The majority of murine V_H12‐expressing B cells are excluded from the peripheral repertoire in adults

Abstract

We have previously demonstrated that at birth most productive (P) VH12 rearrangements in B10.H-2aH-4bp/Wts (2a4b) mice encode a ten-amino acid CDR3, and that a significant fraction of the expected repertoire is absent. We have now examined the adult VH12 CDR3 repertoire involving all four JH gene segments in both peritoneum and spleen. Of the 74 P VH12 rearrangements from these tissues 67 encode a CDR3 of ten amino acids and include a Gly in the fourth position (designated 10/G4). Most of these rearrangements appear to derive from phosphatidylcholine (PtC)-specific B cells, which also have a 10/G4 VHCDR3, since few 10/G4 P rearrangements were present in spleen cells depleted of PtC-specific B cells. Thus, the VH12 B cell repertoire in adult mice is largely restricted to the use of a single CDR3 motif and to a single antigen specificity. This bias results from two selection events: (1) selective exclusion of most VH12 B cells from the peripheral repertoire, and (2) clonal expansion in the periphery of VH12 B cells that have a 10/G4 VHCDR3 and bind PtC. Analysis of VH12-JH1 rearrangements in viable motheaten (mev/mev) mice, which have an abnormal B cell repertoire due to a defective phosphatase (Hcph) and have barely detectable numbers of PtC-specific B cells, indicates that selective exclusion of VH12 B cells from the peripheral repertoire occurs normally, but that clonal expansion of 10/G4 VH12 B cells is minimal. This is evidence that the selective exclusion of VH12 B cells from the peripheral repertoire and the clonal expansion of VH12 B cells with a 10/G4 CDR3 are due to independent signaling events.

Evidence for a Ligand-Mediated Positive Selection Signal in Differentiation to a Mature B Cell

“…This supports the hypothesis that the observed bias in V 4/5H L chain gene use by mature B cells in 6-1 mice (27) is due to an inability of non-V 4/5H L chains to support differentiation to the mature B cell stage. This is the third checkpoint in V H 12 B cell differentiation, after the pre-BI to pre-BII and the pre-BII to immature B checkpoints (26,29,39), that restricts V H 12 B cells primarily to the production of H and L chains that form an anti-PtC BCR and drives differentiation to B-1.…”

B-1 B Cell Development

“…His group has found that the development of B lineage cells with V H 12 rearrangements is constricted at several points, such that B cells with this H chain become progressively restricted to the prototypic anti-PtC BCR, even though the most frequently found V H 12 rearrangement generates a H chain capable of associating with SLC. Thus, at the pre-B stage, rearrangements containing CDR3 segments of 10 aa and with a glycine residue in the fourth position ("10/G4") are much more likely to be found among productive V H 12 sequences (70). A likely explanation for this preference is pre-BCR selection, such that most V H 12 rearrangements generate H chains that fail to associate with SLC, whereas those with the 10/G4 segment do and so can mediate progression to the late pre-B stage where L chain rearrangement takes place.…”

Positive selection focuses the VH12 B‐cell repertoire towards a single B1 specificity with survival function

“…The surviving 5% are enriched in CDR3 sequences of 10 amino acids and include many with a glycine (Gly) in the fourth position (referred to as 10/G4). The 10/G4 CDR3 is used by almost all V H 12‐expressing B1 cells specific for PtC (27). There is no mechanistic bias in V H 12 rearrangement that could account for the enrichment of 10 amino acid CDR3s, beacuse in pre‐B1 cells of µMT mice, where selection cannot occur, there is no enrichment for CDR3 length or sequence (27).…”