Relevance of Tracts of Framework Mutations in IgM Plasma Cells

项目总结 体液免疫的特点是对抗原的长期反应。此响应的持续时间为 依赖于长寿的浆细胞，这些细胞主要位于骨髓中，并处于切换状态 同型。然而，我们最近发现了一群长期存活的IgM浆细胞，它们居住在红色 表现出非典型突变特征的脾脏。我们发现突变主要发生在 VH基因的框架区域与免疫球蛋白原细胞不同，后者的突变主要位于 互补决定区(CDR)。在进一步分析这些突变后，我们发现很强的 这些突变发生在多个IgM浆细胞中并似乎共享的证据 来自单个动物的克隆。此外，这些区域在位置和身份上与其他高度一致的 同源VH基因，因此我们假设这些突变不是随机的，而是以 其他VH基因则通过基因转换的过程发生。我们假设(1)VH基因在 相反的等位基因作为基因转换的供体序列，在表达的VH基因中引入突变 以及(2)这些突变发生在骨架区域，因此它们保持CDR特异性，但改变 CDR构象使CDR能够达到以下漂移和异亚型流感病毒株的靶点 免疫接种。到目前为止，我们已经分析了µ重链，还没有对成对的Heavy和Light进行排序 锁链。在这个项目中，我们的目标是(1)使用高通量配对的IGL分析来表征 数千个IgM浆细胞的重链和轻链使用情况以及每个细胞的突变情况，(2) 分析框架突变在流感抗体结合和交叉保护中的作用 模型，以及(3)确定Balb/c x C57BL/6 F1中的基因转换是否利用另一个等位基因进行基因转换 老鼠。总而言之，从这些研究中获得的知识将确定IgM中框架突变的作用 并可能确定基因转换是重排的多样化的一种机制 免疫球蛋白基因座。

PROJECT SUMMARY Humoral immunity is marked by long-lived responses against antigens. The duration of this response is dependent on long-lived plasma cells that are primarily located in the bone marrow and are of switched isotype. However, we have recently identified a population of long-lived IgM plasma cells that reside in the red pulp of the spleen that display an atypical mutation profile. We find that mutations primarily occur in the framework regions of the VH gene unlike IgG plasma cells, where mutations are predominantly located within the complementarity determining regions (CDRs). Upon further analysis of these mutations, we find strong evidence that these mutations occur in tracts and appear to be shared between multiple IgM plasma cell clones from individual animals. Further, these tracts completely match in position and identity with other highly homologous VH genes and therefore we posit that these mutations are not random but are templated from other VH genes and occur through the process of gene conversion. We hypothesize that (1) VH genes on the opposite allele serve as donor sequences for gene conversion, introducing mutations in expressed VH genes and (2) that these mutations occur in the framework region such that they preserve CDR specificity but alter CDR conformation allowing CDRs to reach targets on drifted and heterosubtypic influenza strains following immunization. To date, we have analyzed the µ heavy chain and have yet to sequence paired heavy and light chains. In this project, our goals are to (1) use a high throughput paired IgH:IgL analysis to characterize the heavy and light chain usage and mutation profile on a per cell basis from thousands of IgM plasma cells, (2) analyze the contribution of framework mutations to antibody binding and cross protection in an influenza model, and (3) identify if gene conversion utilizes the other allele for gene conversion in Balb/c x C57BL/6 F1 mice. Taken together, knowledge gained from these studies will identify the role of framework mutations in IgM plasma cells and could potentially identify gene conversion as a mechanism of diversification of the rearranged IgV loci.