Antiretroviral therapy (ART) sufficiently suppresses HIV replication to reduce plasma viral load (pVL) below the limit of detection, but immune activation (IA) is not normalized and the elevated levels of IA markers- IL-6, TNF, TGFß, C reactive protein (CRP), and D-Dimer remain elevated are associated with increased risk for cardiovascular disease, endothelial disfunction and clotting abnormalities, pulmonary hypertension, interstitial nephritis, development of non-AIDS associated malignancies, and CNS abnormalities. Multiple mechanisms have been proposed to explain the persistence of IA under ART including microbial translocation and herpes virus infections (e.g., HSV, CMV, EBV). However, here we propose that HIV itself is a major cause of persistent IA because of ongoing virus production in lymphoid tissues (LT) while on ART. In this revised proposal, we have one specific aim that tests two hypotheses: 1) Sustained IA during ART is driven by production of virions and/or expression of viral antigens in reactivated latently infected cells with or without persistent low-level virus replication; and 2) that persistent low-level virus replication during ART is the result of intracellular concentrations (IC) of antiretroviral (ARV) drugs in LT that do not completely inhibit replication and virus production. For our first hypothesis we will seek direct evidence of correlations between persistent IA and virus production/antigen expression in LT by identification, at the single cell level, of: 1) virus-producing CD4 T cells lacking markers of activation and proliferation that we have previously shown can sustain low levels of virus production; 2) T follicular helper cells (Tfh) in B cell follicles that have recently been shown to be an independent reservoir for viral persistence; and 3) reactivated latently infected T cells producing virus or p24. We propose investigations of these drivers of IA by our validated and highly sensitive in situ hybridization (ISH) methods to detect, phenotype and locate virus (v) RNA+ and virus-producing cells in LT; by a validated highly sensitive high throughput “envelope detection by induced transcription-based sequencing” (EDITS) assay; and a broadly neutralizing antibody (bNab) method to enrich for HIV-envelope (ENV)+ cells. For the hypothesized correlations of the virus drivers with IA, we will measure IA with standard flow-based antibody assays and with single cell transcriptomic analysis of LT mononuclear cells to identify unique gene signatures associated with IA. Our second hypothesis will be tested by quantification of ARV-intracellular concentrations (ICs) in LT and determine the relationships among ARV-ICs and the frequency of detecting the three putative virus drivers of IA. Establishing that HIV itself is a cause of IA would point future developments to fully suppress virus production in the LT reservoir with benefits both in reducing IA and associated pathologies and as an essential component of HIV Cure Str...