John T. Collins

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.

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.

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.

The Role of Germline Promoters and I Exons in Cytokine-Induced Gene-Specific Class Switch Recombination

Germline transcription precedes class switch recombination (CSR). The promoter regions and I exons of these germline transcripts include binding sites for activation- and cytokine-induced transcription factors, and the promoter regions/I exons are essential for CSR. Therefore, it is a strong hypothesis that the promoter/I exons regions are responsible for much of cytokine-regulated, gene-specific CSR. We tested this hypothesis by swapping the germline promoter and I exons for the murine γ1 and γ2a H chain genes in a transgene of the entire H chain C-region locus. We found that the promoter/I exon for γ1 germline transcripts can direct robust IL-4–induced recombination to the γ2a gene. In contrast, the promoter/I exon for the γ2a germline transcripts works poorly in the context of the γ1 H chain gene, resulting in expression of γ1 H chains that is <1% the wild-type level. Nevertheless, the small amount of recombination to the chimeric γ1 gene is induced by IFN-γ. These results suggest that cytokine regulation of CSR, but not the magnitude of CSR, is regulated by the promoter/I exons.

Oncogenic Myc translocations are independent of chromosomal location and orientation of the immunoglobulin heavy chain locus

Many tumors are characterized by recurrent translocations between a tissue-specific gene and a proto-oncogene. The juxtaposition of the Ig heavy chain gene and Myc in Burkitt’s lymphoma and in murine plasmacytoma is a classic example. Regulatory elements within the heavy chain constant region locus are required for Myc translocation and/or deregulation. However, many genes are regulated by cis-acting elements at distances up to 1,000 kb outside the locus. Such putative distal elements have not been examined for the heavy chain locus, particularly in the context of Myc translocations. We demonstrate that a transgene containing the Ig heavy chain constant region locus, inserted into five different chromosomal locations, can undergo translocations involving Myc. Furthermore, these translocations are able to generate plasmacytomas in each transgenic line. We conclude that the heavy chain constant region locus itself includes all of the elements necessary for both the translocation and the deregulation of the proto-oncogene.

Induced Expression of Murine γ2a by CD40 Ligation Independently of IFN-γ

IgG2a, with γ2a H chains, is important for protection against viruses and other intracellular pathogens. Although a large portion of IgG2a expression is dependent upon IFN-γ, some germline transcription and switch recombination to the murine γ2a H chain gene expression are independent of IFN-γ. We found that agonistic anti-CD40 Abs injected into IFN-γ-deficient mice induce a >200-fold increase in the amount of serum Ig2a, while other Ig isotypes are increased by 16-fold or less. In vitro, ligation of CD40 on B cells, without the addition of other B cell activators or cytokines, results in germline transcription and switch recombination that are largely restricted to the γ2a gene. These results suggest that some immune responses to infectious agents can result in large amounts of IgG2a expression through ligation of CD40, without the expression of IFN-γ by Th1 or other cells.

Transgenes of the mouse immunoglobulin heavy chain locus, lacking distal elements in the 3' regulatory region, are impaired for class switch recombination.

The immunoglobulin heavy (H) chain class switch is mediated by a deletional recombination event between µ and γ, α, or ε constant region genes. This recombination event is upregulated during immune responses by a regulatory region that lies 3′ of the constant region genes. We study switch recombination using a transgene of the entire murine H chain constant region locus. We isolated two lines of mice in which the H chain transgenes were truncated at their 3′ ends. The truncation in both transgenic lines results in deletion of the 3′-most enhancer (HS4) and a region with insulator-like structure and activities. Even though both truncated transgenes express the µ H chain gene well, they undergo very low or undetectable switch recombination to transgenic γ and α constant region genes. For both transgenic lines, germline transcription of some H chain constant regions genes is severely impaired. However, the germline transcription of the γ1 and γ2a genes is at wild type levels for the transgenic line with the larger truncation, but at reduced levels for the transgenic line with the smaller truncation. The dramatic reduction in class switch recombination for all H chain genes and the varied reduction in germline transcription for some H chain genes could be caused by (i) insertion site effects or (ii) deletion of enhancer elements for class switch recombination and transcription, or (iii) a combination of both effects.