Classical non-homologous DNA end-joining (C-NHEJ) is a major mammalian DNA double strand break (DSB) repair pathway. Deficiencies for C-NHEJ factors, such as XRCC4, abrogate lymphocyte development, owing to a strict requirement for C-NHEJ to join V(D)J recombination DSB intermediates1,2. The XRCC4-like factor (XLF) is mutated in certain immunodeficient human patients and has been implicated in C-NHEJ3,4,5,6. Yet, XLF-deficient mice have relatively normal lymphocyte development and their lymphocytes support normal V(D)J recombination5. The Ataxia Telangiectasia-Mutated protein (“ATM”) detects DSBs and activates DSB responses by phosphorylating substrates including histone H2AX7. However, ATM-deficiency causes only modest V(D)J recombination and lymphocyte developmental defects, and H2AX-deficiency does not measurably impact these processes7,8,9. Here, we show that XLF, ATM, and H2AX all have fundamental roles in processing and joining ends during V(D)J recombination; but that these roles have been masked by unanticipated functional redundancies. Thus, combined ATM/XLF-deficiency nearly blocks mouse lymphocyte development due inability to process and join chromosomal V(D)J recombination DSB intermediates. Combined XLF and ATM deficiency also severely impairs C-NHEJ, but not alternative end-joining, during IgH class switch recombination. Redundant ATM and XLF functions in C-NHEJ are mediated via ATM kinase activity and are not required for extra-chromosomal V(D)J recombination, suggesting a role for chromatin-associated ATM substrates. Correspondingly, conditional H2AX inactivation in XLF-deficient pro-B lines leads to V(D)J recombination defects associated with marked degradation of unjoined V(D)J ends, revealing that H2AX indeed has a role in this process.

SUMMARY Mutations in XLF/Cernunnos (hereafter called "XLF") cause lymphocytopenia in humans, and various studies suggest an XLF role in classical non-homologous end joining (C-NHEJ). We now find that XLF-deficient mouse embryonic fibroblasts are ionizing radiation (IR) sensitive and severely impaired for ability to support V(D)J recombination. Yet, mature lymphocyte numbers in XLF-deficient mice are only modestly decreased. Moreover, XLF-deficient pro-B lines, while IR-sensitive, carry out V(D)J recombination at nearly wild-type levels. Correspondingly, XLF/p53-double-deficient mice are not markedly prone to the pro-B lymphomas that occur in previously characterized C-NHEJ/p53-deficient mice; however, like other C-NHEJ/p53-deficient mice they still develop medulloblastomas. Despite nearly normal V(D)J recombination in developing B cells, XLF-deficient mature B cells are moderately defective for IgH class switch recombination. Together, our results implicate XLF as a C-NHEJ factor, but also indicate that developing mouse lymphocytes harbor cell type specific factors/pathways that compensate for absence of XLF function during V(D)J recombination.

V(D)J recombination assembles immunoglobulin (Ig) heavy or light chain (IgH or IgL) variable region exons in developing bone marrow B cells, while class switch recombination (CSR) exchanges IgH constant region exons in peripheral B cells. Both processes employ DNA double strand breaks (DSBs) repaired by non-homologous end-joining (NHEJ). Errors in either V(D)J recombination or CSR can initiate chromosomal translocations, including oncogenic IgH/c-myc translocations of peripheral B cell lymphomas. Collaboration between these processes also has been proposed to initiate translocations. However, occurrence of V(D)J recombination in peripheral B cells is controversial. Here, we report that activated NHEJ-deficient splenic B cells accumulate V(D)J recombination-associated IgL chromosomal breaks, as well as CSR-associated IgH breaks, often in the same cell. Moreover, IgL breaks frequently are joined to IgH breaks to form translocations, a phenomenon associated with specific IgH/IgL co-localization. IgH and c-myc also co-localize in these cells; correspondingly, introduction of frequent c-myc DSBs robustly promotes IgH/c-myc translocations. Our studies reveal peripheral B cells that attempt secondary V(D)J recombination and elucidate a role for mechanistic factors in promoting recurrent translocations in tumors.

Influenza virus hemagglutinin consists of a highly variable and immunodominant head domain and a more conserved but immunosubdominant stalk domain. We introduced seven N-linked glycosylation sites in the hemagglutinin head domain to shield the immunodominant antigenic sites. The hyperglycosylated hemagglutinin enhanced stalk-directed seroreactivity while dampening the head response in immunized mice. Upon influenza virus challenge, mice vaccinated with the hyperglycosylated hemagglutinin were better protected against morbidity and mortality than mice receiving the wild-type hemagglutinin.

Ataxia telangiectasia mutated (ATM) deficiency predisposes humans and mice to T lineage lymphomas with recurrent chromosome 14 translocations involving the T cell receptor α/δ (Tcra/d) locus. Such translocations have been thought to result from aberrant repair of DNA double-strand breaks (DSBs) during Tcra locus V(D)J recombination, and to require the Tcra enhancer (Eα) for Tcra rearrangement or expression of the translocated oncogene. We now show that, in addition to the known chromosome 14 translocation, ATM-deficient mouse thymic lymphomas routinely contain a centromeric fragment of chromosome 14 that spans up to the 5′ boundary of the Tcra/d locus, at which position a 500-kb or larger region centromeric to Tcra/d is routinely amplified. In addition, they routinely contain a large deletion of the telomeric end of one copy of chromosome 12. In contrast to prior expectations, the recurrent translocations and amplifications involve V(D)J recombination–initiated breaks in the Tcrd locus, as opposed to the Tcra locus, and arise independently of the Eα. Overall, our studies reveal previously unexpected mechanisms that contribute to the oncogenic transformation of ATM-deficient T lineage cells.