SUMMARY The use of antiretroviral therapy (ART) in pregnant women living with HIV is an effective way to prevent mother-to-child transmission. However, HIV-exposed, but uninfected (HEU) children experience higher mortality from common infections and severe respiratory disease. However, the independent and combinatorial effects of HIV and ART on fetal immune cell development remain unknown. We propose to utilize the well-established rhesus macaque model of ART-treated simian immunodeficiency virus (SIV) infection to test our hypothesis that maternal SIV and ART exert differential effects on the development and maturation of the fetal immune system, resulting in dysregulated functional responses. To address the differential effects of suppressed SIV infection and ART, we will study three experimental groups of females. In the SIV/ART group, females will be infected with SIV and then suppressed with ART before undergoing timed-mated breeding. We will have two control groups: an ART-only group and an untreated/uninfected healthy pregnancy group. We acknowledge that the ART-only group is not a clinically relevant scenario, but necessary in the context of the proposed studies to distinguish the contribution of ART per se to the fetal immune phenotype exhibited by the offspring of SIV/ART dams. We do not have an untreated SIV-infected group, as this is not relevant to the vast majority of pregnant WLWH. GD130-135 fetuses will be obtained via Caesarean section and studied as outlined in the following Specific Aims. Aim 1. Determine the effects of maternal SIV/ART or ART on fetal immune system function. HEU children exhibit altered lymphocyte function and blunted responses of monocytes to pathogens. However, little is known about the effects of HIV/ART and ART exposure on lymphoid and myeloid cell activation during fetal development. Therefore, we will study the effects of SIV/ART or ART on both the innate and adaptive arms of the fetal immune system in circulation and in fetal tissues using a combination of flow cytometry, functional assays, and single-cell transcriptomics. Aim 2. Determine the effects of maternal SIV/ART or ART on fetal hematopoiesis. SIV/ART or ART may impact fetal immune system development by altering fetal hematopoietic stem and progenitor cells (HSPCs). Therefore, in this aim, we will isolate HSPCs from the fetal liver and fetal bones and determine the transcriptional landscape and functional reconstitution abilities of HSPCs from SIV/ART, ART alone, and control fetuses. Given the increasingly prevalent use of ART in women of reproductive age, this study will further our understanding of the effect of ART on fetal immune development. The ultimate goal of the proposed studies is to identify fetal immune system developmental pathways that can be targeted by therapeutics and interventions to improve immune outcomes for this vulnerable population of infants.