Adenosine deaminase (ADA) deficiency was discovered as a cause of severe combined immuno- deficiency twenty-five years ago and the genetic mechanisms responsible for the disease are well delineated. However, according to the investigator, the mechanism by which the loss of this enzyme causes immunodeficiency, and the relative contributions of the two ADA substrates, adenosine and deoxyadenosine, are still not understood. The investigator proposes to use murine fetal thymic organ culture (FTOC) with either ADA-/-mice or with normal mice and the ADA inhibitor 2'deoxycoformycin to elucidate the molecular mechanism for the failure of T-cell development in ADA deficiency. In this model the thymus is studied in isolation from all other organ systems allowing ADA deficient conditions to be maintained and at the same time eliminating the potential for glucocorticoid-mediated effects secondary to stress caused by multi-system disease. The investigator will first elucidate the stage where T-cell development is blocked (in the thymus) in the absence of ADA (Aim 1). Next, she will examine four potential mechanisms: dATP mediated inhibition of ribonucleotide reductase, suicide inactivation of S-adenosylhomocysteine hydrolase, aberrant adenosine receptor signaling and dATP induced DNA strand breaks (Aim 2). She will also characterize the defects in B-cell differentiation under ADA-deficient conditions using the placentally rescued ADA- knockout mice both in vivo and in a variety of in vitro culture systems (Aim 3). She will apply what she has learned about murine T-cell developments with ADA knock-out mice to human thymocyte differentiation by characterizing residual T- cells in ADA-deficient patients and performing chimeric human/mouse fetal thymic organ cultures (Aim 4). The experiments proposed above will not only give new insight into the pathogenesis of the severe combined immunodeficiency seen in ADA deficiency, but will also yield new knowledge about the regulation of normal lymphocyte development.
