Massively Parallel Sequencing of Peritoneal and Splenic B Cell Repertoires Highlights Unique Properties of B-1 Cell Antibodies

Abstract

B-1 cells are a unique subset of B cells that are positively selected for expressing autoreactive BCRs. We isolated RNA from peritoneal (B-1a, B-1b, B-2) and splenic (B-1a, marginal zone, follicular) B cells from C57BL/6 mice and used 5'-RACE to amplify the IgH V region using massively parallel sequencing. By analyzing 379,000 functional transcripts, we demonstrate that B-1a cells use a distinct and restricted repertoire. All B-1 cell subsets, especially peritoneal B-1a cells, had a high proportion of sequences without N additions, suggesting predominantly prenatal development. Their transcripts differed markedly and uniquely contained V11 and V12 genes, which were rearranged only with a restricted selection of D and J genes, unlike other V genes. Compared to peritoneal B-1a, the peritoneal B-1b repertoire was larger, had little overlap with B-1a, and most sequences contained N additions. Similarly, the splenic B-1a repertoire differed from peritoneal B-1a sequences, having more unique sequences and more frequent N additions, suggesting influx of B-1a cells into the spleen from nonperitoneal sites. Two CDR3s, previously described as Abs to bromelain-treated RBCs, comprised 43% of peritoneal B-1a sequences. We show that a single-chain variable fragment designed after the most prevalent B-1a sequence bound oxidation-specific epitopes such as the phosphocholine of oxidized phospholipids. In summary, we provide the IgH V region library of six murine B cell subsets, including, to our knowledge for the first time, a comparison between B-1a and B-1b cells, and we highlight qualities of B-1 cell Abs that indicate unique selection processes.

Secreted IgM: New tricks for an old molecule

“…Their recent extensive RNA sequencing experiments also provided no evidence that B‐1 cells undergo extensive somatic hypermutation, with significant components being germline encoded. Although an analysis of only IgM‐secreting B‐1 cells was not conducted …”

“…This explains the highly skewed, self‐reactive repertoire of natural IgM and the fact that the antigen‐specificity of B‐1 cells is tightly linked to their unique functions . Given the similarity in the B‐1a cell repertoire of gnotobiotic and conventional‐housed mice, it then appears that self‐antigen recognition shapes the B‐1 cell repertoire through activation and clonal expansion, which explains the strong changes in VH‐usage over the first few months of life . How B‐1 cells can be selected and then differentially activated by self‐antigens to secrete self‐reactive IgM, without this process resulting in autoimmune disease development is a further important but unresolved question.…”

“…61,63,64 Our studies showed that the Blimp-1 neg IgM-secreting cells did not conducted. 58,65 With regard to the specificity of IgM, current evidence suggests that the pool of natural IgM-secreting B-1 cells is positively selected upon the recognition of self-antigens during development. 66 This explains the highly skewed, self-reactive repertoire of natural IgM and the fact that the antigen-specificity of B-1 cells is tightly linked to their unique functions.…”

Dangerous Liaisons: Gammaherpesvirus Subversion of the Immunoglobulin Repertoire

“…Other mouse strains including BALB/c and lupus-prone MRL/lpr express different alleles that belong to that family. Expression of Ighv10 family members is maintained at a low level in naïve, unchallenged and healthy mice [ 216 ].…”

Oncogene-specific T cells fail to eradicate lymphoma-initiating B cells in mice

“…Sequencing analysis detected no somatic mutations in the rearranged IgH genes, but instead V H 11-D H 1/D H 2-J H 1 rearrangements and CDR3 sequences typical for B1a cells (supplemental Table 1). 49 The lymphoma was classified as high-grade B-cell lymphoma accordingly. Further analysis of cell lineage markers characterized the lymphoma primarily as CD19 1 B220 low CD43 1 CD5 1 IgM 1 ( Figure 2C; supplemental Table 2), again suggesting B1a cells as predominant lymphoma origin.…”