Role of the immunoglobulin DQ52 DH gene segment in fetal immunosuppression

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Project Summary Our goal is to address the mechanisms that control the humoral immune response during embryonic and fetal development. During ontogeny, the B cells that are needed to populate the various lymphoid tissues and organs sequentially encounter an increasingly complex array of self-antigens as new cell types and non-lymphoid tissues and organs appear. There is a significant risk that embryonic B cells producing potentially pathogenic autoreactive immunoglobulins (Igs) could pass essential developmental checkpoints and thus survive to be activated after these `neo' antigens are ultimately expressed. Unsurprisingly, there is strong evidence that the humoral immune response is selectively suppressed in mammalian embryos and fetuses. Thus, although in theory Ig and T cell receptor (TCR) repertoires appear to be generated in a strictly stochastic fashion through random VDJ rearrangement and N addition; in practice both VDJ rearrangement and N addition are regulated in utero to restrict Ig and TCR diversity. There are 27 functional DH gene segments in humans and 13 in BALB/c mice. We previously showed that VDJ joins from both human and mouse embryonic B cell progenitors were enriched for use of the DQ52 gene segment (D7-27 in human and D4-01 in mouse). After birth, however, both species use DQ52 sparingly. Consequently, regulation of complementarity-determining region 3 of the Ig heavy (H) chain (CDR-H3), which contains the DQ52 gene segment, does not occur by chance and must be a major element of embryonic and early fetal repertoire control. Our prior research has shown that the sequence of the diversity (D) gene segment both delimits the range of CDR diversity and directs, or even dictates, patterns of epitope recognition and antibody production, thereby influencing host resistance to infection and autoimmune disease. DQ52 is the most highly conserved DH between human in mouse. Interestingly, DQ52 differs dramatically in amino acid content from the rest of the Ds, the most striking difference being the absence of tyrosine and enrichment for glycine. In this regard, DQ52 is more similar to TCR Dβ than the other Ig DH, making the in utero DQ52 CDR-H3 repertoire more like TCRβ CDR-B3 than adult Ig CDR-H3. TCRs function more like polyreactive sensors than monospecific effectors. Thus, using DQ52 in CDR-H3 could have the effect of reducing monospecificity and affinity for antigen, and reducing antibody production. These fundamental observations led us to our hypothesis that the role of Ig DQ52 during ontogeny is to promote the production of B cells while simultaneously serving as an immune suppressant DH. To test this hypothesis and to provide proof-of-concept, we propose to (a) use CRISPR/Cas9 technology to create a mouse whose DH locus contains only DQ52 (ΔD-DQ52), (b) test whether use of DQ52 facilitates B cell production, and (c) test whether use of DQ52 results in reduced antibody production. These studies will fill a major gap in our knowledge about the mechanisms that underlie immune suppression of the fetal humoral immune response, which is a major factor in the increased risk of infection in premature infants and in developing in utero vaccination strategies.