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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ItemEvaluation of influenza-specific antibody-dependent cellular cytotoxicity (ADCC) in human and non-human primates( 2013)Influenza virus is a major cause of morbidity and mortality worldwide. Yearly trivalent influenza vaccination partially reduces the severity of influenza infection. Seasonal influenza vaccines mediate their protective activity through the induction of neutralising antibodies towards the influenza virus surface hemagglutinin (HA) and neuraminidase (NA) glycoproteins. HA- and NA-specific antibodies act by either inhibiting virus entry or preventing the release of new virions from host cells, respectively. However, influenza-specific neutralising antibodies are susceptible to loss of recognition by accumulation of point mutations in the HA and NA over time (antigenic drift), or by the introduction of new viral HA and NA glycoproteins (antigenic shift). Fortunately, antibodies have a number of non-neutralising effector functions including antibody-dependent cellular cytotoxicity (ADCC). Non-neutralising antibody effector functions rely on binding of the antibody fragment antigen-binding (Fab) region to surface antigens, but mediate their effector activity through the fragment crystallisable (Fc) region of the antibody. ADCC in particular may play an important role in protection from influenza infection, but few studies have been undertaken in this area for over 3 decades. Previous studies on influenza-specific ADCC were performed using either chromium-release assays or in in vivo using FcR-knockout mice. Until recently no flow-cytometry based assay was available to measure and characterise influenza-specific ADCC-mediating antibodies in human and non-human primate sera samples. Using newly developed assays we showed that healthy individuals aged between 18-43 years of age did not have neutralising antibodies to a 1968 H3N2 influenza virus, but they did possess binding antibodies. Furthermore, a portion of these non-neutralising antibodies mediated ADCC effector functions including activating NK cells and eliminating virus-infected respiratory epithelial cells (Chapter 2). This highlighted that ADCC-mediating antibodies have broad cross-reactive capacity that may play an important role in protection against influenza virus infections. In April 2009, a swine-origin H1N1 influenza (A(H1N1)pdm09) virus caused a pandemic in the human population. The pandemic was estimated to have resulted in 4 million cases worldwide, but only lead to approximately 106,700-395,699 deaths. This was a comparatively milder influenza pandemic, as seasonal influenza epidemics generally lead to approximately 350,000-500,000 deaths worldwide. Moreover, yearly influenza epidemics generally cause deaths in younger (<5 years old) and older individuals (>65 years old), particularly targeting individuals with major risk factors such as cardiovascular disease. In contrast, the 2009 H1N1 pandemic had a lower mortality in older individuals (>65 years old), with younger healthy individuals experiencing greater morbidity. These two separate observations suggested that there were two levels of protection detected in the population prior to the 2009 H1N1 pandemic: (1) the reduced severity of the 2009 H1N1 pandemic on the population and (2) the lower incidence of mortality in older individuals in the population. The potential role of cross-reactive ADCC-mediating antibodies in providing protection during the 2009 H1N1 pandemic was investigated. To determine if there was ADCC-mediated protection against A(H1N1)pdm09 virus in older individuals, we obtained human sera samples collected from separate individuals (aged between 1-72 years) prior to or following the 2009 H1N1 pandemic. We showed that individuals greater than 45 years of age had a moderate level of cross-reactive A(H1N1)pdm09-specific ADCC-mediating antibodies prior to the 2009 H1N1 pandemic (Chapter 3). To understand the role that ADCC-mediating antibodies may have played in reducing the severity of A(H1N1)pdm09 virus on the whole population, we obtained intravenous immunoglobulin (IVIG) manufactured either prior to the 2009 or post 2009 pandemic. We showed that there was a level of cross-reactive A(H1N1)pdm09-specific ADCC-mediating antibodies in the whole population prior to the 2009 H1N1 pandemic (Chapter 4). Lastly, to examine the mechanism by which cross-reactive A(H1N1)pdm09-specific ADCC-mediating antibodies are induced, we measured ADCC-mediating antibodies in naïve rhesus macaques (Macaca mulatta) infected with seasonal H1N1 influenza virus and subsequently challenged with the A(H1N1)pdm09 virus (Chapter 5). We showed that seasonal H1N1 infection of macaques induced cross-reactive ADCC-mediating antibodies towards the A(H1N1)pdm09 virus. Collectively, of these studies implicate ADCC in the protection observed during the 2009 H1N1 pandemic. The induction of influenza-specific ADCC-mediating antibodies by trivalent vaccines remains unknown. To investigate whether trivalent influenza vaccines can prime or induce ADCC-mediating antibodies we vaccinated influenza naïve pigtail macaques (Macaca nemestrina) with two doses of trivalent influenza vaccine (2012, A/California/07/2009 (H1N1), A/Perth/16/2009 (H3N2) and B/Brisbane/60/2008 (Type B), Sanofi Pasteur) and subsequently serially infected them with either H1N1 or H3N2 influenza strains. Our results showed that ADCC-mediating antibodies and cytotoxic T lymphocytes (CTL) responses were not primed or induced by vaccinations with seasonal trivalent influenza vaccine (Chapter 6). These studies suggested that the induction of ADCC-mediating antibodies by trivalent vaccination most likely relied on a background of previous influenza exposures. The design of new vaccines that induce broad ADCC-mediating antibodies may provide greater protection from divergent influenza strains and should be an important direction for future studies. Together, these studies serve as a basis for future research into influenza-specific ADCC immunity. The association of cross-reactive ADCC mediating antibodies with the protection observed during the 2009 H1N1 pandemic has important implications for the control of potential future influenza pandemics. The induction of broadly cross-reactivity ADCC mediating antibodies in nearly all individuals could be utilised for universal influenza vaccine design.