Relevance of Tracts of Framework Mutations in IgM Plasma Cells

IgM 浆细胞中框架突变区的相关性

• 批准号: 9355461
• 负责人: Gordon Alexander Dale
• 金额: $ 4.9万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-08 至 2019-08-07
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9355461
• 关键词: Acute; Address; Affect; Affinity; Alleles; Animals; Antibodies; Antigens; Binding; Biological Models; Bone Marrow; Cells; Characteristics; Clone Cells; Complementarity Determining Regions; Computer software; Data; Emulsions; Event; Exhibits; Exposure to; Framework Regions; Gene Conversion; Genealogical Tree; Genes; Goals; Humoral Immunities; Immune; Immunity; Immunization; Immunize; Immunoglobulin Class Switching; Immunoglobulin G; Immunoglobulin M; Inbred BALB C Mice; Individual; Infection; Influenza; Influenza A Virus, H1N1 Subtype; Joints; Knowledge; Lead; Life; Light; Link; Linkage Disequilibrium; Modeling; Molecular Conformation; Monoclonal Antibodies; Mus; Mutate; Mutation; Mutation Analysis; Pathway interactions; Pattern; Phase; Plasma Cells; Population; Positioning Attribute; Processed Genes; Puerto Rico; Role; Serological; Silent Mutation; Somatic Mutation; Source; Specificity; Spleen; Splenic Red Pulp; Structure of germinal center of lymph node; Testing; Time; Trees; V(D)J Recombination; Vaccination; Vaccines; Virus; Virus Diseases; Work; base; cell type; influenzavirus; microbial; next generation sequencing; novel; pathogen; response; three dimensional structure

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.

项目摘要 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 依赖于主要位于骨髓中且具有转换功能的长寿浆细胞 同种型然而，我们最近发现了一群长寿的IgM浆细胞，它们位于红细胞中， 脾髓显示出非典型的突变特征。我们发现突变主要发生在 与IgG浆细胞不同，VH基因的框架区突变主要位于 互补决定区（CDR）。通过对这些突变的进一步分析，我们发现 证据表明，这些突变发生在束，似乎是共享之间的多个IgM浆细胞 从动物个体中克隆。此外，这些大片完全匹配的位置和身份与其他高度 同源VH基因，因此我们证实这些突变不是随机的，而是从 其他VH基因，并通过基因转换过程发生。我们假设（1）VH基因在 相反的等位基因用作基因转换的供体序列，在表达的VH基因中引入突变 和（2）这些突变发生在框架区，使得它们保留CDR特异性，但改变 CDR构象允许CDR在以下情况下到达漂移和异亚型流感毒株上的靶标 次免疫到目前为止，我们已经分析了µ重链，还没有对配对的重链和轻链进行测序。 店在这个项目中，我们的目标是（1）使用高通量配对IgH：IgL分析来表征 来自数千个IgM浆细胞的基于每个细胞的重链和轻链使用和突变谱，（2） 分析框架突变对流感中抗体结合和交叉保护的贡献 模型，以及（3）鉴定基因转换是否利用Balb/c x C57 BL/6 F1中的另一个等位基因进行基因转换 小鼠总之，从这些研究中获得的知识将确定IgM中框架突变的作用。 浆细胞，并有可能将基因转换确定为重排细胞多样化的机制 IgV基因座。