PROJECT SUMMARY CD8+ cytotoxic T-lymphocytes (CTLs) transiently control HIV in infected persons, but eventually fail due to viral mutation and other factors, resulting in persistent or chronic infection. However, reports of CTLs suppressing HIV infection in a few patients indicate that overcoming these barriers would allow for successful CTL-mediated control of HIV infection. Adoptive immunotherapy strategies aim to confer directed and enhanced CTL responses via supplementation of ex vivo-expanded autologous CD8+ T cells expressing a desired antigen-specific T cell receptor (TCR). However, these cells are mostly dysfunctional due to decreased proliferative response and limited CTL activity. Moreover, the requirement for a particular human-leukocyte antigen to properly present antigen to the T cell limits exogenous TCR-based approaches. Thus, such approaches have had minimal effects on reducing HIV-viral load. Chimeric antigen receptors (CARs) are artificially engineered receptors that confer a desired specificity onto immune effector T cells. In recent years, CAR immunotherapies have been extensively promoted in anti-cancer clinical trials. Of these, adoptively transferred autologous T cells modified with anti-CD19 CAR showed a dramatic impact on B cell lymphomas in acute and chronic lymphocytic leukemia patients. We hypothesize that CAR immunotherapy against HIV-infected cells can eradicate persistently infected cells and HIV latent reservoirs in patients following reactivation. Regarding an anti-HIV CAR, CD4? CAR consisting of the extracellular domain of the human CD4 molecule linked to the CD3?-chain has been well-studied in vitro and in human clinical trials. This CAR has been shown to mediate highly potent anti-HIV activity in vitro, but had limited effects due to poor survival and functionality of the transduced cells. We considered potential reasons for this failure due to 1) susceptibility of CD4+ T cells and CD8+ T cells expressing CD4? CAR to HIV infection, 2) lack of costimulatory signaling domains required for proper effector and memory response, and 3) massive ex vivo expansion of T cells prior to gene modification leading to cellular aging, resulting in poor effector and memory function. Our overall goal is to confer long-term, enhanced HIV-specific effector and memory responses via transplantation of anti-HIV CAR-engineered T cells. We will 1) develop a new class of anti-HIV CAR using HIV- targeting broadly neutralizing antibodies, 2) protect CAR-transduced T cells from HIV-mediated cytotoxicity by co- transduction with anti-HIV genes, 3) introduce costimulatory-signaling domains into anti-HIV CAR to ensure superior effector and memory responses, and 4) use newly identified memory T cells retaining stem cell properties called ?stem cell memory T (TSCM) cells? as a carrier vehicle for anti-HIV CAR. TSCM cells self-renew in vitro as well as in vivo and differentiate into effector T cells. Thus, TSCM cells engineered to express HIV-specific CAR provide an inexhaustible source of HIV-specific immune cells. These studies will be further modeled in vivo using the humanized bone marrow, liver and thymus (BLT) mouse system.