Analysis of VpreB expression during B lineage differentiation in lambda5-deficient mice

The VpreB/lambda5 surrogate L chain complex is an essential component of the pre-B cell receptor, the expression of which serves as an important checkpoint in B cell development. Surrogate L chains also may serve as components of murine pro-B cell receptors whose function is unknown. We have produced two new mAbs, R3 and R5, that recognize a different VpreB epitope than the one recognized by the previously described VP245 anti-mouse VpreB Ab. These Abs were used to confirm the expression of surrogate L chains on wild-type pro-B and pre-B cell lines. Although undetectable on the cell surface, VpreB was found to be normally expressed within B lineage cells of lambda5-deficient mice. Nevertheless, VpreB expression was extinguished at the B cell stage of differentiation in these mice. The normal pattern of VpreB expression in lambda5-deficient mice excludes an essential role for pro-B and pre-B cell receptors in VpreB regulation.     

Basal Igalpha/Igbeta signals trigger the coordinated initiation of pre-B cell antigen receptor-dependent processes

The pro-B to pre-B transition during B cell development is dependent upon surface expression of a signaling competent pre-B cell Ag receptor (pre-BCR). Although the mature form of the BCR requires ligand-induced aggregation to trigger responses, the requirement for ligand-induced pre-BCR aggregation in promoting B cell development remains a matter of significant debate. In this study, we used transmission electron microscopy on murine primary pro-B cells and pre-B cells to analyze the aggregation state of the pre-BCR. Although aggregation can be induced and visualized following cross-linking by Abs to the pre-BCR complex, our analyses indicate that the pre-BCR is expressed on the surface of resting cells primarily in a nonaggregated state. To evaluate the degree to which basal signals mediated through nonaggregated pre-BCR complexes can promote pre-BCR-dependent processes, we used a surrogate pre-BCR consisting of the cytoplasmic regions of Igalpha/Igbeta that is targeted to the inner leaflet of the plasma membrane of primary pro-B cells. We observed enhanced proliferation in the presence of low IL-7, suppression of V(H)(D)J(H) recombination, and induced kappa light (L) chain recombination and cytoplasmic kappa L chain protein expression. Interestingly, Igalpha/Igbeta-mediated allelic exclusion was restricted to the B cell lineage as we observed normal TCRalphabeta expression on CD8-expressing splenocytes. This study directly demonstrates that basal signaling initiated through Igalpha/Igbeta-containing complexes facilitates the coordinated control of differentiation events that are associated with the pre-BCR-dependent transition through the pro-B to pre-B checkpoint. Furthermore, these results argue that pre-BCR aggregation is not a requirement for pre-BCR function.     

Impaired precursor B cell differentiation in Bruton's tyrosine kinase-deficient mice

Bruton's tyrosine kinase (Btk) is a cytoplasmic signaling molecule that is crucial for precursor (pre-B) cell differentiation in humans. In this study, we show that during the transition of large cycling to small resting pre-B cells in the mouse, Btk-deficient cells failed to efficiently modulate the expression of CD43, surrogate L chain, CD2, and CD25. In an analysis of the kinetics of pre-B cell differentiation in vivo, Btk-deficient cells manifested a specific developmental delay within the small pre-B cell compartment of about 3 h, when compared with wild-type cells. Likewise, in in vitro bone marrow cultures, Btk-deficient large cycling pre-B cells showed increased IL-7 mediated expansion and reduced developmental progression into noncycling CD2(+)CD25(+) surrogate L chain-negative small pre-B cells and subsequently into Ig-positive B cells. Furthermore, the absence of Btk resulted in increased proliferative responses to IL-7 in recombination-activating gene-1-deficient pro-B cells. These findings identify a novel role for Btk in the regulation of the differentiation stage-specific modulation of IL-7 responsiveness in pro-B and pre-B cells. Moreover, our results show that Btk is critical for an efficient transit through the small pre-B cell compartment, thereby regulating cell surface phenotype changes during the developmental progression of cytoplasmic mu H chain expressing pre-B cells into immature IgM(+) B cells.     

Restricted STAT5 activation dictates appropriate thymic B versus T cell lineage commitment

The molecular mechanisms regulating lymphocyte lineage commitment remain poorly characterized. To explore the role of the IL7R in this process, we generated transgenic mice that express a constitutively active form of STAT5 (STAT5b-CA), a key downstream IL7R effector, throughout lymphocyte development. STAT5b-CA mice exhibit a 40-fold increase in pro-B cells in the thymus. As documented by BrdU labeling studies, this increase is not due to enhanced B cell proliferation. Thymic pro-B cells in STAT5b-CA mice show a modest increase in cell survival ( approximately 4-fold), which correlates with bcl-x(L) expression. However, bcl-x(L) transgenic mice do not show increases in thymic B cell numbers. Thus, STAT5-dependent bcl-x(L) up-regulation and enhanced B cell survival are not sufficient to drive the thymic B cell development observed in STAT5b-CA mice. Importantly, thymic pro-B cells in STAT5b-CA mice are derived from early T cell progenitors (ETPs), suggesting that STAT5 acts by altering ETP lineage commitment. Supporting this hypothesis, STAT5 binds to the pax5 promoter in ETPs from STAT5b-CA mice and induces pax5, a master regulator of B cell development. Conversely, STAT5b-CA mice exhibit a decrease in the DN1b subset of ETPs, demonstrating that STAT5 activation inhibits early T cell differentiation or lineage commitment. On the basis of these findings, we propose that the observed expression of the IL-7R on common lymphoid progenitors, but not ETPs, results in differential STAT5 signaling within these distinct progenitor populations and thus helps ensure appropriate development of B cells and T cells in the bone marrow and thymic environments, respectively.     

B-lymphocyte development is regulated by the combined dosage of three basic helix-loop-helix genes, E2A, E2-2, and HEB.

B-lymphocyte development requires the basic helix-loop-helix proteins encoded by the E2A gene. In this study, the control mechanism of E2A was further explored by disruption of the E2A-related genes, E2-2 and HEB. In contrast to E2A, E2-2 and HEB are not essential for the establishment of the B-cell lineage. However, both E2-2 and HEB are required for the generation of the normal numbers of pro-B cells in mouse embryos. Breeding tests among mice carrying different mutations revealed that E2-2 and HEB interact with E2A in many developmental processes including generation of B cells. Specifically, mice transheterozygous for any two mutations of these three genes produced fewer pro-B cells than the singly heterozygous littermates. This study indicates that B-cell development is dependent not only on an essential function provided by the E2A gene but also on a combined dosage set by E2A, E2-2, and HEB.     

Murine pro-B cells require IL-7 and its receptor complex to up-regulate IL-7R alpha, terminal deoxynucleotidyltransferase, and c mu expression

Phenotypic analysis of bone marrow cells from IL-7 knockout (KO) mice revealed that B cell development is blocked precisely at the transition between pro-B cells and pre-B cells. In contrast, the generation of pre-pro-B cells and pro-B cells appeared to be normal, as judged by total cell numbers, proliferative indexes, D-JH and V-DJH gene rearrangements, and mRNA for recombinase-activating gene-1 (RAG-1), RAG-2, TdT, Ig mu, lambda 5, and VpreB. However, upon closer inspection, several abnormalities in pro-B cell development were identified that could be corrected by injection of rIL-7 in vivo. These included the absence of the subset of late pro-B cells that initiates cmu expression for pre-B cell Ag receptor (BCR) formation, and the failure of pro-B cells to up-regulate TdT and the IL-7R alpha (but not the common gamma-chain) chain. Similar defects were present in common gamma-chain and Jak3 KO mice, but not in lambda 5 or (excluding cytoplasmic Ig mu heavy chain (c mu)) RAG-1 KO mice, all of which also arrest at the late pro-B cell stage. Consequently, up-regulation of TdT and IL-7R alpha expression requires signaling through the high affinity IL-7R, but does not require cmu expression or a functional pre-BCR. Taken together, these results suggest that IL-7 and its receptor complex are essential for 1) up-regulating the expression of TdT and IL-7R alpha, 2) initiating the production of cmu and 3) promoting the formation of a functional pre-BCR in/on pro-B cells. These key events, in turn, appear to be prerequisite both for differentiation of pro-B cells to pre-B cells and for proliferation of these cell subsets upon continued stimulation with IL-7.