PROJECT SUMMARY Functional CD4+ T lymphocytes are progressively lost during HIV/Simian Immunodeficiency Virus (SIV) infection and must be continuously replaced. Hematopoietic stem cells (HSCs) are critically important in replenishing T lymphocytes. However, bone marrow dysplasia and HSC dysfunction have been reported in HIV/SIV infection. Surprisingly, a detailed understanding of the impact of HSC dysfunction on T cell generation and their characteristics is elusive. Systematic study of the onset of HSC dysfunction and its impact on immune potential in humans is hampered by infrequent bone marrow sample availability at desired stages of infection. This is further complicated by the lack of an in-vivo system to track T-cell generation from bone-marrow-derived HSCs. Furthermore, a direct viral infection of HSC and distinct thymocyte precursors has been reported. However, these reports are conflicting and lack robust validation. Consistently, productive infection of thymic precursors and their potential contribution to the establishment of latent viral reservoirs is ill-defined. These limitations warrant the development of a system for systematic investigation of HSC dysfunction, its functional consequences on T cell regenerative potential, and their infection. Encouragingly, recent advancements in the thymic organoid culture systems have opened novel possibilities for supporting reproducible and efficient ex-vivo T cell generation from tissue specific-HSCs. This combined with the feasibility of the SIV/NHP model for easy access to the bone marrow and other significant tissue sources of HSCs at the desired stage of SIV infection has opened unprecedented opportunities for HIV/SIV research. Therefore, here we propose to exploit a highly efficient, serum-free, ex-vivo thymic organoid system and utilize the SIV/macaque model of HIV/AIDS to systematically investigate the impact of HSC dysfunction on distinct thymocyte population characteristics and their infection. In this direction, we have successfully established a novel ex-vivo nonhuman primate artificial thymic organoid (nhpATO) model supporting efficient T cell positive selection via established early T cell thymocyte precursor intermediates. Using these systems, we will first perform an in-depth investigation into the HSC dysfunction and its impact on distinct thymocyte population characteristics (Aim 1). In this direction, we will define the stage of the onset of HSC dysfunction, its impact on the kinetics of distinct thymocyte generation, and its impact on other key properties such as TCR-dependent function. Next, we will investigate the host and viral determinants of HSC dysfunction and transcriptional regulation promoting thymopoiesis (Aim 2). Finally, we will investigate the SIV infection of thymic precursors and its potential to establish latent viral reservoirs (Aim 3). Thus, the SIV/NHP model combined with the nhpATO system offers unique opportunities to address key questions and fill crit...