the figure. Data from one representative experiment of three are shown.  40  deletion of DP thymocytes in H-2 2C mice (63). In that same study, it was shown that b  the same level of transgenic CD8 expression in female H-2 H - Y T C R transgenic mice b  did not lead to the deletion of DP thymocytes but instead enhanced positive selection (63). This was taken as evidence that the H - Y T C R was weakly positively selected in female H-2 mice. Here we show that the proportion of DP thymocytes in Fyn"'" H-2 2C b  b  TCR transgenic mice was significantly increased (Fig. 3.1, Table 3.1). This increase in DP thymocytes in Fyn"'" mice was associated with an increase in the number of DP thymocytes that expressed high levels of CD8 in 2C Fyn"'" mice (Fig. 3.1). This result can be explained by assuming that Fyn is involved in the negative selection of thymocytes. In the absence of Fyn, thymocytes that would normally have been deleted will be able to survive. In H-2 Fyn"'" 2C T C R transgenic mice, this translates into a higher proportion of b  DP thymocytes that retained high expression of the CD8 coreceptor molecule. This effect of Fyn is specific for H-2 2C TCR transgenic mice since the Fyn"'" mutation did not lead b  to an increase in CD8 expression in female H-2 H - Y T C R transgenic mice (Fig. 3.1). b  Since Fyn influenced the survival of DP thymocytes in H-2 2C T C R transgenic mice, b  we next determined whether Fyn can alter the course of negative selection in male H-2  b  H - Y TCR transgenic mice. Negative selection in these male transgenic mice is associated with deletion of DP thymocytes, a greatly reduced thymocyte cell yield, and a preponderance of D N thymocytes that expressed the H - Y T C R (53, 116). Thus, i f Fyn is the major tyrosine kinase mediating negative selection, we would expect to see an increase in the survival of DP thymocytes that expressed the H - Y TCR. However, we found that the yield of thymocytes from either male Fyn  +/+  or male Fyn"'" H - Y T C R  transgenic mice was not significantly different (~2 x 10 cells from either Fyn 7  mice), and the surviving population in Fyn  +/+  +/+  or Fyn"'"  or Fyn"'" mice was predominantly of the D N  phenotype (Fig. 3.2). These results indicate that Fyn is not essential for efficient negative selection in these mice, though Fyn did have a subtle effect in male H - Y transgenic mice. We found that the surviving thymocytes in Fyn"'" mice differed from Fyn  +/+  mice in the  expression levels of CD8 and TCR (Fig. 3.2). In Fyn"'" male mice, there were more thymocytes that expressed higher levels of the CD8 co-receptor and the H - Y TCR (Fig. 3.2). Thus, the lack of Fyn enables the survival of thymocytes that expressed slightly higher levels of CD8 and the H - Y TCR.  41  3.1.2.  Deletion of Fyn leads to a less mature population of T cells entering periphery  Functionally immature SP thymocytes are characterized by the expression of high levels of H S A (130, 131). To assess whether the Fyn"'" mutation may have subtle effects on the development of CD8 SP cells, we examined expression of this cell surface molecule on DP and SP thymocytes in Fyn  +/+  and Fyn"'" mice. We found that although  Fyn did not affect the expression of H S A on DP thymocytes from either female H-2 H - Y b  or H-2 2C TCR transgenic mice (Fig. 3.1), it did affect the expression of H S A in CD8 b  SP thymocytes from these mice (Fig. 3.3). In Fyn  +/+  H - Y TCR transgenic mice, the  majority of CD8 SP thymocytes maintained high H S A expression, and a minority of these cells had down-regulated HSA. However, almost all Fyn" " CD8 SP thymocytes 7  from these mice maintained high expression of HSA. Similarly, in Fyn  +/+  2C T C R  transgenic mice, the majority of CD8 SP thymocytes had down-regulated HSA. However, the majority of CD8 SP thymocytes in 2C Fyn"'" mice maintained high expression of HSA. This delayed down-regulation of the H S A maturity marker may reflect a role for Fyn in the development of CD8 SP thymocytes. No significant differences in transgenic TCR a or p chain expression were detected in CD8 SP thymocytes of H-2 2C and b  female H-2 H - Y T C R transgenic mice with the Fyn"'" mutation (Fig. 3.3). b  The expression of the transgenic TCRs and H S A was also determined for peripheral CD8 SP T cells. We found that CD8 SP cells from the lymph nodes of 2C T C R transgenic mice exclusively expressed the 2C TCR, and the Fyn"'" mutation did not affect the expression of the transgenic TCR in these cells (Fig. 3.4). By contrast, CD8 SP cells from the lymph nodes of female H-2 H - Y T C R transgenic mice showed a very b  heterogeneous expression of the T C R a chain. Only a minority of these CD8 SP cells continued to express high levels of the transgenic T C R a chain (Fig. 3.4). The predominant expression of endogenous T C R a chains in peripheral CD8 SP cells has previously been reported and attributed to the preferential expansion of CD8 SP cells that express endogenous T C R a chains in these mice (132). Significantly, the Fyn"'" mutation did not alter the TCR repertoire of peripheral CD8 SP cells in H - Y TCR transgenic mice. This result suggests that the positively selected CD8 SP cells in Fyn  +/+  or Fyn"'" mice,  regardless of whether they expressed transgenic or endogenous T C R a chains, were expanded in a similar fashion in peripheral lymphoid organs. In contrast to CD8 SP  42  % of Total Thymocytes (mean ± SEM) No. of Thymocytes x  CD4 8"  CD4"8  58 ± 3 . 4  8.5 ± 0 . 6  16 ± 1.4  10 ± 1.7  67 ± 2.0  9.1 ± 0 . 6  14 ± 1.7  1.7 ± 0 . 2  45 ± 3.6  22 ± 2.9  14.8 ± 2 . 2  18 ± 2 . 9  4.2 ± 0.6  21 ± 3 . 9  46 ± 3 . 2  6.0 ± 1.2  27 ± 2 . 3  CD4"8"  CD4 8  7.8 ± 1.0  18 ± 2 . 3  H-Y/Fyn "  12.7 ± 2 . 1  2C  Mutation  10" (mean ± SEM^)  H-Y 7  2C/Fyn " 7  +  +  +  +  7  Table 3.1. Effect of Fyn on the proportions of thymocyte subsets in TCR transgenic mice. Six mice (6-12 wk old) were analyzed per group. Thymocytes from H-2 2C T C R b  transgenic mice with or without the Fyn " mutation or female H-2 H - Y T C R transgenic 7  b  mice with or without the Fyn " mutation were stained with PE-labeled anti-CD4 and 7  FITC-labeled anti-CD8 mAbs and analyzed in the FACScan flow cytometer. The total number of viable thymocytes recovered from these transgenic lines is indicated. S E M was calculated from the six mice in each group.  43  H-Y Male  H-Y Fyn ' Male 7  >CD8 14 9 65 11  6 2 82 6  CD 8  F23.1—•  •  T3.70 — •  Figure 3.2. Increased CD8 and H-Y TCR expression by thymocytes from male H-2  b  H-Y TCR transgenic mice with the Fyn' mutation. Thymocytes from male H-2 H - Y TCR transgenic mice with or without the Fyn"'" b  mutation were triply stained with anti-CD4 PE, anti-CD8 FITC, and biotinylated mAb to either the transgenic TCR a (T3.70) or p (F23.1) chain of the H - Y TCR. Binding of the biotinylated mAb was detected by streptavidin-tricolor conjugate. The percentages of thymocytes in each quadrant of the dot plots are indicated beside each figure. The F23.1, T3.70, and CD8 expression levels on all thymocytes from the indicated mouse are indicated in the histograms. The percentages of thymocytes expressing high levels of the CD8 molecule are indicated. Data from one representative experiment of three are shown, 44  2C  2C / Fyrr  H-Y  H-Y / Fyn-/-  Figure 3.3. CD8 SP Fyn"'" thymocytes maintained elevated HSA expression levels. Thymocytes from H-2 2C and female H-2 H - Y TCR transgenic mice with or b  b  without the Fyn"'" mutation were stained as described in Figure 3.1. The percentages of thymocytes in each quadrant of the dot plots are as indicated in Figure 3.1. The expression of the indicated cell surface molecule by gated CD8 SP thymocytes are depicted in the histograms below the dot plots. Data from one representative experiment of three are shown.  45  thymocytes, the vast majority of CD8 SP lymph node cells expressed low levels of H S A (Fig. 3.4). This likely reflects the more functionally mature status of CD8 SP lymph node cells and suggests that Fyn is not essential for the functional maturation of peripheral CD8 SP cells. 3.1.3.  CD8 SP thymocytes and lymph node cells with the Fyn"'" mutation are hyporesponsive to low affinity antigenic ligands  We next sought to determine the responsiveness of cells with or without the Fyn"'" mutation to stimulation by plate bound anti-CD3 mAb. To ensure that any responses we observed were strictly those of CD8 SP cells, we first depleted cell populations of CD4 SP, DP, and D N cells as previously described (133). Once highly purified (>99% CD8 SP) cell populations were obtained, the responsiveness of CD8 SP thymocytes and lymph node cells to anti-CD3 (2C11) mAb stimulation in the presence of 20 U/ml of exogenous IL-2 was determined. As seen in Figure 3.5, thymocytes from female H-2 H - Y T C R b  transgenic mice with the Fyn"'" mutation were hyporesponsive to stimulation by anti-CD3 mAb. This functional defect was not observed in CD8 SP lymph node cells, since no significant difference in proliferative response was observed between the Fyn  +/+  and Fyn"'"  populations. This result agrees with the previously published data (91, 92) in that only Fyn"'" thymocytes, but not Fyn"'" lymph node cells, were refractive to stimulation by antiTCR mAb. Similar experiments with CD8 SP thymocytes and lymph cells expressing the 2C T C R provided different results, however. 2C11 stimulation of 2C CD8 SP thymocytes and lymph node cells (Fig. 3.5) revealed comparable proliferation in both the Fyn"'" and Fyn  +/+  populations. This difference in responsiveness between H - Y and 2C CD8 SP  thymocytes to stimulation by 2C11 likely reflects differences in the functional maturity of these thymocytes.  3.1.4.  CD8 SP thymocytes and lymph node cells with the Fyn' mutation are hyporesponsive to low affinity antigenic ligands  We next determined the effect of Fyn on the proliferative responses of CD8 SP thymocytes and lymph node cells from female H-2 H - Y and H-2 2C T C R transgenic b  b  mice to stimulation by their physiologic ligands. In this situation, the affinity of the T C R for its antigen ligand is not circumvented by the direct stimulation of the CD3e chain.  46  H-Y / Fyrr  CD8 29 2 55 14  3 2 42 54  5 2 62 31  32 2 52 15  Figure 3.4. Down-regulation of HSA by CD8 SP peripheral T cells from Fyn"'" mice. Lymph node cells from H-2 2C and female H-2 H - Y TCR transgenic mice with or b  b  without the Fyn"'" mutation were stained as described in Figure 3.1. The percentages of cells in each quadrant of the dot plots are indicated below each plot. The expression of the indicated cell surface molecule by gated CD8 SP thymocytes (enclosed box in the lower right quadrant) is depicted in the histograms directly below each of the dot plots. Data from one representative experiment of three are shown.  47  H-Y 7000 6000 5000 % 4000 o 3000 2000 1000 0  I  2C 5000' 4000  I  3000 2000 1000  0 [J B 0  Fyn Thymocytes Fyn " Thymocytes Fyn Lymph Node Cells Fyn" " Lymph Node Cells +/+  -7  +/+  7  Figure 3.5. Fyn' thymocytes expressing the H-Y TCR are hyporesponsive to CD3 stimulation. Purified CD8 SP thymocytes or lymph node cells (1 x 10 ; see Materials and 4  Methods) from female H-2 H - Y TCR transgenic mice or 2C TCR transgenic mice were b  stimulated with 1 |^g/ml of 2C11 (anti-CD3 mAb) in the presence of 20 U/ml of murine rIL-2. Cultures were set up in triplicates. Each culture was incubated with 1 uCi of [ H] 3  TdR /well for the final 16 h before harvest. Data from one representative experiment of three are shown.  48  Therefore, this approach provides a more critical evaluation of the requirement of Fyn for the activation of T cells by their cognate ligands. Purified CD8 SP thymocytes and lymph node cells were stimulated with irradiated splenocytes in a standard proliferation assay. Cells transgenic for the H - Y TCR were stimulated with syngeneic (C57BL/6) male splenocytes, whereas 2C TCR cells were stimulated with BDF1 (C57BL/6 x DBA/2) splenocytes. As seen in Figure 3.6, both Fyn" CD8 SP thymocytes and lymph node cells /_  transgenic for the H - Y TCR were hyporesponsive to the male antigen. The response of this TCR to its cognate ligand was ~10-fold lower than the Fyn  +/+  cells. Neither the Fyn  +/+  nor the Fyn" cells were able to respond to female B6 splenocytes, indicating that the /_  observed response was indeed male specific. In contrast to these results, Fyn " CD 8 SP thymocytes or lymph node expressing the _/  2C TCR showed only slightly reduced proliferative responses to stimulation by BDF1 splenocytes relative to their Fyn  +/+  counterpart (Fig. 3.7). These CD8 SP cells did not  respond to B6 or B10.BR splenocytes, indicating that the response was indeed specific for H-2 . Thus, depending on the transgenic TCR, CD8 SP thymocytes or lymph node d  cells display differential requirement for Fyn in optimal proliferative response to stimulation by their specific antigen. One hypothesis to account for the differential requirement for Fyn by CD8 SP thymocytes or lymph node cells expressing either the H - Y or the 2C TCR for optimal responses to stimulation by their specific antigen is that the H - Y TCR is a low affinity TCR and the 2C TCR is a high affinity TCR. We reasoned that a low affinity TCR is more dependent on Fyn than a high affinity TCR for optimal stimulation by antigen. To test this hypothesis we took advantage of the fact that the affinity of the 2C TCR for various antigenic ligands has been determined. We used human T2 cells transfected with either the L or K molecules as APCs (123). Human T2 cells are deficient in peptide d  b  transport and the L or K molecules expressed by T2 cells can be loaded with a specific d  b  peptide, which in our case is p2Ca. The density of the L /p2Ca or the K /p2Ca ligand on d  b  T2 cells can be varied by exposing these APCs to different concentrations of p2Ca. The relative affinity of the 2C TCR for the L /p2Ca and the K /p2Ca ligand has been d  b  determined to be ~2 x 10 M" and ~3 x 10 M" , respectively (113). This approach 6  1  3  1  therefore enabled us to determine whether CD8 2C T C R SP thymocytes or lymph node +  cells can display differential requirements for Fyn in their response to a high affinity (L /p2Ca) or a low affinity (K /p2Ca) ligand. As shown in Figure 3.8, 2C Fyn' d  b  49  A  Thymocytes 10000  1000 CL,  ,.o'  JO"  10  0  -•— F y n -0-- Fyn - B — Fyn" Fyn"  anti-male anti-female anti-male anti-female  +/+  2000  10000  30000  Number of CD4CD8+ Cells  Lymph Node Cells  +/+ A A  10000  1000 S U  -o  10:  2000  10000  30000  Number of CD4CD8+ Cells  Figure 3.6. H-Y Fyn"'" thymocytes and lymph node cells are hyporesponsive to antimale stimulation. The indicated numbers of purified CD8 SP thymocytes or lymph node cells from female H-2 H - Y TCR transgenic mice were cultured in triplicate with 5 x 10 irradiated b  5  male splenocytes in 96-well plates. Irradiated female splenocytes were used as a negative control. Cultures were incubated with 1 uCi of [ H] TdR/well for the final 16 h before 3  harvest. Data from one representative experiment of three are shown.  50  Lymph Node Cells  Thymocytes 100000  100000  10000 a. U  1000  " 10 Number of CD4CD8+ Cells 4  Number of C D 4 C D 8 Cells +  TO  5  2C + BDF1 - D -  2C + B6  —A—  2C + B10.BR 2C/Fyn- " + BDF1 /  2C/Fyn- " + B6 /  2C/Fyn- - + B10.BR /  Figure 3.7. 2C Fyn"" thymocytes and lymph node cells exhibit normal proliferative responses to stimulation by BDF1 spleen cells. The indicated numbers of purified CD8 SP thymocytes or lymph node cells from female H-2 H - Y TCR transgenic mice were cultured in triplicate with 5 x 10 irradiated b  5  BDF1 splenocytes in 96-well plates. Irradiated B6 and B10.BR splenocytes were used as negative controls. Cultures were incubated with 1 uCi of [ H] TdR /well for the final 16 h 3  before harvest. SD, calculated from triplicate samples, was less than 10% of the mean and is therefore not shown. Data from one representative experiment of three are shown.  51  thymocytes required 100- to 1000-fold more p2Ca peptide to respond to the same extent as the Fyn  +/+  thymocytes when stimulated with the T2-L cells and exogenous p2Ca d  peptide. This response was p2Ca specific since no proliferative response was observed in the presence of another L -binding peptide (YPHFMPTNL) (113) (data not shown). d  Thus, even for a high affinity ligand, CD8 SP thymocytes required a relatively high ligand density for optimal proliferation to the specific antigen. These results indicate that the lowering of the avidity of the T cell/APC interaction revealed a requirement for Fyn by CD8 SP thymocytes expressing a high affinity TCR for its specific antigen. By contrast, only minor differences were evident between CD 8 SP Fyn"'" and Fyn  +/+  lymph  node cells in their response to the L /p2Ca ligand, even at the lowest peptide d  concentrations studied (Fig. 3.8). This independence of Fyn by CD8 SP lymph node cells even at low ligand density likely reflects the more functionally mature status of the lymph node cells relative to thymocytes. The results examining the role of Fyn in the response of 2C CD8 SP lymphocytes and lymph node cells to the low affinity K /p2Ca ligand are b  shown in Figure 3.9. As a result of such a low affinity interaction between T cells and APCs, it was necessary to add exogenous IL-2 to these cultures to detect a proliferative response to the K /p2Ca ligand. As seen in Figure 3.9, even in the presence of b  exogenously added IL-2, proliferative responses were only observed at higher peptide concentrations. Significantly, both Fyn" " CD8 SP thymocytes and lymph node cells were 7  hyporesponsive to stimulation by the K /p2Ca ligand relative to their Fyn b  +7+  counterparts.  Not only were the proliferative responses by Fyn" " cells substantially reduced, the 7  responses attained by the Fyn" " cells did not approach that of Fyn 7  +7+  cells, even at high  ligand density. These observations emphasized the importance of Fyn in optimizing the responses of T cells expressing low affinity TCRs to stimulation by their specific antigen. 3.2  Discussion The studies in this chapter were aimed at establishing whether the Fyn P T K has an  essential and unique role in TCR signaling during T cell development and T cell activation. This was done using mice transgenic for TCRs with differing affinity for their antigenic ligands and with the Fyn null mutation. The results indicate that Fyn plays a role in TCR signaling during both thymic development and the proliferation responses of mature T cells and is differentially required for high and low affinity/avidity interactions. A previous study suggests that the positive selection of CD8 SP thymocytes expressing high levels of the H - Y TCR occurs normally even in the absence of Fyn expression (134). Our results are consistent with this report in that we also observed normal production of CD8 SP thymocytes expressing high levels of the H - Y TCR in 52  100000  0  10-  !  IO-  io-  1  ;  10  Concentration of p2Ca ((Xg/ml)  Figure 3.8. 2C Fyn"'" thymocytes are hyporesponsive to stimulation by the high affinity L /p2Ca ligand at low ligand density. d  Purified CD8 SP thymocytes or lymph node cells from H-2 2C TCR transgenic mice b  were cultured in triplicate with mitomycin C-treated T2-L cells. The indicated d  concentrations of the p2Ca peptide were added. Cultures were incubated with 1 uCi of [ H] TdR /well for the final 16 h before harvest. Data from one representative experiment 3  of three are shown.  53  Concentration of p2Ca (fig/ml) Figure 3.9. 2C Fyn"" thymocytes and lymph node cells are hyporesponsive to a low affinity ligand. Purified CD8 SP thymocytes or lymph node cells from H-2 2C TCR transgenic mice b  were cultured with mitomycin C-treated T2-K cells. The indicated concentrations of the b  p2Ca peptide were added. Cultures were incubated with 1 uCi of [ H] TdR /well for the 3  final 16 h before harvest. Data from one representative experiment of three are shown.  54  female H-2 Fyn"'" H - Y T C R transgenic mice. Our study extended this finding by b  demonstrating that Fyn does have subtle effects on positive selection. Our results suggest that although Fyn is not essential for the positive selection of the H - Y TCR, it may facilitate the transition of positively selected DP thymocytes into SP thymocytes. Furthermore, we found that Fyn facilitated the down-regulation of H S A on positively selected CD8 SP thymocytes. Previous reports have provided evidence for the 2C TCR being strongly selected in H-2 2C mice (114), with increases in CD8 expression leading b  to deletion (63, 135). With a recent report indicating that the efficiency of TCR signaling during T cell development is greatly reduced by the abrogation of TCR-^ derived signals (136), it is reasonable to hypothesize that other molecules that contribute to T C R signaling may also influence these processes. Deletion of Fyn is an ideal means to study partial disruption of TCR-£ signaling. The Fyn PTK is constitutively associated with the TCR-^ chain and has been shown to phosphorylate it upon TCR stimulation, allowing for the association of ZAP-70 with the cytoplasmic tail (reviewed in (13). Murine ZAP-70"'" thymocytes arrest at the DP stage (21), suggesting that ZAP-70 signaling is essential for T cell development. As Fyn phosphorylates and activates ZAP-70 (101), it may play a role during thymocyte development. However, this situation is complicated by the finding that another PTK, namely Lck, can also phosphorylate ZAP-70 (101). Thus, the question of whether Fyn is a redundant kinase for T cell development and function has not been resolved. In this study, we found that although Fyn is not essential for negative selection, it alters the cell surface phenotype of surviving thymocytes in either a very strongly positively selecting M H C background (H-2 2C mice) or a negatively selecting M H C b  background (male H-2 H - Y mice). In H-2 2C TCR transgenic mice, introduction of the b  b  Fyn"'" mutation led to a twofold increase in the number of DP thymocytes. In male H-2  b  H - Y TCR transgenic mice, the Fyn"'" mutation enables the survival of thymocytes that expressed higher levels of the CD8 co-receptor and the H - Y TCR. These observations are consistent with the hypothesis that the Fyn"'" mutation may reduce the signaling efficiency of the TCR/CD3 complex during the negative selection process. This hypothesis agrees with previous findings showing that lowering the TCR signaling efficiency by altering the number of £ chains or by the deletion of the C, chain can allow for the survival of thymocytes that would have been expected to have been deleted (5, 136). If the Fyn"'" mutation lowers the effective signaling capabilities of the TCR/CD3 complex and allows for higher avidity interactions before deletion, then one would  55  anticipate higher levels of co-receptor expression on Fyn"'" cells. This was indeed the case in the 2C Fyn"'" thymocyte population, where CD8 expression was higher than that of Fyn  +/+  thymocytes. The reduced deletion and increased CD8 expression in the Fyn"'" 2C  system demonstrate that Fyn does play a role in TCR signaling during thymocyte development. This effect of Fyn was not observed in female H-2 H - Y T C R transgenic b  mice. This can be explained by postulating that the H - Y T C R is positively selected by relatively low affinity/avidity thymocyte/selecting cell interactions in female transgenic mice. This level of interaction leading to the positive selection of H - Y thymocytes in female mice is presumed to be well below the deletion threshold, but above the minimum positive selection threshold. Under these conditions, we do not expect Fyn to have a discernible effect on the survival of DP thymocytes, and this is observed. Previous studies have shown that high cell surface expression of the H S A molecule on CD8 SP thymocytes correlates with positive selection by low affinity/avidity ligands and that these cells are functionally immature (133). Here, we have observed that HSA levels in CD8 SP thymocytes from both H - Y Fyn"'" and 2C Fyn"'" thymocytes are elevated relative to those that expressed Fyn. This observation also argues for the selection of these thymocytes by weaker selecting signals and further implicates a role for Fyn in the positive selection process. However, we noted that although Fyn"'" thymocytes from both TCR transgenic backgrounds had increased H S A expression, only the H - Y Fyn"'" thymocytes were hyporesponsive to stimulation by anti-TCR Abs. This observation may be explained by the hypothesis that the H - Y T C R is positively selected with low efficiency in the first place. The further lowering of this efficiency by introducing the Fyn"'" mutation will lead to the production of CD8 SP thymocytes that are functionally less mature. The Teh lab has recently shown that the 2C TCR, when selected by very weak selecting ligands in an H-2 thymus, also led to the production of CD8 SP H S A k  hlgh  thymocytes that were hyporesponsive to stimulation by anti-TCR antibodies (133). That only minimal differences can be observed in the 2C thymocytes can be explained by the same hypothesis. In this situation, the selecting ligand for these cells is of sufficiently high affinity/avidity that the Fyn"'" mutation does not lower the effective signal enough to slow the development of these cells as significantly as in the H - Y system. Hence, these cells are able to proliferate when stimulated with anti-CD3 mAb (2C11) and IL-2. Stimulation of CD8 SP Fyn"'" cells with their physiologic ligands provided evidence that Fyn is indeed involved in the proliferation responses of both thymocytes and lymph node cells. As these stimulation protocols do not circumvent the natural affinity of the TCR for its ligand, these results provide clear evidence for a role for Fyn in signaling from low affinity TCRs. Using purified CD8 SP cell populations, we saw that both the 56  thymocyte and lymph node cell populations from H - Y Fyn" mice were hyporesponsive A  in an anti-male response. This observation suggested that the low affinity H - Y response was compromised by the Fyn mutation. However, the response of CD8 SP 2C thymocytes or lymph node cells to naturally processed ligands on BDF1 splenocytes did not reveal a role for Fyn in this response. We attribute this to the high affinity of the 2C TCR for the L /p2Ca ligand. Thus, Fyn is not essential for an optimal response by CD8 d  SP thymocytes or lymph node cells, which expressed a high affinity TCR for its cognate ligand. Importantly, we found that we can reveal a role for Fyn in 2C TCR signaling by lowering the affinity/avidity of the 2C/APC interaction by using T2-K or T2-L cells as b  d  APCs and varying the concentrations of the p2Ca peptide. Under these conditions, we observed a role for Fyn in the response of CD8 SP 2C thymocytes to the high affinity L /p2Ca ligand at low ligand density. In the case of the low affinity K /p2Ca ligand, we d  b  were able to demonstrate a role for Fyn even at high ligand density. These observations underscore the importance of Fyn in optimizing responses to low affinity ligands and provide independent evidence supporting the conclusion that the H - Y TCR is indeed a low affinity TCR. The results presented in this chapter provide evidence supporting a role for Fyn in TCR signaling during both thymocyte development and the activation of positively selected thymocytes and peripheral T cells. That the role of Fyn was not as evident in previous reports can be explained by the heterogeneity of the T cell populations that were analyzed. We propose that Fyn is an important player in the positive selection of T cells by low affinity/avidity ligands and in the activation of positively selected T cells by low affinity/avidity ligands. This function of Fyn appears not to be compensated for by other PTKs and argues against Fyn being a redundant kinase in T cell development and T cell activation.  57  Chapter 4  4.0  Introduction The data presented in the previous chapter demonstrate a role for Fyn in the  development and activation of T cells. Another objective for this thesis was to examine the role played by Fyn in T cell anergy. In order to carry out these studies it was first necessary to have a model system. Previous work done in the Teh lab (83) suggested that a population of functionally mature CD4CD8" (herein referred to as DN* cells) T cells that expresses a transgenic ap TCR might provide such a model. Results discussed in this chapter relate to the characterization of these cells. In chapter 5 I will cover work relating to investigations into the role of Fyn in this form of T cell anergy. Our model system for T cell anergy is based on the 2C TCR transgenic receptor (109, 114). The 2C TCR is specific for the p2Ca peptide (derived from the mitochondrial protein 2-oxoglutarate dehydrogenase) presented by L M H C class I molecules (111, d  112) and is positively selected by K M H C class I molecules (110). A substantial b  population of D N cells exists in the peripheral lymph nodes of both H-2 2C mice and H b  2  b/d  2C mice (expressing the p2Ca/L antigenic ligand). It has been shown previously that d  D N cells from both H-2 2C and H-2 b  b/d  2C TCR transgenic mice express equivalent levels  of the 2C TCR (83). However, these two D N populations differ in their responses to stimulation by the p2Ca/L ligand. It was found that D N cells from H-2 2C mice were d  b  able to proliferate in response to the p2Ca/L ligand, to produce IL-2 and to kill (83). In d  contrast, D N cells from the antigen expressing H-2  b/d  2C mice were defective in  proliferation and IL-2 production under identical conditions (83). Such unresponsiveness to antigenic stimulation is characteristic of T cell anergy. D N T cells have been observed in a number of transgenic systems, but they have often been dismissed as artifacts of TCR transgenics. Early studies on male mice transgenic for the H - Y TCR revealed a large population of T cells in the periphery  Please note that these functionally mature D N cells are not equivalent to the thymic population of immature cells mentioned in Chapter 1. 1  58  despite these mice having a very small thymus. These cells were shown to express high levels of the transgenic TCR, but did not express either CD4 or CD8 and were therefore given the double negative name. Other studies have since shown that D N cells are especially prominent in transgenic mice with a negatively selecting background. These cells appear early in the fetal thymus and colonize both epithelial and lymphoid tissues. The origin of these cells has been the subject of much debate. One possibility is that they have somehow escaped negative selection, undergone positive selection, and upon encounter with male specific peptides in the periphery have down-regulated their expression of CD8. A second possibility is that these cells had intrinsically low levels of CD8 expression, allowing them to escape negative selection, and have subsequently expanded in the periphery. A third possibility is that the D N cells represent a distinct lineage of a/p T cells that never expressed the CD4 or CD8 molecules. Development of D N cells in TCR transgenic mice is thymus-dependent (137) but independent of positively-selecting M H C molecules (137, 138). They are also resistant to clonal deletion in antigen expressing mice (116, 138). Evidence from several sources now suggests that the D N cells of TCR transgenic mice may in fact be y8 cells that have been hijacked by the early expression of the aP transgenic TCR (139, 140). In this chapter, I describe further analyses of the anergic state of the H-2  b/d  2C D N cells  and the signaling defects associated with it. The anergic cells were found to exhibit differential requirements in mobilizing intracellular calcium and in the activation of the Erk M A P kinase pathway. These biochemical characteristics were associated with an elevated protein expression level of Fyn but not Lck in the anergic T cells. The inefficient mobilization of intracellular calcium was associated with defects in ZAP-70 and L A T phosphorylation in response to TCR signaling. However, ERK1/2 activation and phosphorylation of SLP-76 were unaffected in anergic D N cells. More interestingly, the anergic D N cells were found to have a lowered activation threshold. It was found that in contrast to non-anergic D N T cells, anergic D N cells upregulated CD25 and CD69 in response to stimulation by a low affinity ligand. Furthermore, anergic D N cells proliferated vigorously in response to stimulation by the low affinity ligand and exogenousIL-2.  59  4.1  Results  4.1.1.  Anergic H-2  b/d  2C DN cells proliferate in response to a low affinity ligand  and exogenous IL-2 The hypo-proliferative response of antigen-stimulated anergic T cells has been reported to be reversible in some systems by the addition of IL-2 (70, 141). In agreement with our previous results (83) non-anergic D N cells from H-2 2C mice exhibit a b  significant proliferative response to stimulation with the high affinity p2Ca/L ligand d  even without the addition of exogenous sources of IL-2 (Fig. 4.1). The addition of exogenous IL-2 to these cultures led to a relatively modest increase in the proliferative response (Fig. 4.1). As previously reported, the anergic D N cells from H-2  b/d  2C mice  showed a minimal proliferative response when stimulated only with high concentrations of the p2Ca/L ligand. Strikingly, when exogenous IL-2 was added to p2Ca/L -stimulated d  d  cultures, they proliferated better than the non-anergic D N cells, particularly at low concentrations of the p2Ca/L ligand (Fig. 4.1). d  The 2C TCR is able to recognize the p2Ca peptide in the context of both the L and d  K M H C class I molecules, but with an ~1000 fold lowering in the affinity for the latter b  interaction (113). Since very low concentrations of the high affinity p2Ca/L ligand were d  able to induce a vigorous proliferative response in anergic D N cells in the presence of exogenous IL-2, we tested the hypothesis that the anergic D N cells may in fact have a lower activation threshold than the non-anergic D N cells. The results in Fig. 4.2 support such a hypothesis. It was found that non-anergic D N cells were unable to proliferate when stimulated with various concentrations of the low affinity p2Ca/K ligand, even in b  the presence of exogenously added IL-2. By contrast, the anergic D N cells already exhibit a small but significant proliferative response to the p2Ca/K ligand even in the b  absence of exogenous IL-2; this small proliferative response was also elicited by the high affinity ligand (Fig. 4.1). In the presence of exogenously added IL-2, these cultures mount a vigorous proliferative response (Fig. 4.2). These results support the hypothesis that the anergic D N cells have a lower activation threshold when compared to nonanergic D N cells.  60  60000  Concentration of p2Ca (|ig/ml) —•—b 2C DN vs. p2Ca/L  d  --^>-b 2C DN vs. p2Ca/L + IL-2 d  —•—b/d 2C DN vs. p2Ca/L  d  b/d 2C DN vs. p2Ca/L + IL-2 d  Figure 4.1. Exogenous IL-2 restores the proliferation response of Ag-stimulated H2  bd  2C DN cells.  Purified D N lymph node cells from H-2 2C and H-2 b  b/d  2C mice were cultured with  mitomycin C-treated T2-L cells and the indicated concentrations of the p2Ca peptide as d  described in Materials and Methods. Where indicated 20 U IL-2/ml were added to the cultures. Cultures were assessed for proliferation at the end of a 72 h culture period as described in Materials and Methods. The error bars represent the standard deviation (SD) of triplicate cultures. Data from one representative experiment of three are shown.  61  80000  60000  40000H  20000H  0.01  0.1  1  10  Concentration of p2Ca (ug/ml)  — • — b 2C DN vs. p 2 C a / k  b  — 0 - - b 2C DN vs. p 2 C a / k + IL-2 b  — • — b / d 2 C DN vs. p 2 C a / k  b  — • - - b / d 2 C DN vs. p 2 C a / k + IL-2 b  Figure 4.2. DN cells from H-2  b/d  2C mice are activated by the low-affinity p2Ca/K  b  ligand. Purified D N lymph node cells from H-2 2C and H-2 b  b/d  2C mice were cultured with  mitomycin C-treated T2-K cells and the indicated concentrations of the p2Ca peptide b  with or without 20 U IL-2/ml. Cultures were assessed for proliferation at the end of a 72h culture period. The error bars represent the standard deviation (SD) of triplicate cultures. Data from one representative experiment of three are shown.  62  We sought independent support for the hypothesis that anergic D N cells have a lower activation threshold than non-anergic D N cells. The expression of activation markers associated with T cell activation following stimulation of these cells with either the high or the low affinity ligand was assessed. D N cells from H-2 or H-2 b  b/d  2C mice were  activated with either the high or the low affinity ligand in the absence of exogenously added IL-2 and the expression of CD25 (the high affinity IL-2 receptor) and CD69 (an early activation marker) on these cells was determined after 40 hours of stimulation. The results in Fig. 4.3 show that the anergic D N cells were able to undergo blastogenesis (increase in forward scatter) and upregulate CD25 and CD69 in response to either the high or the low affinity ligand. By contrast, only the high, but not low, affinity ligand was able to induce blastogenesis, CD25 and CD69 expression in the non-anergic D N cells. These results are consistent with the proliferation data in Figures 4.1 and 4.2 and further support the hypothesis that the anergic D N cells have a lower activation threshold in comparison to the non-anergic D N cells. Furthermore, the vigorous proliferation that ensued when exogenous IL-2 was added to these stimulated cultures indicated that these anergic D N cells retain the biochemical machinery that is required for normal proliferation.  4.1.2.  Impaired tyrosine phosphorylation of L A T in anergic DN cells  The above findings suggest that the anergic D N cells were able to mediate signaling from the TCR, leading to effective induction of CD25 and CD69 expression. The Teh lab has shown in a recent study that antigen-activated anergic D N cells were defective in IL2 production (83). To reconcile these data, whole cell lysate phosphorylation profiles from H-2 2C and H-2 b  b/d  2C D N cells were compared in order to determine the extent of  the signaling defect in the anergic cells. As these experiments were done by stimulating the D N cells with anti-CD3e (2C11 mAb) we repeated the proliferation assays with 2C11 stimulation to confirm the proliferation defect in the anergic cells was not restricted to peptide/MHC stimulation (Fig. 4.4A). In the phosphorylation profiles we observed a  63  T2-L  D  +10 \M  p2Ca  T2-K  B  +10 J I M p 2 C a  FSC  CD69  CD25  Figure 4.3. Induction of CD25 and CD69 by low-affinity ligands in anergic DN cells. Purified D N lymph node cells from H-2 2C and H-2 b  b/d  2C mice were cultured with  mitomycin C treated T2-K or T2-L cells. The indicated concentrations of the p2Ca b  d  peptide were added. After 40h of culture, the cells were washed and stained with either anti-CD25 or CD69-biotinylated mAbs as well as the 1B2 Ab (anti-2C T C R idiotype). lB2-positive cells were gated and analyzed by FACScan. FSC indicates forward light scatter. Data from one representative experiment of three are shown.  64  Figure 4.4. L A T phosphorylation is defective in TCR-stimulated anergic DN cells. A , purified D N lymph node cells (1 x 10 /well) from H-2 2C and H-2 5  b  b/d  2C mice  were cultured with the indicated concentration of immobilized anti-CD3 m A B (2C11) with and without 20 U/ml of IL-2. Cultures were set up in triplicates and were assessed for proliferation at the end of a 72h culture period. The error bars represent the standard deviation (SD) of triplicate cultures. B, Purified H-2 2C and H-2 b  b/d  2C D N cells were  incubated with (+) or without (-) 10 ug/ml 2C11 for 3 min at 37°C. Whole-cell lysates from 1 x 10 cells were resolved by SDS-PAGE and immunoblotted with 4G10 anti6  phosphotyrosine mAb. Numbers indicate molecular weight in kilodaltons. C, H-2 2C b  and H-2  b/d  2C D N cells were stimulated as in B. L A T was immunoprecipitated from  whole-cell lysates of 5 x 10 cells. The precipitates were resolved by SDS-PAGE and 6  L A T phosphorylation was visualized by 4G10 mAb immunoblotting (upper panel). Blots were stripped and reprobed with anti-LAT-specific Abs (lower panel). D, Whole-cell lysates from H-2 2C and H-2 b  b/d  2C were blotted with anti-LAT-specific Abs to determine  L A T protein expression. Data from one representative experiment of three are shown.  65  A. 180000 160000 140000 120000 100000 80000  b2C  DN  b 2C DN + IL-2 b/d 2C D N b/d 2C DN + IL-2  60000 40000 20000 0 0.1  B. Blot Ab:  b  IP Ab: LAT  b/d b b/d Blot Ab: 4G10 LAT  4G10  D.  66  -  Blot Ab: LAT  b  b/d + - + •33 •33  b b/d r-33  major difference in the phosphorylation of a 36 kDa protein (Fig. 4.4B). The apparent molecular mass and rapid phosphorylation of this protein in the H-2 D N cells suggested b  this protein to be the L A T (Linker for the Activation of T cells) adapter molecule (26). Immunoprecipitation studies confirmed the identity of this 36kDa protein to be L A T (Fig. 4.4C) suggesting that impaired phosphorylation of L A T might be important to the defects observed in the H-2  b/d  2C D N cells. Protein expression of L A T , as determined by Western  blotting of whole cell lysates (Fig. 4.4D), showed a 34% reduction in L A T expression in the anergic population compared to the control cells. However, this reduction in protein expression is insufficient to explain the greatly reduced phosphorylation of L A T in the anergic D N cells. Phosphorylation per unit protein was reduced by 60% compared to control cells.  4.1.3.  ZAP-70 phosphorylation is reduced in H-2  b/d  2C DN cells  In order to explain the L A T phosphorylation defect observed in the H-2  b/d  2C D N  cells we examined signaling events known to be upstream of L A T phosphorylation. L A T is a major substrate of ZAP-70 and phosphorylation of L A T by ZAP-70 is a major event in T cell activation (26). We first determined the expression level of ZAP-70 in anergic and non-anergic D N cells and found it to be fairly equivalent (Fig. 4.5A). Immunoprecipitation studies of ZAP-70 found it to be tyrosine phosphorylated in nonanergic D N cells upon TCR ligation (Fig. 4.5B). Previous studies have shown that phosphorylation of ZAP-70 is associated with its activation (13, 17). B y contrast, tyrosine phosphorylation of ZAP-70 upon TCR ligation was found to be impaired in the H-2  b/d  2C  D N cells (Fig. 4.5B). In this experiment, we observed more tyrosine phosphorylation of ZAP-70 despite the fact that slightly less ZAP-70 was precipitated from TCR-stimulated non-anergic cells. This observation strengthens the conclusion that ZAP-70 tyrosine phosphorylation is impaired in anergic D N cells upon TCR stimulation. However, precipitation of less ZAP-70 from TCR-stimulated non-anergic cells was not a consistent finding since in repeat experiments similar amounts of ZAP-70 were precipitated from non-stimulated and stimulated cells (data not shown). Activation of ZAP-70 requires the  67  A .  b/d  Blot Ab: anti-ZAP-70  -70  IP Ab: Z A P - 7 0  B. b  Blot Ab:  b/d  + 4G10  70  anti-ZAP-70  70  Figure 4.5. Hypophosphorylation of ZAP-70 in TCR-stimulated anergic DN cells. A, Whole-cell lysates from purified H-2 2C and H-2 b  b d  2C were resolved by SDS-  P A G E and blotted with anti-ZAP-70-specific Abs to determine ZAP-70 protein expression. B, H-2 2C and H-2 b  b/d  2C D N cells were stimulated as in Fig. 4.4B. ZAP-70  was immunoprecipitated, precipitates were resolved by SDS-PAGE, and ZAP-70 phosphorylation was visualized by 4G10 mAb immunoblotting (upper panel). Blots were stripped and reprobed with anti-ZAP-70 specific Abs (lower panel). The molecular weight in kilodaltons is indicated to the right of each blot. Data from one representative experiment of three are shown.  68  immunoreceptor tyrosine based activation motifs (ITAMs) on the TCR-^ chain to be doubly phosphorylated to allow binding of the tandem SH2 domains present in ZAP-70 (13). These phosphorylation events occur via activation of the Src kinases, Lck and Fyn, and their subsequent recruitment to the TCR-^ chains, their auto-phosphorylation and then subsequently their phosphorylation of the ITAMs within the TCR chains (13, 142). We examined whether the hypophosphorylation of ZAP-70 in anergic D N cells is related to the less efficient phosphorylation of TCR-C, chains upon TCR stimulation. Fig. 4.6 indicates that there are fairly equivalent level of expression of the TCR-^ chain in anergic and non-anergic D N cells. Upon TCR stimulation, phosphorylation of p21 was fairly equivalent in these two cell types. However, the extent of p23 phosphorylation was found to be reduced in anergic D N cells. The reduced induction of the p23, which represents fully phosphorylated TCR-^ chains (143) may explain in part the less efficient recruitment and activation of ZAP-70 to the TCR/CD3 signaling complex in TCRstimulated anergic D N cells. Previous studies have shown that the protein expression level of Fyn is increased in anergic T cell clones (77). Here we showed that the protein expression level of Fyn, as determined by Western blot analysis, was increased in anergic D N cells (Fig. 4.7). Such an increase in expression was not observed for Lck (Fig. 4.7).  4.1.4.  Anergic DN cells have impaired mobilization of intracellular calcium  One major consequence of L A T phosphorylation is the recruitment via the Grb2 adapter molecule of signaling molecules such as P L C - y l , PI3 kinase, and other enzymes such that they are brought into proximity of relevant protein and lipid substrates, resulting in such signaling events as calcium elevation and Ras activation (24, 29). Having observed a defect in the phosphorylation of L A T in TCR-stimulated anergic D N cells, we examined the ability of these cells to mobilize intracellular calcium in response to T C R ligation. In this respect, the anergic D N cells were found to be inefficient in mobilizing intracellular calcium when compared to non-anergic D N cells (Fig. 4.8). This observation supports the notion that one consequence of defective L A T phosphorylation in anergic  69  b/d  Blot Ab:  10  15  4G10 A n t K (G3) Figure 4.6. Phosphorylation and expression levels of TCR£ in DN cells from H-2 2C b  and H-2 H-2 2C and H - 2 b  b/d  bd  2C mice. 2C D N cells were incubated with 10 ug/ml 2C11  for the indicated  number of minutes at 37°C. Whole-cell lysates from 10 cells were resolved by SDS6  P A G E and immunoblotted with 4G10 mAb (upper panel). Blots were stripped and reprobed with anti-^ specific Abs (G3, lower panel). Numbers to the right indicate molecular weight in kilodaltons. Data from one representative experiment of three are shown.  70  Blot Ab:  b  b/d  Figure 4.7. Fyn, but not Lck, is expressed at higher levels in DN cells from H-2 2C b/d  mice. Whole-cell lysates from H-2 2C and H-2 b  b/d  2C were resolved by SDS-PAGE and  blotted with anti-Fyn (upper panel) and anti-Lck (lower panel) specific Abs to determine protein expression. Numbers to the right indicated molecular weight in kilodaltons. Data from one representative experiment of three are shown.  71  D N cells is a failure to mobilize intracellular calcium efficiently in response to T C R ligation. 4.1.5.  Normal phosphorylation of SLP-76 and ERK1/2 activation in TCRstimulated anergic DN cells  The adapter molecule SLP-76, like L A T , is a linker molecule closely associated with proximal TCR signaling (reviewed in (144)). SLP-76 also undergoes tyrosine phosphorylation upon TCR engagement and is also a substrate of ZAP-70 (33). SLP-76 has been shown to associate with the SH3 domain of Grb2 via proline-rich motifs and is essential for the coupling of TCR-regulated PTKs to downstream signaling pathways (145). Previous studies have also shown that tyrosine phosphorylation of P L C - y l and the Ras signaling pathway are defective in SLP-76" T cells (33). The anergic D N cells /_  exhibit efficient induction of CD69 (Fig. 4.3), which has been shown to be dependent on the Ras signaling pathway (33). However, induction of calcium mobilization in TCRstimulated anergic D N cells was shown to be defective (Fig. 4.8). To reconcile these findings, we hypothesize that L A T and SLP-76 may depend differentially on ZAP-70 for their phosphorylation. We also propose that phosphorylated SLP-76 may be sufficient to link TCR signaling pathways to the Ras signaling pathway in anergic D N cells. In order to test this hypothesis, we examined SLP-76 phosphorylation in anergic and non-anergic DN cells upon TCR ligation (Fig. 4.9). SLP-76 was immunoprecipitated from these two cell types before and after TCR stimulation. It was found that similar amounts of SLP-76 were precipitated from H-2 and H-2 b  b/d  2C D N cells and it was similarly phosphorylated  in both cell types after TCR stimulation (Fig. 4.9). This observation is consistent with the notion that normal phosphorylation of SLP-76 is less dependent on activated ZAP-70. Alternatively, it is conceivable that other uncharacterized pathways are responsible for SLP-76 phosphorylation in anergic D N cells. Our observation of efficient CD69 induction in anergic D N cells after antigen stimulation (Fig. 4.3) suggest that the Ras signaling pathway is intact in these cells. Since ERK1/2 phosphorylation and activation occurs downstream of the Ras signaling pathway we sought independent confirmation of activation of the Ras signaling pathway by examining the phosphorylation of ERK1/2 in non-anergic and anergic D N cells upon  72  500  - b 2C D N  c(D co 4 0 0 -  lonomycin  .dded  *=s=t o  '[  i  i  I  0  I  [  i  i  i  i  157  |  i  i  315  i  r  |  i  i  i  i  473  Time (seconds)  |  i  631  789  Figure 4.8. Less efficient mobilization of intracellular calcium in TCR-stimulated H2  b/d  2C DN cells.  Purified H-2 2C and H-2 b  b/d  2C D N cells were loaded with the calcium-binding dye  fiuo-3-acetoxymethyl ester and analyzed at 5 x 10 cells/ml for mobilization of 5  intracellular calcium by FACS analysis using Chronys software. 2C11 (10 (ig/ml) was added at 92s, ionomycin at 470s, and M g C l at 584s. Data from one representative 2  experiment of three are shown.  73  b  Blot Ab: 4G10  U  b/d  +  +  ^70  SLP-76  ^70  Figure 4.9. Normal phosphorylation of SLP-76 in H-2 H-2 2C and H - 2 b  bd  b/d  2C DN cells.  2C D N cells were stimulated as in Fig. 4.4B. SLP-76 was  immunoprecipitated and SLP-76 phosphorylation was visualized by 4G10 mAb immunoblotting (upper panel). Blots were stripped and reprobed with anti-SLP-76 specific Abs (lower panel). Numbers to the right indicated molecular weight in kilodaltons. Data from one representative experiment of three are shown.  74  b/d  b  anti-pErk1/2 anti-Erk1/2 Figure 4.10. Normal activation of ERK1/2 in H-2 H-2 2C and H - 2 b  bd  b/d  2C DN cells.  2C D N cells were incubated with or without 10 ug/ml 2C11 for  the indicated number of minutes at 37°C. Whole-cell lysates from 10 cells were resolved 6  by SDS-PAGE and immunoblotted with anti-phospho-ERKl/ 2 Abs (upper panel). Blots were stripped and reprobed with anti-ERKl/ 2-specific Abs (lower panel). Numbers to the right indicated molecular weight in kilodaltons. Data from one representative experiment of three are shown.  75  TCR ligation. The results in Fig. 4.10 indicate that ERK1/2 phosphorylation occurs normally in anergic D N cells upon TCR stimulation. Quantitation of the data in Fig. 4.10 showed that the level of phosphorylation of ERK1/2 in H-2  b/d  D N cells was within 10%  of that observed in H-2 D N cells. Our data are therefore consistent with the hypothesis b  that the Ras signaling pathway and ERK1/2 are activated normally in TCR stimulated anergic D N cells.  4.2  Discussion In this chapter I have provided functional evidence for the lowering of activation  threshold in a population of in vivo anergized T cells. I found that these anergic cells are able to express CD25 and CD69 and proliferate extensively when stimulated with a low affinity ligand in the presence of an exogenous IL-2 source. By contrast, non-anergic T cells which express equivalent levels of the 2C TCR (83) were not activated by the low affinity ligand. I have also provided biochemical analysis of these in vivo anergized T cells that was aimed at increasing our understanding of the inherent signaling defects of in vivo anergized T cells. The results indicate that TCR induced phosphorylation of L A T is defective in these anergic T cells and that this may be due to their failure to fully activate ZAP-70 upon TCR ligation. The inefficient mobilization of intracellular calcium in TCR-stimulated anergic cells may be a consequence of defective L A T phosphorylation. The defect in L A T phosphorylation is associated with an elevated basal level of the Fyn PTK. In contrast to defective L A T phosphorylation, SLP-76 phosphorylation occurs normally in TCR-stimulated anergic cells. The induction of CD69 in antigen-stimulated cells and the efficient phosphorylation of ERK1/2 M A P kinases also suggest that the Ras signaling pathway is unaffected in these anergic T cells. A critical defining characteristic of T cell anergy is an inability to produce IL-2 upon TCR ligation. The Teh lab has shown previously that antigen-stimulated anergic D N cells are defective in IL-2 production (83). Distinct regulatory regions exist in the 5' promoter region of the IL-2 gene and these distinct regions can be bound by varied nuclear factors to initiate IL-2 transcription. These factors include the AP-1 and N F A T proteins. The A P 1 family of nuclear factors include members of the Fos and Jun families. Fos and Jun can  76  bind D N A as the AP-1 complex and have been implicated in the control of IL-2 transcription. AP-1 dependent D N A binding and IL-2 gene transcription are deficient in some forms of anergy (72). In conjunction with AP-1, N F A T is also involved in IL-2 transcription and can bind the IL-2 promoter at an N F A T site when complexed with A P 1. In contrast to Fos and Jun, which are regulated by Erk and JNK activation (146, 147), N F A T exists in the cytoplasm in an inactive phosphorylated form. TCR mediated activation of calcium signaling leads to activation of calcineurin, a phosphatase able to dephosphorylate NFATp allowing N F A T to enter the nucleus and bind D N A in the presence of AP-1 (148). Thus the nature of IL-2 transcriptional regulation is such that different mechanisms may exist to control the production of IL-2. As mentioned above, a failure to activate the AP-1 complex has been reported in many forms of anergy. As well, a failure to activate N F A T has also been reported (76) and this form of anergy has been referred to as calcium-blocked anergy to contrast it from Ras-blocked anergy (reviewed in (141)). M y findings are consistent with the form of anergy studied here to be calciumblocked and not Ras-blocked. The failure of anergic D N cells to mobilize intracellular calcium upon TCR ligation is likely due to their inability to phosphorylate L A T , and this is most likely due to their failure to optimally activate ZAP-70 (Fig. 4.5). Similar reductions in phosphorylation of a 38-kDa molecule, possibly L A T , have previously been reported in anergic T 1 cells H  (149). L A T is phosphorylated by ZAP-70 upon TCR ligation, leading to recruitment of multiple signaling molecules that culminates in the activation of calcium and Rasdependent pathways (29). In this chapter I have shown that whereas defective L A T phosphorylation affected calcium-dependent pathways in anergic D N cells, it does not seem to affect the Ras signaling pathway in these cells. I also observed efficient tyrosine phosphorylation of SLP-76 upon T C R ligation in the anergic cells (Fig. 4.9). Since SLP-76 is a substrate for ZAP-70 (33) this observation suggests that either sub-optimally activated ZAP-70 is sufficient to phosphorylate SLP-76 but not L A T , or alternatively SLP-76 may be phosphorylated by other mechanisms that remain to be defined. Recent work by Madrenas' group (30) has provided evidence of a mechanism by which SLP-76 can become phosphorylated in the absence of L A T phosphorylation. They demonstrate that upon TCR stimulation with a partial agonist,  77  L A T is not phosphorylated but Grb2 and SOS are recruited to the partially phosphorylated p21 chains of the TCR. By interaction with a Grb2 SH3 domain SLP-76 can perhaps come into contact with Lck to allow for its phosphorylation. Previous studies have shown that SLP-76 tyrosine phosphorylation is required for optimal SLP-76 function including Vav recruitment to SLP-76 (reviewed in (144)). SLP-76 deficient Jurkat T cells exhibit a marked reduction in PLC-yl tyrosine phosphorylation, intracellular calcium mobilization and Erk activation (33). Since Erk activation, but not calcium mobilization, is normal in TCR-stimulated anergic D N cells, these findings support the notion that phosphorylated SLP-76 alone, in the absence of phosphorylated L A T , is sufficient to activate the Ras pathway. However, it is insufficient to activate intracellular calcium mobilization. I have shown that the p23 form of the TCR-£ chain is induced to a lesser extent in TCR-stimulated anergic D N cells. It is unclear whether this is a consequence of the increased expression of Fyn relative to Lck. The less efficient phosphorylation of p23 may lead to less efficient recruitment and activation of ZAP-70. Alternatively, and/or in addition to this mechanism, the failure to fully activate ZAP-70 and hence phosphorylate L A T may be due to the presence or activation of a negative regulator of ZAP-70. Previously, constitutive association of c-Cbl with Fyn has been observed in anergic T cells (74). I found that anergic D N cells have increased basal level of Fyn expression and this may lead to more efficient phosphorylation of c-Cbl. Recent studies have shown that c-Cbl acts as a negative regulator of ZAP-70 (22, 78). Furthermore, tyrosine phosphorylation of L A T and SLP-76 has also been shown to be sustained in TCRstimulated c-Cbl " thymocytes (150, 151). It is therefore conceivable that the sub-optimal 7  phosphorylation of ZAP-70 in TCR-stimulated anergic D N cells is due in part to increased Fyn expression and recruitment of c-Cbl to the TCR signaling complex. In this scenario the hypo-phosphorylation of ZAP-70 is a consequence of negative regulation by c-Cbl. I carried out investigations to explore this possibility. Specifically, I attempted coimmunoprecipitate c-Cbl and Fyn from anergic D N cells. However, these experiments failed to provide evidence for the interaction of these two molecules. Unfortunately, due to the limitations of the system in terms of recoverable material, more detailed investigations were not possible.  78  The elevated levels of Fyn protein expression described in this chapter agree with previously published observations from other groups. Therefore, it was of interest to investigate anergic D N cells with a targeted disruption of thefyn gene to try to discover what role, i f any, the elevated expression levels of Fyn may be playing. These experiments will be presented in the following chapter.  79  Chapter 5  5.0  Introduction In the previous chapter I presented a biochemical analysis of anergy in D N T cells.  Significantly, I found that the H - 2 D N T cells expressed elevated levels of Fyn protein b/d  (Figure 4.7). This result agrees with previously published data (77, 106). Other results that suggest Fyn may play an important role in anergy models are based on recent observations from Jurkat cells lacking Lck demonstrating that preferential TCR signaling via Fyn results in reduced ZAP-70 activation, reduced phosphorylation of L A T , normal SLP 76 phosphorylation and CD69 induction, and impaired IL-2 production and cell growth (108). Such a signaling phenotype agrees closely with what we have observed in the anergic H-2  b/d  D N cells and therefore provide further rationale for investigating the  role of Fyn in T cell anergy. In addition, Fyn has been reported to preferentially associate with the TCR^ chain (87) and this preferential association has been shown to be important for T cell anergy (107). I addressed questions regarding the importance of Fyn in T cell anergy by using Fyn null mice and the anergy model discussed in the previous chapter. I found that anergic cells lacking Fyn proliferate nearly normally in response to antigen stimulation, in contrast to the impaired proliferation of Fyn expressing anergic T cells. This recovery can not be explained by ameliorated production of IL-2 and the improved proliferation correlates with an enhanced ability of the Fyn" anergic T cells to upregulate the high /_  affinity IL-2 receptor. In antibody blocking experiments I found that CD25 was indeed responsible for the enhanced proliferation of the H-2  b/d  Fyn" D N cells. We also observe A  that anergic cells have a heightened survival ability that is partially dependent on the elevated levels of Fyn and IL-2R(3 expressed by these cells. The enhanced survival correlates with an increased capacity of the anergic cells to respond to IL-15. These studies support the hypothesis that Fyn participates in the induction and/or maintenance  80  of T cell anergy. Furthermore, Fyn promotes the survival of anergic T cells by a I1-15/IL2R.P dependent mechanism.  5.1  Results  5.1.1.  The deletion of Fyn from anergic DN cells leads to partial recovery of their proliferation defect.  T cell anergy is generally defined as the failure of T cells to proliferate and produce IL-2 in response to stimulation with their cognate antigenic ligand (82). We compared the proliferative responses of H-2 and H-2 b  D N cells with and without Fyn to H-2L and  b/d  d  the p2Ca peptide in a standard proliferation assay (Fig. 5.1). The response of H-2 and H b  2 F y n D N cells reveals that cells lacking Fyn were at a disadvantage in the proliferation b  /_  assay, requiring between 100 and 1000 times more peptide to yield the same counts per minute (CPM) as their Fyn expressing counterparts, a result similar to what we have observed for C D 4 C D 8 1 B 2 T cells (Chapter 3 and (152)). Conversely, the H-2 +  +  cells clearly outperform the Fyn expressing H-2  b/d  b/d  Fyn " 7  cells. The phenomenon also held true  when the cells were stimulated with the low affinity p2Ca/K ligand. These results b  demonstrate that Fyn does play a role in in vivo induced T cell anergy. To address whether the role of Fyn in T cell anergy is restricted to TCR signaling or i f it extends to other aspects of T cell biology, we designed the following experiments.  5.1.2.  H-2  b/d  Fyn"'" DN cells do not have improved IL-2 production  The Teh lab has previously demonstrated that the H-2  b/d  D N cells have impaired IL-2  production compared to D N cells from H-2 mice (83). One possible explanation for the b  improved proliferation of the H-2  b/d  Fyn" DN cells could be an improved ability to /_  produce IL-2 compared to their Fyn expressing counterpart. Therefore, I examined the ability of the H-2  b/d  Fyn " cells to produce IL-2 mRNA using a competitive RT-PCR _/  method (125). However, contrary to this prediction, I was unable to detect any difference in IL-2 transcription between the H-2  b/d  cells with or without Fyn. Interestingly, I did  observe a reduction in the ability of the H-2 Fyn cells to produce IL-2 mRNA which b  81  A  may, at least in part, explain their decreased proliferation observed in Fig. 5.1. As we were unable to detect any increased production of IL-2 by the H-2 Fyn" " D N cells I b7d  7  sought alternative explanations for their enhanced proliferation. 5.1.3.  H-2  b/d  Fyn' DN cells display enhanced upregulation of the high affinity IL-2  receptor Expression of the high affinity IL-2 receptor is essential for optimal T cell proliferation, and upregulation of the high affinity IL-2 receptor is a critical step in the T cell activation process. Naive T cells express a low affinity heterodimeric receptor consisting of the common gamma chain (yc) and the IL-2 receptor beta chain (IL-2RP) (reviewed in (153)). After activation-induced expression of the alpha chain (CD25), it pairs with yc and IL-2RP, and forms the high affinity IL-2 receptor. This heterotrimeric receptor has a 100 fold greater affinity for IL-2 than the IL-2Rp/yc pair (K =10pM vs. d  K = l n M ) . As IL-2R.0C is a key component of the high affinity IL-2R and is only d  expressed on activated T cells, it seems reasonable to propose that activated cells that are able to rapidly upregulate IL-2Ra would outgrow cells that do not. Interestingly, IL-2RP has a dual function, serving also as the beta subunit of the IL-15 receptor and binding IL15 (154). Previously, we have observed that CD25 and CD69 are upregulated more rapidly by H-2  b/d  than H-2 D N cells following antigen-stimulation (Chapter 4 and (155)). To b  address whether Fyn has a role in these processes, I stimulated H-2 D N cells with or b/d  without Fyn with the antigenic ligand and assayed by FACS their ability to upregulate these two activation markers. As seen in Fig. 5.3 (top panel), the H-2  b/d  Fyn" " D N cells 7  were able to upregulate CD25 to maximal levels with p2Ca/L at 1 |JM, whereas the H d  2  b/d  D N required 10 flM p2Ca/L to maximally express CD25. Interestingly, with the low d  affinity p2Ca/K ligand, the H-2 b  u M p2Ca dose but the H-2  b/d  b/d  Fyn" " D N achieved maximal upregulation with the 1 7  D N were unable to maximally upregulate CD25 even with  the 10 (iM p2Ca dose. These observations suggest that the H-2 even lower threshold for CD25 upregulation than the H-2  b7d  b/d  Fyn  Fyn" " D N cells have an  +7+  7  D N cells. In contrast to  CD25 upregulation, CD69 upregulation did not appear to be influenced by the expression of Fyn, with rapid increases in surface expression being observed in both the H-2  82  b7d  and  B  20000 -m- H-2b/d 15000  -•--H-2b/d Fyn-/ APC=T2-I_d  10000 500 0 0*  C  0.01  0.1 1 p2Ca (uM)  2000 0 -•—H-2b/d  ^ 1500 0  -o--H-2b/d Fyn-' APC=T2-Kb  1000 0 500 0 0*  0.01  10  0.1 1 p2Ca (uM)  Figure 5.1. Deletion of Fyn allows enhanced proliferation of anergic H-2 DN cells. b/d  D N cells from H-2 , H-2 Fyn"'", H-2 b  b  b/d  and H-2  b/d  Fyn " mice were stimulated in a 7  standard proliferation assay for 72 hours and assayed for H thymidine incorporation. 3  Stimulation was done either with p2Ca presented by mitomycin C treated T2-L cells (A d  and B) or p2Ca presented by T2-K cells (C) (see Materials and Methods for details). The b  error bars represent standard deviations of triplicate cultures. One representative experiment of three is shown.  83  A. H-2b  H-2b/d  + /+  Fyn[ Competitor 10  1  -/10  +/+ 1 10  mm **mmm****-**  r*r\M A —  "  Competitor cDNA  1 10  1  mm  IL-2 RT-PCR H-2 + -  H-2b/d + - ] Fyn  b  p  -/-  HPRT RT-PCR  Figure 5.2. Defective IL-2 production by H-2  b/d  DN cells is not rescued by deletion of  Fyn. Competitive RT-PCR (see Materials and Methods for details) was used to assess IL-2 production by D N cells after 9 hours of stimulation with mitomycin C treated T2-L cells d  and 1 |0JV1 p2Ca. Panel B shows HPRT cDNA normalization for the amount of template. One representative experiment of three is shown.  84  o a 3 Z • 1  H-2  H-2b/d Fyn " 7  b/d  T-2L  H-2  b/d  T-2K  d  H  _ b/d 2  F y n  -/-  b  u ,j>  «i  mil  11  m  CD25 H-2  b/d  Fyn-/-  CD69  H-2  H-2 / Fynb  b/d  d  0 |iM p2Ca 1 |iM p2Ca -10 mM p2Ca  Figure 5.3. Efficient upregulation of CD25 on antigen-stimulated H-2  D/a  Fyn"'" DN  cells. Purified D N cells were stimulated with mitomycin C treated T2-L or T2-K cells, d  b  with the indicated concentrations of p2Ca peptide. After 18 hours of culture the cells were washed and stained with either anti-CD25 (top panel) and anti-CD69 (bottom panel), as well as the 1B2 mAb (specific for the 2C TCR). The expression of CD25 and CD69 on 1B2 cells are as indicated. Data from one representative experiment of three +  are shown.  85  H-2  b/d  Fyn" " samples (Fig. 5.3, bottom panel). These observations suggest that Fyn may 7  have some specific negative effect on aspects of TCR signaling related to the upregulation of CD25 but not CD69. They also provide an explanation for the higher proliferative responses of antigen-stimulated H-2 2  b/d  Fyn  +/+  Fyn"'" D N cells as compared to the H -  b/d  D N cells (Fig. 5.1). This increased proficiency of expressing high affinity IL-2  receptors by H-2  b/d  Fyn" D N cells may support their greater proliferative response by /_  allowing them to more effectively utilize the small amounts of IL-2 they produce (83). To further investigate the importance of the rapid upregulation of CD25 in the enhanced proliferation of the H-2  b/d  Fyn"'" D N cells we repeated the proliferation assays described in  Figure 5.1, but with the addition of blocking antibodies for either IL-2 or CD25 (Fig. 5.4). With the addition of anti-CD25 antibodies, the proliferation of the H-2  b/d  Fyn" " D N 7  cells was reduced by half. The addition of anti-IL-2 antibodies completely eliminated the improvement in the H-2 proliferation of the H-2  b/d  b/d  Fyn" " D N cells. These results suggest that the enhanced 7  Fyn" " D N cells is due an enhanced ability to respond to IL-2, 7  and that this is at least partially mediated by CD25.  5.1.4.  Elevated Expression of IL-2Rp on H-2 DN cells and their enhanced b/d  survival in culture The Teh lab has previously shown that the H-2  b/d  D N cells have an activated/memory  phenotype in terms of expression of some surface antigens, notably CD44 (83). Here I examined the expression of IL-2Rp, another cell surface antigen upregulated on memory cells (156). Examinations of IL-2RP expression on ex-vivo cells revealed elevated levels on the H-2  b/d  D N relative to H-2 D N (Fig. 5.5A). Increased expression was also detected  on the H-2  b/d  Fyn" " D N cells. The high level of IL-2Rp expression by immediately ex-vivo  H-2  b7d  b  7  D N cells decreased to near basal levels after 18 h of culture without stimulation  (Fig. 5.5B). Interestingly, in H-2  b/d  D N cells stimulated with L /p2Ca (Fig. 5.5B) (or d  K /p2Ca, data not shown), the expression of IL-2RP did not decrease to base level after b  18 h of culture. This observation suggests that TCR signaling can at least partially block this decrease. By contrast, antigen stimulation of H-2  b7d  D N Fyn" " cells did not prevent the 7  downregulation of IL-2RP to base levels in these cells. This finding suggests that Fyn is  86  14000 -H- -2b/d H  12000  -•—H-2b/d + anti-CD25 —_H-2b/d +anti-IL-2 - - • — H-2b/d Fyn-/-  10000  —0  —0— H-2b/d Fyn-/- +anti-CD25 8000 1 —O— H-2b/d Fyn-/- + anti-IL-2  j  6000 4000 2000  0  0.1  1  10  p2Ca (uM) Figure 5.4. Enhanced responsiveness to IL-2 accounts for the reversal of the proliferation defect in H-2 Fyn' DN cells. b/d  H-2  b/d  and H-2 Fyn ' D N cells were stimulated with T2-L cells as in Figure 5.1, but b/d  7  d  with either no addition, anti-CD25 (10 |ig/ml) or anti-IL-2 (10 |ig/ml).  87  A  IL-2Rp H-2  I  I  H-2 '  b  1  b  H-2 ' Fyn-/-  d  b  13  d  II  IL-2RJ3  — 0 uM p2Ca ---1 uM p2Ca — 10 uJVI p2Ca IL-2Rp MFI (M±SD)  Cell type  Time 0  0 uM p2Ca at 18 hours  1 \iM p2Ca at 18 hours  10 uM p2Ca at 18 hours  H-2>>  274±5  231±4  227±15  222±11  H-2t"d  424±10  297±5  351±21  324±33  404±6  286±10  242±1  238±1  H-2  b/d  Fyn-/-  Figure 5.5. Immediately ex-vivo H-2  b/d  D N have elevated IL-2RP expression.  A, purified D N cells from the indicated mouse lines were stained with anti-IL-2R(3 mAb and assayed by FACS. B, D N cells were stimulated with mitomycin C treated T2-L  d  cells plus the indicated concentration of p2Ca peptide and assayed at 18 hours for surface expression of IL-2Rp\ The level of IL-2R(3 expression by the various cell types is expressed as mean fluorescence intensity (MFI) values and the means are averages of triplicate cultures. Data from one representative experiment of three are shown.  88  necessary in preventing the decrease in IL-2R(3 expression of antigen-stimulated H-2  b/d  D N cells. As the H-2  b/d  D N cells demonstrated increased expression of IL-2R(3,1 sought to  attach a functional significance to this finding. IL-2Rp has been shown to play an important role in promoting the survival of CD8 memory cells (157) and has been +  demonstrated to associate with Fyn and Lck (158, 159). Activation of both of these kinases are important events for the activation of PI3K via IL-2Rp (102, 160, 161). PI3K activation from IL-2Rp/yc is believed to play a critical role in supporting survival (162). Without the addition of any exogenous growth factors, H-2  b/d  D N cells displayed  enhanced survival compared to H-2 D N cells (Fig. 5.6, top panel). This enhanced b  survival is Fyn-dependent considering H-2  Fyn"'" D N cells did not have a survival  b/d  advantage compared to H-2 Fyn"'" D N cells (Fig. 5.6, bottom panel). Another indication b  that Fyn may confer a survival advantage on H-2 recover about twice as many D N cells from H-2  b/d  b/d  D N cells is that we consistently  2C mice than from H-2  b/d  Fyn"'" 2C  mice, suggesting that the enhanced survival of anergized D N T cells in vivo is also dependent on Fyn.  5.1.5.  IL-15 or IL-2 interacts with IL-2RP to promote the survival of H-2 DN BD  cells Next, I determined whether the increase in IL-2RP expression is reflected in increased responsiveness to IL-2 and IL-15, two cytokines known to signal through the IL-2Rp/yc complex (154). With the addition of IL-2 I observed an improvement of cell survival by both wild type and Fyn-deficient H-2 and H-2 b  b/d  D N cell types (Fig. 5.7A).  Therefore, the effects of IL-2 seem to be independent of the level of IL-2Rp expression by these cells. IL-15 also confers survival benefits on all four cell types but with one notable difference (Fig. 5.7B). D N from H-2 concentrations of IL-15 as compared to H-2  b/d  mice were able to respond better to low Fyn-deficient cells. After three days of  b/d  culture in medium containing 1 ng/ml of IL-15, the percentage survival in the H-2 group was 55% compared to 29% for the H-2  b/d  b/d  DN  Fyn"'" D N group (Fig. 5.7B). After two  days of culture the percentage survival for these two groups of cells were 69% and 50%,  89  • H-2 • H-2  b  Day 1  b/d  Day 3  Day 2  • H-2 Fyn-/• H-2 Fyn-/ b  b/d  Day 1  Figure 5.6. Enhanced survival of cultured H-2 D N cells from Fyn  +/+  Day 3  Day 2 b/d  DN cells is dependent on Fyn.  (top panel) and Fyn " (bottom panel) H-2 and H-2 _/  b  b/d  mice were  cultured without stimulation and their survival was assessed by 7-AAD staining and FACS analysis. The error bars represent standard deviations of triplicate cultures. Data from one representative experiment of three are shown.  90  T T  H-2  b  H-2  H-2b/d  Fyrr  b  1  t / /  V /'/  /  1  H-2b/d Fyn-  90 i 80 70 60 50 40 30 20 10 0 J  H-2b  H-2b Fyn-/-  Figure 5.7. IL-2 and IL-15 enhance the survival of cultured DN cells. D N cells were cultured with 0, 1 or 10 ng/ml IL-2 (A) or IL-15 (B). After 72 hours cells were washed and stained with 7-AAD and assayed by FACS. The error bars represent standard deviations of triplicate cultures. Data from one representative experiment of five are shown.  91  B  Day 2  • H-2 • H-2  Day 3  Day 2 Day 3  b/d  b/d  Fyr^-  Figure 5.8. Blastogenic response of H-2 DN cells to IL-15 impaired by Fyn"'" b/d  mutation. D N cells from the indicated mouse line were cultured with 10 ng/ml IL-2 (A) or 10 ng/ml IL-15 (B). After the indicated time cell size was determined by forward scatter using FACS analysis. Annotation with * indicates the H-2  b/d  Fyn"'" sample were  determined to be statistically different from the corresponding H-2  b/d  sample (see  Materials and Methods for details). The error bars represent standard deviations of triplicate cultures. Data from one representative experiment of three are shown.  92  respectively (values are averages of triplicate cultures). Additionally, H-2  b/d  D N cells  were more responsive to IL-15 as determined by cell size after culture in IL-15containing medium (Fig. 5.8). The cell size of H-2  b/d  D N cells cultured in 10 ng/ml of IL-  15 for two or three days was larger than similarly cultured H-2  b/d  Fyn" D N cells. This /_  effect was specific for IL-15 as the cell size of these two groups of cells increased to a similar extent when cultured in 10 ng/ml of IL-2 (Fig. 5.8). To determine whether IL-2Rp7lL-15 and/or IL-2R(3/IL-2 interactions contributed to the survival of H - 2 D N cells, these cells were cultured with blocking anti-IL-2Rp or b/d  anti-CD25 antibodies. The presence of anti-IL-2Rp, but not anti-CD25, reduced the survival of the H-2  b/d  D N cells (Fig. 5.9A). Furthermore, the enhanced survival in  response to exogenously added IL-2 or IL-15 was specifically blocked by the anti-IL2RP, but not the anti-CD25 antibody (Fig. 5.9B). These results suggest that the enhanced survival of the H - 2 D N cells is mediated by interaction of either IL-15 or IL-2 with ILb/d  2Rp.  5.2  Discussion In this chapter I addressed questions regarding the importance of Fyn in an in vivo  model of T cell anergy. Anergized T cells lacking Fyn exhibited a recovery of their proliferative capacity and exhibited an enhanced ability to upregulate CD25. In addition, cultured anergic D N cells displayed a survival advantage over Fyn"'" anergic D N cells. The increased survival of anergic D N cells correlated with elevated IL-2RP expression and an enhanced ability to respond to IL-15. As this survival advantage was lost in Fyndeficient anergic cells, and since Fyn associates with IL-2RP, a potential explanation for these observations is that Fyn is required for efficient signaling through IL-2Rp. I have previously shown that anergic D N cells express elevated levels of Fyn (Chapter 4 and (155)). The enhanced proliferative ability of the H-2  b/d  Fyn" " D N cells 7  suggests that the elevated level of Fyn in anergic cells may negatively regulate T cell activation. Fyn has been shown to associate with the CD3^ chains in anergic T cells (107). It has also been demonstrated that Fyn and c-Cbl constitutively associate in anergic T cells (74). c-Cbl has also been shown to be an inhibitor of ZAP-70 kinase activity (22)  93  A 110  I  100  \  Day  1  Day 2  Day 3  B 110  ,  1  +IL-15  +IL-2  Figure 5.9. Enhanced survival of H-2 DN cells is blocked by anti-IL-2Rp antibody. b/d  H-2 D N cells were cultured with no stimulation in the presence of the indicated b/d  antibodies (A). Cells were also cultured with 10 ng/ml IL-2 or 10 ng/ml IL-15 and with the indicated antibodies for 72 hours (B). At the indicated time cells were washed and stained with 7-AAD and assayed by FACS. The error bars represent standard deviations of triplicate cultures. Data from one representative experiment of two are shown.  94  and we have demonstrated previously that ZAP-70 phosphorylation is significantly decreased in H-2  b/d  D N cells upon TCR stimulation (Chapter 4 and (155)). Consistent  with this observation we have seen L A T phosphorylation to be reduced in H-2 D N cells b/d  upon TCR stimulation (Chapter 4 and (155)). The role Fyn plays in in vivo induced T cell anergy may be to bring c-Cbl to the TCR-£ chains and provide a constitutive negative regulation of TCR signaling (74). According to this model, L A T phosphorylation is expected to be increased in anergic Fyn " D N cells after T C R stimulation. However, my 7  data are inconsistent with this hypothesis as I failed to detect increases in L A T phosphorylation upon TCR stimulation of H-2  b/d  Fyn " cells (unpublished observations). 7  One explanation for this discrepancy regarding L A T phosphorylation is that the cell types used for the analysis of T cell anergy are of diverse origin. We have used D N cells that express a transgenic TCR for our studies while others have used anergic alloantigenspecific human T cells for their studies (74). Furthermore, these D N cells may be of the y8 T cell lineage as a result of the early expression of the transgenic a(3 TCR during ontogeny (139, 140). It is possible that Fyn may perform distinct functions in different T cell lineages and in this way contributes to different observations. An alternative explanation for the enhanced proliferative response of Fyn " anergic 7  D N cells to antigen stimulation is that the Fyn " anergic cells have not been anergized to 7  the same extent as their Fyn expressing counterparts due to less efficient TCR signaling. This hypothesis is supported by my observation that Fyn is required for the optimal antigen-induced proliferative responses of non-anergic D N cells (Figure 5.1). The observation that D N cells from male mice expressing the low affinity transgenic malespecific TCR are not anergic (83) also supports the hypothesis that the strength of the autoreactive stimulus is important for the induction of the anergic state. It is therefore possible that the lack of Fyn may reduce the magnitude of autoreactive signaling, thereby reducing the severity of the anergic state of the Fyn " D N cells. However, it is clear that 7  the Fyn " D N cells receive anergizing stimulus since H-2 7  b/d  Fyn " D N cells are defective 7  in producing IL-2 in response to antigen stimulation. Furthermore, they possess a lower activation threshold for the induction of CD25 when compared to anergized Fyn  +/+  DN  cells. CD25 expression is induced either by antigens or cytokines and is controlled by at least 3 positive regulatory regions within the 5' regulatory region of the IL-2Ra gene  95  (163, 164). Regions I and II are for mitogen induced IL-2Ra expression and region III is essential for IL-2 induced IL-2Ra expression. The deletion of Fyn may affect the signaling pathways responsible for mitogen stimulated upregulation of CD25. The removal of Fyn-mediated negative regulation allows for more efficient upregulation of CD25 (Fig. 5.3, top panel) and hence the observed increases in proliferative capacity (Fig. 5.IB and C). The fact that upregulation of CD69 is not influenced by the presence or absence of Fyn (Fig. 5.3, bottom panel) suggests that signaling to upregulate this molecule is not subject to the same regulation. The enhanced survival of the anergic H-2  b/d  D N cells in culture correlated with  elevated expression of IL-2R(3 on these cells. Other studies have shown that memory CD8 T cells express elevated levels of IL-2Rp and their enhanced survival in vivo is +  likely mediated by IL-15 and not IL-2 (157). IL-15 is ubiquitously expressed in mouse tissues including placenta, skeletal muscles, kidney, lung, heart, fibroblasts, epithelial cells and monocytes (165). Subsequently, we have detected expression of IL-15 in H-2 and H-2 H-2  b/d  b/d  b  D N cells by RT-PCR (our unpublished observations). I propose that since the  D N cells express elevated levels of IL-2R(3, they may be responding to IL-15 in an  auto- or paracrine manner and that this may explain their enhanced survival. I have provided evidence that the enhancement of survival by IL-15 is mediated by IL-2RP (Fig. 5.9). Although ex vivo H-2  b/d  Fyn" " D N cells exhibit elevated IL-2Rp expression, they did 7  not exhibit a survival advantage in culture implicating a role for Fyn in this increased longevity (Fig. 5.6, bottom panel). Furthermore, I noted that H-2  b7d  Fyn" " D N cells were 7  less responsive to IL-15 in cell survival and blastogenesis assays as compared to H-2  b/d  D N cells. These observations suggest that IL-15/IL-2RP signaling may be Fyn-dependent and this function of Fyn is not compensated for by Lck in Fyn" " cells. 7  In our in vitro assays, IL-2 can perform the same function as IL-15 in promoting the survival of anergic D N cells. The effects of IL-2 are also mediated by IL-2Rp. As I observed enhanced survival of both wild type and Fyn" " H-2 or H-2 7  b  b7d  D N cells in  response to IL-2, it is clear that the enhanced survival promoted by IL-2 is not Fyndependent. It is conceivable that Lck may compensate for the lack of Fyn under these conditions. Anergic D N cells have been shown to be defective in the production of IL-2, a cytokine primarily made by activated T cells ((83) and Fig. 5.2). It is therefore likely  96  that IL-2 is generally not available to anergic D N cells under physiological conditions. By contrast, IL-15 is ubiquitously expressed (165) and therefore readily accessible to the D N cells under physiological conditions. I have shown that the anergic D N cells express high levels of the IL-2Rp receptor allowing them to respond to low concentrations of IL15 in a Fyn-dependent manner. Furthermore, the higher recovery of anergic D N cells from Fyn  +/+  mice also supports the hypothesis that Fyn is important for the survival of  anergic D N cells under physiological conditions. In summary, in this chapter I have provided data that support the hypothesis the Fyn plays a dual role in T cell anergy: one in negatively regulating aspects of TCR signaling and the other in promoting cell survival.  97  Chapter 6 Final Discussion and Future Studies  In this chapter I will recap some of the key findings reported in this thesis, discuss how they are relevant to other recent work in the literature, and suggest future avenues of investigation.  6.0  Fyn and low affinity interactions From the data presented in Chapter 3 it can be concluded that Fyn plays a significant  role in T cell function and regulation. I presented data arguing that Fyn functions in the selection of the T cell repertoire. The influence of the Fyn"'" mutation was observed in the extreme selection environments of the male H - Y mouse and the H-2 2C mouse. In both b  of these conditions, T cells that would have otherwise been deleted survived the selection process. As well, I observed that T cells maturing in both the H - Y and the 2C transgenic mice matured more slowly when they lacked Fyn, as demonstrated by their continued expression of the developmental marker H S A as recent thymic emigrants. An example from the literature of an alteration in TCR signaling leading to an altered TCR repertoire is the deletion of the phosphatase CD45 (166). In this study, these researchers demonstrated that the elimination of CD45 (and hence positive regulation of Src kinases like Lck and Fyn) leads to decreased positive selection of what would be naturally selected TCRs, and to increased selection of TCRs that may have been deleted. Other work in mice with deletions in the TCR-^ ITAMs reveals that mice with increasingly hampered TCR signaling have increasingly impaired T cell selection (5). B y the same rationale, from my data it appears that Fyn allows for the selection of a more diverse range of TCR affinities. The increased range of TCR affinities selectable in the presence of Fyn may come into play once thymocytes exit to the peripheral lymphoid system, where they may be called upon to respond to all types of pathogens. The strength of the interaction of the TCR with M H C presenting peptides from these various pathogens will determine whether there is a T cell response or not. Indeed it has been argued that the ability for 98  each cell to be stimulated is based on the TCR affinity for antigen and the cell's ability to compete for limited resources in terms of stimulation by dendritic cells and cytokines (167). My studies into how the deletion of Fyn influences responses with differing affinities revealed that Fyn is important for low affinity interactions. M y results may have important implications for in vivo situations as many biologically relevant interactions fall in the low affinity range of T C R / M H C interactions that I found Fyn to be important for. Besides the question of whether or not a T cell responds to a given ligand, the affinity of an interaction can also influence numerous aspects of the T cell response. These include the skewing of T helper cell responses to either T 1 or T 2. Though the cytokine H  H  milieu plays a major role, strong evidence points to the conclusion that signaling strength influences the generation of T 1 and T 2 responses (168, 169). This suggests that fine H  H  tuning of the TCR signal may be important to the outcome of the T C R / M H C interaction: although Fyn" T cells can still signal, their signals may be missing important information A  or be misinterpreted for the type of antigen that is initiating the T cell response. Low affinity interactions are also believed to be important for the long term maintenance of T cells (170). Some researchers have found that in the absence of M H C , T cells can not survive (171, 172). These results have been attributed to low affinity interactions between the TCR and M H C molecules presenting self-peptides, referred to as "TCR tickling". In fact, evidence is emerging which suggests that the presence of selecting ligands is required for the maintenance of T cells in the periphery (173). From these observations it is clear that even if no proliferation response is detectable, important weak interactions are happening all the time. These results suggest that the very ligands that select thymocytes are required to maintain T cells once they exit to the periphery. The nature of these low affinity interactions suggests that Fyn may play an important role in T cell homeostasis. Of note, recent work in mice expressing Lck under the control of an inducible promoter has demonstrated that although Lck is required for homeostatic expansion, it does not appear to play a role in T cell survival (174). This result suggests that other kinases (potentially Fyn) may mediate the TCR derived signals essential to T cell survival. A possible area for future work may be investigations into the importance of Fyn in T cell survival and homeostatic expansion.  99  The results I presented in Chapter 5 regarding the role of Fyn in T cell anergy relate to this hypothesis. I found that anergic cells lacking Fyn exhibited a reduced survival capacity. This result was attributed to the observed importance of Fyn in IL-15 responses mediated by IL-2RP (Figure 5.7 and 5.8). Though this observation was made for anergic cells, it may be interesting to follow up this work with memory cells lacking Fyn, as memory cells also exhibit a IL-2R(3  hlgh  phenotype and have been reported to require IL-15  for their maintenance (157). The predicted outcome of such an experiment would be that memory cells lacking Fyn would have a reduced survival ability. These experiments may address the importance of Fyn in signaling from IL-2RJ3 which mediates T cell survival.  6.1  Fyn and the upregulation of IL-2 receptors In Chapter 5 I also presented data pertaining to the ability of anergic cells lacking Fyn  to proliferate. Though anergic cells lacking Fyn did not produce more detectable IL-2 than Fyn expressing cells, I found that Fyn"'" cells upregulated CD25 more rapidly and that this could to account for their enhanced proliferative capacity (Figure 5.4). At the suggestion of my Ph.D. advisory committee, I examined the production of transcription products of the CD25 gene upon mitogenic stimulation but found no evidence for enhanced mRNA production from the CD25 gene by the Fyn"'" anergic cells (data not shown). This analysis of CD25 transcription was done after 9 hours of stimulation of T2L APCs and peptide. It is known from the literature that signaling for CD25 upregulation d  is dichotomous: upregulation can be induced either by mitogenic stimulation or by cytokine stimulation. The transcription of the CD25 gene is similar in the Fyn"'" and Fynexpressing anergic cells at the 9 hour time point but we observed a difference in surface expression at 18 hours. It is possible that early transcription is driven by mitogenic stimulation. This could be the same mitogenic stimulation that leads to the equal upregulation of CD69 by these two cell types. The differences observed in CD25 protein expression at 18 hours could be due to differences in upregulation of CD25 via the cytokine pathway. These could either be due to minor differences in IL-2 production between the two cell types, or to enhanced signaling from CD25 in the Fyn"'" anergic cells. As IL-2 production by the anergic D N cells is negligible, it is perhaps more likely  100  that the deletion of Fyn removes negative regulation of CD25 signaling and allows CD25 upregulation to proceed more rapidly. As discussed above, we have found IL-2R(3 expression to be elevated in the anergic cells. Though IL-2R|3 expression was elevated immediately ex vivo on the H-2  b/d  Fyn'  A  D N cells, this expression was not maintained in response to antigenic stimulation. Interestingly, ex vivo maintenance of elevated IL-2R(3 levels required the expression of Fyn. Perhaps the role of Fyn in survival is to maintain expression of IL-2Rp. Further investigations into the survival of memory cells may provide insight into this area.  6.2  Signaling in anergic DN cells In Chapter 4,1 demonstrated that TCR mediated signaling in the anergic D N cells is  perturbed at the level of ZAP-70 activation/phosphorylation and L A T phosphorylation. The precise cause of this signaling defect is not known. Though the signaling pattern is similar to that seen in Lck deficient Jurkat (JCaMl)upon predominantly Fyn mediated signaling (108), the simple explanation that these effects are purely Fyn controlled is not valid as anergic D N cells lacking Fyn demonstrated a similar signaling phenotype (data not shown). The signaling profile observed in the anergic D N cells is also similar to that observed upon the stimulation of T cell clones with altered peptide ligands. The importance of maintaining self tolerance likely dictates that there is more than one mechanism of regulation in place to control the proliferation of the anergic D N cells I have studied and perhaps all anergic cells. From my proliferation data demonstrating an increased proliferation in anergic D N cells lacking Fyn, it is clear that Fyn does play a role in this regulation. It would be interesting to uncover what other regulatory mechanisms are in place and investigations to this effect could be a follow up to the work presented here. A large body of recent work has outlined the importance of lipid rafts to TCR signaling (reviewed in (175)). It is possible that the assembly of signaling complexes in lipid rafts may be impaired in the anergic D N cells and that this may explain their altered signaling phenotype. Though the limited number of cells recoverable in this system precluded their analysis by density gradient centrifugation, it may be interesting to analyze these cells by other techniques as they become available. Also of  101  interest would be investigations into the nature of signaling defects in in vivo anergized CD8 or CD4 cells. These studies would be interesting to demonstrate similarities +  +  between the anergic D N cells and more conventional cell types.  6.3  Double negative cells A large proportion of the results I have discussed in this thesis relate to experiments  done on the anergic D N cells. The exact nature of these D N cells is still in question. Some groups have proposed that D N cells are in fact an artifact of transgenic T C R systems brought about by the premature expression of rearranged a and (3 genes in y8 lineage cells (139). A recent report has examined several transgenic systems and reported that earlier expression of TCRP genes leads to more pronounced populations of D N cells (140). These D N cells were believed to be of the y8 T cell lineage, and that expression of the aP TCR has "hijacked" these cells. Indeed, in systems in which the transgenic TCR is expressed later during development, cells were obtained that expressed both an aP and a y5 TCR. We have examined the D N cells in our 2C system for y8 TCR expression, but we were unable to detect any by FACS analysis. This result does not preclude the possibility that our D N cells may be of the y8 lineage. Recent work done in the Teh lab, largely by Dr. John Priatel, has provided evidence that the anergic D N cells may actually have regulatory properties. Dr. Priatel has made several interesting findings regarding the biological importance of these cells. Dr. Priatel found that the proliferation of anergic D N cells can be rescued by the presence of bystander T cells responding to antigen, and producing cytokines such as IL-2. This finding is of significance in that the anergic D N cells express a TCR that can recognize self. The fact that they may be able to proliferate in response to IL-2 produced during an immune response and therefore cause an autoimmune reaction is significant. 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