Although it is expressed very transiently and at very low cell surface density, the pre-BCR plays a critical role in early B cell development (1, 2). The proteins that are required to assemble the pre-BCR, the signal transduction molecules Igα and Igβ, and the components of the surrogate L chain, λ5 and VpreB, escort the μ H chain to the cell surface as soon as an effective VDJ rearrangement has been completed. What happens then is less clear. Characterization of the signaling events that are controlled by the pre-BCR continues to be an active area of investigation. Since the discovery of the pre-BCR, studies in both murine and human models have generated some synergistic perspectives as well as some provocative controversies. The article by Kitamura et al. (3), chosen for this month's Pillars of Immunology, puts the focus firmly on the contradictory aspects of the pre-BCR.

The pre-B cell stage in B cell differentiation was first recognized in the mid-1970s when Melchers et al. (4), using the incorporation of radioactive leucine followed by immunoprecipitation, showed that cells from the murine fetal liver produced Ig molecules before IgM could be detected on the cell surface. A different approach was taken by Raff et al. (5), who found that by fixing and thus permeabilizing fetal liver cells prior to immunofluorescence staining, they could identify cells that expressed intracellular μ H chain 2 to 3 d before surface IgM+ cells could be detected. They coined the term “pre-B cell” for this stage of differentiation.

In that pre-mAb era, it was difficult to make polyclonal Abs that were L chain-specific with no cross-reactivity to the μ H chain, so it was not clear whether the pre-B cells made L chains as well as H chains. This question was answered in 1979 when Burrows et al. (6), using the new hybridoma technology, fused murine fetal liver cells with a plasma cell line that made neither H nor L chains. The resulting hybridomas made only μ H chains with no κ or λ L chains.

The clinical importance of the pre-B cell stage of differentiation was highlighted by the observation that the phenotype of leukemic cells in the majority of children with acute lymphoblastic leukemia was that of a pre-B cell (7, 8). Analysis of these leukemic cells and murine pre-B cell hybridomas indicated that not only was μ H chain protein expressed before L chain, but also H chain gene rearrangement generally preceded L chain rearrangement (8, 9).

Another piece of the puzzle was provided by several groups who showed that there were two different types of μ H chain transcripts that differed at their 3′ ends (1012). One form encoded the membrane form of μ H chain and the other the secreted form. Both forms could be identified in pre-B cell lines (13). This supported the hypothesis that cell surface expression of μ H chain generated a signal that was essential for progression of B cell development. But how did μ H chain get to the cell surface in the absence of L chain? The search was on.

An excellent candidate was found in 1986 by Sakaguchi and Melchers (14). They used subtractive cDNA libraries to identify genes that were preferentially expressed in pre-B cell lines. Sequence homology demonstrated that one particular gene, a gene they called λ5, encoded a protein with homology to the J segment and constant domain of λ L chains. Attempts to find regulatory regions or additional coding exons upstream of λ5 led Kudo and Melchers (15) to the discovery of VpreB1 and VpreB2, genes with homology to L chain variable regions. These authors suggested the possibility that VpreB and λ5 might form a noncovalent complex that could transport μ H chain to the cell surface (15).

Clear evidence that μ H chain could be expressed on the surface of pre-B cells in a complex with a “surrogate light chain” was provided in 1987 by Pillai and Baltimore (16), who described a small protein that coprecipitated with μ H chain and was specific to pre-B cell lines. Cell surface iodination followed by immunoprecipitation using an anti-μ H chain Ab brought down a tetramer containing two μ H chains and two covalently linked proteins with an m.w. of 18,000. They hypothesized that the 18-kDa protein was the product of the λ5 gene.

Was λ5 essential for B cell development? Kitamura et al. (3), in their Pillars article, used the relatively new experimental approach of knocking out the λ5 gene to determine its function. The λ5-deficient mice demonstrated a block in B cell differentiation that was similar to that seen in μMT mice, a strain of mice made the previous year. The μMT mice were unable to make the transmembrane form of μ H chain because the exons encoding this portion of the molecule had been replaced with a neomycin cassette (17). Both the λ5−/− and μMT mice had a profound block at the stage in B cell development at which the pre-BCR is first expressed on the cell surface. The big surprise was that the λ5 defect was leaky. Unlike the μMT mice, the λ5-deficient mice did make a small number of B cells, and, with age, the number of B cells increased to ∼20% of normal. The serum concentration of most Ig isotypes was close to normal, and Ab responses to both T cell-dependent and T cell-independent Ags could be detected in the λ5 knockout mice. Several possible explanations for the leakiness were suggested. Perhaps there was a protein that could inefficiently substitute for λ5. Maybe VpreB could bind to μ H chain in the absence of λ5 and escort μ H chain to the cell surface. The authors favored a third alternative, that early rearrangement of a conventional L chain gene could rescue the λ5-deficient precursor.

Later studies ruled out the possibility that VpreB could provide some protection from λ5 deficiency. Mice that were null for λ5 and both copies of VpreB were indistinguishable from mice that lacked λ5 alone (18). The idea that premature L chain rearrangement was responsible for the leakiness of the λ5 defect took hold. However, not all of the data were consistent with this explanation. Unlike the λ5-deficient mice, humans with mutations in λ5 have a severe block in B cell development that is not leaky (19). Their bone marrow contains normal numbers of pro-B cells, like the λ5-deficient mice, but they have <1% of the normal number of B cells in the peripheral circulation and almost complete absence of all Ig isotypes. This cannot be attributed to the absence of L chain rearrangement before H chain in humans. Earlier studies in EBV-transformed human pre-B cell lines showed that L chain sometimes rearranges before H chain (20), and bone marrow from a λ5-deficient patient showed a marked increase in rearranged κ transcripts when compared with marrow from patients unable to make μ H chain or Btk (21). This suggested that the B cell precursors from the λ5-deficient patient had somehow received a signal that is usually given by the pre-BCR, a signal that led to the rearrangement of the L chain genes, but which did not lead to further B cell differentiation.

Transcripts encoding an unusual type of μ H chain that might elicit this signal were found in the bone marrow of the λ5-deficient patient and in pro-B cells but not in pre-B cells or mature B cells from normal controls (21). The μ H chain encoded by these transcripts could be expressed on the surface of Jurkat T cells with Igα and Igβ in the absence of surrogate or conventional L chains. The fact that these L chain-independent μ H chains induced cell activation and apoptosis helped explain their absence in pre-B cells and mature B cells. The CDR3 regions of these peculiar μ H chains contained a high proportion of positively charged residues, particularly arginine, and the D regions were often in rarely used reading frames.

Later studies showed that similar μ H chain proteins could be found in mice (2224). Mice that were null for both RAG2 and λ5, and transgenic for an inducible μ H chain, were able to develop pre-B cells, although inefficiently (22). Expression of the inducible μ H chain resulted in events typical of pre-BCR signaling, including downregulation of TdT and RAG (24). In another model, mice that were null for BLNK and λ5 developed pre-B cells with polyclonal μ H chains (23). Although it was argued that the pre-BCRs lacking the surrogate L chain in mice cannot be attributed to the use of the unusual subset of L chain-independent μ H chains (23), the H chain repertoire of λ5-deficient mice was not examined in detail.

More recent studies have shown that λ5−/− mice have an increased proportion of B cells that produce autoantibodies (25). Similar to the L chain-independent μ H chains seen in humans, the CDR3 regions of these autoantibodies are enriched for arginines and often include D regions that are encoded by infrequently used reading frames. Why are these unusual μ H chains seen in mature B cells from λ5−/− mice but not humans? Studies by Su et al. (23) may provide a clue. These investigators found that pre-B cell lines derived from mice that were BLNK−/−/λ5−/− demonstrated markedly increased tyrosine phosphorylation and calcium flux after cross-linking of surface IgM when compared with BLNK−/− pre-B cell lines. Instead of generating a decreased pre-BCR signal, the L chain-independent μ H chains may evoke an increased signal, a signal that in the human results in negative selection but is somewhat better tolerated in the mouse. This suggests a complex role for the pre-BCR in control of the Ig repertoire. In addition to positively selecting rearranged μ H chains that bind well to surrogate L chain (or negatively selecting those that do not), the pre-BCR guards against receptors that give too strong a signal. The window of signal intensity that is tolerated in the mouse may be a little wider than the same window in humans. How the pre-BCR does all of the things it does is still not well understood.

We thank Maureen McGargill and Vanessa Howard for critically reviewing the manuscript.

Disclosures The authors have no financial conflicts of interest.

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