Analysis of factors influencing CD8+ T cell responses after influenza A virus infection

© 2013 Dr. Tania CukalacCD8+ T cell responses to pathogens are characterised by the clonal expansion of cells expressing T cell receptors (TCRs) of unique specificity for antigenic peptide (p) determinants presented by major histocompatibility complex (MHC) class I molecules. Following infection, the magnitude of various antigen-specific T cell responses often fall into a highly reproducible immunodominance hierarchy. Evidence suggests that the smaller, subdominant CD8+ T cell responses are important in the control of many viral infections, thus a better understanding of how these responses are generated may have implications for augmenting typically subdominant responses. Furthermore, epitope-specific CD8+ T cell populations expressing a diverse array of TCRs have been suggested to provide enhanced immunity to virus infection and reduced risk of immune evasion through viral escape mutations. However, there has been limited analysis of the usage and diversity of complete TCRαβ repertoires with previous examination generally restricted the TCRβ chain. It is vital to include the TCRα chain as there is increasing evidence to suggest that this chain is important in providing pMHC specificity. A better knowledge of the usage and diversity of complete TCRαβ repertoires 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 roles of several determinants of subdominant epitope status and examined the usage and diversity of epitope-specific TCRαβ repertoires in response to viral infection. The role of naïve CD8+ T cell precursor (CTLp) frequency and epitope abundance as determinants of subdominant epitope status were examined. These studies demonstrated that the numbers of CTLps may control the early immunodominance hierarchy, with the level of epitope abundance on the surface of CD8+ T cells and the role of inefficient cell recruitment and clonal expansion a possible mechanisms for determining acute phase subdominant epitope status. Importantly, the role of the various determinants of immunodominance may contribute differently for each epitope specificity. The importance of the TCRα and TCRβ chains in determining TCR specific and repertoire diversity was investigated though the examination of the complete TCRαβ repertoire of epitope-specific CD8+ T cell populations. This investigation highlighted the necessity for complete characterisation of the responding TCRαβ heterodimers, rather than just the individual TCRα or TCRβ chains. This was due to the TCRβ repertoire of an epitope-specific CD8+ T cell population (the influenza-derived DbNP366-specific set) not reflecting the characteristics of the complete TCRαβ repertoire. The TCRαβ chains were shown to be vital in conferring pMHC specificity. This high degree of specificity is generally achieved through the biased usage of particular V and J gene segments which appear to offer the appropriate structural characteristics for optimal recognition, whilst diversity is maintained thought the use of varying amino acid sequences in the complementarity determining region-3 (CDR3). Though the examination of the selection of particular T cells during various stages of an immune response the responding epitope-specific CD8+ T cell response was shown to select T cells that are capable of optimal antigen recognition and may offer the greatest protection against infection. However, the overall extent of TCR diversity was maintained when selection of T cells from the memory pool into the recall response was examined. This preservation of diversity suggests this selection process may be an effective strategy for enabling immune control and subsequent protection as well as maintaining the ability to respond to viral escape mutants. Overall, the findings in this thesis expanded our understanding of the factors that determine the size and TCR complexity of the responding CD8+ T cell immune response to acute viral infection. It is only through the increase in knowledge of the process by which CD8+ T cell immune responses are generated from the small numbers of naïve CD8+ T cells that can we improve the strategies for vaccine design and development of therapeutics that target CD8+ T cell mediated immunity for viruses and tumour immunotherapy

Multiplexed combinatorial tetramer staining in a mouse model of virus infection

Use of fluorescently labelled multimers, particularly tetramers of peptide and MHC class I glycoprotein (pMHC-I) complexes, is essential for the analysis of CD8+ T cell immunity in basic research and clinical settings. A recently described combinatorial approach using pMHC-I multimers coupled to a unique combination of distinct fluorochromes has facilitated the simultaneous screening of multiple T cell specificities within a single human blood sample. The present analysis establishes that this multiplexed tetramer staining protocol can also be applied in mouse models of a disease to detect multiple subdominant CD8+ T cell specificities in the presence of prominent immunodominant T cell sets at different stages of infection. We have established a modified protocol that concurrently identified influenza-specific CD8+ T cells at the acute and long-term memory phases of influenza virus infection in B6 mice. Highly dominant (DbNP366+CD8+ and DbPA224+CD8+) and subdominant (KbPB1703+CD8+, DbPB1-F262+CD8+ and KbNS2114+CD8+) T cell responses can be detected simultaneously at levels comparable to the conventional tetramer staining with this combinatorial approach. The technique proved particularly useful with aged mice, where we used 5-fold fewer animals, making the detection of multiple T cell specificities more cost-effective and less time-consuming. Overall, our study establishes that this comprehensive concurrent analysis of multiple T cell specificities is of value for analysing mouse models of disease, especially in situations where sample size and/or response magnitude is limiting. © 2010 Elsevier B.V.Link_to_subscribed_fulltex

Altered CD8+ T cell immunodominance after vaccinia virus infection and the naive repertoire in inbred and F1 mice

Previous studies of CD8+ T cell immunodominance after primary virus infection of F1 mice compared with their inbred parents have generally concluded that no dramatic changes occur. In this study, we revisit this issue using vaccinia virus (VACV), which has a large genome, a recently defined immunodominance hierarchy in mice, and is a candidate vector for vaccines.We found that immunogenicity of VACV peptides defined using inbred mice was highly variable in F1 progeny: some peptides were equally immunogenic in F1 and inbred, whereas others elicited responses that were reduced by >90% in F1 mice. Furthermore, the dominance of a peptide in the relevant inbred parent did not predict whether it would be poorly immunogenic in F1 mice. This result held using F1 hybrids of MHC-congenic mice, suggesting that MHC differences alone were responsible. It was also extended to foreign epitopes expressed by an rVACVvaccine. F 1 mice were less able to mount responses to the poorly immunogenic peptides when used as a sole immunogen, ruling out immunodomination. In addition, conserved TCR Vβ usage between inbred and F1 mice did not always correlate with strong responses in F1 mice. However, direct estimation of naive precursor numbers showed that these were reduced in F1 compared with inbred mice for specificities that were poorly immunogenic in the hybrids. These data have implications for our understanding of the extent to which MHC diversity alters the range of epitopes that are immunogenic in outbred populations

Primary CTL response magnitude in mice is determined by the extent of naive T cell recruitment and subsequent clonal expansion

CD8+ T cell responses to viral infection are characterized by the emergence of dominant and subdominant CTL populations. The immunodominance hierarchies of these populations are highly reproducible for any given spectrum of virus-induced peptide-MHCI complexes and are likely determined by multiple factors. Recent studies demonstrate a direct correlation between naive epitope-specific CD8+ T cell precursor (CTLp) frequency and the magnitude of the response after antigen challenge. Thus, the number of available precursors in the naive pool has emerged as a key predictor of immunodominance. In contrast to this, we report here no consistent relationship between CTLp frequency and the subsequent magnitude of the immune response for 4 influenza virus–derived epitopes following intranasal infection of mice with influenza A virus. Rather, the characteristic, antigen-driven T cell immunodominance hierarchy was determined by the extent of recruitment from the available pool of epitope-specific precursors and the duration of their continued expansion over the course of the infection. These findings suggest possibilities for enhancing protective immune memory by maximizing both the size and diversity of typically subdominant T cell responses through rational vaccine design

Heightened self-reactivity associated with selective survival, but not expansion, of naïve virus-specific CD8⁺ T cells in aged mice

In advanced age, decreased CD8(+) cytotoxic T-lymphocyte (CTL) responses to novel pathogens and cancer is paralleled by a decline in the number and function of naïve CTL precursors (CTLp). Although the age-related fall in CD8(+) T-cell numbers is well established, neither the underlying mechanisms nor the extent of variation for different epitope specificities have been defined. Furthermore, naïve CD8(+) T cells expressing high levels of CD44 accumulate with age, but it is unknown whether this accumulation reflects their preferential survival or an age-dependent driver of CD8(+) T-cell proliferation. Here, we track the number and phenotype of four influenza A virus (IAV)-specific CTLp populations in naïve C57BL/6 (B6) mice during aging, and compare T-cell receptor (TCR) clonal diversity for the CD44hi and CD44lo subsets of one such population. We show differential onset of decline for several IAV-specific CD8(+) T-cell populations with advanced age that parallel age-associated changes in the B6 immunodominance hierarchy, suggestive of distinct impacts of aging on different epitope-specific populations. Despite finding no evidence of clonal expansions in an aged, epitope-specific TCR repertoire, nonrandom alterations in TCR usage were observed, along with elevated CD5 and CD8 coreceptor expression. Collectively, these data demonstrate that naïve CD8(+) T cells expressing markers of heightened self-recognition are selectively retained, but not clonally expanded, during aging