T cell homeostasis is the maintenance of peripheral T cells at a relatively constant level. When total T cell numbers are reduced, the remaining T cells proliferate to restore the normal T cell number. Similarly, naive T cells proliferate when a small number is transferred into lymphopenic hosts such as mice deficient in recombination activating gene-1 (RAG1-/-). The homeostatic T cell proliferation requires the recognition of cognate self-peptide/MHC (pepMHC) complexes by T cell receptor (TCR) and a lymphopenic condition, or "space." Recent studies have further shown that as naive CD8 T cells proliferate in lymphopenic hosts, they progressively acquire cell surface markers and functional properties of antigen-induced memory CD8 T cells. The long-term goal of the proposed studies is to understand molecular mechanisms that promote T cell proliferation and differentiation in lymphopenic hosts. Findings from the proposed studies may help to better understand T cell homeostasis and mechanisms underlying the increased incidence of autoimmune diseases in lymphopenic individuals, as in those with AIDS and in patients given radiation, or chemotherapy and bone marrow transplants. Specific aim #1 will identify the molecular basis of "space" for homeostatic T cell proliferation. Using a novel T cell-dendritic cell (T-DC) cocuture system in which T cell proliferation mimics homeostatic proliferation in mice, dendritic cells were found to produce soluble factors that stimulate T cell proliferation. The stimulatory factors secreted by dendritic cells will be identified by screening candidate factors and by biochemical purification. Once they are identified, their role in homeostatic T cell proliferation in mice will be evaluated. In addition, whether "space" involves relief of inhibitory signals among T cells or stimulatory cell-cell contacts mediated by receptors other than TCR will be investigated m the T-DC coculture system. Specific aim #2 will characterize a fraction of polyclonal CD4 and CD8 T cells that undergo exceptionally rapid proliferation in syngeneic lymphopenic hosts. The activation status, repertoire diversity and the ability to respond to environmental antigens by the rapidly proliferating T cells will be analyzed. Specific aim #3 will investigate the role of cell proliferation in homeostasis-driven differentiation and maintenance of memory-like T cells. Naive TCR transgenic T cells expressing the Coxsackie and adenovirus receptor (CAR) will be infected with adenovirus expressing a cyclin dependent kinase inhibitor gene to evaluate the effects of inhibiting cell division on homeostasis-driven differentiation of memory-like cells. Proliferation of homeostatic memory CD8 T cells will be similarly inhibited to evaluate the effects on the maintenance of their surface phenotype and functional characteristics. In addition, the role of cell division in facilitating changes in chromatin structure at the IFN-g locus will be investigated by measuring the extent of DNA methylation, histone acetylation and factor binding.