No abstract

Murine fetal thymic organ culture was used to investigate the mechanism by which adenosine deaminase (ADA) deficiency causes T-cell immunodeficiency. C57BL/6 fetal thymuses treated with the specific ADA inhibitor 2′-deoxycoformycin exhibited features of the human disease, including accumulation of dATP and inhibition of S-adenosylhomocysteine hydrolase enzyme activity. Although T-cell receptor (TCR) Vβ gene rearrangements and pre-TCR-α expression were normal in ADA-deficient cultures, the production of αβ TCR + thymocytes was inhibited by 95%, and differentiation was blocked beginning at the time of β selection. In contrast, the production of γδ TCR + thymocytes was unaffected. Similar results were obtained using fetal thymuses from ADA gene-targeted mice. Differentiation and proliferation were preserved by the introduction of a bcl-2 transgene or disruption of the gene encoding apoptotic protease activating factor-1. The pan-caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone also significantly lessened the effects of ADA deficiency and prevented the accumulation of dATP. Thus, ADA substrates accumulate and disrupt thymocyte development in ADA deficiency. These substrates derive from thymocytes that undergo apoptosis as a consequence of failing to pass developmental checkpoints, such as β selection.

CD73 is a glycosyl phosphatidylinositol-anchored protein with both ecto-enzyme activity (ecto-5'-nucleotidase) and signal transducing capabilities for human T lymphocytes. We now report an analysis of the distribution and function of CD73 in murine lymphoid tissues made possible by the development of the first monoclonal antibodies (mAb) specific for murine CD73. Subsets of T and B lymphocytes are CD73+ and the level of expression increases with lymphocyte maturation in both species. Among B cells, CD73 is largely restricted to cells which have undergone isotype switching. The signal transmitting function of CD73 is also conserved, as splenic T cells treated with anti-CD73 mAb plus phorbol 12-myristate 13-acetate proliferate and secrete IL-2. Fyn-/- mice are unresponsive to CD73 ligation, however, demonstrating the requirement for this tyrosine kinase in CD73-mediated signal transduction. CD73 is down-regulated after mAb plus cross-linking, suggesting that expression may be controlled by interaction with a ligand. Only small numbers of thymocytes are CD73+, so CD73 receptor functions are unlikely to be important for developing T cells. However, immunohistochemical analysis reveals that reticular and vascular cells throughout the thymus and other lymphoid tissues are markedly CD73+. Therefore, CD73 might mediate lymphocyte-stromal cell interactions or condition the local microenvironment to facilitate lymphocyte development and/or function.

The adenosine producing enzyme ecto-5 Ј -nucleotidase (5 Ј -NT) is not normally expressed during thymocyte development until the medullary stage. To determine whether earlier expression would lead to adenosine accumulation and/or be deleterious for thymocyte maturation, thymic purine metabolism, and T cell differentiation were studied in lck NT transgenic mice overexpressing 5 Ј -NT in cortical thymocytes under the control of the lck proximal promoter. In spite of a 100-fold elevation in thymic 5 Ј -NT activity, transgenic adenosine levels were unchanged and T cell immunity was normal. Inosine, the product of adenosine deamination, was elevated more than twofold, however, indicating that adenosine deaminase (

Mutations in the gene encoding adenosine deaminase (ADA), a purine salvage enzyme, lead to immunodeficiency in humans. Although ADA deficiency has been analyzed in cell culture and murine models, information is lacking concerning its impact on the development of human thymocytes. We have used chimeric human/mouse fetal thymic organ culture to study ADA-deficient human thymocyte development in an “in vivo-like” environment where toxic metabolites accumulate in situ. Inhibition of ADA during human thymocyte development resulted in a severe reduction in cellular expansion as well as impaired differentiation, largely affecting mature thymocyte populations. Thymocyte differentiation was not blocked at a discrete stage; rather, the paucity of mature thymocytes was due to the induction of apoptosis as evidenced by activation of caspases and was accompanied by the accumulation of intracellular dATP. Inhibition of adenosine kinase and deoxycytidine kinase prevented the accumulation of dATP and restored thymocyte differentiation and proliferation. Our work reveals that multiple deoxynucleoside kinases are involved in the phosphorylation of deoxyadenosine when ADA is absent, and suggests an alternate therapeutic strategy for treatment of ADA-deficient patients.