Hypomorphic Rag1 mutations alter the preimmune repertoire at early stages of lymphoid development.

Hypomorphic RAG1 mutations allowing residual T- and B-cell development have been found in patients presenting with delayed-onset combined immune deficiency with granulomas and/or autoimmunity (CID-G/AI) and abnormalities of the peripheral T- and B-cell repertoire. To examine how hypomorphic Rag1 mutations affect the earliest stages of lymphocyte development, we used CRISPR/Cas9 to generate mouse models with mutations equivalent to those found in patients with CID-G/AI. Immunological characterization showed partial development of T and B lymphocytes, with persistence of naïve cells and preserved serum immunoglobulin but impaired antibody responses and presence of autoantibodies, thereby recapitulating the phenotype seen in patients with CID-G/AI. By using high-throughput sequencing, we identified marked skewing of Igh V and Trb V gene usage in early progenitors, with a bias for productive Igh and Trb rearrangements after selection occurred and increased apoptosis of B-cell progenitors. Rearrangement at the Igk locus was impaired, and polyreactive immunoglobulin M antibodies were detected. This study provides novel insights into how hypomorphic Rag1 mutations alter the primary repertoire of T and B cells, setting the stage for immune dysregulation frequently seen in patients.

Ku70 is required for late B cell development and immunoglobulin heavy chain class switching.

Immunoglobulin (Ig) heavy chain (HC) class switch recombination (CSR) is a late B cell process that involves intrachromosomal DNA rearrangement. Ku70 and Ku80 form a DNA end-binding complex required for DNA double strand break repair and V(D)J recombination. Ku70(-/-) (K70T) mice, like recombination activating gene (RAG)-1- or RAG-2-deficient (R1T or R2T) mice, have impaired B and T cell development at an early progenitor stage, which is thought to result at least in part from defective V(D)J recombination (Gu, Y., K.J. Seidl, G.A. Rathbun, C. Zhu, J.P. Manis, N. van der Stoep, L. Davidson, H.L. Cheng, J.M. Sekiguchi, K. Frank, et al. 1997. Immunity. 7:653-665; Ouyang, H., A. Nussenzweig, A. Kurimasa, V.C. Soares, X. Li, C. Cordon-Cardo, W. Li, N. Cheong, M. Nussenzweig, G. Iliakis, et al. 1997. J. Exp. Med. 186:921-929). Therefore, to examine the potential role of Ku70 in CSR, we generated K70T mice that carry a germline Ig HC locus in which the JH region was replaced with a functionally rearranged VH(D)JH and Ig lambda light chain transgene (referred to as K70T/HL mice). Previously, we have shown that B cells from R1T or R2T mice carrying these rearranged Ig genes (R1T/HL or R2T/HL mice) can undergo CSR to IgG isotypes (Lansford, R., J. Manis, E. Sonoda, K. Rajewsky, and F. Alt. 1998. Int. Immunol. 10:325-332). K70T/HL mice had significant numbers of peripheral surface IgM+ B cells, which generated serum IgM levels similar to those of R2T/HL mice. However, in contrast to R2T/HL mice, K70T/HL mice had no detectable serum IgG isotypes. In vitro culture of K70T/HL B cells with agents that induce CSR in normal or R2T/HL B cells did lead to the induction of germline CH transcripts, indicating that initial signaling pathways for CSR were intact in K70T/HL cells. However, treatment with such agents did not lead to detectable CSR by K70T/HL B cells, and instead, led to cell death within 72 h. We conclude that Ku70 is required for the generation of B cells that have undergone Ig HC class switching. Potential roles for Ku70 in the CSR process are discussed.

Class switching in B cells lacking 3' immunoglobulin heavy chain enhancers.

The 40-kb region downstream of the most 3' immunoglobulin (Ig) heavy chain constant region gene (Calpha) contains a series of transcriptional enhancers speculated to play a role in Ig heavy chain class switch recombination (CSR). To elucidate the function of this putative CSR regulatory region, we generated mice with germline mutations in which one or the other of the two most 5' enhancers in this cluster (respectively referred to as HS3a and HS1,2) were replaced either with a pgk-neor cassette (referred to as HS3aN and HS1,2N mutations) or with a loxP sequence (referred to as HS3aDelta and HS1,2Delta, respectively). B cells homozygous for the HS3aN or HS1,2N mutations had severe defects in CSR to several isotypes. The phenotypic similarity of the two insertion mutations, both of which were cis-acting, suggested that inhibition might result from pgk-neor cassette gene insertion rather than enhancer deletion. Accordingly, CSR returned to normal in B cells homozygous for the HS3aDelta or HS1,2Delta mutations. In addition, induced expression of the specifically targeted pgk-neor genes was regulated similarly to that of germline CH genes. Our findings implicate a 3' CSR regulatory locus that appears remarkably similar in organization and function to the beta-globin gene 5' LCR and which we propose may regulate differential CSR via a promoter competition mechanism.

Integrative analysis reveals 53BP1 copy loss and decreased expression in a subset of human diffuse large B-cell lymphomas.

p53-Binding protein 1 (53BP1) encodes a critical checkpoint protein that localizes to sites of DNA double-strand breaks (DSBs) and participates in DSB repair. Mice that are 53bp1 deficient or hemizygous have an increased incidence of lymphoid malignancies. However, 53BP1 abnormalities in primary human tumors have not been described. By combining high-density single nucleotide polymorphism (HD SNP) array data and gene expression profiles, we found 9 of 63 newly diagnosed human diffuse large B-cell lymphomas (DLBCLs) with single copy loss of the chromosome 15q15 region including the 53BP1 locus; these nine tumors also had significantly lower levels of 53BP1 transcripts. 53BP1 single copy loss found with the HD SNP array platform was subsequently confirmed by fluorescence in situ hybridization. These studies highlight the role of 53BP1 copy loss in primary human DLBCLs and the value of integrative analyses in detecting this genetic lesion in human tumors.

Ig heavy chain class switching in Rag-deficient mice.

To investigate potential roles of the RAG-1 and RAG-2 gene products in Ig heavy chain class recombination (CSR), we have generated RAG-1(-/-) and RAG-2(-/-) mice which contain both a rearranged Ig HC V(D)J gene (referred to as B1-8) inserted into the endogenous Ig heavy chain (HC) locus in place of the JH segments, and a rearranged lambda1 light chain (LC) transgene (which are referred to as RAG-1(-/-)B1-8lambda and RAG-2(-/-)B1-8lambda mice respectively). The B1-8 HC gene and lambda LC genes encode proteins that associate to form a complete Ig molecule, the expression of which leads to substantial reconstitution of the peripheral B cell compartments of RAG-1(-/-)B1-8lambda and RAG-2(-/-)B1-8lambda mice. Both RAG-1(-/-)B1-8lambda and RAG-2(-/-)B1-8lambda mice have relatively normal levels of the various IgG isotypes, but greatly reduced levels of serum IgM and IgA compared to normal littermates. Furthermore, RAG-1(-/-)B1-8lambda and RAG-2(-/-)B1-8lambda B cells activated in vitro with lipopolysaccharide (LPS) or LPS plus IL-4 responded similarly to control B cells with respect to surface expression and secretion of IgG3, IgG1, IgG2b, IgG2a and IgE, but again were deficient in the secretion of IgM. Together, these findings indicate that neither RAG-1 nor RAG-2 expression is required for efficient class switching to most HC isotypes in B cells.

Interplay of p53 and DNA-repair protein XRCC4 in tumorigenesis, genomic stability and development.

XRCC4 is a non-homologous end-joining protein employed in DNA double strand break repair and in V(D)J recombination. In mice, XRCC4-deficiency causes a pleiotropic phenotype, which includes embryonic lethality and massive neuronal apoptosis. When DNA damage is not repaired, activation of the cell cycle checkpoint protein p53 can lead to apoptosis. Here we show that p53-deficiency rescues several aspects of the XRCC4-deficient phenotype, including embryonic lethality, neuronal apoptosis, and impaired cellular proliferation. However, there was no significant rescue of impaired V(D)J recombination or lymphocyte development. Although p53-deficiency allowed postnatal survival of XRCC4-deficient mice, they routinely succumbed to pro-B-cell lymphomas which had chromosomal translocations linking amplified c-myc oncogene and IgH locus sequences. Moreover, even XRCC4-deficient embryonic fibroblasts exhibited marked genomic instability including chromosomal translocations. Our findings support a crucial role for the non-homologous end-joining pathway as a caretaker of the mammalian genome, a role required both for normal development and for suppression of tumours.

Position-dependent inhibition of class-switch recombination by PGK-neor cassettes inserted into the immunoglobulin heavy chain constant region locus.

The Ig heavy chain (IgH) constant region (CH) genes are organized from 5' to 3' in the order Cmicro, Cdelta, Cgamma3, Cgamma1, Cgamma2b, Cgamma2a, Cepsilon, and Calpha. Expression of CH genes downstream of Cdelta involves class-switch recombination (CSR), a process that is targeted by germ-line transcription (GT) of the corresponding CH gene. Previously, we demonstrated that insertion of a PGK-neor cassette at two sites downstream of Calpha inhibits, in cultured B cells, GT of and CSR to a subset of CH genes (including Cgamma3, Cgamma2a, Cgamma2b, and Cepsilon) that lie as far as 120 kb upstream. Here we show that insertion of the PGK-neor cassette in place of sequences in the Igamma2b locus inhibits GT of and CSR to the upstream Cgamma3 gene, but has no major effect on the downstream Cgamma2a and Cepsilon genes. Moreover, replacement of the Cepsilon exons with a PGK-neor cassette in the opposite transcriptional orientation also inhibits, in culture, GT of and CSR to the upstream Cgamma3, Cgamma2b, and Cgamma2a genes. As with the PGK-neor insertions 3' of Calpha studied previously, the Cgamma1 and Calpha genes were less affected by these mutations both in culture and in mice, whereas the Cgamma2b gene appeared less affected in vivo. Our findings support the existence of a long-range 3' IgH regulatory region required for GT of and CSR to multiple CH genes and suggest that PGK-neor cassette insertion into the locus short circuits the ability of this region to facilitate GT of dependent CH genes upstream of the insertion.

DNA ligase IV deficiency in mice leads to defective neurogenesis and embryonic lethality via the p53 pathway.

DNA ligase IV (LIG4) is a nonhomologous end-joining (NHEJ) protein used for V(D)J recombination and DNA repair. In mice, Lig4 deficiency causes embryonic lethality, massive neuronal apoptosis, arrested lymphogenesis, and various cellular defects. Herein, we assess potential roles in this phenotype for INK4a/ARF and p53, two proteins implicated in apoptosis and senescence. INK4a/ARF deficiency rescued proliferation/senescence defects of Lig4-deficient fibroblasts but not other phenotypic aspects. In contrast, p53 deficiency rescued embryonic lethality, neuronal apoptosis, and fibroblast proliferation/senescence defects but not lymphocyte development or radiosensitivity. Young Lig4/p53 double null mice routinely died from pro-B lymphomas. Thus, in the context of Lig4 deficiency, embryonic lethality and neuronal apoptosis likely result from a p53-dependent response to unrepaired DNA damage, and neuronal apoptosis and lymphocyte developmental defects can be mechanistically dissociated.

Growth retardation and leaky SCID phenotype of Ku70-deficient mice.

Ku70, Ku80, and DNA-PKcs are subunits of the DNA-dependent protein kinase (DNA-PK), an enzyme implicated in DNA double-stranded break repair and V(D)J recombination. Our Ku70-deficient mice were about 50% the size of control littermates, and their fibroblasts were ionizing radiation sensitive and displayed premature senescence associated with the accumulation of nondividing cells. Ku70-deficient mice lacked mature B cells or serum immunoglobulin but, unexpectedly, reproducibly developed small populations of thymic and peripheral alpha/beta T lineage cells and had a significant incidence of thymic lymphomas. In association with B and T cell developmental defects, Ku70-deficient cells were severely impaired for joining of V(D)J coding and recombination signal sequences. These unanticipated features of the Ku70-deficient phenotype with respect to lymphocyte development and V(D)J recombination may reflect differential functions of the three DNA-PK components.