Tracking human CD8+ and γδ T cell receptor repertoire dynamics to understand the impact on immune responses towards influenza viruses

Abstract

© 2018 Dr. Sneha Ashok SantSeasonal IAV epidemics cause severe morbidity and mortality, resulting in up to 250,000 -600,000 deaths worldwide annually, especially in the young, elderly, immunocompromised, pregnant and those with co-morbidities. Given the segmented nature of the viral genome and a rapid mutational rate, newly-emerging influenza viruses have the potential to cause pandemics. Current seasonal vaccination regimens elicit neutralizing antibodies (nAbs), which require yearly updates to account for the antigenic evolution of influenza viruses. Alternative strategies such as the development of a universal vaccine that can provide broad protection by eliciting immune responses across different strains of influenza viruses are ugently needed. CD8+ T cells recognize internal conserved segments of the viral protein and thus have the potential to provide cross-strain immunity. The efficient immune response of a CD8+ T cell is dictated by the recognition of peptide-MHC complex by its T cell receptor (TCR). However, the response magnitude varies with age and immunogenetics. Similarly, innate γδ T cells are potently activated by stress-induced ligands, independently of classical antigen-presenting molecules and could provide immediate effector function for novel influenza immunotherapies and vaccines. However, much of human γδ T cell biology remains understudied. Therefore, this PhD aimed to determine TCR dynamics of both γδ T cells and influenza-specific CD8+ T cells across different age groups, anatomical locations and influenza infection. Aim 1 explored the diversity of the human γδ T cell TCR repertoire and how γδ T cells are actived by influenza viruses. We implemented a single-cell RT-PCR for paired analysis of the TCRγ and TCRδ chains and developed an in vitro infection model of influenza-infected lung epithelial cells co-cultured with peripheral blood mononuclear cells (PBMCs). We performed a thorough repertoire analysis of ex vivo γδ T cells from cord blood, young adult, elderly adult and human tissues (spleen, lung and lymph node). Our analyses found diverse and private γδ T cells in cord blood and spleen, while those in young adults and lungs were highly focused towards invariant γ9δ2 TCRs. Elderly adult γδ T cells displayed expansion of private or the invariant γ9δ2 clonotypes. Using the in vitro infection model of influenza, we next investigated γδ TCRs which produced IFNγ during an in vitro influenza infection and PBMC co-culture. Our results demonstrated that the majority of responding γδ T cells harbored γ9δ2 TCRs. We observed heterogeneity in the influenza response between cord blood, young adult and elderly adults. γδ T cells within cord blood and the elderly adults had minimal IFN- production in the absence of γ9δ2 TCRs. Thus, this study provided an understanding on how γδ T cells contribute to immune protection during influenza infection and which TCRs are important to elicit across all age groups vulnerable to influenza virus infection. In Aim 2 and 3, we tracked the repertoire dynamics of influenza specific CD8+ T cells across age groups and tissue locations. Since HLA-A*02:01-restricted M158-66 viral peptide has high sequence conservation and elicits immunodominant CD8+ responses, we focused our analyses on HLA-A*02:01-M158+CD8+ TCRs. A robust response elicited by HLA-A*02:01-M158+CD8+ TCRs is governed by the presence of the public TCR signature, TRBV19/TRAV27 (CDR3 motif “GGSQGNL”/“SIRSYEQ”). Our study demonstrated the loss of this public TCR and presence of large private clonotypes in the HLA-A*02:01-M158+CD8+ TCRs isolated from the elderly donors, as compared to young adults who maintained high frequencies of public TCRs. Our study showed, for the first time, HLA-A*02:01-M158+CD8+ T cells were present in human tissues (spleen, lung and lymph nodes) obtained from young adults. Furthermore, lung tissue-resident HLA-A*02:01-M158+CD8+ T cells and those isolated from spleen and lymph nodes displayed a prominent presence of public TCRs. Overall, we showed a loss of public TCRs with aging and we speculate that this is a mechanism underlying reduced immune responsiveness during influenza infection with aging. Moreover, the presence of public TCRs in distal tissues could provide a reservoir to replenish “optimum” TCRs at the site of infection. The magnitude of antigen-specific CD8+ T cells can be influenced by the different Human Leukocyte Antigens (HLAs) expressed by an individual, thus contributing to the phenomenon of CD8+ T cell immunodominance hierarchy. Using the known highly conserved immunodominant epitopes restricted by 6 HLAs that have broad coverage towards influenza viruses across different ethnicities, we compared the response magnitude of these epitope-specific CD8+ T cells to that of HLA-A*02:01-M158+CD8+ T cells. We showed that individuals co-expressing HLA-B*27:05 and HLA-A*02:01 had higher magnitude of B*27:05-NP383+CD8+ T cell responses compared to that of A*02:01-M158+CD8+ T cells. Our findings showed that B27/NP383+CD8+ T cells had higher functional capacity as compared to A02/M158+CD8+ T cells. Moreover, TCRs of A02/M158+CD8+ T cells from heterozygous donors showed a reduction or complete loss of the public TCR present in A*02:01+B*27:05- individuals. This suggested that the reduction in the observed magnitude of response was partly attributed to changes within the A02/M158 TCR repertiore. Overall, this PhD contributes to our understanding of innate and adaptive T cell compartments during influenza virus infection. It provides evidence that influenza-specifc γδ and CD8+ T cell immune responses are affected by age, HLA genotype and alterations in the TCR repertoire. These findings form an important foundation for future research developing universal vaccines against influenza viruses and immunotherapies against viral infections or cancer