The intestinal and peripheral plasma cell antibody repertoire in mice
Final Report Abstract
Using single cell immunoglobulin (Ig) gene sequencing and antibody cloning we characterized the intestinal IgA+ plasma cell (PC) antibody repertoire and aimed to determine the degree of clonal relationship with bone marrow and spleen PCs. Ig gene usage showed no overlap between intestinal IgA PCs and peritoneal B1 cells, a population of B cells that had previously been thought to contribute to the intestinal IgA repertoire. Instead, we observed clonal overlap with spleen and bone marrow plasma cells and identified PC clusters that were shared between spleen and bone marrow. This included IgA as well as IgG cells. Germ-free mice were analyzed to determine the role of bacterial colonization on the intestinal IgA antibody repertoire. As previously reported, intestinal PCs of germ-free mice were unmutated and therefore showed no signs of antigenmediated selection although class-switching was not abolished. To determine the degree of specificity of intestinal PC antibodies, we generated recombinant monoclonal antibodies from individual cells. Sequence analysis of individual LP PCIgA and PerC B1a cells showed that LP PCIgA were hypermutated, showed signs of antigen-mediated selection and substantial expansion of clonally related events. Repertoire analysis revealed a frequent usage of canonical BCRs (VH11/Vκ9 and VH12/Vκ4) in PerC B1a, which were not detected in the LP PCIgA. Antibody expression and reactivity testing showed that the microflora shapes the intestinal antibody repertoire based on antibody reactivity as we observed a higher level of polyreactivity in the LP PC compartment than in bone marrow or spleen, whereas the level of polyreactivity was lower than for PerC B1a cells. Polyreactivity included anti-bacteria reactivity, but we did not identify bacteria-specific antibodies using ELISA with a selected set of commensal bacteria. In summary, our results provide basic insight in the origin and clonal relationship of intestinal PCIgA compared to systemic B cells.
