Microbiology & Immunology - Theses
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ItemTranscriptional regulation and co-stimulatory signaling in antiviral T cell immunity( 2018)Special AT-rich binding protein-1 (SATB1) is a global chromatin organizer, promoting or repressing gene transcription in mice and human. In this PhD thesis, SATB1 expression was examined in humans across ages and tissues (Chapter 3). The molecular (Chapter 4) and functional (Chapter 5) role of SATB1 was investigated during anti-viral immunity in mice using an influenza (IAV) infection model. Additionally, the effect of CD27-mediated co-stimulation was studied in the context of HIV-1 infection (Chapter 6). SATB1 has pivotal roles during T cell development and maturation, with lineage fate decision in hematopoietic stem cells and gradual changes in SATB1 expression contributing to T cell development in the thymus in mice. In Chapter 3, SATB1 expression was analyzed across lymphocyte compartments from different human tissues and correlated with PD-1 expression in virus-specific CD8+ T cells. SATB1 expression in pediatric and adult donors showed that SATB1 expression was highest in the human thymus with differential expression levels from DN to DP thymocytes and down-regulation of SATB1 in peripheral T cells. Chapter 3 shows that SATB1 expression in the periphery is not static but follows fine-tuned expression dynamics with downregulation from naïve to antigen-specific CD8+ T cells, likely to be antigen- and tissue-dependent. These data led to the hypothesis that fine-tuned SATB1 expression is necessary for maintaining fate-potential in developing and mature, peripheral T cells. Several molecular mechanisms have been identified for gene regulation by SATB1 with wide-range impacts on the overall chromatin landscape. Previous studies in our laboratory showed that SATB1 mRNA levels are high in naïve, but low in effector CD8+ T cells. The impact of SATB1 in repressing transcriptional programs in naïve CD8+ T cells, prior to its downregulation in effector T cells, was addressed in Chapter 4 of this study. ChIP-Sequencing analysis was performed to decipher genomic binding sites of SATB1 in naïve and effector CD8+ T cells. SATB1 ChIP-Seq data demonstrated that SATB1 binding sites were predominately distal to transcriptional start sites, likely to harbor transcriptional enhancer sites, with reduced SATB1 binding sites in effector over naïve CD8+ T cells. To understand the effects of SATB1 on the transcriptional regulation in naïve and IAV-specific CD8+ T cells, SATB1 imposter mice (SATB1imp/imp) were used in this PhD study. In these mice, Satb1 contains a point mutation in the DNA-binding domain encoding position. SATB1 protein expression in SATB1imp/imp mice persists but is dysfunctional with reduced DNA-binding capability. CD8+ T cells from SATB1imp/imp mice showed up-regulation of certain gene profiles, especially at the naïve stage, such as Pdcd1, Ctla4 and Ccl5, characteristic of activated or exhausted T cells. In Chapter 5, an IAV infection model was used, to examine the effects of dysfunctional SATB1 in IAV-specific CD8+ T cell response generation. CD8+ T cell numbers were consistently reduced in SATB1imp/imp mice with significantly reduced IAV-specific CD8+ T cell numbers in lungs on d10 post-infection. SATB1imp/imp CD8+ T cells exhibited an early overexpression of PD-1 from the naïve stage and reduced polyfunctionality within IAV-specific SATB1imp/imp CD8+ T cells. Using a bone marrow chimera approach, in which mice were reconstituted with a mixture of wildtype and SATB1imp/imp-derived lymphocytes, data showed that reduced T cell numbers and PD-1 overexpression are T cell intrinsic in SATB1imp/imp mice. Immunotherapies, including anti-PD-1, anti-CD27 and histone deacetylase inhibitors, are often used in clinical trials to manipulate activation of T cells. In Chapter 6, we used CD27-mediated stimulation to understand the effect on CD4+ T cells with and without HIV-1 infection. CD27 is a co-stimulatory receptor of the TNF-family, expressed on naïve and central memory T cells. Non-permanent stimulation via CD27 leads to increased primary and memory antiviral CD8+ T cell responses in mice. Here, in humans, CD27-mediated stimulation of CD4+ T cells via its ligand CD70 exhibited profound activation potential in vitro, with high CD4+ T cell proliferation and GzmB production. To examine whether this high activation potential could trigger re-activation of viral transcription in latently infected CD4+ T cells, we re-stimulated CD4+ T cells with conventional α-CD28 or CD27-mediated co-stimulation in an in vitro latency model. Unexpectedly, re-stimulation via CD27 of CD4+ T cells led to reduced viral reactivation compared to α-CD28 stimulation of CD4+ T cells. However, similar transcriptional reactivation levels were obtained when CD4+ T cells isolated from HIV+ individuals on ART were re-stimulated with the two protocols. Strikingly, pre-stimulation of CD4+ T cells prior to in vitro HIV-1 infection showed a trend towards reduced HIV-DNA integration and overall infection. This suggests that CD27-mediated stimulation could lead to activation of antiviral mechanisms that reduces CD4+ T cells HIV-1 infection. Overall, this PhD study provides an in-depth understanding of the transcriptional and co-stimulatory regulations of T cell differentiation in response to viral infections. SATB1’s ability to regulate immune checkpoint molecules, such as PD-1 by its DNA-binding capability in antiviral immunity highlights its significance in future PD-1-related cancer and HIV-1 immunotherapy trials used to reverse T cell exhaustion.
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ItemNo Preview AvailableTracking human CD8+ and γδ T cell receptor repertoire dynamics to understand the impact on immune responses towards influenza viruses( 2018)Seasonal 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.