Microbiology & Immunology - Theses
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ItemUnderstanding CD8+ T cell responses to seasonal and pandemic influenza viruses( 2015)In the absence of neutralizing antibodies, CD8+ T cells can provide protection and ameliorate disease severity during influenza A virus (IAV) infection. Thus, there is great interest in the development of a CD8+ T cell-mediated IAV vaccine directed towards the relatively conserved internal proteins of IAV. The rational design of a CD8+ T cell mediated vaccine requires a greater understanding of factors, which govern the development of an optimal CD8+ T cell response. However, despite the breadth and depth of murine studies, little is known about human immune responses towards IAV. This PhD thesis focuses on the identification and characterisation of IAV-specific CD8+ T cell responses towards IAV to determine key factors required for the development of an optimal immune response. In particular, this PhD thesis examines the importance of the CD8+ T cell receptor (TCR) in inter-species but not intra-species cross-reactivity. A systematic screening approach was utilised for the unbiased detection of immunodominant IAV-specific CD8+ T cell responses regardless of donor HLA profile. Nucleoprotein (NP) was the most immunogenic protein of IAV, in which seven novel and highly conserved CD8+ T cell epitopes restricted by a range of HLA alleles were identified. Interestingly, these were clustered within the carboxyl terminal 2/3 of NP, suggesting that the epitope-rich regions within NP present a promising target for a CD8+ T cell mediated cross-strain protective influenza vaccine. IAV replicates using an RNA-dependant RNA polymerase, therefore rapidly evolves, and it is unknown how human CD8+ T cells respond to distinct IAV strains. The potential for inter-strain cross-reactivity was assessed using the HLA-B*37:01-restricted NP338 peptide in which two major variants bearing mutations at positions 6 and 7 were identified. In vitro assays revealed that CD8+ T cells stimulated with one NP338 peptide could recognise and respond to the NP338 variants. Human single-cell multiplex RT-PCR for detection of TCRαβ repertoire revealed that this strain cross-protection was attributable to diverse and cross-reactive TCRαβ repertoires, which can recognise the subtle differences within the distinct NP338-pHLA complexes. This suggests that diverse and cross-reactive TCRαβ repertoires are important in the recognition of variant IAV peptides and may provide protection in the face of novel IAV strains emerging in the human population. Having identified inter-species cross-reactivity, intra-species cross-reactivity (heterologous cross-reactivity) was investigated between two sets of HLA-A*02:01 restricted peptides, IAV-M158 and EBV-BMLF-1280 or IAV-NA231 and HCV-NS31073. Despite the sequence homology of the peptide pairs, no cross-reactivity was observed either ex vivo or after in vitro expansion in healthy or HCV-infected donors. Structural analysis revealed that each pHLA complex displays a distinct conformation and TCRαβ analysis showed that the TCRαβ repertoire used for the recognition of the different pHLA complexes was unique, demonstrating that CD8+ T cells are highly specific due to their TCRαβ and although they display some inter-species cross-reactivity, it is unlikely that intra-species cross-reactivity is common. CD27 is constitutively expressed on the majority of memory human T cells at any given stage of life, however, little is known about the effect of CD27 co-stimulation in human lymphocytes. CD27-negative cells were superior in function and cytotoxic potential directly ex vivo, suggesting the importance of CD27 co-stimulation on CD27-expressing lymphocytes. Using a plate bound CD70 (pCD70) monoclonal antibody (mAb) the role of CD27 co-stimulation was investigated. Co-stimulation with pCD70 was required for naïve CD27hiCD45RA+CD8+ T cell proliferation, accelerated Granzyme B acquisition and enhanced the phenotypic plasticity of CD27-expressing CD8+ T cells. This suggests that CD27 co-stimulation is vital during CD8+ T cell priming and enhances recall, similar to murine data and thus might be manipulated for future vaccines or immunotherapies. Overall, this PhD thesis has expanded our understanding of human CD8+ T cell responses towards IAV, in particular the importance of a cross-reactive and diverse TCR repertoire for protection against distinct IAV strains. Furthermore, my PhD work discusses characteristics that govern an “optimal” CD8+ T cell responses, thus providing important foundations for future research into universal immunity against influenza viruses.