A VH12 transgenic mouse exhibits defects in pre-B cell development and is unable to make IgM+ B cells.

V(H)12 B cells undergo stringent selection at multiple checkpoints to favor development of B-1 cells that bind phosphatidylcholine. Selection begins with the V(H) third complementarity-determining region (CDR3) at the pre-B cell stage, in which most V(H)12 pre-B cells are selectively eliminated, enriching for those with V(H)CDR3s of 10 aa and a fourth position Gly (designated 10/G4). To understand this selection, we compared B cell differentiation in mice of two V(H)12 transgenic lines, one with the favored 10/G4 V(H)CDR3 and one with a non-10/G4 V(H)CDR3 of 8 aa and no Gly (8/G0). Both H chains drive B cell differentiation to the small pre-BII cell stage, and induce allelic exclusion and L chain gene rearrangement. However, unlike 10/G4 pre-B cells, 8/G0 pre-B cells are deficient in cell division and unable to differentiate to B cells. We suggest that this is due to poor 8/G0 pre-B cell receptor expression and to an inability to form an 8/G0 B cell receptor. Our findings also suggest that V(H)12 H chains have evolved such that association with surrogate and conventional L chains is most efficient with a 10/G4 CDR3. Thus, selection for phosphatidylcholine-binding B-1 cells is most likely the underlying evolutionary basis for the loss of non-10/G4 pre-B cells.

Identification of a precursor to phosphatidyl choline-specific B-1 cells suggesting that B-1 cells differentiate from splenic conventional B cells in vivo: cyclosporin A blocks differentiation to B-1.

The origin of B-1 cells is controversial. The initial paradigm posited that B-1 and B-2 cells derive from separate lineages. More recently it has been argued that B-1 cells derive from conventional B cells as a result of T-independent Ag activation. To understand B-1 cell differentiation, we have generated Ig transgenic (Tg) mice using the H and L chain genes (VH12 and Vkappa4) of anti-phosphatidyl choline (anti-PtC) B cells. In normal mice anti-PtC B cells segregate to B-1. Segregation is intact in VH12 (6-1) and VH12/Vkappa4 (double) Tg mice that develop large numbers of PtC-specific B cells. However, if B-1 cell differentiation is blocked, anti-PtC B cells in these Tg mice are B-2-like in phenotype, suggesting the existence of an Ag-driven differentiative pathway from B-2 to B-1. In this study, we show that double Tg mice have a population of anti-PtC B cells that have the phenotypic characteristics of both B-2 and B-1 cells and that have the potential to differentiate to B-1 (B-1a and B-1b). Cyclosporin A blocks this differentiation and induces a more B-2-like phenotype in these cells. These findings indicate that these cells are intermediate between B-2 and B-1, further evidence of a B-2 to B-1 differentiative pathway.

Interdependence of N nucleotide addition and recombination site choice in V(D)J rearrangement.

Diversity in the Ag binding receptors of B and T cells is achieved through a process of genomic rearrangement involving selection of recombination sites and, in adult mice, addition of nontemplated (N) nucleotides. We have analyzed 543 Ig heavy chain nonproductive rearrangements, involving a single variable region gene segment, from adult and perinatal mice. We infer several fundamental and novel features of the recombination mechanism. N regions are formed predominantly from the DNA plus strand or from the DNA minus strand polymerizations, rather than as a concatenation of the two. Homologous overlaps of as few as one nucleotide between gene segments cause significant skewing of recombination sites. The V(H) recombination site spectrum differs in perinatal and adult mice, with sites representing overlap between V(H) and D over-represented in the perinatal mice, and sites representing overlaps between V(H) and the N strand polymerized onto the D segment over-represented in the adult mice. Thus, in V(D)J joining, N nucleotide addition and recombination site choice are highly interdependent events.

The transition of pre-BI to pre-BII cells is dependent on the VH structure of the mu/surrogate L chain receptor.

We have demonstrated previously that the majority ( > 90%) of VH12 B cells are absent from the adult peripheral repertoire, and that most that remain have the fourth position at the D-J function (designated 10/G4). We report here that most VH 12-expressing pre-B cells are lost during the transition from the pre-BI to the pre-BII cell stage in normal mice, and that pre-BII cell productive (P) rearrangements ar enriched in 10/G4 CDR3. This coincides with the initial expression of H chain and the generation of the mu/surrogate L chain (SL) receptor. In contrast, there is not enrichment for 10/G4 CDR3 in mu MT mice, and the frequency of P rearrangements is as expected from a random rearrangement mechanism, ruling out a biased rearrangement mechanism unique to VH12. We have also demonstrated that non-10/G4 mu chains can associate with SL and be expressed on the cell surface, suggesting that they are available on the cell surface for selection. Thus, transition of pre-BI to pre-BII cells is dependent on the structure of the VH domain.

The majority of murine VH12-expressing B cells are excluded from the peripheral repertoire in adults.

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.

Development of B-1 cells: segregation of phosphatidyl choline-specific B cells to the B-1 population occurs after immunoglobulin gene expression.

Adult mice have two easily recognizable subsets of B cells: the predominant resting population of the spleen, called B-2, and those called B-1, which predominate in coelomic cavities and can express CD5. Some antibody specificities appear to be unique to the B-1 population. Cells expressing antibody specific for phosphatidyl choline (PtC) are the most frequent, comprising 2-10% of peritoneal B cells in normal mice. To understand the basis for the segregation of the anti-PtC specificity to this population, we have produced transgenic (Tg) mice expressing the rearranged VH12 and V kappa 4 genes of a PtC-specific B-1 cell lymphoma. We find that VH12-Tg and VH12/V kappa 4 double-Tg mice develop very high numbers of PtC-specific peritoneal and splenic B cells. These cells have the characteristics of B-1 cells; most are CD5+, and are all IgMhi, B220lo, and CD23-. In the peritoneum these cells are also CD11b+. In addition, adult mice have many splenic B cells (up to one third of Tg+ cells) that express the VH12 Tg but do not bind PtC, presumably because they express a V kappa gene other than V kappa 4. These cells appear to be B-2 cells; they are CD23+, CD11b-, IgMlo, B220hi, and CD5-. Thus, mice given either the VH12 Tg alone or together with the V kappa 4 Tg develop a large population of PtC-specific B cells which belong exclusively to the B-1 population. Since B-2 cells can express the VH12 and V kappa 4 gene separately, we interpret these data to indicate that the events leading to the segregation of PtC-specific B cells to the B-1 population in normal mice are initiated after Ig gene rearrangement and expression. These data are discussed with regard to hypotheses of the origin of B-1 cells. We also find that VH12-Tg mice have a marked decrease in the generation of Tg-expressing B cells in adult bone marrow, but not newborn liver. We speculate that this may be related to positive selection of VH12-expressing B cells during differentiation.

VH CDR3-dependent positive selection of murine VH12-expressing B cells in the neonate.

Five to fifteen percent of peritoneal B1 (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 VH/V kappa gene combinations, VH12/V kappa 4 or VH11/V kappa 9. Both the VH11 and VH12 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 VH12-D-JH1 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 VHCDR3. 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 VH12 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 VHCDR3. We suggest further that the majority of VH12-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 VH12-expressing cells begins before birth.