Metabolites from apoptotic thymocytes inhibit thymopoiesis in adenosine deaminase–deficient fetal thymic organ cultures
Linda F. Thompson, … , Michael S. Hershfield, Regina Resta
Linda F. Thompson, … , Michael S. Hershfield, Regina Resta
Published November 1, 2000
Citation Information: J Clin Invest. 2000;106(9):1149-1157. https://doi.org/10.1172/JCI9944.
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Article

Metabolites from apoptotic thymocytes inhibit thymopoiesis in adenosine deaminase–deficient fetal thymic organ cultures

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.

Authors

Linda F. Thompson, C. Justin Van De Wiele, Aletha B. Laurent, Scott W. Hooker, James G. Vaughn, Hong Jiang, Kamayani Khare, Rodney E. Kellems, Michael R. Blackburn, Michael S. Hershfield, Regina Resta

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Inhibitors of apoptosis abrogate the effects of ADA deficiency in FTOC. ...
Inhibitors of apoptosis abrogate the effects of ADA deficiency in FTOC. FTOCs were performed with bcl-2 transgenic mice (a), Adam1/m1 mice (b), or Apaf-1–/– mice and control littermates at day 15 of gestation. The ADA inhibitor dCF (5 μM) was added to cultures performed with bcl-2 transgenic mice and Apaf-1–/– mice and control littermates. The pan-caspase inhibitor z-VADfmk or a control peptide, z-YVADfmk, which was demonstrated to be ineffective in abrogating the effects of ADA deficiency in C57BL/6 FTOCs (data not shown), was added at 100 μM to FTOCs performed with Adam1/m1 mice. After 2 days, the cultures were harvested and thymocytes were counted and stained with FITC-anti-CD4 plus PE-anti-CD8. Dead cells were excluded by PI staining. The viable cell recoveries per lobe are shown beneath the dot plots. Representative results are shown from two to four independent experiments. In a separate experiment (d), dATP was measured by HPLC in extracts of 30–35 fetal thymic lobes from normal C57BL/6 mice cultured for 2 days under control conditions or in the presence of z-VADfmk (100 μM), dCF (5 μM), or a combination of both. All cultures contained equivalent concentrations of DMSO (0.25%). The data are representative of three independent experiments.