The study of HIV rebound potential necessitates rigorous viral and immunological characterization directly within tissues in people with HIV (PWH) on ART with minimal comorbidities. As a result, this project leverages the longitudinal San Francisco POstmortem Systematic InvesTigation of Sudden Cardiac Death (POST SCD) Study, a postmortem study to bank samples and autopsy data on PWH and uninfected controls who were victims of sudden cardiac death (SCD). To date we have collected extensive tissue samples, including brain, multiple lymph node chains, liver, spleen, heart, pulmonary vasculature and other tissues of interest from 47 HIV-infected and >500 uninfected individuals who experienced SCD. Importantly, ~80% of HIV+ SCD cases were on ART and died suddenly of non-HIV (i.e. cardiac) causes. As a result, the HIV POST SCD cohort is a one-of-kind resource for the study of tissue HIV persistence. This highly innovative project involves in situ hybridization and cutting-edge tissue-based transcriptomic/proteomic nanoString Digital Spatial Profiling (DSP) to clearly define how the reservoirs of intact and HIV-expressing proviruses and biomarker expression differ between tissues and determine how tissue-specific differences in the transcriptionally active HIV reservoir relate to in situ host cell gene and protein expression. Our central hypothesis is that infected CD4 T cells and various myeloid lineage cells within immune privileged histologic environments express full-length, intact HIV RNA transcripts and be capable of rapid viral recrudescence following ART withdrawal. We expect to observe lower expression of host antiviral factors and higher expression of pro-survival factors in cell clusters expressing HIV transcripts. Furthermore, we posit that transcriptional activity and immune states of reservoir cells that are identified as predictors of viral rebound in Project 3 will be visualized within specific lymphoid tissue regions of interest. Our aims are to: 1) measur