Homologous recombination (HR) and nonhomologous end-joining (NHEJ) are mechanistically distinct DNA repair pathways that contribute substantially to double-strand break (DSB) repair in mammalian cells. We have combined mutations in factors from both repair pathways, the HR protein Rad54 and the DNA-end-binding factor Ku80, which has a role in NHEJ. Rad54 −/− Ku80−/− mice were severely compromised in their survival, such that fewer double mutants were born than expected, and only a small proportion of those born reached adulthood. However, double-mutant mice died at lower frequency from tumors than Ku80 single mutant mice, likely as a result of rapid demise at a young age from other causes. When challenged with an exogenous DNA damaging agent, ionizing radiation, double-mutant mice were exquisitely sensitive to low doses. Tissues and cells from double-mutant mice also showed indications of spontaneous DNA damage. Testes from some Rad54 −/− Ku80−/− mice displayed enhanced apoptosis and reduced sperm production, and embryonic fibroblasts from Rad54 −/− Ku80−/− animals accumulated foci of ␥-H2AX, a marker for DSBs. The substantially increased DNA damage response in the double mutants implies a cooperation of the two DSB repair pathways for survival and genomic integrity in the animal.[Keywords: DNA double-strand break; homologous recombination; nonhomologous end-joining; Ku80, Rad54] Supplemental material is available at http://www.genesdev.org.

In an attempt to provide a global picture of the TCR repertoire diversity of a chronic T cell response against a common Ag, we performed an extensive TCR analysis of cells reactive against a dominant HLA-A2-restricted EBV epitope (hereafter referred to as GLC/A2), obtained after sorting PBL or synovial fluid lymphocytes from EBV-seropositive individuals using MHC/peptide multimers. Although TCR β-chain diversity of GLC/A2+ T cells was extensive and varied greatly from one donor to another, we identified in most cell lines several recurrent Vβ subsets (Vβ2, Vβ4, and Vβ16 positive) with highly conserved TCRβ complementarity-determining region 3 (CDR3) length and junctional motifs, which represented from 11 to 98% (mean, 50%) of GLC/A2-reactive cells. While TCR β-chains expressed by these subsets showed limited CDR1, CDR2, and CDR3 homology among themselves, their TCR α-chains comprised the same TCRAV region, thus suggesting hierarchical contribution of TCR α-chain vs TCR β-chain CDR to recognition of this particular MHC/peptide complex. The common occurrence of T cell clonotypes with public TCR features within GLC/A2-specific T cells allowed their direct detection within unsorted PBL using ad hoc clonotypic primers. These results, which suggest an unexpectedly high contribution of public clonotypes to the TCR repertoire against a dominant epitope, have several implications for the follow-up and modulation of T cell-mediated immunity.

The T-cell receptor (TCR) a locus is thought to undergo multiple cycles of secondary rearrangements that maximize the generation of ab T cells. Taking advantage of the nucleotide sequence of the human Va and Ja segments, we undertook a locus-wide analysis of TCRa gene rearrangements in human ab T-cell clones. In most clones, VaJa rearrangements occurred on both homologous chromosomes and, remarkably, resulted in the use of two neighboring Ja segments. No such interallelic coincidence was found for the position of the two rearranged Va segments, and there was only a loose correlation between the 5¢ or 3¢ chromosomal position of the Va and Ja segments used in a given rearrangement. These observations question the occurrence of extensive rounds of secondary Va®Ja rearrangements and of a coordinated and polarized usage of the Va and Ja libraries. Fluorescence in situ hybridization analysis of developing T cells in which TCRa rearrangements are taking place showed that the interallelic positional coincidence in Ja usage cannot be explained by the stable juxtaposition of homologous Ja clusters.

Rag2 plays an essential role in the generation of antigen receptors. Mutations that impair Rag2 function can lead to severe combined immunodeficiency (SCID), a condition characterized by complete absence of T and B cells, or Omenn syndrome (OS), a form of SCID characterized by the virtual absence of B cells and the presence of oligoclonal autoreactive T cells. Here, we present a comparative study of a panel of mutations that were identified in the noncanonical plant homeodomain (PHD) of Rag2 in patients with SCID or OS. We show that PHD mutant mouse Rag2 proteins that correspond to those found in these patients greatly impaired endogenous recombination of Ig gene segments in a Rag2-deficient pro-B cell line and that this correlated with decreased protein stability, impaired nuclear localization, and/or loss of the interaction between Rag2 and core histones. Our results demonstrate that point mutations in the PHD of Rag2 compromise the functionality of the entire protein, thus explaining why the phenotype of cells expressing PHD point mutants differs from those expressing core Rag2 protein that lacks the entire C-terminal region and is therefore devoid of the regulation imposed by the PHD. Together, our findings reveal the various deleterious effects of PHD Rag2 mutations and demonstrate the crucial role of this domain in regulating antigen receptor gene assembly. We believe these results reveal new mechanisms of immunodeficiency in SCID and OS. Introduction V(D)J recombination is the site-specific DNA rearrangement process that assembles the B cell receptor and TCR genes during lymphoid development. Recombination is initiated by the lymphoid-specific Rag1 and Rag2 recombinase (1, 2), which introduces double-strand DNA breaks at recombination signal sequences (RSSs) flanking variable (V), diversity (D), and junction (J) gene segments spread along the Ig and TCR loci. Subsequently, the ubiquitous nonhomologous end-joining (NHEJ) machinery, which includes Ku70/Ku80, DNA-PKcs, Artemis, XRCC4, LigaseIV, and Cernunnos-XLF, repairs those lesions to form V(D)J coding joints and signal joints (3, 4). The recombination process is tightly regulated, occurring at specific stages of development and in specific cell types (e.g., Ig and TCR genes are rearranged in B and T cells, respectively). This process takes place in a temporal manner, with Ig heavy chain rearrangements preceding Ig light chain rearrangements and D-to-J rearrangements preceding V-to-DJ rearrangements (5).V(D)J recombination is critical for proper immune function and, when impaired, generally results in the arrest of both B and T cell development, leading to severe combined immunodeficiency (SCID). Accordingly, mutations in Rag1, Rag2, or NHEJ factors have been described in patients characterized by a complete absence

Expression of a β-chain, as a pre-TCR, in T cell precursors prevents further rearrangements on the alternate β allele through a strict allelic exclusion process and enables precursors to undergo differentiation. However, whether allelic exclusion applies to the TCRδ locus is unknown and the role of the γδ TCR in γδ lineage commitment is still unclear. Through the analysis of the rearrangement status of the TCRγ, δ, and β loci in human γδ T cell clones, expressing either the TCR Vδ1 or Vδ2 variable regions, we show that the rate of partial rearrangements at the δ locus is consistent with an allelic exclusion process. The overrepresentation of clones with two functional TCRγ chains indicates that a γδ TCR selection process is required for the commitment of T cell precursors to the γδ lineage. Finally, while complete TCRβ rearrangements were observed in several Vδ2 T cell clones, these were seldom found in Vδ1 cells. This suggests a competitive αβ/γδ lineage commitment in the former subset and a precommitment to the γδ lineage in the latter. We propose that these distinct behaviors are related to the developmental stage at which rearrangements occur, as suggested by the patterns of accessibility to recombination sites that characterize the Vδ1 and Vδ2 subsets.