Wesley A. Dunnick

A Murine Model of Nijmegen Breakage Syndrome

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by microcephaly, immunodeficiency, and predisposition to hematopoietic malignancy. The clinical and cellular phenotypes of NBS substantially overlap those of ataxia-telangiectasia (A-T). NBS is caused by mutation of the NBS1 gene, which encodes a member of the Mre11 complex, a trimeric protein complex also containing Mre11 and Rad50. Several lines of evidence indicate that the ataxia-telangiectasia mutated (ATM) kinase and the Mre11 complex functionally interact. Both NBS and A-T cells exhibit ionizing radiation (IR) sensitivity and defects in the intra S phase checkpoint, resulting in radioresistant DNA synthesis (RDS)-the failure to suppress DNA replication origin firing after IR exposure. NBS1 is phosphorylated by ATM in response to IR, and this event is required for activation of the intra S phase checkpoint (the RDS checkpoint). We derived a murine model of NBS, the Nbs1(DeltaB/DeltaB) mouse. Nbs1(DeltaB/DeltaB) cells are phenotypically identical to those established from NBS patients. The Nbs1(DeltaB) allele was synthetically lethal with ATM deficiency. We propose that the ATM-Mre11 complex DNA damage response pathway is essential and that ATM or the Mre11 complex serves as a nexus to additional components of the pathway.

Mutations, duplication, and deletion of recombined switch regions suggest a role for DNA replication in the immunoglobulin heavy-chain switch

The heavy-chain switch from immunoglobulin M (IgM) expression to IgA expression is mediated by a recombination event between segments of DNA called switch regions. The switch regions lie two to six kilobases upstream of the mu and alpha constant region coding segments. Switch recombination to IgA expression results in a recombinant mu-alpha switch region upstream of the expressed alpha constant region gene. We have characterized the products of switch recombination by a lymphoma cell line, I.29. Two sets of molecular clones represent the expected products of simple mu to alpha switches. Five members of a third set of molecular clones share the same recombination site in both the mu and the alpha switch regions, implying that the five molecular clones were derived from a single switch recombination event. Surprisingly, the five clones fall into two sets of sequences, which differ from each other by several point mutations and small deletions. Duplication of switch region sequences are also found in these five molecular clones. An explanation for these data is that switch recombination involves DNA synthesis, which results in nucleotide substitutions, small deletions, and duplications.

Switch recombination and somatic hypermutation are controlled by the heavy chain 3′ enhancer region

Both class switch recombination (CSR) and somatic hypermutation (SHM) require transcription and the trans-acting factor activation-induced cytidine deaminase (AID), and must be up-regulated during antigen-dependent differentiation of B lymphocytes. To test the role of the heavy chain 3′ enhancers in both CSR and SHM, we used a BAC transgene of the entire heavy chain constant region locus. Using Cre-loxP recombination to delete a 28-kb region that contains the four known 3′ heavy chain enhancers, we isolated lines of BAC transgenic mice with an intact heavy chain locus and paired lines in the same chromosomal insertion site lacking the 3′ enhancers. Intact heavy chain transgenes undergo CSR to all heavy chain genes and mutate their transgenic VDJ exon. In paired transgenes lacking the 3′ enhancer region, CSR to most heavy chain genes is reduced to ∼1% of the levels for intact heavy chain loci; SHM is also reduced. Finally, we find that in B cells with a transgene lacking the 3′ enhancers, interchromosomal recombination between the transgenic VDJ exon and the endogenous heavy chain C genes is more easily detected than CSR within the transgene.

Enhancement of humoral immunity by interleukin-12.

We have found that IL-12 treatment of mice leads to long-lasting enhancement in production of most antibody isotypes in conventional B-cell responses. Initial recruitment of new B-cell clones into the response is mediated by IFN-gamma, but subsequent enhancement of Ig secretion appears to be IFN-gamma-independent. We have further found that activated B cells can directly bind IL-12. Taken together, our results suggest a two-step model for the role of IL-12 in enhancement of humoral immunity. Initially, IL-12 induces production of IFN-gamma from Th1 and NK cells. Enough cytokine can be produced from either cell type to then mediate gamma 2a heavy chain isotype switching as well as temporary suppression of IgG1 production. IL-12 further stimulates post-switched cells, including cells producing IgG1, to secrete greatly increased amounts of antibody. This step is not mediated by IFN-gamma but might be due to direct IL-12 binding to activated B lymphocytes. Depletion of B1 cells by IL-12 may further enhance antibody responsiveness since B1 cells are known to competitively inhibit Ig secretion by conventional B cells. The end result is that IL-12 causes a generalized upregulation in production of all antibodies and therefore acts as a strong adjuvant for humoral as well as cellular immunity.

Copy choice mechanism of immunoglobulin heavy-chain switch recombination.

The immunoglobulin heavy-chain switch is mediated by a recombination event between DNA switch regions associated with donor and recipient constant-region genes. We have determined that the mutations which can be found in some switch regions after recombination appear to arise on only one strand of DNA. This result suggests that switch recombination involves error-prone synthesis of one DNA strand and ligation of the other strand from preexisting DNA.

The 3′ end of the heavy chain constant region locus enhances germline transcription and switch recombination of the four γ genes

The switch in immunoglobulin (Ig) heavy chain class is preceded by germline transcription and then mediated by a DNA recombination event. To study germline transcription and class switch recombination we used transgenic mice with a 230-kilobase bacterial artificial chromosome that included a rearranged VDJ gene and the entire heavy chain constant region locus. In addition to several lines with intact transgenes, we identified two lines in which the heavy chain locus transgene lacked Cα and everything 3′ of it, including the regulatory elements HS3a, HS1-2, HS3b, and HS4. B cells from both lines with the truncated transgenes make abundant transgenic (Tg) VDJCμ transcripts and IgM protein. Deletion of the 3′ end of the locus results in dramatically reduced expression of both germline transcripts and switched VDJCH transcripts of the γ3, γ2b, γ2a, and ɛ genes. In addition, the transgenes lacking the 3′ end of the locus express reduced amounts of γ1 germline transcripts and 2–3% of the amount of Tg IgG1 in tissue culture compared with intact transgenes. Finally, switch recombination to γ1 is undetectable in the transgenes lacking the 3′ elements, as measured by digestion circularization–polymerase chain reaction or by the expression of VDJCγ1 transcripts.

Receptors and Counterreceptors Involved in NK-B Cell Interactions

In addition to the well-documented effect of NK cells on B cell differentiation via their ability to secrete IFN-γ, NK cells can also induce, via direct cell-cell interactions, germline transcripts (Iγ2a) necessary for switch recombination to IgG2a. Analysis of the ligand-receptor pairs that could be involved in this induction revealed that the expression of CD48 on B cells is crucial for the induction. NK cells from mice with targeted deletions of either the CD2 or the CD244 gene, both of which encode ligands for CD48, are compromised in their ability to induce B cell Iγ2a expression. Interestingly, although CD244 can bind to CD48 with a higher affinity, the ability of NK cells from CD244−/− mice to stimulate Iγ2a is not as compromised as NK cells from CD2−/− mice. Despite the difference between cell surface receptors that are stimulated by NK cells vs those stimulated by the combination of LPS and IFN-γ, we show in this study that the initiation of γ2a germline transcription is regulated by similar cis-acting elements located at the 3′ end of the IgH locus. However, NK cells cannot induce the final steps of switch recombination resulting in the production of mature mRNA from recombined DNA. Our findings suggest that these different signaling pathways converge on regulatory elements that are common to germline transcription; however, because NK induction does not result in the final steps of switch recombination, some signals initiated by LPS plus IFN-γ are not induced by NK cells.

Germline Transcription and Switch Recombination of a Transgene Containing the Entire H Chain Constant Region Locus: Effect of a Mutation in a STAT6 Binding Site in the γ1 Promoter

The switch (S) in H chain class is preceded by germline transcription and then mediated by a DNA recombination event. One of the impediments toward understanding the mechanism is the lack of a system in which a recombinant DNA molecule undergoes cytokine-regulated class S recombination. To study class S recombination, we used transgenic mice with a 230-kb bacterial artificial chromosome that included a rearranged VDJ gene and the entire murine H chain constant region locus. We found that both germline transcription and S recombination to the transgenic γ1 H chain gene were regulated by IL-4 like that of the endogenous genes. In mice with two or more copies of the H chain locus transgene, both germline transcripts and S recombination took place at levels comparable to those from the endogenous loci. We also prepared a version of the transgene with a 4-bp mutation in a STAT6 binding site in the γ1 promoter region. On the average, this mutation reduced germline transcription by 80%, but did not change the amount of S recombination in vitro. Among both the wild-type and mutant transgenes, we found no significant correlation between the amount of germline transcripts and the amount of S recombination. We infer that the physiologic level of germline transcription of the γ1 gene is in excess over the amount required for efficient S recombination.

Interruption of two immunoglobulin heavy-chain switch regions in murine plasmacytoma P3.26Bu4 by insertion of retroviruslike element ETn.

A number of moderately reiterated murine genetic elements have been shown to have structures like those of retroviral proviruses. These elements are thought to be transposons, although little evidence of their transposability exists. Two members of one of these families of reiterated elements, the ETn family, have inserted into separate immunoglobulin heavy-chain switch regions in the plasmacytoma P3.26Bu4. Switch regions are those DNA segments associated with each immunoglobulin heavy-chain gene in which the somatic recombinations that accompany the heavy-chain switch occur. This role in somatic recombination may be relevant to the ETn insertions into the switch regions in P3.26Bu4 DNA. P3.26Bu4 and a number of other B-lineage cells contain ETn transcripts.

Enhancement of Antibody Class-Switch Recombination by the Cumulative Activity of Four Separate Elements

Class-switch recombination of Ab isotype is mediated by a recombinational DNA deletion event and must be robustly upregulated during Ag-driven differentiation of B cells. The enhancer region 3′ of the Cα gene is important for the upregulation of switch recombination. Using a transgene of the entire H chain C region locus, we demonstrate in this study that it is the four 3′ enhancer elements themselves (a total of 4.7 kb) that are responsible for the upregulation rather than the 24 kb of DNA in between them. Neither allelic exclusion nor transgenic μ expression is reduced by deletion of the four 3′ enhancers. We also test deletions of two or three of the 3′ enhancers and show that deletion of more 3′ enhancers results in a progressive reduction in both switch recombination and germline transcription of all H chain genes. Nevertheless, we find evidence for special roles for some 3′ enhancers; different H chain genes are affected by different 3′ enhancer deletions. Thus, we find that the dramatic induction of class-switch recombination during Ag-driven differentiation is the result of an interaction among four separated regulatory elements.