Therapeutic strategies that enhance anti-viral immunity and reduce the viral reservoir are critical to achieving durable remission of HIV. The co-inhibitory receptor programmed death-1 (PD-1) regulates CD8+ T cell dysfunction during chronic HIV and SIV infections 1-4. In addition, PD-1+ CD4+ T cells constitute a significant fraction of the HIV/SIV viral reservoir5-7. We previously demonstrated that in vivo blockade of PD-1 during chronic SIV infection improves the function of anti-viral CD8+ T cells and B cells 4,8. However, the immunological effects of PD-1 blockade during anti-retroviral therapy (ART) and the potential to destabilize the persistent HIV/SIV reservoir have not been studied. Here, we show that administration of anti-PD-1 antibody (PD-1 Ab) 10 days prior to initiation of ART rapidly enhanced anti-viral CD8+ T cell function and diminished interferon stimulated genes (ISGs) that resulted in markedly improved viral suppression in plasma and better Th17 cell reconstitution in the rectal mucosa following ART initiation. In addition, PD-1 blockade during suppressive ART resulted in transient increases in plasma viremia, induction of gene signatures associated with effector CD8+ T cell function and ISGs, and lower levels of cell associated replication-competent virus suggesting destabilization of the viral reservoir. Following ART interruption, PD-1 Ab treated animals showed markedly higher expansion of proliferating CXCR5+ Perforin+ Granzyme B+ effector CD8+ T cells and lower regulatory T cells that resulted in delayed viral rebound and better control of viremia. PD-1 blockade administered only during suppressive ART however was only partially effective. Our results indicate an optimal regimen of PD-1 blockade administered in combination with ART, that augments anti-viral CD8+ T cell function and reduces the viral reservoir leading to improved control of viral rebound after ART interruption. These results have important implications for developing novel therapeutic interventions to achieve durable remission of HIV. Overall design: To determine the effects of PD-1 blockade in combination with ART and its potential to enhance anti-viral immunity and reduce the viral reservoirs, we performed PD-1 blockade in two phases: I) prior to initiation of ART and II) during suppressive ART. In phase I of the study, we administered 5 doses of PD-1 Ab (3 mg/kg/dose) intravenously on days 0, 3, 7, 10, and 14 to 10 SIVmac251-infected rhesus macaques (RMs) between 24-30 weeks after infection (Fig. 1A). As a control, we treated 10 SIVmac251-infected RMs with saline. We initiated ART in all animals at day 10 after the initiation of PD-1 blockade. To determine the effects of PD-1 blockade on a global scale, we performed RNASeq to compare gene signatures in the blood of 10 PD-1 Ab treated and 5 saline treated animals at day 0 and day 10 of PD-1 blockade. To determine the additive biological effects of PD-1 blockade on anti-viral immunity and the persistent viral reservoirs when administered during suppressive ART (phase II), the 10 RMs given PD-1 Ab during phase I were again treated with PD-1 Ab (double treated) at 26-30 weeks following ART initiation. Three monthly infusions of PD-1 Ab were administered at 10mg/kg/dose (Fig. 3A). To test the influence of PD-1 blockade administered only during suppressive ART, we split the 10 RMs from the saline group into two groups and gave either PD-1 Ab (single treated group; n=5) or saline (saline control group; n=5). RNASeq was only performed on the double treated and single treated groups, not the saline control group. We again looked to compare gene signatures in the blood of PD-1 Ab treated RMs from day 0 of first PD-1 Ab infusion under ART and 7 days after.