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 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.
Following vaccination, we observe an atypical pattern of mutation in a novel cell type that secretes antibodies for a lifetime. Upon further analysis, it appears that these cells undergo a novel pathway of mutation. In this project, we investigate the contribution of these mutations to protection against influenza virus and also the mechanism by which these cells mutate.
