Follicular helper T cells (Tfh) are believed to be major contributors to the viral reservoir that persists in HIV-infected individuals, even when on antiretroviral therapy. We will explore the novel hypothesis that cytokines IL-10 and TGF-b, which play important roles in lymph node biology and are upregulated post HIV infection, are critically important for the formation of such a reservoir and represent potential targets for intervention. Specifically, we hypothesize that IL-10/TGF-b promote the differentiation of HIV-susceptible Tfh cells, with reduced antiviral defenses, that favor integration of intact proviruses into open chromatin gene targets downstream of IL-10/TGF-b signaling pathways. We will examine multiple aspects of this model, including how IL-10 and TGF-b suppress the intrinsic antiviral machinery in Tfh cells and how chromatin accessibility in genes downstream of IL10/TGF-b signaling, promoted by STAT3/SMADs activation, allows infecting virions to integrate preferentially in these open loci. As a result of these mechanisms, infected Tfh cells provide a reservoir of actively transcribing intact proviruses, even under ART. In Aim 1, we will investigate these hypothesized roles of IL-10 and TGF-b in Tfh differentiation and HIV integration/transcription using biobanked samples from lymph nodes of HIV-infected individuals at different stages post HIV infection (i.e. Fiebig IV/V, untreated chronic infection, and ART-treated). In Aim 2, we will mechanistically validate our model, using tools such as CRISPR gene knockouts in ex vivo tonsil isolated CD4 T cells, to examine how the major pathways identified in Aim 1 contribute to the differentiation of Tfh cells, the suppression of anti-viral machinery, and the promotion of integration of intact proviruses into active transcription sites. Finally, in Aim 3, we will take advantage of lymph node specimens available from a cohort of rhesus macaques that previously controlled SIV replication post-ART interruption (VL<1000 cps/mL), subsequent to in vivo blockade of the IL-10 and PD-1 pathways. We will dissect mechanistically how this treatment led to SIV viral DNA decay in their LNs. This proposal has several innovative aspects. Additionally to the available biobanked human and macaque samples, we will use cutting-edge methods, including multiome, MIP-seq, multiplexed confocal imaging with vDNA/RNA scope, and spatial transcriptomics. The project builds on our expertise with HIV pathogenesis, Tfh biology, and the modulation of immune pathways through both in vitro models and in vivo interventions in rhesus macaques. Additionally, we have the support of strong Co-Investigators with expertise in virology (Vandekerckhove), tissue imaging (Petrovas), and data analysis, integration and interpretation (Kamaleswaran and Sekaly). With such multidisciplinary approaches, synergies across the Aims, and a highly collaborative group of established and early-stage investigators (proposing PI), we are confid...