THE ROLE OF CD4+ T MEMORY SUBSETS IN HIV INFECTION: IMMUNE RESPONSE IN PEOPLE WITH HIV AND LATENCY ESTABLISHMENT

T lymphocytes comprise diverse subsets which differ in phenotype, function and maturation level. Among them, T memory stem cells (TSCM) represent the more developmentally immature subpopulation of the memory pool, endowed with longevity, self-renewing capacity and multipotency, other than recall capabilities. These features make them pivotal for maintaining durable immune memory and controlling persistent infections. In the context of vaccination, the induction of vaccine-specific TSCM is a key determinant of long-term protection. In the context of human immunodeficiency virus (HIV), CD4⁺ TSCM, serve as target of infection as well as reservoir, sustaining long-lived, self-renewing pools of latently infected cells despite antiretroviral therapy (ART). Thus, TSCM are central to both protective immunity and the persistence of HIV reservoir. In people with HIV (PWHIV), vaccine-induced immune responses are generally attenuated as a consequence of residual inflammation and immune dysfunction that might persist under ART treatment. HIV infection profoundly disrupts the homeostasis of T cell subsets, leading to a marked depletion of developmentally immature T lymphocytes. However, the effects of this chronic immune dysregulation on TSCM remain poorly defined, thus elucidating the functional characteristics of TSCM in PWHIV is of particular importance. By taking advantage of the longitudinal sampling during the COVID-19 vaccination campaign, we examined the humoral and cellular immune response of PWHIV, with a focus on the T memory phenotype and vaccine-induced TSCM. To this purpose, we enrolled 22 PWHIV and a control group of 14 healthy donors (HDs) who received mRNA-1273 vaccine. PWHIV were stratified according to CD4+ T cell count and CD4/CD8 ratio, two markers of immune reconstitution after ART initiation. The humoral and cellular immune responses to the vaccine were studied using pseudoviruses-based neutralization assay, Activation-Induced Markers (AIM) assay and intracellular Cytokine Staining (ICS). We observed a decrease in SARS-CoV-2 Spike-reactive CD4+ TSCM in PWHIV with an incomplete immune reconstitution (defined as CD4+ T count below 500 cells/µL and a CD4/CD8 ratio below 0.4). This functional impairment was accompanied by a stronger commitment to exhaustion. Indeed, we measured a higher expression of immune checkpoint molecules by CD4+ TSCM and T central memory (TCM) and we measured a higher frequency of T progenitors committed to exhaustion (defined as TSCM and TCM co-expressing PD-1 and TIGIT) in PWHIV with incomplete immune reconstitution. In these participants, TCF-1, a master regulator of T cell stemness, was less expressed across all T memory subsets. It has been recently shown that GSK3β inhibition could arrest CD8+ T cell differentiation, trigger the formation of stem cell-like memory T cells, and a more functional state of CD8+ T cells. Therefore, we analyzed the effects of GSK3β inhibition on the homeostasis and function of TSCM from the participants of our cohort. GSK3β inhibition restored TCF-1 expression in CD4+ TSCM from PWHIV which led to a partial rescue of their functionality. To investigate the mechanisms determining CD4⁺ T memory cells differentiation and HIV latency establishment, we analyzed the transcriptome landscape of primary human CD4⁺ T cells exposed to γc-cytokines or T cell receptor (TCR) agonists prior to infection with a dual-reporter HIV (HIV-pMorpheus) that allows detection of both productively and latently infected cells. The distribution of HIV latent and productive infection across the CD4+ T memory subsets and their propensity to latency was examined through flow cytometry. TCR stimulation drove proliferation, metabolic activation, and apoptosis, leading to expansion of differentiated subsets and a bias toward productive infection. Conversely, γc-cytokines preserved subset homeostasis and viability while favoring latent infection in the less differentiated memory populations. In conclusion, our findings reveal impaired vaccine-induced CD4+ TSCM responses and enrichment of exhausted T progenitor populations in PWHIV with incomplete immune reconstitution, highlighting the need for tailored vaccination strategies in this vulnerable population. Furthermore, modulation of the GSK3β–TCF-1 axis emerges as a promising avenue to restore T cell stemness and enhance TSCM functionality, although context-specific effects must be carefully considered. Finally, our mechanistic dissection of T cell stimulation pathways highlights the influence of subsets differentiation on the outcome of HIV infection, with less differentiated memory cells exhibiting greater propensity to latency. Collectively, these studies provide novel insights into the interplay between HIV persistence, TSCM biology, and immune modulation strategies.