Selection of Individual V_H Genes Occurs at the Pro-B to Pre-B Cell Transition

Abstract

B cells are subjected to selection at multiple checkpoints during their development. The selection of Ab H chains is difficult to study because of the large diversity of the CDR3. To study the selection of individual Ab H chain V region genes (V_H), we performed CDR3 spectratyping of ∼75–300 rearrangements per individual V_H in C57BL6/J mice. We measured the fraction of rearrangements that were in-frame in B cell DNA. We demonstrate that individual V_Hs have different fractions of in-frame rearrangements (IF fractions) ranging from 10 to 90% and that these IF fractions are reproducible in different mice. For most V_Hs, the IF fraction in pro-B cells approximated 33% and then shifted to the nearly final (mature) B cell value by the cycling pre-B cell stage. The frequency of high in-frame (IF) V_H usage increased in cycling pre-B cells compared with that in pro-B cells, whereas this did not occur for low IF V_Hs. The IF fraction did not shift as much in BCR-expressing B cells and was minimally affected by L chain usage for most V_H. High IF clan II/III V_Hs share more positively charged CDR2 sequences, whereas high IF clan I J558 CDR2 sequences are diverse. These data indicate that individual V_Hs are subjected to differential selection, that V_H IF fraction is mainly established through pre-BCR–mediated selection, that it may operate differently in clan I versus II/III V_Hs, and that it has a lasting influence on the Ab repertoire.