The development and function of lymphocytes is critically dependent upon regulation of their survival by apoptosis. Studies 'carried-out over the last several years have shown that members of the TNF receptor superfamily (e.g., TNF receptor 1 and Fas) function as key inducers of apoptosis in activated mature lymphocytes. However, it is not known whether these apoptotic pathways 'are also important for regulating survival of developing lymphocytes. The goal of this investigation is to provide a better understanding of pro-apoptotic and anti-apoptotic mechanisms involved in developing lymphocyte survival. Specifically, we will study the role of the apoptosis-regulating NF-kB transcription factor in regulating survival of developing B-lymphocytes, using knock-out mouse models. 1) We show here an impairment of lymphopoiesis following adoptive transfer of ReIA-/- fetal liver cells into irradiated lymphocyte-deficient Rag1-/- mice. Impaired lymphocyte generation was largely rescued in the combined absence of Re1A and TNF receptor 1 (TNFR1), indicating a key function of Re1A may be to prevent TNFR1-induced killing of developing lymphocytes. Here we will investigate the mechanisms by which TNF induces killing of RelA-/- developing B cells, including the potential involvement of caspase proteases and reactive oxygen species (ROS) in TNF killing.2) To understand mechanisms responsible for susceptibility of RelA-/- B cells to TNF, we have studied regulation of potentially important anti-apoptotic genes. We have found that constitutive expression of one key anti-apoptotic gene, BcI-2, was 'significantly reduced in Re1A-/- B cells. Here we will further investigate a possible role for Re1A in regulation of potentially important anti-apoptotic genes. Using a retroviral transduction system, we will re-express anti-apoptotic genes showing impaired expression in RelA-/- B cells to determine their effect on TNF killing. The ability of retrovirus-transduced RelA-/- fetal liver hematopoietic precursors in restoring lymphocyte generation in Rag1-/- mice will also be determined.3) We have also identified a potentially important role for mature T cells in regulating survival of developing lymphocytes. Our results suggest that mature T cells may provide signals, which can counteract the cytotoxic effects of TNFR1. Here we will identify T cell subsets involved, and the nature of T cell derived factors responsible for this protective effect. Of particular interest as mediators of this T cell protective effect are CD4OL and IL-3. Here we will determine the role of CD4OL and IL-3 in inhibition of TNF killing of Re1A-/- developing B cells in vitro, and in rescuing Iymphopoiesis in vivo. These studies may also provide insights into development of strategies for enhancing lymphopoiesis under therapeutic settings.
