Abstract Virus-specific CD8 T cells exert potent antiviral activity against HIV-1 and SIV. Despite abundant CD8 T cell responses, these are unable to fully suppress virus replication. This is likely due to the majority of viral replication occurring in CD4+ T cells concentrated in specific areas in the body, such as B-cell follicles in secondary lymphoid tissues and in intestinal tissues. Viral reservoir studies indicate that the majority of SIV and HIV replication may occur in the intestinal mucosa: After ART interruption, the location, abundance, and phenotype of recrudescing virally infected cells are not well understood. However, emerging data suggest that both CD4 T cells, as well as myeloid cells, in lymph node (LN) and the intestine are important contributors. Thus, there is a need to develop methods to reduce viral load in mucosal tissues, as well as in LN, and to more fully understand the source of viral reservoirs in non-lymphoid tissues. CAR T cells can be potent mediators of immune control and these are being explored as an HIV therapy. We have developed and begun testing an HIV/SIV targeting CAR. In this study, we have developed and propose to test autologous T cells engineered to express the HIV-targeting CAR and CCR9, a molecule that will target cells to the intestinal mucosa. We will test our hypothesis that targeting HIV-specific T cells to the intestinal mucosa, as well as B cell follicles, will lead to durable remission of HIV. We propose two aims to test the hypothesis. Aim 1: to determine the in vivo localization, persistence and antiviral efficacy and impact on intestinal mucosa, lymphoid tissue, and other tissue viral reservoirs in SIV infected ART suppressed rhesus macaques infused with autologous CAR/CCR9 T cells. Aim 2: to determine whether combination therapy using both intestine- and lymphoid follicle-homing CAR T cells is superior in promoting sustained remission of SIV after antiretroviral drug treatment interruption. If successful, this work may lead to an immunotherapy that leads to long-term suppression of HIV.