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IL-7 plays many fundamental roles during murine B lineage development. One reported function is to maintain progenitors in a developmentally immature state by preventing differentiation to the surface IgM (sIgM)(+) stage. Withdrawal of IL-7 from cultures has been shown to lead to increases in mature traits such as RAG expression, IgL rearrangements and expression of sIgM. These observations have been interpreted as an inductive event promoting the differentiation of progenitor cells. In contrast to this, we reproducibly observe sIgM(+) cells that have differentiated in cultures containing IL-7. We find that sIgM(+) cells arise as a normal consequence when B lineage cells are cultured in the presence of IL-7. However, these cells are short-lived and are quickly replaced by newly emerging sIgM(+) cells that differentiate from sIgM(-) progenitors. Withdrawal of IL-7 from these cultures only prevents the survival and proliferation of CD2(-)sIgM(-) pro-B cells but does not change the number of cells that differentiate to the sIgM(+) stage. This changes the ratio of sIgM(-):sIgM(+) cells and results only in an apparent maturation of the culture as a whole. Therefore withdrawal of IL-7 from these cultures acts as a selection event, not an induction event, for populations that are normally present.

Efficient generation of mature B lineage cells requires the participation of the BCR, the pre-BCR, accessory coreceptors, and growth factor receptors. Together these receptors integrate cell intrinsic signals with regulatory pathways initiated by surrounding cells and structures. CD45 is a receptor tyrosine phosphatase expressed at high levels on all hemopoietic cells, and has been shown to modulate many signaling cascades in both positive and negative manners. In the absence of B220, the B lineage isoform of CD45, differentiation to the mature B cell stage is incomplete. We demonstrate that CD45-deficient mice also accumulate pro-B cells in the bone marrow. In vitro differentiation is altered in that B lineage populations exhibit prolonged survival in the presence of high concentrations of IL-7. Cell lines derived from CD45-deficient animals experience prolonged JAK/STAT activation in response to IL-7 stimulation, and constitutively elevated levels of phosphorylated src kinases. Aberrant IL-7Ralpha expression is observed in vivo, and may be responsible for the skewed development present in CD45(-/-) animals. Demonstrating that CD45-deficient pro-B cells are affected by the absence of B220 highlights a previously unrecognized parallel in B and T lineage precursors, and emphasizes that the presence of normal numbers of peripheral B cells does not assure that the bone marrow compartment is intact.

Many of the stromal-derived signals and factors that regulate B lymphopoiesis have been identified. We review recent evidence from our laboratory that shows that there are at least three phases during B-cell development when cells direct their own maturation, independent of stromal cells. Following the expression of the preB-cell receptor (preBCR), cells acquire the ability to proliferate in low levels of interleukin-7 (IL-7), which acts as a self-selecting mechanism to expand cells that have successfully expressed a preBCR in environments that are non-permissive to preBCR- cells. Second, the preBCR is required for a contact-mediated event between B-cell progenitors. Disruption at this stage prevents the further maturation of progenitors to the lipopolysaccharide (LPS)-responsive stage. Finally, the transition from IL-7 receptor to mature antigen receptor-based signaling is enhanced by a novel member of the tachykinin family, hemokinin-1. This series of maturation, survival, and differentiation signals is generated by B-lineage cells as they progress through developmental checkpoints on the way to becoming functionally mature cells.

Hematopoiesis is regulated by a variety of signals that either originate within a developing cell or are supplied by the surrounding environment in secreted- or contact-dependent forms. This review discusses the effects of one secreted factor, interleukin-7, on the development of B lymphocytes. We describe a molecular mechanism for a crucial checkpoint during B lineage maturation, based on the integration of signals mediated by the pre-B cell receptor, the interleukin-7 receptor, and the environment in which these signals are received.

B lymphocyte development is regulated at multiple checkpoints, mediated by signals originating both inside and outside the cell. Two signaling pathways known to be essential in this process are interleukin-7 (IL-7) and the pre-B cell receptor (pBCR). We have shown previously that these signaling pathways intersect functionally. Specifically, response to low concentrations of IL-7 requires pBCR expression. In this report, we identify the ERK/MAP kinase pathway as a key regulatory component of this response. We propose a molecular mechanism for the selective expansion of pBCR(+) precursors and for the culling of inappropriately rearranged pro-B cells.

Numerous studies have demonstrated that B lymphopoiesis is dependent upon a stromal cell microenvironment. Many of the stromal cell-derived factors and cell surface interactions that regulate B cell development have been identified; however, little consideration has been paid to the intimate interactions known to occur among B cell precursors themselves in both the fetal liver and marrow microenvironments. In this study we show that homotypic interactions between B cell precursors play an important role in promoting the development of mature B cells. We used an in vitro assay system to demonstrate that the function of stromal cells can be replaced by culturing B cell precursors in proximity. B cell precursors isolated from bcl-2 transgenic mice were used to rule out the possibility that improved survival, hypothesized to result from culturing precursors in proximity, solely accounted for the observed increase in B cell maturation. The putative maturation signal(s) were shown to be dependent upon direct contact between precursors rather than the release of soluble factors from nearby cells. Upon examination of the potential role of several known cell surface proteins, we found that blocking mu heavy chains with monovalent Fab antibody fragments dramatically inhibited maturation, in a stage-specific manner. Together these results suggest that a major function of stromal cells in vivo may be to act as a docking site to promote critical preB-preB homotypic interactions and ensuing signals. Further, the antibody blocking experiments raise the interesting possibility that interactions between B cell precursors themselves may promote and/or regulate preB cell receptor-driven signals.

Considerable progress has been made in defining intermediate stages in the process leading from stem cells to mature B cells. Cell-bound and secreted molecules direct the progression through these stages and regulate the selection of clones from which the immune repertoire emerges. In fact, a myriad of signals derived from B-cell progenitors themselves and the microenvironment in which they develop direct the differentiation process. These signals are provided by B-cell antigen receptors (BCR) and their surrogates, and by adhesion and cytokine receptors. The co-operation of these receptors to control survival, expansion, and differentiation of early B-cell progenitors is the topic of this review. Specifically, we will summarize recent findings from our laboratory demonstrating that preBCR expression lowers the threshold for interleukin (IL)-7 responsiveness. How signals initiated by these receptors may intersect at this critical point of B-cell selection will be discussed. At the stage following IL-7 responsiveness we have shown that interactions between B-cell progenitors themselves promote their differentiation to immunoglobulin-secreting B cells. We propose that one function of stromal cells, known to be central to B lymphopoiesis, is to promote critical preB-preB homotypic interactions and ensuing signals.

The IL-7R and the pre-B cell receptor (pre-BCR) each provide critical signals during differentiation of B cell precursors. In this study we examine the interplay between signals dependent upon these receptors. We demonstrate that pre-BCR-deficient pro-B cells differ significantly from controls in their ability to use the IL-7R. We show that this difference, characterized by a failure to proliferate in response to IL-7, is narrowly restricted to IL-7 concentrations in the picogram per milliliter range and can be overcome with increasing amounts of IL-7. Restoration of Ig heavy chain to recombinase-activating gene-2-deficient pro-B cells leads to a restored response to picogram per milliliter levels of IL-7, providing strong evidence that modulation of the IL-7 dose-response threshold is dependent on pre-BCR. Culture of normal pro-B cells under low IL-7 conditions leads to selective outgrowth of cells expressing mu heavy chain, suggesting that modulation of IL-7 dose-response thresholds can allow for selective expansion of pre-BCR+ cells under conditions where IL-7 is limiting. We also provide evidence that expression of pre-BCR on pro-B cells limits the duration of IL-7 responsiveness by causing differentiation to an IL-7-unresponsive pre-B cell stage. Thus, the pre-BCR-dependent modulation of IL-7 responsiveness affects both the dose-response threshold and the duration of IL-7-induced clonal expansion. Our results suggest that positive selection of pre-BCR+ pro-B cells may be achieved through the fine tuning of IL-7 responses.