V(H) CDR3-DEPENDENT POSITIVE SELECTION OF MURINE V(H)12-EXPRESSING B-CELLS IN THE NEONATE

Five to fifteen percent of peritoneal B 1 (CD5+) cells from unmanipulated mice produce antibodies that bind bromelain-treated mouse red blood cells and the hapten phosphatidylcholine (PtC). The majority of these B cells express either of two V(H)/Nkappa gene combinations, V(H)12/Vkappa4 or V(H)11/Vkappa9. Both the V(H)11 and V(H)12 genes are rearranged to JH1 and encode third complementarity determining regions (CDR3) of restricted length and sequence. These and other observations argue strongly that PtC-specific B1 cells are antigen selected. To determine when selection of PtC-specific B1 cells begins in mice we have used the polymerase chain reaction to amplify V(H)12-D-J(H)I rearrangements from livers of fetal and neonatal mice, and determined the CDR3 encoding sequences of individual clones. We find an unusually low ratio of productive (P) to non-productive (NP) rearrangements (0.4 - 1.0) at both developmental stages. P rearrangements in day 1 neonates are biased in D gene use and in the sequence and length of their deduced V(H) CDR3. These biases are similar to those of PtC-specific B1 cells in the adult peritoneum. D gene use and CDR3 length and sequence are significantly less biased among V(H)12 P rearrangements 2 to 3 days earlier in the day 18 fetal liver. We suggest that this rapid change in repertoire is due to positive ligand selection that is dependent on the sequence of VH CDR3.We suggest further that the majority Of V(H)12-expressing cells are not ligand selected and consequently undergo programmed cell death. The evidence of restriction in day 1 neonatal livers and the low P/NP ratio in the fetus suggests that selection Of V(H)12-expressing cells begins before birth.