PROJECT SUMMARY Gut dysfunction, dysbiosis, and residual inflammation are major factors contributing to the high prevalence of comorbidities such as metabolic, cardiovascular, kidney, and liver diseases in people living with HIV under antiretroviral therapy (ART). However, the immune mechanisms underlying the process of gut dysfunction and persistent inflammation in the setting of long-term viral suppression with ART remain poorly understood. We have recently reported in the nonhuman primate model of HIV with ART that following initial resolution of circulating leaky gut biomarkers during early ART, dysregulation of IL-17/IL-22 functions of intestinal Gamma delta (γδ) T cells are correlated with resurgence of intestinal epithelial barrier damage (IEBD) and systemic inflammation during long-term ART. Further, intestinal Vδ2T cell frequencies significantly correlated with the loss of specific gut microbial species during long-term ART. Based on this, we propose to test the hypothesis that specific dysregulation of the IL-17/IL-22 pathway in γδT cells, particularly the Vδ2 subset, contributes to breakdown of the gut epithelial barrier, resulting in microbial translocation (MT) and systemic inflammation during chronic treated HIV infection. Here, we propose to assess the role of Vδ2 γδT cells in the repair and maintenance of gut homeostasis through a direct in vivo intervention. We will evaluate the effect of in vivo Vδ2T-stimulation and expansion on IEBD, MT, and inflammation in long-term ART suppressed SIV-infection via treatment with the aminobisphosphonate drug, Zoledronate (ZOL) in combination with IL-2 and IL-15 cytokines. Second, we propose to test the hypothesis that modulation of gut microbiome during chronic SIV+ART with microbial supplementation combined with in vivo Vδ2T cell stimulation will have a synergistic effect on improving gut barrier functions. We will administer fecal microbial transplant (FMT) supplemented with anti-inflammatory bacterial species, with/without ZOL treatment to assess normalization of systemic inflammatory markers and gut immune functions in FMT-only vs. FMT with immunomodulation. Finally, we will determine the mechanisms by which γδT cells and microbiome modulate intestinal epithelial homeostasis during long-term treated SIV infection. We will longitudinally assess the transcriptional and functional signatures of intestinal IL-17/IL-22 producing cells and evaluate epithelial barrier functions in precisely timed gut biopsies and in vitro coculture assays to identify novel cellular/molecular mechanisms involved. By exploring the beneficial effects of combined immune/microbiome modulation on epithelial barrier-protective function and systemic inflammation, this study will have the critical impact of opening new avenues for the continued development of combinatorial approaches to target enteropathy and chronic inflammation in people living with HIV.