INTEGRATED ANALYSIS OF PHENOTYPIC AND METABOLIC REMODELING IN HIV ANTIGEN-SPECIFIC T AND B CELLS

Human Immunodeficiency Virus (HIV) infection remains a major global health challenge due to persistent viral reservoirs, chronic immune activation, and progressive immune dysregulation that endures despite suppressive antiretroviral therapy (ART). While traditional research has primarily focused on numerical CD4+ T-cell depletion, emerging evidence highlights the pivotal role of cellular immunometabolism in driving T- and B-cell differentiation, effector functions, and immune exhaustion. This thesis aimed to investigate the phenotypic, functional, and metabolic remodeling of HIV-specific T and B lymphocytes across different infection stages in people living with HIV (PLWH) to define metabolic signatures associated with antiviral efficacy versus dysfunction. Peripheral blood mononuclear cells (PBMCs) were isolated from cohorts of PLWH on ART and stimulated in vitro with HIV-derived peptide pools (Gag, Pol, and Env). High-dimensional single-cell metabolic regulome profiling (scMEP) via mass cytometry (CyTOF) was integrated with unsupervised clustering (FlowSOM/UMAP) to map metabolic regulomes, while intracellular cytokine staining (ICS) assessed functional responses (via IL-2, IL-17, IFN-γ, TNF, CD107a, and Granzyme B production). The overall frequencies of antigen-specific (Ag+) T cells and total B cells were comparable across PLWH cohorts, indicating that disease duration does not alter response magnitude. However, scMEP revealed profound immunometabolic heterogeneity. Within Ag+ CD4+ T cells, distinct clusters emerged, including hyper-metabolic central memory FoxP3+ regulatory T cells (Tregs) relying on glycolysis, the TCA cycle, and fatty acid oxidation (FAO), contrasting with metabolically quiescent effector memory Tregs. Among Ag+ CD8+ T cells, subsets spanned from highly active glycolytic/proliferative effector memory cells to quiescent naive and TEMRA populations. Notably, double-negative (DN) T cells contained a highly activated, hyper-metabolic TEMRA-like subset. In the B-cell compartment, HIV-envelope-specific (gp140+) B cells displayed marked metabolic activation consistent with sustained antigen exposure. The overall B-cell landscape was heavily dominated by glycolytic atypical B cells (CD21-CD20-CD27-CD38-) and hyper-metabolic DN2/age-associated B cells (ABCs) engaged in oxidative, glycolytic, and mTOR/HIF-1α signaling. Functional evaluation via ICS showed similar polyfunctional profiles across cohorts, though individuals with longer disease duration exhibited selective reductions in Env- and Pol-stimulated cytokine production (such as IL-2 and IL-17). Overall, this study demonstrates that persistent viral antigen exposure and chronic inflammation drive distinct metabolic rewiring in HIV-specific T and B cell subsets. Unraveling these immunometabolic alterations provides critical insights into the mechanisms of immune exhaustion and points toward novel metabolic targets for restoring immune function and controlling viral persistence.