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.

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.

Defining subsets of naive and memory B cells based on the ability of their progeny to somatically mutate in vitro.

The increased affinity of memory antibody responses is due largely to the generation and selection of memory B cells that accumulate somatic mutations after initial antigenic stimulation. Further affinity maturation and mutation also accompany subsequent immunizations. Previous studies have suggested that, like primary antibody-forming cell (AFC) clones, secondary AFC do not accumulate further mutations and, therefore, the origins of progressive affinity maturation remain controversial. Here, we report the generation of somatically mutated memory B cell clones in vitro. Our findings confirm the existence of a naive B cell subset whose progeny, rather than generating AFC, somatically mutate and respond to subsequent antigenic stimulation. Interestingly, upon stimulation, a subset of memory B cells also generates antigen-responsive cells that accumulate further somatic mutations.