Role of the immunoglobulin DQ52 DH gene segment in fetal immunosuppression

项目摘要 我们的目标是解决胚胎和胎儿期间体液免疫反应的控制机制。 发展。在个体发育过程中，各种淋巴组织和器官所需的B细胞 作为新的细胞类型和非淋巴样细胞，顺序地遇到日益复杂的自身抗原阵列 组织和器官出现了。胚胎B细胞产生潜在致病作用的风险很大 自身反应性免疫球蛋白(IGs)可以通过重要的发育检查点，从而存活到 在这些‘neo’抗原最终表达后被激活。不出所料，有强有力的证据表明 体液免疫反应在哺乳动物胚胎和胎儿中被选择性地抑制。因此，尽管在 理论免疫球蛋白和T细胞受体(TCR)谱似乎是以严格随机的方式产生的，通过 随机VDJ重排和N加成；在实践中，VDJ重排和N加成都是在 子宫限制免疫球蛋白和TCR的多样性。在人类和BALB/c中分别有27个和13个功能基因片段 老鼠。我们之前已经证明，来自人类和小鼠胚胎B细胞前体细胞的VDJ是 富含DQ52基因片段(人的D7-27和小鼠的D4-01)。然而，在出生后，两者 物种对DQ52的使用较少。因此，对Ig Heavy互补决定区3的调节 (H)包含DQ52基因片段的链(CDR-H3)不是偶然出现的，必须是一个主要的 胚胎和早期胎儿控制曲目的元素。我们之前的研究已经表明，这些序列 多样性(D)基因片段既界定了CDR多样性的范围，又指导甚至决定了CDR多样性的模式 表位识别和抗体产生，从而影响宿主对感染和自身免疫的抵抗力 疾病。DQ52是人与小鼠之间高度保守的水解酶。有趣的是，DQ52不同 与其他D的氨基酸含量显著不同，最显著的区别是没有 酪氨酸和甘氨酸的浓缩。在这一点上，DQ52与TCRDβ更相似，而不是另一个IGDH，使 宫内DQ52CDR-H3谱系更像TCRCDR-B3，而不是成人βCDR-H3。TCR的功能更像 多反应传感器比单特异性效应器。因此，在CDR-H3中使用DQ52可能具有以下效果 降低对抗原的单特异性和亲和力，并减少抗体的产生。这些基本原则 观察结果引导我们提出假设，即Ig DQ52在个体发育过程中的作用是促进生产 同时作为一种免疫抑制剂。来检验这一假设，并 为了提供概念验证，我们建议(A)使用CRISPR/Cas9技术来创建其DH基因座的小鼠 只包含DQ52(ΔD-DQ52)，(B)测试DQ52的使用是否有助于B细胞的产生，以及(C)测试 使用DQ52会导致抗体产生减少。这些研究将填补我们对 胎儿体液免疫反应的免疫抑制的机制，这是一个主要的 导致早产儿感染风险增加和制定宫内疫苗接种策略的因素。

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.