Gene-specific signal joint modifications during V(D)J recombination of TCRAD locus genes in murine and human thymocytes.

V(D)J recombination assembles functional T-cell receptor (TCR) genes from V, D and J components in developing thymocytes. Extensive processing of V, D and J extremities before they are ligated creates a high degree of junctional diversity which results in the generation of a large repertoire of different TCR chains. In contrast, the extremities of the intervening DNA segment, which bear the recombination signal sequences, are generally held to be monomorphic, so that signal joints (SJs) consist of the perfect head-to-head juxtaposition of recombination signal extremities. We analyzed the structure of SJs generated during the recombination of TCRAD locus genes in murine and human thymocytes. Junctional diversity resulting from N nucleotide additions or from N nucleotide additions and base loss was found for each type of SJ examined. Different patterns of processing/modification were found, suggesting that different enzymatic activities operate during recombination of TCRA and TCRD genes, although they are located within the same genetic locus. Recombination of the deltaRec-1 element generates a diverse repertoire of SJs exhibiting both combinatorial and junctional diversity in murine and human thymocytes. Therefore, SJ diversity appears to be an intrinsic feature of V(D)J recombination in unmanipulated thymocytes

p53-dependent and p53-independent pathways for radiation-induced immature thymocyte differentiation

International audienceThe pre-T-cell receptor (TCR) delivers essential survival/differentiation signals to the developing thymocytes. Severe combined immunodeficient (SCID) and recombination-activating gene (RAG)-deficient mice are unable to assemble antigen receptor genes, and therefore cannot express a pre-TCR. Consequently, T lymphocyte differentiation is arrested at an early stage in the thymus of these animals, and immature thymocytes are eliminated through apoptotic processes. This maturation arrest can be relieved and thymocyte differentiation rescued after the exposure of these mice to whole-body gamma-irradiation. Whereas the promotion of immature thymocyte survival/differentiation was shown to require p53 activity in irradiated SCID mice, it was suggested, on the other hand, that p53 activation prevents immature thymocytes survival/differentiation in irradiated RAG-deficient mice. However, SCID mice have impaired responses to ionizing radiation. In this paper, we analysed p53 requirement in radiation-induced thymocyte differentiation in CD3epsilon(Delta5/Delta5) mice, where pre-TCR deficiency also results in an early block of lymphocyte development. Our results show at the cellular and molecular levels that, in this DNA repair-proficient model, irradiation-induced thymocyte differentiation proceeds either by a p53-dependent or by a p53-independent pathway, which differ in their sensitivity to the radiation dose delivered

Gene-specific signal joint modifications during V(D)J recombination of TCRAD locus genes in murine and human thymocytes

International audienceV(D)J recombination assembles functional T-cell receptor (TCR) genes from V, D and J components in developing thymocytes. Extensive processing of V, D and J extremities before they are ligated creates a high degree of junctional diversity which results in the generation of a large repertoire of different TCR chains. In contrast, the extremities of the intervening DNA segment, which bear the recombination signal sequences, are generally held to be monomorphic, so that signal joints (SJs) consist of the perfect head-to-head juxtaposition of recombination signal extremities. We analyzed the structure of SJs generated during the recombination of TCRAD locus genes in murine and human thymocytes. Junctional diversity resulting from N nucleotide additions or from N nucleotide additions and base loss was found for each type of SJ examined. Different patterns of processing/modification were found, suggesting that different enzymatic activities operate during recombination of TCRA and TCRD genes, although they are located within the same genetic locus. Recombination of the deltaRec-1 element generates a diverse repertoire of SJs exhibiting both combinatorial and junctional diversity in murine and human thymocytes. Therefore, SJ diversity appears to be an intrinsic feature of V(D)J recombination in unmanipulated thymocytes

Analysis of the TCR alpha-chain rearrangement profile in human T lymphocytes.

doi : 10.1016/j.molimm.2007.02.017International audienceThe size of the available human alphabeta T cell repertoire is difficult to determine and is open to debate. Empirical analysis of TCR beta-chain diversity reveals approximately 10(6) different beta chains in peripheral blood. Due in part to locus complexity, comparable information for TCR alpha is lacking. Rather, current estimates for human TCR alpha diversity, and hence, total repertoire diversity, are based on theoretical analyses that assume equal probabilities of rearrangement between any V alpha gene and J alpha gene. Here, we report on a systematic locus-wide rearrangement analysis of the TCR alpha-chain in human T cells. We first demonstrate that the V-J alpha recombination in the thymus is not random but depends on the reciprocal V alpha and J alpha position within the locus. Characterization of the frequency of gene usage combined with identification of five previously unrecognized pseudogenes enables us to empirically estimate the human TCR alpha combinatorial repertoire. The number of V-J alpha combinations achieved is approximately 44-56% of the total combinatorial possibilities, significantly lower than theoretical estimates. We also demonstrate that TCR alpha-chain diversity in peripheral T lymphocytes mimics the same general patterns of rearrangement as observed in the thymus, and these patterns appear conserved among different individuals. This unexpected observation indicates that, unlike the TCR beta locus, the human TCR alpha-chain repertoire is primarily predetermined by genetic recombination and its size is restricted by limits on the combinatorial repertoire rather than post-thymic selection

Differential chronology of TCRADV2 gene use by α\alpha and δ\delta chains of the mouse TCR

International audienceThe genes coding for TCR alpha and delta chains share the same genetic locus (TCRA/D). The rules governing the utilization of a V gene with the alpha and delta chains have not been established. More specifically, it is not known whether the position of a gene within the locus influences its utilization in alpha and delta TCR. To elucidate these points, we mapped ADV2 genes in the TCRA/D locus of BALB/c mice and analyzed their utilization in TCR alpha and delta transcripts from thymi isolated from mice of different ages. Our results show that all ADV2 genes can be used by the two chains, but with strikingly different patterns. Moreover, ADV2 utilization by the alpha chain proceeds in successive concentric waves during development, suggesting a progressive regulation of gene accessibility and utilization. These results support independent control of TCRA and TCRD gene assembly