Mutations in the gene for adenosine deaminase (ADA) cause severe combined immunodeficiency in humans. ADA-deficient patients exhibit a profound lymphopenia and are susceptible to a wide array of infectious agents. Although this disease has been actively investigated, the exact mechanism by which a loss of ADA inhibits lymphocyte development is still not firmly established. In the first funding period, we exploited murine fetal thymic organ cultures (FTOCs) to delineate this mechanism. Under ADA-deficient conditions, thymocyte development was blocked at the CD4-CD8CD441xCD25 + stage and was accompanied by the accumulation of dATP derived from the ADA substrate deoxyadenosine. ADA-deficient FTOCs were rescued by an adenosine kinase inhibitor that blocked the accumulation of dATP. The cultures were also rescued by a pan-caspase inhibitor, deletion of apoptosis protease activating factor-1, or expression of a bcl-2 transgene, strongly implicating mitochondrial-dependent apoptosis in the inhibition of thymocyte production in ADA-deficient FTOCs. Our data are consistent with this block being caused by dATP-induced mitochondrial cytochrome c release followed by apoptosis. However, other mechanisms involving proapoptotic bcl-2 family members are also feasible. Distinguishing between these alternatives is a goal of the next funding period (Aim I). We will also investigate the mechanism by which ADA-deficiency impacts human thymocyte development using chimeric human/mouse thymic organ cultures (Aim II). Important differences in the consequences of ADA inhibition in murine vs. human thymic organ cultures provide compelling rationale for this Aim. Finally, we will examine the effects of ADA deficiency upon both murine and human B cell differentiation (Aim III), a virtually unexplored area, following up on exciting initial observations that the ADA substrate adenosine, as well as deoxyadenosine, may play an important role. In addition to providing new insights into the pathogenesis of severe combined immunodeficiency caused by ADA deficiency, the above studies will increase our understanding of the molecular events in normal murine and human lymphopoiesis. Our data will contribute to the growing knowledge base needed to understand and control the vulnerability of the developing human immune system to environmental threats and infectious agents.
