Throughout history, human populations have continuously been challenged by new and emerging infectious diseases. For the past 26 years, sub-Saharan Africa and other countries around the world have been dealing with a pandemic caused by a relatively new pathogen called the human immunodeficiency virus (HIV). Although antiretroviral (ARV) therapies effectively reduce morbidity and mortality rates, the long term use in those who have access to treatment inevitably leads to drug-related toxicity and resistance. Even with a strong commitment from governments to expand and finance prevention and treatment programs, transmission rates continue to outpace the benefits of these efforts. Therefore to effectively eradicate the disease, research is focusing on the design of protective and therapeutic vaccines. The first major step in designing these alternative therapies is to define correlates of immune protection. / The research presented in this thesis focuses on characterizing the quantitative and qualitative features of T cell immune responses in individuals who spontaneously control viral replication and exhibit a benign course of disease while remaining off ARV therapy. A comprehensive analysis of HIV-specific IFN-gamma secreting immune responses revealed that neither the breadth nor the magnitude of responses directed against the entire HIV proteome accurately predicts the viral load or rate of CD4 decline. Subsequent analyses showed that the preferential targeting of Gag was associated with reduced rates of CD4 decline and was later confirmed in a cohort of individuals in primary infection whereby the relative breadth and magnitude of Gag p24 was inversely correlated with viral load set point. / The maintenance of polyfunctional immune responses in HIV-infected subjects with a benign course of disease prompted us to develop a method that could comprehensively assess the breadth, magnitude and specificity of three functionally distinct subsets of HIV-specific lymphocytes (single IFN-gamma, single IL-2 and dual IFN-gamma/IL-2 secretors). Survey of immune responses in chronically infected individuals revealed that only the breadth and magnitude of dual IFN-gamma/IL-2 secreting lymphocytes correlated with reduced viral loads and increased CD4 counts suggesting that secretion of IFN-gamma alone was a poor correlate of protection. We also showed that the contribution of polyfunctional lymphocytes to the total response was greater for epitopes restricted by major histocompatibility complex (MHC) class I alleles associated with slow disease progression compared to those restricted by alleles associated with rapid or neutral rates of HIV disease progression. / Taken together, this work supports the view that immune monitoring of infected and vaccinated individuals should include methodologies capable of detecting both IFN-gamma and IL-2 secretion from responding T lymphocytes. The studies presented here have furthered our understanding of what constitutes protection from disease progression emphasizing that both specificity and polyfunctionality are features of effective control of viral replication.

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O citomegalovírus (CMV) é uma séria ameaça aos receptores de transplante de medula óssea. A reativação está associada com uma imunidade mediada por células TCD8+ defeituosa. Nosso objetivo foi correlacionar as diferentes subpopulações de células TCD8+ com a reconstituição imunológica dos pacientes, especificamente a imunidade anti-CMV, analisando as subpopulações de células T infundidas nas diferentes modalidades de transplante de medula óssea. Receptores de transplante alogênico de células tronco mobilizadas para o sangue periférico (n=16) ou coletadas diretamente da medula óssea (n=28) e receptores de transplante autólogo de células tronco mobilizadas para o sangue periférico (n=22) foram avaliados. Verificamos que as transferências de células mobilizadas para o sangue periférico dos doadores, tanto nos transplantes alogênicos como autólogos, são proporcionalmente enriquecidas por subpopulações de células memória efetora e efetora, comparadas às transferências de células procedentes diretamente da medula óssea. Este enriquecimento por subpopulações de células TCD8+ mais diferenciadas foi também correlacionado com maior número de células contendo altos níveis de granzima B, considerado um marcador para linfócitos citotóxicos, sendo também encontrado em maior número nas transferências de células do sangue periférico. Entretanto, no pós-transplante, observou-se que somente os receptores de transplante autólogo de células tronco mobilizadas para o sangue periférico, e não os das outras modalidades de transplante, exibiam números elevados de células T CD8+ de memória-efetora e efetora. Ao mesmo tempo, estes receptores apresentaram menos freqüentemente episódios de reativação pelo CMV, e mais freqüentemente produziram IFN-gama em resposta ao CMV. Portanto, a transferência de células do sangue periférico, desde que em ambiente autólogo, está associada não só com a transferência de células TCD8+ com um fenótipo mais maduro, mas também com uma persistência mais prolongada das mesmas, podendo proporcionar uma resposta imunológica antiviral mais rápida e eficiente, como esperado para as células de memória versus naïve. / Cytomegalovirus (CMV) is a serious threat to the recipients of bone marrow transplantation. Reactivation is associated with defective CD8+ T cell-mediated immunity. We aimed to correlate the different subsets of CD8+ T cells with the patients\' immune reconstitution, specifically anti CMV immunity, by analyzing the CD8+ T cell subsets infused in the different types of bone marrow transplantation. Recipients of allogeneic transplant of peripheral blood stem cells (n=16) or bone marrow (n=28) and recipients of autologous transplant of peripheral blood stem cells (n=22) were evaluated. We show that infusions of stem cells derived from donor\'s peripheral blood, either allogeneic or autologous, are proportionally enriched for the memory-effector and effector phenotypes, compared to the infusions of stem cells of bone marrow origin. This increased number of more differentiated subsets of CD8+ T cells was also correlated with an increased number of cells containing high levels of granzyme B, which is another reliable marker of cytotoxic lymphocyte, and which was also more evident in autologous recipients. However, post-transplant, we observed that only the recipients of autologous peripheral blood cells, and not the recipients of the other transplant modalities, exhibited very high numbers of memory-effector and effector TCD8+ cells. At the same time, they less frequently presented CMV reactivation, and more frequently produced IFN-gama in response to CMV antigens. Thus, transfer of stem cells from peripheral blood, provided in an autologous setting, is associated with transfer and prolonged survival of CD8+ T cells with a more mature phenotype, which may provide a more rapid and efficient anti-viral immune response, as expected for memory versus naïve cells.

O citomegalovírus (CMV) é uma séria ameaça aos receptores de transplante de medula óssea. A reativação está associada com uma imunidade mediada por células TCD8+ defeituosa. Nosso objetivo foi correlacionar as diferentes subpopulações de células TCD8+ com a reconstituição imunológica dos pacientes, especificamente a imunidade anti-CMV, analisando as subpopulações de células T infundidas nas diferentes modalidades de transplante de medula óssea. Receptores de transplante alogênico de células tronco mobilizadas para o sangue periférico (n=16) ou coletadas diretamente da medula óssea (n=28) e receptores de transplante autólogo de células tronco mobilizadas para o sangue periférico (n=22) foram avaliados. Verificamos que as transferências de células mobilizadas para o sangue periférico dos doadores, tanto nos transplantes alogênicos como autólogos, são proporcionalmente enriquecidas por subpopulações de células memória efetora e efetora, comparadas às transferências de células procedentes diretamente da medula óssea. Este enriquecimento por subpopulações de células TCD8+ mais diferenciadas foi também correlacionado com maior número de células contendo altos níveis de granzima B, considerado um marcador para linfócitos citotóxicos, sendo também encontrado em maior número nas transferências de células do sangue periférico. Entretanto, no pós-transplante, observou-se que somente os receptores de transplante autólogo de células tronco mobilizadas para o sangue periférico, e não os das outras modalidades de transplante, exibiam números elevados de células T CD8+ de memória-efetora e efetora. Ao mesmo tempo, estes receptores apresentaram menos freqüentemente episódios de reativação pelo CMV, e mais freqüentemente produziram IFN-gama em resposta ao CMV. Portanto, a transferência de células do sangue periférico, desde que em ambiente autólogo, está associada não só com a transferência de células TCD8+ com um fenótipo mais maduro, mas também com uma persistência mais prolongada das mesmas, podendo proporcionar uma resposta imunológica antiviral mais rápida e eficiente, como esperado para as células de memória versus naïve. / Cytomegalovirus (CMV) is a serious threat to the recipients of bone marrow transplantation. Reactivation is associated with defective CD8+ T cell-mediated immunity. We aimed to correlate the different subsets of CD8+ T cells with the patients\' immune reconstitution, specifically anti CMV immunity, by analyzing the CD8+ T cell subsets infused in the different types of bone marrow transplantation. Recipients of allogeneic transplant of peripheral blood stem cells (n=16) or bone marrow (n=28) and recipients of autologous transplant of peripheral blood stem cells (n=22) were evaluated. We show that infusions of stem cells derived from donor\'s peripheral blood, either allogeneic or autologous, are proportionally enriched for the memory-effector and effector phenotypes, compared to the infusions of stem cells of bone marrow origin. This increased number of more differentiated subsets of CD8+ T cells was also correlated with an increased number of cells containing high levels of granzyme B, which is another reliable marker of cytotoxic lymphocyte, and which was also more evident in autologous recipients. However, post-transplant, we observed that only the recipients of autologous peripheral blood cells, and not the recipients of the other transplant modalities, exhibited very high numbers of memory-effector and effector TCD8+ cells. At the same time, they less frequently presented CMV reactivation, and more frequently produced IFN-gama in response to CMV antigens. Thus, transfer of stem cells from peripheral blood, provided in an autologous setting, is associated with transfer and prolonged survival of CD8+ T cells with a more mature phenotype, which may provide a more rapid and efficient anti-viral immune response, as expected for memory versus naïve cells.

We have previously shown that chronic Hepatitis C virus (HCV) infection can induce DNA damage and immune dysfunctions with excessive oxidative stress in T cells. Furthermore, evidence suggests that HCV contributes to increased susceptibility to metabolic disorders. However, the underlying mechanisms by which HCV infection impairs cellular metabolism in CD4 T cells remain unclear. In this study, we evaluated mitochondrial mass and intracellular and mitochondrial reactive oxygen species (ROS) production by flow cytometry, mitochondrial DNA (mtDNA) content by real-time qPCR, cellular respiration by seahorse analyzer, and dysregulated mitochondrial-localized proteins by Liquid Chromatography-Mass Spectrometry (LC-MS) in CD4 T cells from chronic HCV-infected individuals and health subjects. Mitochondrial mass was decreased while intracellular and mitochondrial ROS were increased, expressions of master mitochondrial regulators peroxisome proliferator-activated receptor 1 alpha (PGC-1α) and mitochondrial transcription factor A (mtTFA) were down-regulated, and oxidative stress was increased while mitochondrial DNA copy numbers were reduced. Importantly, CRISPR/Cas9-mediated knockdown of mtTFA impaired cellular respiration and reduced mtDNA copy number. Furthermore, proteins responsible for mediating oxidative stress, apoptosis, and mtDNA maintenance were significantly altered in HCV-CD4 T cells. These results indicate that mitochondrial functions are compromised in HCV-CD4 T cells, likely the deregulation of several mitochondrial regulatory proteins.