Targeted disruption of the Artemis murine counterpart results in SCID and defective V(D)J recombination that is partially corrected with bone marrow transplantation.

Artemis is a mammalian protein, the absence of which results in SCID in Athabascan-speaking Native Americans (SCIDA). This novel protein has been implicated in DNA double-strand break repair and V(D)J recombination. We have cloned the Artemis murine counterpart, mArt, and generated a mouse with a targeted disruption of mArt. Artemis-deficient mice show a similar T-B- NK+ immunodeficiency phenotype, and carry a profound impairment in coding joint rearrangement, while retaining intact signal ends and close to normal signal joint formation. mArt-/- embryonic fibroblasts show increased sensitivity to ionizing radiation. Hemopoietic stem cell (HSC) transplantation using 500-5000 enriched congenic, but not allogeneic mismatched HSC corrected the T cell and partially corrected the B cell defect. Large numbers (40,000) of allogeneic mismatched HSC or pretreatment with 300 cGy of radiation overcame graft resistance, resulting in limited B cell engraftment. Our results suggest that the V(D)J and DNA repair defects seen in this mArt-/- mouse model are comparable to those in humans with Artemis deficiency, and that the recovery of immunity following HSC transplantation favors T rather than B cell reconstitution, consistent with what is seen in children with this form of SCID.

Restoration of T cell-specific V(D)J recombination in DNA-PKcs(-/-) mice by ionizing radiation: The effects on survival, development, and tumorigenesis.

DNA-dependent protein kinase (DNA-PK) is a DNA-activated nuclear serine/threonine protein kinase. DNA-PK consists of a heterodimeric Ku subunit (composed of a 70 and 86 kD subunit) which binds DNA ends and targets the catalytic subunit DNA-PKcs to DNA strand breaks. DNA-PK plays a major role in the repair of double-strand breaks (DSB) induced in DNA after exposure to ionizing radiation. To better understand the nature of DNA repair defect associated with DNA-PKcs deficiency, we have established DNA-PKcs(-/-) mouse embryo fibroblast cell lines and DNA-PKcs(-/-) null mice, and investigated the response of these mutant cells and mice to DNA damage. DNA-PKcs(-/-) cells are hypersensitive to gamma-irradiation, as evidenced by their low survival as assayed by colony formation efficiencies. Consistent with the radiation hypersensitive phenotype of the cell lines, DNA-PKcs(-/-) mice also display an extreme radiosensitivity, characterized by enhanced mortality after gamma-irradiation. Treatment of newborn DNA-PKcs(-/-) mice with sublethal doses of ionizing radiation restores T cell receptor (TCR) beta recombination and T cell maturation. However, radiation does not restore B cell development. All these mice eventually developed thymic lymphoma. These observations suggest an interrelationship between DSB repair, V(D)J recombination and lymphomagenesis, and provide an in vivo model to elucidate the critical pathways between the regulation of DNA DSB repair, V(D)J recombination, and the molecular mechanism of lymphoid neoplasia.

Irradiation promotes V(D)J joining and RAG-dependent neoplastic transformation in SCID T-cell precursors.

Defects in the nonhomologous end-joining (NHEJ) pathway of double-stranded DNA break repair severely impair V(D)J joining and selectively predispose mice to the development of lymphoid neoplasia. This connection was first noted in mice with the severe combined immune deficient (SCID) mutation in the DNA-dependent protein kinase (DNA-PK). SCID mice spontaneously develop thymic lymphoma with low incidence and long latency. However, we and others showed that low-dose irradiation of SCID mice dramatically increases the frequency and decreases the latency of thymic lymphomagenesis, but irradiation does not promote the development of other tumors. We have used this model to explore the mechanistic basis by which defects in NHEJ confer selective and profound susceptibility to lymphoid oncogenesis. Here, we show that radiation quantitatively and qualitatively improves V(D)J joining in SCID cells, in the absence of T-cell receptor-mediated cellular selection. Furthermore, we show that the lymphocyte-specific endonuclease encoded by the recombinase-activating genes (RAG-1 and RAG-2) is required for radiation-induced thymic lymphomagenesis in SCID mice. Collectively, these data suggest that irradiation induces a DNA-PK-independent NHEJ pathway that facilitates V(D)J joining, but also promotes oncogenic misjoining of RAG-1/2-induced breaks in SCID T-cell precursors.

Irradiation-mediated rescue of T cell-specific V(D)J recombination and thymocyte differentiation in severe combined immunodeficient mice by bone marrow cells.

In SCID (severe combined immunodeficient) mice, proper assembly of immunoglobulin and T cell receptor (TCR) genes is blocked by defective V(D)J recombination so that B and T lymphocyte differentiation is arrested at an early precursor stage. Treating the mice with gamma irradiation rescues V(D)J rearrangement at multiple TCR loci, promotes limited thymocyte differentiation, and induces thymic lymphomas. These effects are not observed in the B cell lineage. Current models postulate that irradiation affects intrathymic T cell precursors. Surprisingly, we found that transfer of irradiated SCID bone marrow cells to unirradiated host animals rescues both TCR rearrangements and thymocyte differentiation. These data indicate that irradiation affects precursor cells at an earlier stage of differentiation than was previously thought and suggest new models for the mechanism of irradiation rescue.

[Radiation and immunity. Interference of ionizing radiation with key immune processes]. / Radiatsiia i immunitet. Vmeshatel'stvo ioniziruiushchikh izluchenii v kliuchevye immunnye protsessy.

Recent evidences of the interference of ionizing radiation into the intimate immune processes are presented in the review. gamma-irradiation induces some events in V-gene rearrangement in T cells. Low-dose irradiation can result in the thymocyte differentiation and activation. Immediately after irradiation stromal thymic cell activity are stimulated and in the later stages it is depressed. Irradiation induces an expression of some functionally important molecules on a surface of the cells of immune system and thus influences the processes of cell interaction, costimulation, adhesion and transvascular migration. It is shown that many events of the signal transduction pathways resulting in radiation-induced and activation-induced apoptosis of lymphocyte are general. This data evidence that the possibility of the choice between the death and activation exists for the cells which are undergone to action of ionizing radiation.

Variations in the frequency of T-cell receptor beta/gamma-interlocus recombination in long-term cultures of non-irradiated and X- and gamma-irradiated human lymphocytes.

PURPOSE: The increased level of illegitimate V(D)J recombination at the T-cell receptor (TCR) loci in lymphoid tumours as well as in T lymphocytes of ataxia telangiectasia patients and humans exposed to carcinogens in vivo suggest that site-specific interlocus recombination events could serve as markers of genomic instability and early genetic changes associated with carcinogenesis. The purpose of this study was to investigate the ability of ionizing radiation to induce TCRbeta/gamma-interlocus rearrangements in human lymphocytes in vitro. MATERIALS AND METHODS: Peripheral blood lymphocytes (PBL) from two healthy donors were exposed to 3 Gy of either X- or gamma-irradiation in vitro. Growth factor-stimulated cell cultures were established, and cell samples for DNA extraction were taken immediately after exposure and at several time points during long-term growth. A PCR-based method was used to measure the frequency of variant cells with Vgamma-Jbeta1 TCR rearrangements. RESULTS: The frequency of TCRbeta/gamma-variant cells was not significantly different in the irradiated and control cultures at any time studied up to 55 days after PHA-stimulation, indicatin that V(D)J-mediated Vgamma-Jbeta1 rearrangement is not induced by X- or gamma-irradiation under these conditions. However, in both irradiated and non-irradiated cultures, the frequency of TCRbeta/gamma variants increased approximately fourfold after mitogen stimulation, from a normal background level of 0.3-0.4 x 10(-5) to 1.3-1.6 x 10(-5) at days 4-9. These levels then gradually declined during prolonged cultivation, and after 2-4 weeks the frequency of variant cells was below the detection limit ( < 0.13 x 10(-5)). CONCLUSIONS: These results provide no evidence that TCRbeta/gamma gene rearrangements can be induced by X- or gamma-irradiation in vitro. However, in contrast with cells with normal TCR receptors, TCRbeta/gamma-variant cells display a relative growth advantage for 1-2 weeks, followed by gradual loss of proliferative capacity. Eventually, they are eliminated from the cell population or outnumbered by cells with normal TCR. If there are similar differences in vivo between cells with hybrid and normal TCR, this may explain the previously reported time- and season-dependent changes in the frequency of cells with hybrid TCR in occupationally exposed populations and individuals receiving cytostatic treatment.

Thymocyte differentiation in gamma-irradiated severe-combined immunodeficient mice: characterization of intermediates and products of V(D)J recombination at the T cell receptor alpha locus.

Treatment with DNA-damaging agents promotes rescue of V(D)J recombination, limited thymocyte differentiation, and development of thymic lymphomas in severe-combined immunodeficient (SCID) mice. One intriguing aspect of this system is that irradiation rescues rearrangements at the T cell receptor (TCR) beta, gamma and delta loci, but not at the TCR alpha locus. Current models posit that only those loci that are recombinationally active at the time of irradiation can be rescued. Here, we employ sensitive, semiquantitative ligation-mediated polymerase chain reaction assays to detect a specific class of recombination intermediates, hairpin coding ends, at the TCR alpha locus. We found that J alpha-coding ends are undetectable in unirradiated SCID thymocytes, but accumulate after irradiation at times coincident with the emergence of a CD4+ CD8+ thymocyte population. Coding joints produced by joining of these ends, however, are extremely rare. To test whether the presence of hairpin coding ends at TCR alpha is sufficient for irradiation-mediated rescue of coding joint formation, we administered a second dose of gamma-irradiation after abundant CD4+ CD8+ thymocytes and hairpin TCR alpha coding ends had accumulated. This treatment failed to stimulate rescue of TCR alpha coding joints. Thus, the presence of hairpin coding ends at the time of irradiation, while perhaps necessary, is not sufficient for rescue of V(D)J rearrangements. These results support a refined model for irradiation-mediated rescue of TCR rearrangements in SCID mice.

p53 is required for both radiation-induced differentiation and rescue of V(D)J rearrangement in scid mouse thymocytes.

The murine scid mutation affects both V(D)J recombination and DNA repair. This mutation has been mapped to the gene encoding the catalytic subunit of the DNA-dependent protein kinase (DNA-PK), which is activated by DNA damage in normal cells. In scid mice, antigen receptor gene rearrangements are initiated normally, but impaired joining of coding ends prevents assembly of functional receptor genes, resulting in arrest of B- and T-cell development. Others have shown that exposure of scid mice to genotoxic agents such as gamma-irradiation rescues rearrangement at the T-cell receptor (TCR) beta locus and promotes thymocyte development. Here we demonstrate that irradiation rescues rearrangements at multiple TCR loci, suggesting a general effect on the recombination mechanism. Furthermore, our data show that p53 is required for irradiation-mediated rescue of both thymocyte development and V(D)J recombination. We also find that thymocyte proliferation and differentiation in the absence of DNA damage do not require p53 and are not sufficient to rescue V(D)J recombination. These results suggest that exposure to ionizing radiation facilitates a partial bypass of the scid defect, perhaps by inducing p53-dependent DNA damage response pathways.

Recombination sequence-binding protein in thymocytes undergoing T-cell receptor gene rearrangement.

Rearrangement of T-cell antigen receptor and immunoglobulin genes occurs in immature lymphoid cells by an unknown mechanism. To identify components of the rearrangement machinery, we isolated a population of murine thymocytes enriched for rearranging pre-T cells. In the nuclear fraction of these cells, we detected a protein that specifically bound the recombination sequences that flank T-cell receptor and immunoglobulin genes and are required for their rearrangement. This protein recognized both heptamer and nonamer motifs of the recombination sequence, separated by either 12 or 23 bp. The protein complexed with the recombination sequence oligonucleotide had an apparent molecular mass of 30 kDa. The binding characteristics of the protein and its presence in rearranging thymocytes and cell lines suggest that it could serve as the recognition unit of a recombinase complex.