PROJECT SUMMARY/ABSTRACT Persons living with HIV infection (PWH) on antiretroviral therapy (ART) are succumbing earlier to many of the same complications that plague the HIV-uninfected elderly. This elevated CVD risk in PWH is at least partly due to persistently increased inflammation and activation of CD8 T cells and monocytes that our group has identified and characterized. Many elderly and nearly all PWH are coinfected with cytomegalovirus (CMV), which we have shown is associated with inflammation and dramatic expansion of CD8 T cells in PWH, and has been independently linked to CVD. Activated CD8 T cells accumulate at sites of endothelial dysfunction in vivo, promote atherosclerotic CVD (ASCVD) in animal models, and can trigger a proatherogenic phenotype on monocytes in vitro – yet whether CD8 T cells infiltrating the vasculature recognize cognate antigenic peptides has never been definitively shown in vivo, nor have antigen-nonspecific activating pathways been rigorously evaluated. In this proposal, we seek to identify how CMV and activated T cells conspire to exacerbate CVD beyond traditional risk factors in HIV-uninfected persons, and to understand if and how activated T cells further promote CVD risk in PWH with CMV coinfection. We hypothesize that active CMV replication within atherosclerotic lesions provides antigen for infiltrating CMV-reactive T cells and that additional antigen- nonspecific signals (including IL-15 and the CD2/LFA-3 axis) further shape the T cell response. We propose that these signals exacerbate ASCVD in HIV-uninfected persons and that they are further elevated in those with HIV infection. We will test these hypotheses with the following specific aims: Aim 1: To define the mechanisms of plaque CD8 T cell activation in vivo. We will use bulk and single cell RNA-sequencing coupled with barcoded- antibody and multimer labeling to test the hypothesis that CD8 T cells recovered from atherosclerotic plaques of CMV+ donors exhibit a more proinflammatory transcriptional program than plaque CD8 T cells from CMV- donors, and we will identify the activating pathways involved. We will then use immunofluorescence imaging and fluorescence in situ hybridization to confirm spatial relationships with activating signals such as IL-15, LFA-3, and CMV. Aim 2: To define the effect of HIV infection on the activation and in situ localization of vascular CD8 T cells. We will use RNA-sequencing to identify and characterize the mechanisms of activation and antigen- specificity of vascular CD8 T cells. We will also use immunofluorescence imaging to test the hypothesis that aortas from PWH will have increased activating signals and additional pro-inflammatory factors that bolster T cell activation compared to aortas from age- and gender-matched controls. Our studies will define mechanisms whereby chronic viral infection drives CD8 T cell-mediated vascular pathology and may identify novel targets beyond traditional risk factors to prevent/treat...