Browsing by Author "Riou, Catherine"

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    Open Access
    Characterization of Mycobacterium tuberculosis-specific Th22 cells in HIV-TB co-infection
    (2020) Makatsa, Mohau Steven; Burgers, Wendy A; Riou, Catherine
    Tuberculosis (TB) remains the infectious disease causing the greatest global mortality, with an estimated 10 million incident cases of TB and 1.45 million deaths in 2018. Although there is a cure for TB, the success of the treatment is hampered by multidrug resistant TB and HIV infection. There is an urgent need for an effective TB vaccine to prevent ongoing transmission. The development of a new and efficacious TB vaccine will likely be dependent on our understanding of protective immunity to TB. Although it is well established that Th1 cells are crucial in the response against Mycobacterium tuberculosis (Mtb), Th1 cytokines may not be sufficient to control Mtb infection. A major focus of this thesis is the contribution of an understudied Th subset in Mtb immunity, namely Th22 cells, producing the cytokine IL-22. IL-22 functions to preserve mucosal barriers and induce antimicrobial peptides, contributing to protective immunity to a range of extracellular and intracellular bacteria. A recent study in IL-22-deficient mice described a protective role for IL-22 during the development of TB. In humans, soluble IL-22 has been detected at sites of extra-pulmonary tuberculosis (TB), and a polymorphism in the IL-22 promoter has been linked to TB susceptibility. However, much remains to be understood about Th22 cells and their role in protective immunity to Mtb. In this study, we investigated the contribution of Th22 cells to TB immune responses by providing a detailed characterisation of Mycobacterium tuberculosis-specific Th22 cells in latent TB infection (LTBI), TB disease and HIV co-infection, using flow cytometric techniques. In Chapter 2, we optimised detection of IL-22 and determined the factors that contribute to Mtb-specific IL-22 production by CD4+ T cells, as well as characterising some aspects of Th22 cell biology. In Chapter 3, we examined the impact of TB disease and HIV infection on Th22 cells, compared to Th1 and Th17 cells. Finally, in Chapter 4, we explored Mtb-specific cytokine production by CD8+ T cells and CD4+ T cells following Mtb peptide stimulation, and the effect of TB disease and HIV infection. We detected significant IL-22 production from CD4+ T cells in healthy individuals following whole blood stimulation with Mtb whole cell lysate (MtbL). However, IL-22 responses were poorly detectable when peripheral blood mononuclear cells (PBMC) were stimulated with MtbL. Therefore, we sought to investigate conditions that influence IL-22 detection in whole blood and PBMC, and characterise Th22 cells further. We found that PBMC are able to produce IL-22 in response to Mtb but appear to lack the physiological environment for optimal induction of IL-22. We also discovered that TCR blocking inhibited Mtb-specific IL-22 production, suggesting that responses are stimulated through recognition of Mtb antigen by the TCR, rather than through bystander activation. IL-22 is produced by CD4+ T cells that appear to be conventional, rather than MAIT, γδ or iNKT cells. Indeed, analysis of the TCR clonality using vβ repertoire typing revealed similar repertoire usage between IL-22, IFN-γ-producing CD4+ T cells, and total CD4+ T cells. Overall, these data shed more light on the biology of IL-22-producing CD4+ T cells. Next, we examined the effects of HIV infection and TB disease on the magnitude, memory profile and activation phenotype of Mtb-specific Th22 cells, compared them to Th1 and Th17 cells. Blood samples were collected from 72 individuals classified into four groups based on their HIV-1 and TB status, namely HIV-/LTBI, HIV+/LTBI HIV-/active TB and HIV+/active TB. Blood was stimulated with MtbL and analysed for cytokine production using multiparameter flow cytometry. We observed similar frequencies of IL-22 to IFN-γ-producing CD4+ T cells in LTBI. Mtb-specific Th22 cells were reduced to a greater extent than Th1 cells by a combination of HIV infection and TB disease. Th22 cells demonstrated differences in their memory and activation phenotype compared to Th1 and Th17 cells. In the context of active TB, Th1 cells were characterised by a high expression of the activation marker HLA-DR. In contrast, Th22 cells did not demonstrate activation using this marker during TB disease. Similarly, Th1 cells were more differentiated in TB disease irrespective of HIV status, while there was no difference in the memory phenotype of Th22 cells during different disease states. Finally, we characterised Mtb peptide-specific CD4+ and CD8+ T cell responses in LTBI, active TB and HIV infection. CD4+ T cells did not produce detectable IL-22 when blood was stimulated with Mtb peptides, and there was also no IL-22 response from CD8+ T cells. Th1 cytokines IFN-γ and TNF-α were detectable from CD4+ and CD8+ T cells in response to Mtb peptides. Consistent with previous studies, there was a higher proportion of individuals with detectable CD8+ responses during active TB and HIV co-infection compared to HIV-infected LTBI individuals, but no difference is the magnitude of response was observed. Interestingly, HIV infection and TB disease induced similar levels of activation in Mtb-specific CD8+ compared to CD4+ T cells. Moreover, active TB and HIV co-infection impaired memory differentiation of Mtb-specific CD8+ T cells towards a less differentiated profile, compared to LTBI. These results confirm that both CD4+ and CD8+ T cells contribute to TB immune responses. In summary, we confirm that Th22 cells constitutes a substantially portion of CD4+ T cell response to Mtb . IL-22 appears to be produced by conventional CD4+ T cells but may require specific antigen presentation requirements to optimally induce its production. Interestingly, HIV infection during TB disease led to a near absence of Th22 cells in blood. Our results warrant further study of the role of Th22 cells in TB immunity, which may lead to insights that could assist the development of an effective vaccine against TB.
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    Open Access
    Characterizing the cellular latent reservoir of HIV-1 and the effect of immune activation on characteristics of the reservoir
    (2022) Ismail, Sherazaan Dineo; Burgers, Wendy A; Williamson, Carolyn; Riou, Catherine; Abrahams, Melissa-Rose
    Since the advent of antiretroviral therapy (ART) and the resultant suppression of viraemia in the majority of people living with HIV-1 (PLWH) on ART, HIV-1 infection has become manageable and PLWH have similar life expectancies as uninfected persons. However, ART is not curative, is needed lifelong, and its cessation leads to the recrudescence of viraemia. This is due to the formation of a latent reservoir that is long-lived and stable over time, precluding HIV-1 cure. The factors affecting reservoir formation, establishment, and kinetics are not fully understood. Furthermore, differences exist at the population level in disease progression in PLWH depending on ethnicity, biological sex, and infecting viral subtype. Similarly, differences in the latent reservoir of HIV-1 have been described, although less extensively. Understanding what shapes the latent HIV-1 reservoir is critical for developing strategies for cure. Furthermore, it is imperative that cure research is undertaken in diverse populations to ensure coverage of knowledge across different demographics. The latter will ensure that a cure strategy can be developed that will be globally implementable. In the Introductory chapter of this thesis, I provide a detailed review of the current literature and address the need for cure research in low-and middle- income countries. If a global cure is to be achieved, the burden of HIV-1 will need to be addressed in many different populations, most notably African women, as women bear the burden of HIV-1 globally. In South Africa, the country in the sub-Saharan African region with the highest prevalence of HIV-1, women are roughly twice as likely to be living with HIV than men (aged 15 to 49), with a prevalence rate 6% higher than the national average of 19%. Since women are underrepresented in HIV-1 research in general and more specifically in cure studies due to the paucity of research in countries outside of the global North, reservoirs and cure strategies ii need to be characterized in this context. Furthermore, while early treatment is the WHO standard of care for people diagnosed with HIV, a large majority of PLWH only initiated treatment in chronic infection. Since early ART is known to restrict formation of the latent reservoir of HIV-1, research in both early and late ART initiators is necessary. This research focused on characterising the viral reservoir in South African women in a well-established cohort of women who were recruited during acute HIV infection and followed until treatment initiation (which occurred during chronic infection) and beyond. Overall, this thesis focuses on characterising immune activation and inflammation during the course of both untreated and treated HIV-1 infection in a cohort of South African women and subsequently determining whether clinical or immune measures influence characteristics of the latent reservoir of HIV-1. T cell activation and the levels of soluble inflammatory cytokines in plasma were determined in forty-six women in the CAPRISA 002 Acute infection cohort. Chapter 2 describes the cellular immune activation and inflammation profiles of these participants throughout the course of infection at the following timepoints: acute infection, oneyear post-infection, and within a year preceding ART initiation, and two- and four- years postART initiation. T cell activation peaked in chronic infection and reduced dramatically after ART initiation. CD4+ and CD8+ T cell activation reached a post-treatment nadir by two years after ART initiation. Cytokine measures were within the ranges reported in the literature for PLWH. Notably CXCL-10 levels in plasma decreased significantly between two- and four years post-ART, indicating that it may be a sensitive marker of ongoing systemic inflammation in people on ART. In short, the T cell activation and inflammation profiles of the women in this study reflected what has been observed in other cohorts. iii The size of the replication-competent HIV-1 reservoir, measured by quantitative viral outgrowth assay after 5 years of suppressive antiretroviral therapy (ART), was quantified in twenty women of the cohort. In Chapter 3, the clinical and immunological correlates of reservoir size were investigated. Predictive modelling showed that the size of the replicationcompetent reservoir is directly related to viral load and CD4+ T-cell counts over the course of infection, although these measures do not fully predict reservoir size. We found that, in addition to viral load and CD4+ T-cell count, CD8+ T-cell activation within the year preceding ART, nadir CD4+ T-cell count, and baseline as well as on-treatment CD4:CD8 ratio at the time of sizing was associated with replication-competent reservoir size. We provide evidence that the late CD8+ T-cell activation level before treatment, together with viral loads and CD4+ T-cell counts, are directly related to the size of the replication-competent reservoir of HIV-1. Our results are consistent with the hypothesis that the host immune milieu near the time of ART initiation plays an important role in shaping the durable reservoir of HIV infection that persists on ART. Another characteristic of the HIV-1 reservoir is persistence: the presence of all forms of HIV1 within cells and tissues that contribute to pathogenesis, including defective, non-induced, and non-integrated forms of HIV-1. In Chapter 4, total HIV-1 DNA levels were measured as a proxy for viral persistence in thirty-one participants, and the correlates thereof investigated. The HIV-1 DNA levels in this cohort were similar to those reported in the literature for other cohorts where participants initiated therapy in late chronic infection. HIV-1 DNA levels did not differ significantly between two- and four years post-ART, but there was a trend to lower HIV-1 DNA when measuring pol versus gag gene frequencies in peripheral blood mononuclear cells (PBMC). These findings indicate that HIV-1 DNA decay rates may differ depending on the gene being measured, even when using the same assay. A weak significant correlation was iv found between CD4+ T cell counts at ART initiation and the change in HIV-DNA levels between two-and four years on ART. There was a significant correlation between residual CD4+ T cell activation at four years post-ART initiation and gag copies per million PBMC. A trend towards a correlation was found between CD4+ T cell activation and pol copies per million PBMC at the same timepoint. Finally, we found significant correlations between several cytokines at one-year post-infection and within one year pre-ART. These findings further solidify the hypothesis that the immune milieu around the time of ART initiation and after may play a complex role in formation of the viral reservoir of HIV-1. Our studies show a significant link between chronic immune activation and replication competent reservoir size, and also ongoing immune activation and viral persistence on ART. Further studies into whether these immune measures affect the timing of establishment and clonality of the reservoir in this cohort are ongoing and will inform the field about whether differences in cure strategies will need to be explored for those PLWH who had high levels of chronic immune activation before treatment initiation and subsequent shaping for the long-lived viral reservoir.
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    Effect of standard tuberculosis treatment on plasma cytokine levels in patients with active pulmonary tuberculosis
    (Public Library of Science, 2012) Riou, Catherine; Peixoto, Blas Perez; Roberts, Lindi; Ronacher, Katharina; Walzl, Gerhard; Manca, Claudia; Rustomjee, Roxana; Mthiyane, Thuli; Fallows, Dorothy; Gray, Clive M
    BACKGROUND: Sputum Mycobacterium tuberculosis (Mtb) culture is commonly used to assess response to antibiotic treatment in individuals with pulmonary tuberculosis (TB). Such techniques are constrained by the slow growth rate of Mtb, and more sensitive methods to monitor Mtb clearance are needed. The goal of this study was to evaluate changes in plasma cytokines in patients undergoing treatment for TB as a means of identifying candidate host markers associated with microbiologic response to therapy. METHODS: Twenty-four plasma cytokines/chemokines were measured in 42 individuals diagnosed with active pulmonary TB, 52% were HIV co-infected. Individuals, undergoing a 26-week standard TB treatment, were followed longitudinally over 18 months and measurements were associated with HIV status and rates of sputum culture conversion. RESULTS: Plasma concentrations of interferon-inducible protein-10 (IP-10) and vascular endothelial growth factor (VEGF) were significantly reduced upon TB treatment, regardless of HIV status. By the end of treatment, IP-10 concentrations were significantly lower in HIV negative individuals when compared to HIV-positive individuals (p = 0.02). Moreover, in HIV negative patients, plasma VEGF concentrations, measured as early as 2-weeks post TB treatment initiation, positively correlated with the time of sputum conversion (p = 0.0017). No significant changes were observed in other studied immune mediators. CONCLUSIONS: These data suggest that VEGF plasma concentration, measured during early TB treatment, could represent a surrogate marker to monitor sputum culture conversion in HIV uninfected individuals.
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    Investigating the molecular pathogenesis of tuberculosis-associated immune reconstitution inflammatory syndrome
    (2025) Moseki, Raymond; Meintjes, Graeme; Wilkinson, Robert J.; Riou, Catherine; Lai, Rachel
    Thesis summary This thesis comprises a series of studies that adapted a reverse translational approach to investigate the mechanisms underlying the immunopathogenesis of paradoxical tuberculosis associated immune reconstitution inflammatory syndrome (TB-IRIS). Paradoxical TB-IRIS is an aberrant immune response targeted against Mycobacterium tuberculosis or its residual antigens in patients with advanced human immunodeficiency virus (HIV) infection shortly after commencement of antiretroviral therapy (ART). TB-IRIS is characterized by hyperinflammation and tissue damage. Pulmonary and lymph node involvement are the most common clinical manifestations. The incidence of TB-IRIS can reach up to 54% in settings where both HIV and TB are endemic, such as the sub-Saharan-Africa region of the African continent. Approximately 25% of patients with paradoxical TB-IRIS will require hospitalization for syndromic management. The management and prevention of paradoxical TB-IRIS rely on the use of anti-inflammatories such as immunosuppressive corticosteroid intervention, which is associated with an increased risk of Kaposi sarcoma. Paradoxical TB-IRIS is potentially fatal, with cases of central nervous system (CNS) involvement having the highest fatalities (up to 56%). The underlying cause of paradoxical TB-IRIS is thought to arise from a dysregulated hyperinflammatory immune response during ART-mediated immune recovery, although the causal mechanisms and associated molecular pathways are incompletely defined. This knowledge gap compromises efforts to develop new diagnostic and prognostic modalities as well as safer and more efficacious therapeutic interventions. This served as the rationale for undertaking the studies reported in this thesis. Using clinical samples from the PredART intervention trial and the TB-ART observational study, this thesis investigated the cellular and molecular determinants of paradoxical TB-IRIS. Conventional scientific hypothesis-based approaches as well as unbiased hypothesis generating approaches were used, with major experimental approaches including ribonucleic acid (RNA) sequencing, flow cytometry and soluble analyte analyses. RNA extracted from whole blood of consenting participants from the Pred-ART intervention trial was sequenced to profile global gene expression. Cross-sectional and longitudinal analyses were performed to compare transcriptomic profiles in participants who developed paradoxical TB-IRIS to those who did not, at week 0, 2 and 12 on ART. Using pathway analysis, samples collected prior to the initiation of ART (week 0) in patients who later developed paradoxical TB-IRIS (cases) were characterized by the upregulation of transcripts encoding neutrophil-derived antimicrobial peptides and defensins and associated with hypoxemia compared to the TB-non-IRIS controls. These largely reflected biological processes involved in neutrophilic responses and adaptation to metabolic stress and dysfunction. Furthermore, pathways that were significantly downregulated in patients who later developed paradoxical TB-IRIS compared to those who did not, included chondroitin sulfate biosynthesis and interleukin (IL)-6. These finding suggest that paradoxical TB-IRIS is preceded by increased expression of antimicrobial peptides and the impairment of immune cellular function controlled by the extracellular matrix. Pathway analysis at week 2 on ART, which coincided with the median time of clinical manifestations in patients that developed paradoxical TB-IRIS, revealed the significant enrichment of neutrophil degranulation, interleukin (IL)-1 signalling, as well as type I and II interferon (IFN). Moreover, biological pathways that were significantly downregulated in cases as opposed to controls at week 2 on ART were largely overrepresented by epigenetic biological processes. These data suggested that neutrophil degranulation preceded and characterized the onset of paradoxical TB-IRIS. To validate these findings, we evaluated the associations between absolute neutrophil counts and neutrophil soluble markers, with paradoxical TB-IRIS at week 0 and week 2 on ART. No significant differences in absolute neutrophil counts were reported in participants who later developed paradoxical TB-IRIS compared to those who did not at week 0. However, azurocidin measured in plasma samples by enzyme-linked immunosorbent assay (ELISA) was significantly lower in samples from participants who later developed paradoxical TB-IRIS at week 0. This suggests that participants who are likely to develop paradoxical TB-IRIS might have impaired innate immune responses at week 0 compared to those who did not develop the syndrome. Absolute neutrophil counts were higher in cases compared to controls at week 2 on ART. Soluble neutrophil markers including human neutrophil peptide 1-3, myeloperoxidase, and neutrophil elastase were significantly higher in cases compared to controls at week 2 on ART. These data suggest that patients who develop paradoxical TB-IRIS have heightened neutrophil degranulation at the time of symptoms onset. Longitudinal perturbation of gene expression was investigated between week 0 and week 2 in people who developed paradoxical TB-IRIS and TB-non-IRIS controls, respectively. Among the top significantly upregulated biological pathways in samples of participants that developed paradoxical TB-IRIS were neutrophil degranulation, type I and II interferon signaling, IL-1 cytokine signalling, and pyroptosis. Biological pathways that were significantly downregulated in these patients included heme biosynthesis and oxygen-carbon dioxide gaseous exchange signalling metabolic dysfunction. In TB-non-IRIS controls, biological pathways that were significantly upregulated included epigenetic modulation of gene expression, collagen biosynthesis and extracellular cytoskeleton remodelling indicating anabolic processes that restore or maintain proper cellular functionality such as cellular signalling. Type I interferon signalling and immune responses to viral pathogens were among biological pathways that were significantly downregulated in people who did not develop paradoxical TB-IRIS at week 2 on ART compared to week 0, likely indicating decrease in antiviral responses as HIV viral load declined. The management and prevention of paradoxical TB-IRIS rely on the use of corticosteroids which is associated with an increased risk of Kaposi Sarcoma and herpes reactivations. Thus, there is a need to explore other anti-inflammatory but non-immunosuppressive drugs that can be repurposed for the management of IRIS-related pathologies. Gene expression data at week 2 on ART and previous published data indicated that aberrant inflammasome activation represent one of the molecular mechanisms that underpins the pathogenesis of paradoxical TB IRIS. We proposed that chemical inhibition of the inflammasome would result in a targeted reduction of inflammation. Two candidate drugs (anakinra and parthenolide) were evaluated and benchmarked against the standard of care (prednisolone) in an ex vivo mycobacterial cell stimulation model using patient samples. Both anakinra and parthenolide significantly reduced several markers of inflammation compared to prednisolone. However, parthenolide was the more potent in reducing cytokine production including IL-1 among others. These findings provide experimental evidence of in vitro use of novel anti-inflammatory agents. Anakinra has been approved for clinical use in inflammatory conditions such as rheumatoid arthritis and could potentially be repurposed for use in the context of paradoxical TB-IRIS. A hallmark of paradoxical TB-IRIS includes the partial reconstitution of Mtb-specific CD4 T cell responses within 3 months of initiating ART while on effective antituberculosis therapy. The phenotype and transcription factors of antigen-specific interferon-gamma producing (IFN+) CD4+ T cells and their potential involvement in the pathogenesis of paradoxical TB IRIS were characterized in an experimental mouse model of IRIS (by collaborators) as well as in human peripheral blood mononuclear cells (PBMC). In mice, antigen-specific CD4 T cells knocked out for Eomes showed significantly higher proportion of survival compared to wildtype mice. No significant differences in Eomes expression were observed in Mtb-300 specific, IFN+ CD4 T cells in PBMC from paradoxical TB-IRIS cases compared to controls at the onset of clinical symptoms. However, Eomes expression was higher in cases compared to controls in bulk CD4 T cells at week 2 on ART. HLA-DR expression was higher in cases compared to controls in Mtb300-specific IFN+ CD4 T cells at week 2 on ART. Additionally, Granzyme B producing, Mtb300-specific IFN+ CD4 T cells co-expressing Eomes and Tbet were significantly higher in cases compared to controls at week 2 on ART. These data indicate that HLA-DR expression on Mtb300-specific CD4 T cells producing IFN+ can potentially be developed as a diagnostic tool to identify individuals with paradoxical TB-IRIS. Collectively, these findings highlight the key contribution of neutrophils and Mtb-specific CD4 lymphocytes in the pathogenesis of paradoxical TB-IRIS. The gene expression findings suggest that the underlying mechanism involves interferon stimulated genes which trigger the secretion of proinflammatory cytokines and the aberrant activation of the inflammasome. Members of the IL-1 family of cytokines are secreted as catalytically inactive proenzymes that require the action of mature inflammasome for activation. IL-1a is the only exception since its proform and mature form are both fully functional. Active IL-1 family of cytokines are potent modulators of inflammation which is a defining feature in people who develop paradoxical TB-IRIS at the time of clinical symptom onset. In addition, the pathogenesis of paradoxical TB-IRIS appears to be mediated by the heightened degranulation of neutrophils which may cause acute inflammation and tissue damage.
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    Investigating the T cell-specific role of Interleukin-4 receptor alpha (IL-4Rα) in tuberculosis (TB)
    (2024) Rousseau, Robert Pierre; Parihar, Suraj P; Riou, Catherine; Brombacher, Frank
    Protective immunity to tuberculosis (TB) is dependent on the ability of the host to mount a robust T cell response. The main effector T cells which contribute to protection in TB include T helper (Th) Th1 and Th17 T cells. The Th2 response, associated with IL-4Rα mediated signalling, remains largely overlooked in TB. The role of IL-4Rα in TB appears to be different according to cell type. Deletion of IL-4Rα on macrophages has no effect on disease, but deletion of the receptor on B cells, leads to better control. This thesis aimed to explore the T cell-specific role of IL-4Rα in TB disease by making use of T cell specific knockout model, iLCKCreIL-4Rα-/lox. We found that absence of IL-4Rα on T cells results in a delay in the recruitment of T cells to the lung. This was demonstrated by decreased CD4 and CD8 T cell numbers during acute infection compared to littermate controls. Consequently, the bacilli are able to better establish infection and proliferate in the lung, shown by increases in lung mycobacterial burden at both acute and chronic stages of infection. However, no differences are observed in the spleen, indicating deletion of IL-4Rα does not have a role in the dissemination of TB. In the absence of IL-4Rα, T cells express higher amounts of T-bet or RORγt transcription factors, indicating stronger Th1 and Th17 responses, respectively. The stronger pro-inflammatory responses do not clear the pathogen, and instead contribute to immunopathology. During chronic infection, we observed higher amounts of IL-17 as well as a corresponding increase in neutrophils, which in turn lead to a decrease in alveolar free space. The promotion of increased Th1 CD4 T cells resulted in greater amounts of terminally differentiated (CD44+KLRG1+ ) which have a poor proliferative capacity despite secreting large amounts of IFN-γ. KLRG1 expression is also associated with a reduced ability to migrate into the lung parenchyma, the site of disease. We also found that these (CD44+KLRG1+ ) expressed reduced amounts of CXCR3, CD69, and CD103, which are all markers associated with poorer migration into the lung parenchyma. Importantly, these factors result in decreased survival in T cell-specific IL-4Rα mice. In conclusion this study demonstrates that absence of IL-4Rα on T cells promotes a predominant Th1/Th17 response, that results in delayed recruitment into the lung tissue, which ultimately proves detrimental for the host.
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    Restoration of cellular immunity in HIV-infected individuals on antiretroviral therapy
    (2017) Fatime Ramla, Tanko; Burgers, Wendy A; Riou, Catherine; Soares, Andreia
    During the course of HIV pathogenesis, the virus induces multiple defects in immune cells, altering their functional ability to efficiently control HIV itself and other infections. Whilst the widespread implementation of antiretroviral therapy (ART) has led to reduced morbidity and mortality in most HIV-infected individuals having access to treatment, we still do not know whether full restoration of immune function occurs. The aim of this study was to assess the extent to which ART restores both phenotypic and functional T and B cell immunity. HIV-infected women were studied before and 1 year after ART initiation. In Chapter 2, the effect of ART on T cell activation and differentiation profiles was evaluated in HIV-infected individuals (n=28; pre- and post-ART), and compared to HIVuninfected age- and sex-matched controls (n=23). In Chapter 3, the restoration of copathogen specific CD4+ T cells was determined by comparing their cytokine secretion ability and memory differentiation profiles in response to Mycobacterium tuberculosis and cytomegalovirus in HIV-infected (n=15; pre- and post-ART), compared to uninfected (n=9) individuals. Finally, Chapter 4 examined changes in B cell activation and memory profiles in HIV-infected persons (n=19; pre- and post- ART), and compared profiles to HIV-uninfected individuals (n=19). Multiparameter flow cytometry was performed to address the study objectives. T cell activation, as measured by CD38 and HLA-DR expression, was significantly reduced one year after ART for both CD4+ and CD8+ T cells, but normalisation to levels in HIV-uninfected individuals did not occur, despite suppression of viral load. In addition, skewed CD4+ and CD8+ T cell memory profiles were not completely restored. Furthermore, no change in the cytokine production capacity and memory profile of pathogen-specific CD4+ T cells was found before and after ART, but pathogen-specific CD4+ T cells exhibiting a late differentiated profile (CD27- CD45RO+) had a lower ability to replenish (p=0.02; r = -0.5) compared to cells with an early differentiated profile (CD27+CD45RO+; p=0.04; r = 0.45) prior to ART. Similar to T cells, activated B cells (CD40+CD86+) were only partially normalised post-ART, and remained significantly higher than B cells of HIV-uninfected individuals. The frequency of all B cell memory subsets were comparable between HIV-treated and uninfected individuals, with the exception of plasmablasts, whose frequency was still significantly higher than in HIV-uninfected subjects. In summary, these results demonstrate that HIV-infected women on suppressive ART show a substantial but only partial normalisation of T cell and B cell memory subsets, and lower levels of T cell and B cell activation. In addition, restoration of co-pathogen specific memory CD4+ T cells upon treatment was dependent on their memory profile before ART. Understanding the impact of HIV on T and B cell dysfunction and restoration upon ART may provide important insights into the mechanisms of HIV pathogenesis in the era of ART.
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    Studies on CD4+ T cell subsets in Mycobacterium tuberculosis immunity during HIV co-infection
    (2023) Nesamari, Rofhiwa; Burgers, Wendy; Riou, Catherine
    Tuberculosis (TB) is a global pandemic which resulted in 5.8 million disease cases and approximately 1.5 million deaths worldwide in 2020. TB disease is the leading cause of death worldwide from a single infectious agent, Mycobacterium tuberculosis (M.tb). It is estimated that a one fourth of the world's population is latently infected with TB and of these 5-10% will go on to develop active TB in their lifetime. Human immunodeficiency virus (HIV) infection is the greatest risk factor for developing active TB, with people living with HIV (PLWH) up to 19 times more likely to develop active TB compared to HIV uninfected individuals. Epidemiological studies show that there is increased risk of developing active TB (either reactivation of latent TB or new M.tb infection) throughout the course HIV infection, with the highest risk observed within the first year of infection, even when CD4+ T cell counts are still high. Previous studies have demonstrated that M.tb-specific CD4+ T cell responses are depleted early after HIV infection, within 3-12 months. This early defect in M.tb-specific CD4+ T cells may explain the elevated risk of TB even within the first year of HIV infection, prior to substantial immunosuppression. Antiretroviral therapy (ART) is an important strategy in preventing TB in PLWH. Earlier studies done in sub-Saharan Africa show that ART is associated with a 70-90% reduction in TB risk and a 52% decrease in TB related mortality. Several studies have also shown the importance of early ART initiation for HIV pathogenesis and transmission. However, despite ART intervention studies in sub-Saharan Africa, as well as Europe and North America, show that in these regions rates of TB remain high in PLWH. These high TB rates are most likely linked to the incomplete restoration of TB immune responses during ART. Overall, this thesis focused on characterising M.tb-specific CD4+ T cell responses during the course of HIV infection, from the acute phase of infection to post ART initiation. In Chapter 3, we aimed to confirm and extend previous findings that show there is a rapid depletion of M.tb-specific CD4+ T cells soon after HIV infection. We examined the kinetics of M.tb-specific CD4+ T cell responses over the course of HIV infection in two cohorts, a cross sectional cohort (n=58) and a longitudinal cohort (n=17). Consistent with previous findings, our results showed that there is a significant decrease in the frequency of M.tb responders 3 months after HIV infection. However, not all participants experienced M.tb-specific CD4+ T cell loss after HIV infection, with half the cohort losing 50% or more of their responses and the other half maintaining their responses. In the group that maintained their M.tb responses after HIV infection, the majority had very little fluctuations in their responses during the acute and chronic phase of infection. When comparing the clinical characteristics and the function and phenotype of M.tb-specific CD4+ T cells between individuals who maintained their M.tb-specific response and those who don't, no significant differences were found. In Chapter 4, to investigate the impact of early ART on M.tb immunity, we characterised M.tb-specific CD4+ T cell responses in individuals who initiated ART at an early stage of HIV infection (median 7.5 months post infection, n=16) in comparison to those who started ART during the chronic phase of HIV infection (median 66 months post infection, n=22). In this study, we examined multiple parameters between the two groups including their clinical characteristics, as well as the magnitude, function and phenotype of their M.tb-specific CD4+ T cells. We show that 2 years after ART initiation (irrespective of its timing) induced a significant immune reconstitution, marked by an increase in CD4 T cell count and restoration of the C4/CD8 ratio, but had no significant impact on the magnitude, function or phenotype of M.tb-specific CD4+ T cells. In Chapter 5, we sought to characterise M.tb-specific T cell responses in a small (n=13) cohort of participants who developed active TB during the CAPRISA 002 study. We performed a descriptive study examining the magnitude and phenotype of M.tbspecific T cells longitudinally over the course of TB treatment: before TB treatment/diagnosis, during treatment and after successful treatment. Despite the limited number of participants in this sub-study, we showed that after TB treatment there was a decreasing trend of activated M.tb-specific CD4+ T cells coincident with an increase in IFN-+ IL-2+ dual functional cells. Overall, our data confirms previous findings that there is an early depletion of M.tbspecific CD4+ T cells within a year HIV infection. However, in our study we found that not all HIV infected individuals lose their M.tb-specific responses. Instead, there was a variation in M.tb-specific responses after HIV infection, with some individuals maintaining their responses and others completely losing them. Results of our study also showed there were no major differences between participants who initiated ART early and those who initiated later during chronic HIV infection. To our knowledge this is the first study which looks at the impact of ART timing on M.tb-specific immunity in PLWH. The study therefore provides further insight, for future research, into whether early ART preserves TB immunity and therefore reduce the early, elevated risk of TB in HIV infected individuals.
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    A subset of circulating blood mycobacteria-specific CD4 T cells can predict the time to Mycobacterium tuberculosis sputum culture conversion
    (Public Library of Science, 2014) Riou, Catherine; Gray, Clive M; Lugongolo, Masixole; Gwala, Thabisile; Kiravu, Agano; Deniso, Pamela; Stewart-Isherwood, Lynsey; Omar, Shaheed Vally; Grobusch, Martin P; Coetzee, Gerrit
    We investigated 18 HIV-negative patients with MDR-TB for M. tuberculosis (Mtb)- and PPD-specific CD4 T cell responses and followed them over 6 months of drug therapy. Twelve of these patients were sputum culture (SC) positive and six patients were SC negative upon enrollment. Our aim was to identify a subset of mycobacteria-specific CD4 T cells that would predict time to culture conversion. The total frequency of mycobacteria-specific CD4 T cells at baseline could not distinguish patients showing positive or negative SC. However, a greater proportion of late-differentiated (LD) Mtb- and PPD-specific memory CD4 T cells was found in SC positive patients than in those who were SC negative (p = 0.004 and p = 0.0012, respectively). Similarly, a higher co-expression of HLA-DR + Ki67 + on Mtb- and PPD-specific CD4 T cells could also discriminate between sputum SC positive versus SC negative (p = 0.004 and p = 0.001, respectively). Receiver operating characteristic (ROC) analysis revealed that baseline levels of Ki67 + HLA-DR + Mtb- and PPD-specific CD4 T cells were predictive of the time to sputum culture conversion, with area-under-the-curve of 0.8 (p = 0.027). Upon treatment, there was a significant decline of these Ki67 + HLA-DR + T cell populations in the first 2 months, with a progressive increase in mycobacteria-specific polyfunctional IFNγ + IL2 + TNFα + CD4 T cells over 6 months. Thus, a subset of activated and proliferating mycobacterial-specific CD4 T cells (Ki67 + HLA-DR + ) may provide a valuable marker in peripheral blood that predicts time to sputum culture conversion in TB patients at the start of treatment.