PROJECT SUMMARY Studies in animal models have suggested 98% of the HIV reservoir to reside within the gut, yet most studies of reservoir dynamics derive from peripheral blood. Unique challenges likely exist for purging the gut reservoir as an HIV cure strategy. One of these is the high concentrations of the cytokine TGF in this tissue, as TGF can have the combined undesired effects of reinforcing latency and dampening effector CD8+ T cells that could otherwise recognize and eliminate reactivated cells. A second challenge for the design of a universal HIV cure is latency-promoting effects of estrogen, which may necessitate different approaches for HIV cure in infected men vs. women. In this study, we will recruit, among virally-suppressed people living with HIV (PLWH), both pre- and post-menopausal women (who harbor different endogenous estrogen levels) and clinically-matched men, for donation of gut biopsy and paired blood specimens. In Aim 1, we will characterize at the single-cell level the transcriptomes, surface proteomes, and clonal expansion histories of the gut reservoir cells, to better understand the mechanisms allowing for their persistence and to try to identify novel biomarkers of these cells. In Aim 2, we will test ex vivo combinations of next-generation latency-reversal agents (LRAs) - ones that have documented in vivo latency reversal activity and are promising agents to achieve HIV cure through "shock and kill" - and use CyTOF and bioinformatics approaches to determine the phenotypes of gut reservoir cells that do vs. do not reactivate in response to these treatments. These studies will not only reveal how effective the various LRA combinations are against the gut reservoir, but also reveal features of gut reservoir cells resistant to reactivation by these treatments which can be targeted using adjunctive approaches. In Aim 3, we will develop and test "kill" approaches designed to eliminate reservoir cells in the TGF -rich environment of the gut, by 1) blocking TGF signaling during recognition of infected cells by HIV-specific CD8+ T cells, and 2) by transforming TGF signal from an immunosuppressive to an activating one, via engineering HIV-specific CART cells co-expressing a TGF -41BB chimera. In addition to establishing a better understanding of the gut reservoir and its maintenance (including through extensive comparison with its blood counterpart) in both men and women living with HIV, this study will test multiple novel aspects of "shock and kill" designed to eliminate the most abundant HIV reservoir in PLWH.