ABSTRACT/SUMMARY 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 NK cell- mediated control of viral replication in the BCF and T-cell zone of secondary lymphoid tissues (SLT). In SIVagm-infected AGMs, we 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 showed high cytolytic activity against target cells presenting SIV-Env peptides in the context of MHC-E. In contrast, the formation of NKTD cells was blocked in pathogenic SIVmac infection in rhesus macaques (RMs), and not improved with ART, in favor of a subset (NKG2ahiCD16+) with pro- inflammatory function, such as production of IFN-γ. Remarkably, we demonstrated in SIVmac-infected, ART- treated RMs that AGM-like profiles of adaptive NKTD cells are rescued in blood via treatment with interleukin-21 (IL-21) as a monotherapy following ART initiation. In IL-21-treated RMs, the frequency and responsiveness of blood 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 and of replication-competent virus in SLT. Yet, we were not able to determine if IL-21 induces NKTD cells in tissues and we showed that, after analytical ART treatment interruption (ATI) in absence of IL-21 administration, the NKTD cells rapidly vanished, and virus then concomitantly rebounded in all RMs. The main objectives of this proposal are (i) to define at an unprecedented level the function and distribution of NK cells in tissues from SIV-infected, ART-treated RMs after IL-21 administration; an analysis which in parallel will be extended to other immune cells that either produce (e.g. TFH) or are sensitive to (e.g. antigen-presenting cells and CD8+ T cells) IL-21 signaling (Aim 1); (ii) to evaluate if the levels of NKTD cells that are induced by early IL-21 administration can be durably prolonged after ATI in the presence of IL-21, bNAbs, or both (Aim 1 and 2); and (iii) to test if IL-21-induced NKTD cells will synergize with a cocktail of SIV-Env targeted broadly neutralizing antibodies (bNAbs) to induce ADCC-mediated clearance of infected cells and to durably control viral rebound after ATI (Aim 2). Based on a large set of preliminary data, we hypothesize that the proposed approach will allow differentiation into NKTD cells with IL-21 followed by improved bNAb targeting and ADCC-mediated killing of those infected cells that support viral rebound following ART cessation. Using a pre-clinical model reproducing the comple...