The recognition of MHC-peptide ligands by the T cell receptor (TCR) is central to the induction of the adaptive immune response. This thesis describes the development of a bioassay for TCR recognition which was then used to undertake a molecular analysis of the TCR/MHC-peptide interaction. 1. A TCR-CD3ϛ chimeric receptor was stably expressed in the cell line RBL-2H3 to give the transfectant RBL-008. RBL-008 was shown to exhibit MHC-restricted peptide-specific responses to both cellular and multimerised recombinant HLA-A2-pol peptide targets (Chapter 3). 2. By competitively inhibiting the response of RBL-008 to HLAA2 pol complexes with monovalent soluble recombinant MHCpeptide complexes it was confirmed that the TCR makes significant contact with both the MHC and peptide parts of its ligand. Furthermore it was found that only a few peptides in a random mixture can prevent contact between the TCR and HLA-A2. This has implications for positive selection since it supports evidence suggesting that some TCRs can be selected on a wide range of unrelated peptides (Chapter 4). 2. The bioassay was used to examine the flexibility of TCRpeptide interactions using a panel of variant peptides designed on the basis of the previously published HLA-A2-pol peptide structure (Chapter 5). Several variant peptides were recognised by the TCR and interestingly one of these altered peptide ligands was actually recognised better than the index peptide, raising the prospect of designing 'improved epitopes'. 3. By mutating the β chain of TCR-CD3ϛ chimeric receptor it was shown that allelic variation in the TCR genes can have a significant effect on antigen recognition and may therefore be disease susceptibility candidates genes (Chapter 6). 4. The structural relationship between the V and C domains of the TCR was examined and found to be of considerable functional significance since disruption of this relationship resulted in loss of expression of the TCR-CD3ϛ receptor.

No description available.

Human cytotoxic T lymphocytes (CTL) recognize antigen in the form of short peptide fragments presented by Human Leukocyte Antigen (HLA) class 1 molecules. The HLA B27-restricted CTL response to Influenza A virus is directed against a single nine amino acid epitope within the viral nucleoprotein NP383-391. Influenza A-specific CTL lines and clones, generated from the peripheral blood of three unrelated individuals, exhibited remarkable fine specificity for NP383-391, failing to recognize synthetic variants containing single amino acid substitutions at positions 1, 4, 7 or 8. The sequences of rearranged T cell receptor (TCR) alpha and beta chain genes showed remarkable conservation of variable and joining gene segment usage, and also of non germline-encoded B chain residues proposed to interact with peptide, even between clones from unrelated individuals. Correlation of fine specificity for peptide with TCR alpha chain usage suggested a possible orientation for the TCR/HLA/peptide ternary complex in this immune response. The finding of restricted TCR usage in the HLA B27-restricted CTL response to influenza A virus has implications for the study and potential therapy of HLA B27 associated autoimmune disease. The TCR heterodimer from one clone was expressed in a rat basophil cell line and shown to confer specificity for HLA B27 + NP383-391. Influenza A-specific CTL were also shown to recognize bacterial superantigens in an MHC class II dependent but unrestricted manner. A rapid screening assay for candidate superantigens was developed and used to identify a novel superantigen produced by Clostridium perfringens. This superantigen was found to have a unique specificity for human TCRβ chains.

Chemokines are responsible for directing leukocyte migration and triggering firm arrest by activating integrins on leukocytes. It is now apparent that chemokines have critical biological roles beyond chemo-attraction. Throughout this thesis, I describe the importance of the CCL5/CCR5 axis in the context of the immune response and cancer biology. Specifically, CCL5 invokes dose-dependent distinct signalling events downstream of CCR5 activation in T cells. I show that nM concentrations of CCL5 mediate CD4+ T cell migration that is partially dependent on mTOR activation. CCL5 induces phosphorylation and de-activation of the repressor 4E-BP1, resulting in its dissociation from the eukaryotic initiation factor-4E to initiate protein translation. I provide evidence that CCL5 initiates rapid translation of cyclin D1 and MMP-9, known mediators of cell migration. The data demonstrated that up-regulation of chemotaxis-related proteins may “prime” T cells for efficient migration. During an immune response, recently recruited T cells are exposed to high CCL5 concentrations. The propensity of CCL5 to form higher-order aggregates at high, µM concentrations, prompted studies to investigate their effects on T cell function. I show that at these high doses, CCL5 induces apoptosis in PM1.CCR5 and MOLT4.CCR5 T cell lines. CCL5-induced cell death involves the cytosolic release of cytochrome c and caspase-9/-3 activation. Furthermore, I identified Tyrosine-339 as a critical residue within CCR5, suggesting that tyrosine phosphorylation signalling events are important in CCL5-mediated apoptosis. Our data suggest that CCL5-induced cell death, in addition to Fas/FasL mediated events, may contribute to clonal deletion of T cells during an immunological response. I subsequently examined the possible pathological consequence of aberrant CCL5/CCR5 signalling in breast cancer. Exogenous CCL5 enhances MCF-7.CCR5 proliferation, which is abolished by anti-CCR5 antibody and rapamycin. CCL5 induces the formation of the eIF4F translation initiation complex, and mediates a rapid up-regulation of cyclin D1, c-Myc and Dad-1 protein expression. Thus, our data demonstrate the potential for breast cancer cells to exploit downstream CCL5/CCR5 signalling pathways for their proliferative and survival advantage. Taken altogether, each of these studies reinforces the notion that chemokines are not only potent chemotactic mediators, but are key effectors in diverse developmental, immunological and pathological processes.

Neisseria gonorrhoeae infections are of major concern in areas of low socioeconomic status in both developed and developing nations. N. gonorrhoeae colonizes the genital tract by adhering to mucosal tissues through a number of adhesins, including the colony opacity-associated (Opa) proteins. Despite the random phase-variable expression of Opa proteins, 95% of clinical isolates express Opa variants that bind to the carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1), suggesting an essential role in vivo. Interestingly, even though gonorrhea is characterized by an intense inflammatory response, the organism is capable of evading the adaptive immune response. In previous studies by the Gray-Owen group, it has been established that certain gonococcal Opa variants bind CEACAM1 expressed by CD4+ T helper lymphocytes and, thereby, reduce their activation and proliferation. Since T cells are essential in establishing immune memory, inhibition of T cell function could explain the deficit in immune memory following gonococcal infection. In this thesis, I describe my studies to elucidate how CEACAM1 inhibits T cell activation on a molecular level. In Chapter 2, I demonstrate that outer membrane vesicles (OMVs) naturally shed by OpaCEA-expressing Neisseria sp. effectively inhibit CD4+ T cell activation, implicating a role for OMVs during infection and establishing that the Opa proteins do not have to be expressed in the context of the bacterium in order to elicit an inhibitory effect. In Chapter 3, I describe early steps in the CEACAM1-dependent inhibitory signaling cascade elicited when N. gonorrhoeae binds to CD4+ T cells. Finally, in Chapter 4, I show that a dynamic monomer-dimer equilibrium controls CEACAM1 function in epithelial cells and lymphocytes. Combined, the results presented in this thesis allow the derivation of a model to explain how CEACAM1 controls CD4+ T cell function at a molecular level.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (MScMed)--University of Stellenbosch, 2006. / Bibliography. Also available via the Internet.

Thesis (Ph.D.)--Drexel University, 2007. / Includes abstract and vita. Includes bibliographical references (leaves 112-129).

Thesis (Ph. D. in Microbiology and Immunology)--Vanderbilt University, May 2009. / Title from title screen. Includes bibliographical references.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2008. / Includes bibliographical references (leaves 57-69). Also available in electronic version.