PROJECT SUMMARY Nearly 40 million people world-wide are infected with HIV, an infection for which there is no cure. Many strategies that aim to cure HIV focus on harnessing HIV-specific T cells to control the virus that rebounds after antiretroviral therapy (ART) is discontinued because of their ability to specifically recognize and kill HIV-infected cells. However, HIV-specific T cell therapies will not be effective if they cannot overcome the T cell exhaustion that occurs in the setting of chronic antigen stimulation. In mouse models of chronic infection and cancer, T cells enriched for a stem cell memory (TSCM) phenotype or stem-like transcriptional program robustly expand and differentiate into effector cells while resisting exhaustion. We have found that the expression of the stem/memory-promoting transcription factor, TCF-1, is associated with high in vitro proliferative capacity in HIV- specific CD8+ T cells in individuals who naturally control HIV infection and that TCF-1 overexpression leads to better HIV-specific T cell expansion after in vitro peptide stimulation. While T cells that are enriched for stemness programs proliferate and control cancer better in animal models, it is currently unknown whether stem-like T cells (versus, for example, effector-differentiated T cells) are optimal for controlling HIV after ART is stopped. The overarching goal of this proposal is to directly establish the role of T cell stemness in promoting the ability of HIV- specific T cells to expand a functional effector population to control HIV infection after discontinuation of ART. In Aim 1, we will use multi-modal high-dimensional single cell phenotypic and transcriptional profiling to establish whether the proportion of stem-like virus-specific CD8+ T cells on ART correlates with their in vivo expansion and subsequent control of HIV in humans or SIV in macaques after treatment interruption. In Aim 2, we will use envelope (Env)-targeting HIV-specific CAR-T cells with potent anti-HIV activity (duoCAR-T cells) as a model to ask how precisely tuning the level of stemness influences the ability of CAR-T cells to suppress HIV long-term in the HIV Participant-Derived Xenograft (PDX) mouse model. In Aim 3, we will leverage blood and tissue samples from a highly unique clinical trial of people with HIV on ART who receive an infusion of anti-HIV duoCAR- T cells and undergo an ATI. We hypothesize that CAR-T cells that are more stem-like prior to infusion (i.e., either the CAR-T cells in the total pre-infusion product or sub-populations that we identify pre-infusion and then track in vivo post-infusion using single cell paired α/β TCR sequences as “barcodes”) will expand a larger, less- exhausted effector population. These studies will test for the first time in the context of HIV infection the compelling hypothesis that T cells enriched for stemness have greater proliferative capacity and durability and are able to give rise to effector T cells that are better abl...