Further differentiation of murine double-positive thymocytes is inhibited in adenosine deaminase-deficient murine fetal thymic organ culture

J Immunol. 2006 May 15;176(10):5925-33. doi: 10.4049/jimmunol.176.10.5925.


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(low) stage in ADA-deficient FTOC 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 FTOC when substantial numbers of CD4(+)CD8(+) thymocytes were already present. dATP was highly elevated in ADA-deficient cultures, and the recovery of alphabeta 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 or by the use of apoptotic protease-activating factor-1-deficient mice. Rescue was even more dramatic, with 60- to >200-fold increases in the numbers of CD4(+)CD8(+) cells, when FTOC 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 carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone 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 positive/negative selection checkpoint.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adenosine Deaminase / deficiency*
  • Adenosine Deaminase / genetics*
  • Animals
  • Apoptotic Protease-Activating Factor 1
  • Cell Differentiation / genetics
  • Cell Differentiation / immunology*
  • Fetus
  • Genetic Predisposition to Disease*
  • Immunologic Deficiency Syndromes / enzymology
  • Immunologic Deficiency Syndromes / genetics
  • Immunologic Deficiency Syndromes / immunology
  • Intracellular Signaling Peptides and Proteins / deficiency
  • Intracellular Signaling Peptides and Proteins / genetics
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Mice, Transgenic
  • Organ Culture Techniques
  • Proteins / genetics
  • T-Lymphocytes / enzymology*
  • T-Lymphocytes / immunology
  • T-Lymphocytes / pathology
  • Thymus Gland / embryology
  • Thymus Gland / enzymology*
  • Thymus Gland / pathology


  • Apaf1 protein, mouse
  • Apoptotic Protease-Activating Factor 1
  • Intracellular Signaling Peptides and Proteins
  • Proteins
  • Adenosine Deaminase