PROJECT ABSTRACT The HIV-1 reservoir is thought to contain cells that vary in their susceptibility to clearance by the host immune system. Identifying mechanisms that allow infected cells to avoid clearance could inform the development of cure strategies that are able to more effectively eliminate HIV-infected cells in the long-lived reservoir. Persistence during long periods of untreated infection, when immune surveillance for HIV-infected cells is highest and the immune environment is highly inflammatory, may select for infected cells with an enhanced ability to evade detection and clearance by the host immune system. This study will focus on two groups of reservoir cells that carry intact proviral genomes and likely differ in susceptibly to clearance. The first are "early" reservoir cells that were infected (or are a clone of a cell infected) early in untreated infection. The second are "late" reservoir cells that were infected (or are a clone of a cell infected) near the time of ART initiation. These "late" reservoir cells have persisted in the reservoir for less time and have primarily persisted during antiretroviral therapy when HIV- specific immune responses are blunted and HIV-infected cells typically have very long half-lives. In contrast, "early" reservoir cells persisted for long periods of untreated infection when most HIV-infected cells have a short half-life. We hypothesize that early proviruses will have epigenetic features that make them resistant to T cell stimulation and less susceptible to clearance. In this study we will identify intact "early" and "late" proviruses in CD4+ T cells isolated from the blood of 15 participants on suppressive ART (Aim 1a). We will then examine phenotypes predicted to impact reservoir clearance. Specifically, we will examine whether "early" and "late" proviruses differ in their sensitivity to T cell stimulation (Aim 1b) and/or have different genetic (proviral sequence and integration site) or epigenetic features (Aim 1c). To test the hypothesis that "early" proviruses are more difficult to clear, we will measure the susceptibility of "early" and "late" proviruses to killing by autologous CD8 T cells (Aim 2a) and assess whether disruption of epigenetic regulators alters their susceptibility to killing by autologous CD8 T cells (Aim 2b). Our focus on "early" and "late" viruses allows us to explore potential mechanisms associated with clearance in populations that likely differ in their susceptibility to clearance, however, the proposal work will also reveal if features other than proviral age are predictive of susceptibility to clearance. We will then explore strategies to improve antigen presentation in infected cells (Aim 3a) and assess whether improved antigen presentation can enhance killing by autologous CD8 T cells in populations typically refractory to clearance (Aim 3b). The overarching goal of this project is to define a population of cells with an enhanced ability to evade clearance, identi...