Microbiology & Immunology - Theses
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ItemA structural and functional analysis of T cell receptor recognition and its consequences for virus infection( 2011)CD8+ T cell responses to pathogens are characterised by the clonal expansion of cells expressing T cell receptors (TCRs) that are specific for epitopes of pathogen-derived peptide (p) presented by major histocompatibility complex class I (MHCI) molecules. Evidence suggests that epitope-specific CD8+ T cell populations expressing a diverse array of TCRs provide enhanced immunity to virus infection and reduced risk of immune evasion through viral escape mutations. However, the factors that determine epitope-specific TCR repertoire diversity are poorly understood. A better knowledge of how diverse epitope-specific TCR repertoires are generated and maintained will assist in the design of vaccine and immunotherapy protocols that make optimal use of the available T cell repertoire and provide effective immune protection. This thesis therefore assessed the role of several factors in shaping epitope-specific TCR repertoire diversity in response to virus infection. The impact of MHC diversification and thymic cross-tolerance on the selection of epitope-specific TCR repertoires was investigated using MHC homozygous and heterozygous mice. These studies demonstrated that whilst the expression of different MHC alleles can lead to shifts in intrathymic selection of the TCR repertoire to maintain self-tolerance, this does not necessarily lead to the deletion of particular TCR specificities, enabling some epitope-specific TCR repertoires to be maintained in genetically diverse individuals. Thus, a broad array of epitope-specific T cell responses are possible under conditions of increased MHC diversity, which may assist in immune control of virus infection. The importance of TCR α- and β-chain pairing in determining TCR specificity and repertoire diversity was investigated using retrogenic mice engineered to express single α- or β-chains from influenza virus-specific TCRs, paired with endogenously rearranged TCR chains. Analysis of epitope-specific TCR repertoires selected within retrogenic mice following influenza virus infection showed that particular TCRαβ heterodimers were selected for recognition of pMHC epitopes. This demonstrates that pairing of particular α- and β-chains is key for determining optimal TCR specificity, and that requirements for appropriate αβ pairing could severely restrict diversity within epitope-specific TCR repertoires. Interestingly, antigen exposure induced preferential expansion of T cells expressing certain TCRαβ heterodimers, suggesting that a mechanism of clonal selection shapes T cell repertoires during infection, and could potentially focus immune repertoires towards TCR clonotypes that are capable of optimal antigen recognition. To investigate how structural requirements for antigen recognition influence TCR diversity, the crystal structure of a prototypic TCR complexed to the influenza-derived DbPA224 epitope was analysed. In this structure, amino acid side-chains within the complementarity determining region-3α (CDR3α) loop played a key role in determining peptide specificity, whilst the CDR3β loop only made main-chain interactions with peptide. Using retrogenic mice with fixed expression of either the α- or β-chain from the prototypic TCR, it was shown that use of main-chain interactions and a conserved structure enabled CDR3β sequence diversity to be maintained within the DbPA224-specific TCRβ repertoire. Meanwhile, requirements for specific side-chain interactions with peptide resulted in limited CDR3α sequence diversity within the DbPA224-specific TCRα repertoire. This provides a molecular explanation for how CDR3 sequence diversity can be maintained within epitope-specific TCR repertories. Interestingly, retroviral-mediated expression of mutant DbPA224-specific TCRs in mice demonstrated that certain CDR3α residues were required for efficient thymic selection of CD8+ T cells. This provides the first direct evidence that CDR3 loops are involved in positive selection events, giving new insight into the molecular basis of positive selection and how this could shape the peripheral TCR repertoire. Overall, the findings in this thesis demonstrate that T cell developmental processes play an important role in shaping the naïve TCR repertoire. However, structural requirements for optimal recognition ultimately determine the spectrum of TCRs selected in response to a given pMHC epitope. The findings provide a molecular basis for differences in TCR diversity within epitope-specific responses and suggest a potential for vaccine strategies to incorporate peptide antigens that prime memory T cell populations with a high level of TCR diversity, which could provide the best possible immune protection against pathogen infection.