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.

Aggregation-resistant domain antibodies selected on phage by heat denaturation.

We describe a method for selecting aggregation-resistant proteins by heat denaturation. This is illustrated with antibody heavy chain variable domains (dAbs), which are prone to aggregate. The dAbs were displayed multivalently at the infective tip of filamentous bacteriophage, and heated transiently to induce unfolding and to promote aggregation of the dAbs. After cooling, the dAbs were selected for binding to protein A (a ligand common to these folded dAbs). Phage displaying dAbs that unfold reversibly were thereby enriched with respect to those that do not. From a repertoire of phage dAbs, six dAbs were characterized after selection; they all resisted aggregation, and were soluble, well expressed in bacteria and could be purified in good yields. The method should be useful for making aggregation-resistant proteins and for helping to identify features that promote or prevent protein aggregation, including those responsible for misfolding diseases.

Gamma-irradiation directly affects the formation of coding joints in SCID cell lines.

SCID mice have a defect in the catalytic subunit of the DNA-dependent protein kinase, causing increased sensitivity to ionizing radiation in all tissues and severely limiting the development of B and T cell lineages. SCID T and B cell precursors are unable to undergo normal V(D)J recombination: coding joint and signal joint products are less frequently formed and often will exhibit abnormal structural features. Paradoxically, irradiation of newborn SCID mice effects a limited rescue of T cell development. It is not known whether irradiation has a direct impact on the process of V(D)J joining, or whether irradiation of the thymus allows the outgrowth of rare recombinants. To investigate this issue, we sought to demonstrate an irradiation effect ex vivo. Here we have been able to reproducibly detect low-frequency coding joint products with V(D)J recombination reporter plasmids introduced into SCID cell lines. Exposure of B and T lineage cells to 100 cGy of gamma irradiation made no significant difference with respect to the number of coding joint and signal joint recombination products. However, in the absence of irradiation, the coding joints produced in SCID cells had high levels of P nucleotide insertion. With irradiation, markedly fewer P insertions were seen. The effect on coding joint structure is evident in a transient assay, in cultured cells, establishing that irradiation has an immediate impact on the process of V(D)J recombination. A specific proposal for how the DNA-dependent protein kinase catalytic subunit influences the opening of hairpin DNA intermediates during coding joint formation in V(D)J recombination is presented.

Molecular and cellular basis of the altered immune response against arsonate in irradiated A/J mice autologously reconstituted.

The humoral immune response to arsonate (Ars) in normal A/J mice is dominated in the late primary and particularly in the secondary response by a recurrent and dominant idiotype (CRIA) which is encoded by a single canonical combination of the variable gene segments: VHidcr11-DFL16.1-JH2 and Vkappa10-Jkappa1. Accumulation of somatic mutations within cells expressing this canonical combination or some less frequent Ig rearrangements results in the generation of high-affinity antibodies. By contrast, in partially shielded and irradiated A/J mice (autologous reconstitution) immunized with Ars-keyhole limpet hemocyanin (KLH), both the dominance of the CRIA idiotype and the affinity maturation are lost, whereas the anti-Ars antibody titer is not affected. To understand these alterations, we have analyzed a collection of 27 different anti-Ars hybridomas from nine partially shielded and irradiated A/J mice that had been immunized twice with Ars-KLH. Sequence analysis of the productively rearranged heavy chain variable region genes from those hybridomas revealed that (i) the canonical V(D)J combination was rare, (ii) the pattern of V(D)J gene usage rather corresponded to a primary repertoire with multiple gene combinations and (iii) the frequency of somatic mutations was low when compared to a normal secondary response to Ars. In addition, immunohistological analysis has shown a delay of 2 weeks in the appearance of full blown splenic germinal centers in autoreconstituting mice, as compared to controls. Such a model could be useful to understand the immunological defects found in patients transplanted with bone marrow.

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.

A DNA repair abnormality specific for rearranged immunoglobulin variable genes in germinal center B cells.

The somatic hypermutation mechanism produces high-rate mutagenesis specifically targeted to rearranged immunoglobulin (Ig) variable (V) gene segments during the germinal center (GC) stage of B lymphocyte differentiation. The mechanism of this process remains uncertain, partly due to the lack of a direct assay for hypermutation activity. In this study, a gene-specific DNA repair assay was used to compare the rate and quality of DNA repair in the mantle zone (MZ) and GC B cells at rearranged and unrearranged Ig V genes. GC B cells were distinguished from MZ B cells by a retarded repair rate specific for rearranged Ig V genes. In addition, a unique feature of GC cells after DNA repair was the appearance of predominant mutations in rearranged Ig VH5 gene PCR products. These predominant mutations also occurred in natural mutants of VH5 genes. However, repair-associated mutations reflected, at least in part, "template-jumping" during amplification of the residually damaged genomic template. Overall, these findings reflect a repair abnormality associated with the hypermutation process by the criteria of sequence- and B cell stage-specificity. We conclude that locus-specific retardation of DNA repair is a component of the hypermutation mechanism. RFLP or SSCP analysis provides a simple assay to monitor this repair abnormality as a surrogate biochemical marker for hypermutation during B cell differentiation.