Project Summary: Human immunodeficiency virus (HIV) is associated with persistent immune activation and dysfunction, even during suppressive antiretroviral therapy (ART) that was initiated early post-infection. Perinatally acquired HIV (PHIV) infection and lifelong ART likely alter the development and function of the immune system. The exposure of HIV and ART in utero, the decades of ART therapy, the lasting effects of older toxic ART with mitochondrial toxicity and the long-term sub-optimal adherence known to occur with prolonged ART use, heighten concern that sequelae of HIV and ART in children and adolescents may be more frequent and potentially more devastating than in adults. Better understanding of immune dysfunction in PHIV is crucial, as it is often easier to limit or even reverse comorbidities at an early stage. We are exploring the consequences of PHIV and its treatment with ART in a cohort of adolescents in Uganda and have reported significant differences in the immune profiles of HIV- and HIV+ children. These differences may be driven by trained immunity, a process by which cells of the innate immune system (e.g. monocytes, macrophages and natural killer cells) are reprogrammed to respond differently to subsequent exposure to microbial products and proinflammatory lipids. Recent studies, including our own, demonstrate that ART exposure may also contribute to alterations in immune cell activation, potentially through decreased mitochondrial function and through modulation of intracellular signaling cascades. The knowledge gained from this proposal could be substantial, and from a translational perspective will lay the foundation to identify key pathways with biological, clinical, and prognostic relevance for adolescents who are advancing into adulthood. These results could inform intervention trials to mitigate the development of comorbidities associated with immune activation. We propose: Aim 1: To measure innate immune profiles (i.e monocytes and NK cells) in adolescents with and without HIV in Uganda and the United States. Aim 2: To identify the role of trained immunity in innate immune modulation in adolescents with and without HIV infection in Uganda. Aim 3: To evaluate how mitochondrial function plays a role in innate immune profiles longitudinally in adolescents with and without HIV in Uganda. This proposal combines a well-characterized cohort of children with and without PHIV with ex vivo and in vitro assessments of immune cell phenotype and function. We will use high-dimensional flow cytometry analyses, single cell transcriptional profiling, and an in-depth analytical approach to define the potential mechanisms whereby chronic exposure to ART, microbial products, and clinical and environmental factors may contribute to innate immune cell activation or dysfunction. This is a continuation of a highly successful collaboration among the study team members and we are well positioned to perform this innovative project.