The long range goal of this proposal is to understand the selective events involved in B-1 and B-2 repertoire formation and focuses on two selection steps. The first is mediated by the mu/surrogate L chain (mu/SL) receptor of pre-B cells, and acts on all pre-B cells regardless of whether they contribute to the B-1 or B-2 repertoires. Ligand binding by the mu/SL receptor rescues pre-B cells from an otherwise inevitable progression to cell death. We propose that ligand binding of this receptor is dependent on the structure of the VH region, and therefore, that this selective event affects the expressed VH repertoire of mature B cells. We base this on our observation that VH12 pre-B cells are restricted in CDR3 sequence, suggesting that most VH12 pre-B cells die for failure to bind ligand. The second selection step to be examined is mediated by the Ig receptor of mature B cells and results in the segregation of B cells to the B-l and B- 2 subsets. We propose that specificity determines inclusion in the B-1 subset. This is based on the observation that mature VH12 B cells that bind phosphatidyl choline (PtC) belong exclusively to the B-l subset. We hypothesize that only those VH12 B cells that bind PtC segregate to the B- l subset. In the first aim we will test three predictions of the hypothesis that selective rescue of VH12 pre-B cells is dependent on the structure of VH CDR3; l) VH12 CDR3 diversity is unrestricted before selection. 2) VH12 R chains, regardless of CDR3 structure can associate with surrogate L chain, and 3) the restriction in CDR3 results from loss of most pre-B cells by programmed cell death. In the second aim we will test whether the diminished VH12 pre-B cell production in the adult BM of VH12 Tg mice is due to interference of the selective rescue of pre-B cells. In the third aim we will test the hypothesis that specificity determines inclusion in the B-1 subset. VH12 transgenic (Tg) mice will be crossed with Vkappa Tg mice and Vlambda restricted mice to generate double Tg mice that can generate B cells of only a single non-PtC specificity and thus will be under the same selective pressure. If specificity determines inclusion in the B-1 subset, then all B cells should segregate to either the B-1 or B-2 subsets, but not both. If specificity is irrelevant, then mice will have both B-1 and B-2 cells.
