ABSTRACT Even with successful viral control, HIV-infected individuals exhibit co-morbidities associated with older age, including osteoporosis, stroke, dementia, and cancer. In aviremic HIV+ individuals and the general geriatric population, age-associated diseases and mortality correlate with plasma markers of inflammation and intestinal permeability. The gut is a major reservoir of latently HIV-infected cells, and HIV enteropathy, defined as pathologic processes in the small intestine and colon, is a hallmark of HIV infection. Our preliminary data implicate gamma delta T cells as an inflammatory driver in ART-suppressed HIV infection and with normal aging. gamma delta T cells are a non-conventional T cell lineage that comprise ≤10% of circulating T cells yet are found in considerably higher proportions in the epithelium of the intestine. Also, there is evidence that this unique T cell population regulates intestinal barrier function during normal conditions, and that gamma delta T pro-inflammatory activity causes damage at epithelial sites and to epithelial barriers. Therefore, we hypothesize that with virally suppressed HIV infection and with normal aging, gastrointestinal gamma delta T cells are stimulated via directly harboring HIV and/or exposure to inflammatory factors and this aberrant activation leads to breakdown of tight junctions of the intestinal epithelial barrier, causing increased release of microbial products and inflammatory gamma delta T cells into the circulation. Further, we predict that aged gamma delta T cells exhibit functional profiles skewed towards inflammatory cytokines/cytotoxicity in response to either direct HIV infection and/or stimulatory factors. In this application, we propose to test these hypotheses using advanced and innovative approaches, including 25-color flow cytometry, 31-color imaging mass cytometry of recto-sigmoid biopsies, 19- and 33-plex analyses of plasma and cell culture supernatants, respectively, and multiple algorithms for multivariate analysis of collected datasets. Our bioinformatic data analysis plan will enable identifying novel gamma delta T cell subsets and parsing the differential impacts of age with and without HIV infection. In Aim 1 we will perform a cross-sectional study of our HIV and Aging cohort to determine the links between circulating gamma delta T cell subsets, plasma inflammatory and intestinal permeability markers, and intestinal architecture and cellular composition. In Aim 2 we will determine the temporal links between gamma delta T cell subsets, plasma markers, and the onset and/or progression of geriatric outcomes via a longitudinal study of older subjects with and without ART-suppressed HIV. In Aim 3, we will perform in vitro assays to determine how age and HIV infection impact gamma delta T cell functions, including the capacity to breakdown intestinal epithelial cell monolayers. We predict that our proposed experiments will identify the biological mechanisms that drive...