The V-J recombination of T cell receptor-gamma genes is blocked in interleukin-7 receptor-deficient mice

Abstract

IL-7R-deficient mice have severely impaired expansion of early lymphocytes and lack gamma delta T cells. To elucidate the role of IL-7R on gamma delta T cell development, we analyzed the rearrangements of TCR-alpha, beta, gamma, and delta genes in the thymus of the IL-7R-deficient mice. Southern blot analysis with a J gamma 1 probe revealed that more than 70% of J gamma 1 and J gamma 2 alleles are recombined to form distinct V gamma 1.2-J gamma 2 and V gamma 2-J gamma 1 fragments in control mice. On the contrary, no such recombination was detected in the mutant mice. The rearrangements in the TCR-alpha, beta, and delta loci were comparably observed in control and mutant mice. PCR analysis indicated that the V-J recombination of all the V gamma genes is severely hampered in the mutant mice. The mRNA of RAG-1, RAG-2, Ku-80, and terminal deoxynucleotidyl transferase (TdT) genes was equally detected between control and mutant thymi, suggesting that the expression of common recombination machinery is not affected. These data demonstrated that the V-J recombination of the TCR gamma genes is specifically blocked in the IL-7R-deficient mice and suggested the presence of highly specific regulation for TCR gamma gene rearrangement.

Figure 1

TCR gene rearrangements in the thymus of IL-7R-deficient mice. Lane 1, thymocytes from IL-7R +/− mice; lane 2, thymocytes from IL-7R −/− mice; lane 3, E14.1 ES cells. The position of HindIIIdigested phage λ DNA fragments was shown on the right. (A) Thymocyte DNA was digested with HindIII. A Southern blot was sequentially hybridized with the Jγ1 (left), the Jβ2 (middle), and the RAG-2 (right) probes. (B) Thymocyte DNA was digested with EcoRI. A Southern blot was sequentially hybridized with the Jδ1 (left), the Jα1 (middle), and the RAG-2 (right) probes.

Figure 1

TCR gene rearrangements in the thymus of IL-7R-deficient mice. Lane 1, thymocytes from IL-7R +/− mice; lane 2, thymocytes from IL-7R −/− mice; lane 3, E14.1 ES cells. The position of HindIIIdigested phage λ DNA fragments was shown on the right. (A) Thymocyte DNA was digested with HindIII. A Southern blot was sequentially hybridized with the Jγ1 (left), the Jβ2 (middle), and the RAG-2 (right) probes. (B) Thymocyte DNA was digested with EcoRI. A Southern blot was sequentially hybridized with the Jδ1 (left), the Jα1 (middle), and the RAG-2 (right) probes.

Figure 2

TCR gene rearrangements in the thymocytes detected by PCR. Rearrangement of TCR γ (A), δ (B), and β (C) genes. The DNA from thymocytes of fetuses at day 17 of gestation (for Vγ3–Jγ1, Vγ4– Jγ1, and Vδ1–Jδ1 rearrangements) and at 4 wk old (for Vγ1.1–Jγ4, Vγ1.2–Jγ2, Vγ2– Jγ1, Vγ5–Jγ1, Vδ4–Jδ1, Vδ5– Jδ1, and all the β gene rearrangements) was amplified by PCR, and the Southern blots of the products were hybridized with oligonucleotide probes. Combination of primers used are shown on the left side (A and B). Dβ–Jβ2 and Vβ8.2–Dβ–Jβ2 rearranged fragments are shown on the left side (C).

Figure 3

Expression of V–(D)–J recombination-associating genes in the thymus of the IL-7R-deficient mice. cDNA prepared from 4-wk-old adult thymocytes was amplified by PCR using RAG-1, RAG-2, TdT, Ku-80, and HPRT primers, and the Southern blots of PCR products were hybridized with each probe.