PROJECT SUMMARY/ABSTRACT Lymph nodes and the gastrointestinal tract are major tissue viral reservoirs in people living with HIV and treated with antiretroviral treatment (ART). A distinguishing feature of SIV infection in African green monkeys (AGMs), a natural host species for SIV, is the absence of viral dissemination in the lymphoid B-cell follicle (BCF) and the presence of strong NKcell-mediated control of viral replication in the T-cell zone and BCF of secondary lymphoid tissues (SLT). In SIVagm-infected AGMs, we recently identified the expansion of NK cells with a terminally-differentiated phenotype expressing (i) high levels of CD16, an activating Fc receptor that mediates antibody-dependent cellular cytotoxicity (ADCC), and (ii) low levels of NKG2a, an inhibitory receptor, that modulates NK cell education via interactions with MHC-E. This terminally-differentiated NK cell (NKTD) subset (NKG2alowCD16+) exhibited adaptive-like properties and, due at least in part to reduced NKG2a expression, showed high cytolytic activity against target cells presenting SIV-Env peptides in the context of MHC-E. The formation of adaptive NKTD cells was blocked in SIVmac-infected rhesus macaques (RMs) in favor of a subset (NKG2ahiCD16+) with pro-inflammatory function, such as production of IFN, a cytokine also known for its capacity to increase MHC-E expression on target cells. Remarkably, in a separate cohort of SIVmac-infected, ART-treated RMs, we demonstrated that AGM-like profiles of adaptive NKTD cells are rescued via treatment with interleukin-21 (IL-21). In IL-21 treated RMs, the frequency and responsiveness of the adaptive NKTD cells to target cells presenting SIV peptides in the context of MHC-E strongly correlated with a reduction of SIV DNA in the gut, lower levels of replication competent virus in SLT, and a delay in viral rebound following ART interruption. In Aim 1 of this proposal we will define (I) the mechanisms driving the formation and activity of adaptive NKTD cells, such as cytokine environment and lymphoid inflammation; (II) their tissue distribution; and (III) ADCC capacity. In addition to NK cells, the impact of IL-21 will be investigated in T-cells and other immune cells that can respond to IL-21. In Aim 2, we will assess whether and to what extent the combination of IL-21-induced NKTD cells and a cocktail of anti-SIV broadly neutralizing antibodies (bNAbs) facilitates, through ADCC-mediated clearance of infected cells, a reduction of viral burden and control of viral rebound following ART interruption. Specifically, in SIVmac-infected, ART-treated RMs, the “differentiate, target, and kill” approach will allow differentiation of the NKTD cells with IL-21 followed by targeting of cells harboring reactivated virus with bNAbs. Using a model reproducing the complex virus-host interactions occurring in people living with HIV, we expect that the rescue of NK cell terminal differentiation will promote robust ADCC activity, which will synerg...