The initiation of B cell clonal expansion occurs independently of pre-B cell receptor formation.

Current models of B cell development posit that clonal expansion occurs as a direct result of Ig H chain expression. To test this hypothesis, we isolated a population of early B cells in which H chain recombination is initiated and assessed V(H)DJ(H) rearrangements in both cycling and noncycling cells. We found that actively dividing cells within this population are enriched for H chain rearrangements that are productive when compared with their counterparts in G(0)/G(1), apparently supporting a role for H chain expression in initiating early B cell division; entrance into the cell cycle was accompanied by V(H) gene-dependent H chain selection. However, we also identified a phenotypically identical population of actively cycling early B cells in the absence of H chain expression in recombination activating gene knockout mice. In addition, actively cycling early B cells could be detected in pre-B cell receptor (pBCR)-negative lambda5 knockout mice, but we found no evidence for V(H)-dependent H chain selection in this population. Given these results, we suggest that the initiation of clonal expansion, at this early stage in B cell development, occurs independently of H chain expression. Although the cycling cell pool is enriched for pBCR-positive cells in mice expressing surrogate L chain, pBCR formation is not required for the initiation of cell division.

B cell maintenance in aged mice reflects both increased B cell longevity and decreased B cell generation.

In aged mice the population of mature peripheral B cells is maintained despite a severalfold decrease in the population of bone marrow B cell progenitors. The analysis of the rate of accumulation of 5'-bromo-2-deoxyuridine (BrdU)-labeled splenic B cells in mice fed BrdU for 8 days to 8 wk demonstrated a severalfold increase in the half-life of mature B cells in aged mice. Consistent with a role for decreased B cell turnover in maintaining the mature B cell population of aged mice, several findings indicate that fewer newly generated B cells enter the spleen from the bone marrow in aged vs young adult mice. These include 1) a fourfold decrease in the population of relatively immature splenic B cells, defined as cells that express high levels of heat-stable Ag and accumulate BrdU within 8 wk of labeling; and 2) an equivalent decrease in the population of bone marrow cells representative of later stages of B cell maturation (sIgD-sIgM(int-high)). Surprisingly, despite a four- to sixfold decrease in pre-B cells, the population of least mature bone marrow B cells (IgD-sIgM(very low)) remains intact. Because this population accumulates BrdU-labeled cells more slowly in aged mice than in younger mice, and bone marrow B cells at more mature developmental stages are diminished, it appears that in aged mice B cell development beyond the sIgM(very low) stage may be retarded and that cells, therefore, accumulate within this population.

Pre-B cell receptor-mediated selection of pre-B cells synthesizing functional mu heavy chains.

Ig gene rearrangements could generate V(H)-D-J(H) joining sequences that interfere with the correct folding of a mu-chain, and thus, its capability to pair with IgL chains. Surrogate light (SL) chain might be the ideal molecule to test the capacity of a mu-chain to pair with a L chain early in development, in that only pre-B cells that assemble a membrane mu-SL complex would be permitted to expand and further differentiate. We have previously identified two SL chain nonpairing V(H)81X-mu-chains with distinct V(H)-D-J(H) joining regions. Here, we show that one of these V(H)81X-mu-chains does not rescue B cell development in J(H) knock-out mice, because flow cytometric analysis of bone marrow cells from V(H)81X-mu transgenic J(H) knock-out mice revealed normal numbers of pro-B cells, but essentially no pre-B and surface IgM+ B cells. Immunoprecipitation analysis of transfected pre-B and hybridoma lines revealed that the same mu-chain fails to pair not only with SL chain but also with four distinct kappa L chains. These findings demonstrate that early pre-B cells are selected for maturation on the basis of the structure of a mu-chain, in particular its V(H)-D-J(H) joining or CDR3 sequence, and that one mechanism for this selection is the capacity of a mu-chain to assemble with SL chain. Therefore, we propose a new function of SL chain in early B cell development: SL chain is part of a quality control mechanism that tests a mu-chain for its ability to pair with conventional L chains.

The B-cell biology of aging.

Although both the number and responsiveness of peripheral B cells in aged mice remain relatively intact, there are dramatic changes in B-cell generation. Alterations in B-cell development include both a skewing of V-gene utilization, especially in cells responsive to phosphorylcholine, and a decrease in the generation of various developmental B-cell subsets. The altered representation of these subsets appears to be the consequence of two developmental blocks. The first developmental block occurs during the maturation of pro-B cells and is evidenced by a decrease in the number of pre-B cells. The second developmental block occurs at the earliest stage of sIg(+)-cell maturation (sIgMvery lo). Because of this block in B-cell maturation, in spite of a decrease in incoming pre-B cells, the number of sIgMvery lo cells appears to increase in aged mice. Additionally, the time of residence of cells within this maturational stage increases dramatically, while the proportion of cells in more mature (sIgMhi) stages of bone marrow development are decreased. In addition to the decreased number of maturing bone marrow B cells, the population of splenic B cells that represent recent bone marrow émigrés (HSAvery hi) is markedly decreased. In the face of this decrease in newly emerging cells from the bone marrow, the population of mature splenic B cells is maintained by their increased longevity.

Heavy chain V gene-specific elimination of B cells during the pre-B cell to B cell transition.

As developing B cells acquire their surface Ig (sIg) receptors, they become highly susceptible to sIg-mediated negative selection, a process best exemplified by tolerance induction. Recent studies with sIg transgenic mice have suggested that B cells may become inactivated by tolerogens only after a developmental stage wherein they express low levels of sIgM and during the course of up-regulating their expression of sIgM. To determine whether inactivation of B cells of conventional mice occurs at this or other maturational stages, we have analyzed the ratio of productive vs nonproductive rearrangements of VH81X gene segments in developmental subsets of adult bone marrow cells. Earlier studies had demonstrated that cells whose productively rearranged H chain V region contained a VH81X gene segment were selectively disfavored both during pre-B cell development and subsequent to sIg expression. Contrary to the expectations for elimination by tolerance, no decrease in the proportion of cells expressing productive rearrangements of VH81X was observed as cells matured from the sIgMlow to the sIgMhigh maturational stage. However, a significant decrease in the proportion of productively rearranged VH81X gene segments was observed following the transition from sIg- pre-B cells to sIgMlow immature B cells. Additionally, the proportion of productively rearranged VH81X gene segments was significantly higher in sIgMhigh bone marrow cells than in splenic B cells. These findings demonstrate that B cells are susceptible to H chain-specific elimination at two developmental stages other than that wherein B cells are generally assumed to be negatively selected by tolerance.