Further Differentiation of Murine Double Positive Thymocytes Is Inhibited in Adenosine Deaminase-Deficient Murine Fetal Thymic Organ Culture

摘要

Murine fetal thymic organ culture (FTOC) was used to investigate the mechanism by which a lack of adenosine deaminase (ADA) leads to a failure of T cell production in the thymus. We previously showed that T cell development was inhibited beginning at the CD4⁻CD8⁻CD25⁺CD44^lo stage in ADA-deficient FTOCs initiated at day 15 of gestation when essentially all thymocytes are CD4⁻CD8⁻. In the present study, we asked whether thymocytes at later stages of differentiation would also be sensitive to ADA inhibition by initiating FTOCs when substantial numbers of CD4⁺CD8⁺ thymocytes were already present. dATP was highly elevated in ADA-deficient cultures and the recovery of αβ TCR⁺ thymocytes was inhibited by 94%, indicating that the later stages of thymocyte differentiation are also dependent upon ADA. ADA-deficient cultures were partially rescued by the pan-caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone (z-VADfmk) or by the use of Apaf-1-deficient mice. Rescue was even more dramatic, with 60- to >200-fold increases in the numbers of CD4⁺CD8⁺ cells, when FTOCs were performed with an inhibitor of adenosine kinase, the major thymic deoxyadenosine phosphorylating enzyme, or with bcl-2 transgenic mice. dATP levels were normalized by treatment with either z-VADfmk or an adenosine kinase inhibitor, but not in cultures with fetal thymuses from bcl-2 transgenic mice. These data suggest that ADA deficiency leads to the induction of mitochondria-dependent apoptosis as a consequence of the accumulation of dATP derived from thymocytes failing the positiveegative selection checkpoint.