Regulation of reactive oxygen species by Atm is essential for proper response to DNA double-strand breaks in lymphocytes.

The ataxia telangiectasia-mutated (ATM) gene plays a pivotal role in the maintenance of genomic stability. Although it has been recently shown that antioxidative agents inhibited lymphomagenesis in Atm(-/-) mice, the mechanisms remain unclear. In this study, we intensively investigated the roles of reactive oxygen species (ROS) in phenotypes of Atm(-/-) mice. Reduction of ROS by the antioxidant N-acetyl-l-cysteine (NAC) prevented the emergence of senescent phenotypes in Atm(-/-) mouse embryonic fibroblasts, hypersensitivity to total body irradiation, and thymic lymphomagenesis in Atm(-/-) mice. To understand the mechanisms for prevention of lymphomagenesis, we analyzed development of pretumor lymphocytes in Atm(-/-) mice. Impairment of Ig class switch recombination seen in Atm(-/-) mice was mitigated by NAC, indicating that ROS elevation leads to abnormal response to programmed double-strand breaks in vivo. Significantly, in vivo administration of NAC to Atm(-/-) mice restored normal T cell development and inhibited aberrant V(D)J recombination. We conclude that Atm-mediated ROS regulation is essential for proper DNA recombination, preventing immunodeficiency, and lymphomagenesis.

Quantitation of aberrant interlocus T-cell receptor rearrangements in mouse thymocytes and the effect of the herbicide 2,4-dichlorophenoxyacetic acid.

Small studies in human populations have suggested a correlation between the frequency of errors in antigen receptor gene assembly and lymphoid malignancy risk. In particular, agricultural workers exposed to pesticides have both an increased risk for lymphoma and an increased frequency of errors in antigen receptor gene assembly. In order to further investigate the potential of such errors to serve as a mechanistically based biomarker of lymphoid cancer risk, we have developed a sensitive PCR assay for quantifying errors of V(D)J recombination in the thymocytes of mice. This assay measures interlocus rearrangements between two T-cell receptor loci, V-gamma and J-beta, located on chromosomes 13 and 6, respectively. The baseline frequency in four strains of mice was determined at several ages (2-8 weeks of age) and was found to be stable at approximately 1.5 x 10(-5) per thymocyte. Strain AKR, which has a high susceptibility to T-cell lymphomas, did not show an elevated frequency of aberrant V(D)J events. We used this assay to examine the effects of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) on the frequency of these events. Female B6C3F1 mice, 27 days of age, were exposed to 2,4-D by gavage at doses of 0, 3, 10, 30, and 100 mg/kg/day for 4 successive days and sacrificed on day 5. Thymus DNA was isolated and examined for illegitimate V(D)J recombination-mediated gene rearrangements. In addition, pregnant mice were exposed to 2,4-D and thymocytes from the offspring examined at 2 weeks of age. No significant increase in aberrant V(D)J rearrangements was found, indicating that under these conditions 2,4-D does not appear to effect this important mechanism of carcinogenesis.

Functional and structural similarity of V gamma 9V delta 2 T cells in humans and Aotus monkeys, a primate infection model for Plasmodium falciparum malaria.

Gammadelta T cells are implicated to play crucial roles during early immune responses to pathogens. A subset of human gammadelta T cells carrying the Vgamma9Vdelta2 TCR recognize small, phosphorylated nonpeptidic Ags. However, the precise role of these cells and the ligands recognized in human immune responses against pathogens remains unclear because of the lack of suitable animal models. We have analyzed the reactivity of spleen cells of the New World monkey Aotus nancymaae against isopentenyl pyrophosphate (IPP), a phosphorylated microbial metabolite selectively activating Vgamma9Vdelta2 T cells. Spleen cells were stimulated by IPP and the expanding cell population expressed the Vgamma9 TCR. TRGV-J and TRDV-D-J rearrangements expressed by IPP-stimulated cells of Aotus were analyzed by RT-PCR and DNA sequencing. The TRGV-J and TRDV-D-J rearrangements expressed by IPP-stimulated Aotus and human gammadelta T cells were similar with respect to 1) TCR gene segment usage, 2) a high degree of germline sequence homology of the TCR gene segments used, and 3) the diversity of the CDR3 regions. Phylogenetic analysis of human, Pan troglodytes, and A. nancymaae TRGV gene segments showed that the interspecies differences are smaller than the intraspecies differences with TRGV9 gene segments located on a distinct clade of the phylogenetic tree. The structural and functional conservation of Vgamma9Vdelta2 T cells in A. nancymaae and humans implicates a functionally important and evolutionary conserved mechanism of recognition of phosphorylated microbial metabolites.

Control of chromatin accessibility for V(D)J recombination by interleukin-7.

IL-7 is a key factor for lymphoid development, and it contributes to V(D)J recombination at multiple loci in immune-receptor genes. IL-7 signal transduction, involving gamma(c) and Jak3, is required for successful recombination at the TCR-gamma locus. IL-7 signaling controls the initiation phase of V(D)J recombination by controlling access of the V(D)J recombinase to the locus. In the absence of IL-7, the TCR-gamma locus is methylated and packaged in a repressed form of chromatin consisting of hypoacetylated histones. IL-7 signaling likely increases the acetylation state of the nucleosomal core histones resulting in an "open" form of chromatin. This opening leads to a higher accessibility for the transcription machinery and increased accessibility of the Rag heterodimer that performs the cleavage of DNA.

Interleukin 7 receptor control of T cell receptor gamma gene rearrangement: role of receptor-associated chains and locus accessibility.

VDJ recombination of T cell receptor and immunoglobulin loci occurs in immature lymphoid cells. Although the molecular mechanisms of DNA cleavage and ligation have become more clear, it is not understood what controls which target loci undergo rearrangement. In interleukin 7 receptor (IL-7R)alpha-/- murine thymocytes, it has been shown that rearrangement of the T cell receptor (TCR)-gamma locus is virtually abrogated, whereas other rearranging loci are less severely affected. By examining different strains of mice with targeted mutations, we now observe that the signaling pathway leading from IL-7Ralpha to rearrangement of the TCR-gamma locus requires the gammac receptor chain and the gammac-associated Janus kinase Jak3. Production of sterile transcripts from the TCR-gamma locus, a process that generally precedes rearrangement of a locus, was greatly repressed in IL-7Ralpha-/- thymocytes. The repressed transcription was not due to a lack in transcription factors since the three transcription factors known to regulate this locus were readily detected in IL-7Ralpha-/- thymocytes. Instead, the TCR-gamma locus was shown to be methylated in IL-7Ralpha-/- thymocytes. Treatment of IL-7Ralpha-/- precursor T cells with the specific histone deacetylase inhibitor trichostatin A released the block of TCR-gamma gene rearrangement. This data supports the model that IL-7R promotes TCR-gamma gene rearrangement by regulating accessibility of the locus via demethylation and histone acetylation of the locus.

Interleukin 7 induces TCR gene rearrangement in adult marrow-resident murine precursor T cells.

Rearrangement of the T cell antigen receptor genes is a complex, highly regulated process. To gain a better understanding of the extracellular factors involved in the regulation of TCR beta and gamma gene rearrangement in adult murine bone marrow-resident precursor T cells, several cytokines were tested for their ability to induce gene recombination. A selected population of C58/J bone marrow cells (Thy 1(low), CD3, CD8, B220) that is enriched for pre-T cell activity was propagated in vitro in medium supplemented with IL-3 and mast cell growth factor (MGF, also referred to as stem cell factor, Steele factor and c-kit ligand). These cytokines were required for the maintenance of pre-T cell activity in culture, but had no effect on TCR gene expression. Several additional cytokines were added to the culture medium. Of all those tested, only IL-7 induced complete rearrangement of the TCR gamma locus. Complete rearrangement of the TCR beta locus was not induced under any of the culture conditions analysed here. The bone marrow cells cultured in IL-3, MGF and IL-7 did not begin to express mature T cell proteins and maintained their in vivo progenitor potential. Furthermore, IL-7 cultured bone marrow cells were capable of differentiation in vivo into all phenotypic subpopulations of T cells, without an apparent bias toward the gammadelta lineage. The data presented here suggest that TCR gamma gene rearrangement in adult pre-T cells is regulated by IL-7, but that the TCR beta locus requires additional or alternative signals for the induction of complete rearrangement.

Interleukin 7-induced expression of specific T cell receptor gamma variable region genes in murine fetal liver cultures.

We previously reported that culture of murine fetal liver (FL) cells with interleukin 7 (IL-7) results in expression of high levels of T cell receptor (TCR) gamma transcripts by a population of cells expressing Thy-1 and Pgp-1, suggesting that IL-7 promotes the growth and/or differentiation of pre-T cells. We demonstrate herein that culture of FL cells for 7 d with IL-7 caused the rearrangement and expression of TCR gamma variable (V) region genes V gamma 4 and V gamma 6, but not V gamma 5 or V gamma 7. Since this effect was not blocked by hydroxyurea, it appeared to represent induction of expression of these genes by IL-7 rather than expansion of a preexisting positive population. We also show that IL-7 induced RAG-1 and RAG-2 mRNA expression by FL cells. These data provide evidence that specific TCR gamma/delta V region genes can be rearranged and expressed by T lineage cells before their migration to the thymus, in response to IL-7.