Truncated immunoglobulin Dmu causes incomplete developmental progression of RAG-deficient pro-B cells.

Early stages of B cell development are dependent on the expression of a pre-B cell receptor (BCR), composed of a mu heavy chain (HC) in association with surrogate light chain (SLC) proteins and the signaling molecules, Igalpha and Igbeta. During the formation of the variable region of the mu chain by somatic gene rearrangement, a truncated form of the mu protein (called Dmu) is sometimes produced by the rearrangement of a D(H) segment to a J(H) segment using one of three reading frames (designated rf2). When a Dmu protein is formed, subsequent B cell development is blocked by down-regulation of further HC rearrangements, so that a full-length muHC cannot be formed. In this study, we demonstrate that in recombinase activating gene (RAG)-2-deficient B220(+) CD43(+) pro-B cells in which B lymphopoiesis has been arrested at fraction C, transgenic expression of Dmu promoted partial developmental progression to fraction C', but was unable to mediate the pro-B to pre-B cell transition to fraction D effected by full-length muHC protein. These data suggest that the intracellular signaling pathways engaged by the Dmu pre-BCR are insufficient to facilitate the expansion and/or survival of pre-B cells, and are distinct from those engaged by the pre-BCR-containing full-length muHC.     

Conventional light chains inhibit the autonomous signaling capacity of the B cell receptor

Signals from the B cell antigen receptor (BCR), consisting of mu heavy chain (muHC) and conventional light chain (LC), and its precursor the pre-BCR, consisting of muHC and surrogate light chain (SLC), via the adaptor protein SLP-65 regulate the development and function of B cells. Here, we compare the effect of SLC and conventional LC expression on receptor-induced Ca(2+) flux in B cells expressing an inducible form of SLP-65. We found that SLC expression strongly enhanced an autonomous ability of muHC to induce Ca(2+) flux irrespective of additional receptor crosslinking. In contrast, LC expression reduced this autonomous muHC ability and resulted in antigen-dependent Ca(2+) flux. These data indicate that autonomous ligand-independent signaling can be induced by receptor forms other than the pre-BCR. In addition, our data suggest that conventional LCs play an important role in the inhibition of autonomous receptor signaling, thereby allowing further B cell differentiation.     

Involvement of SLP-65 and Btk in tumor suppression and malignant transformation of pre-B cells

Signals from the precursor-B cell receptor (pre-BCR) are essential for selection and clonal expansion of pre-B cells that have performed productive immunoglobulin heavy chain V(D)J recombination. In the mouse, the downstream signaling molecules SLP-65 and Btk cooperate to limit proliferation and induce differentiation of pre-B cells, thereby acting as tumor suppressors to prevent pre-B cell leukemia. In contrast, recent observations in human BCR-ABL1(+) pre-B lymphoblastic leukemia cells demonstrate that Btk is constitutively phosphorylated and activated by the BCR-ABL1 fusion protein. As a result, activated Btk transmits survival signals that are essential for the transforming activity of oncogenic Abl tyrosine kinase.     

Three-dimensional modeling of a pre-B-cell receptor

Signals delivered by the immunoglobulin (Ig)-like pre-B-cell receptor (pre-BCR) are critical for efficient maturation of early precursor B (pre-B) cells. A pre-BCR contains two immunoglobulin mu-heavy chains (muHC), two surrogate light chains (SLC) consisting of the non-covalently associated polypeptides, VpreB and lambda5, and the heterodimeric signaling transducer Igalpha/beta. Although, it is generally accepted that signals initiated from the pre-BCR are required for efficient expansion and differentiation of pre-B cells, the three-dimensional structure of this receptor has not yet been determined by either NMR or X-ray spectroscopy. Therefore, we used indirect computer-assisted molecular modeling techniques to predict for the first time three-dimensional coordinates of the pre-BCR, the conformation of the SLC components, VpreB and lambda5, and the position and flexibility of the so-called non-Ig-like unique tails at the C-terminus of VpreB and the N-terminus of lambda5. Structure prediction revealed that these unique tails of VpreB and lambda5 protrude from the SLC at the position where the CDR3 of a conventional IgLchain would be located. Thus, the unique tails are accessible for ligand binding, which supports the recent finding that the lambda5 unique tail is required for pre-BCR/stroma cell interaction. Further, the non-covalent interaction of the extra beta-strand of lambda5 (beta8) with VpreB is predicted to result in a stabilization of the tertiary structure of VpreB. In summary, three-dimensional computer modeling suggests that the structure of a pre-BCR resembles that of a conventional B-cell receptor (BCR) and that the lambda5 unique tail could be a major binding site for pre-BCR ligands.     

Powered by pairing: the surrogate light chain amplifies immunoglobulin heavy chain signaling and pre-selects the antibody repertoire

Selective expansion of functional pre-B cells is accomplished by the assembly of a signaling-competent pre-B cell receptor (pre-BCR) consisting of immunoglobulin mu heavy chains (muHC), surrogate light chains (SLC) and Igalpha/Igbeta. Here, we review recent data showing that muHCs, in the absence of SLC, deliver autonomous differentiation signals. However, enhanced signaling necessary for pre-B cell expansion requires cross-linking of pre-BCRs via the non-immunoglobulin tail of SLC's subunit lambda5. We also discuss how SLC's ability to modulate the strength of pre-BCR signals is controlled by a muHC's idiotype and its affinity to the chaperone BiP. In this model, BiP in concert with SLC functions as a pre-selector of the antibody repertoire.     

Signal transduction through mu kappa B-cell receptors expressed on pre-B cells is different from that through B-cell receptors on mature B cells.

We introduced kappa light chain genes into pre-B cells to increase the surface mu HC expression, and established transfectants expressing mature type of B-cell receptors (BCR) on pre-B-cell surfaces. Since the cytoplasmic conformations of the reconstituted BCR and intrinsic pre-B-cell receptor (pre-BCR) are identical, they would be connected with the identical signal transduction pathways in pre-B cells. By using the transfectants, we revealed that the reconstituted BCR on pre-B cells was functionally equivalent to BCR on mature B cells in terms of the induction of intracellular Ca++ mobilization. However, we found that the signal-transduction pathways through BCR on pre-B cells were quantitatively different from those mature B cells in two ways. First, cross-linkage of the reconstituted BCR on pre-B cells induced preferential tyrosine phosphorylation of p120 and p100, which was not observed when BCR on mature B cells was cross-linked. Second, BCR in pre-B cells was physically associated with a larger amount of phosphatidylinositol-3 kinase (PI-3K) than BCR in mature B cells in spite of the fact that both pre-B and B cells expressed a similar amount of PI-3K in cytoplasm. Signals through pre-BCR and BCR are known to cause distinct biological effects in B-cell development. The biochemical features in the downstream of reconstituted BCR on pre-B cells, which we revealed in this study, will be of help in understanding the mechanism of functional differences between pre-BCR and BCR.     

Internalization of B cell and pre-B cell receptors is regulated by tyrosine kinase and phosphatase activities

Prior to the expression of the B cell antigen receptor, the μ heavy chain associates with two non-polymorphic polypeptides, λ like and VpreB, which form a pseudo-light chain complex in pre-B cells and pre-B cell lines. Surface expression of the so-called pre-B cell receptor (pre-BCR) occurs only in the presence of Igα and Igβ, known to be involved both in B cell antigen receptor (BCR) signaling and trafficking. Although the pre-BCR organization is consistent with an efficient transport to the cell surface, most of the newly synthesized receptor remains within the cells, and so far, no data are available concerning the rate of exit from the endoplasmic reticulum. Using the human pre-B cell line Nalm-6, we found that only a small fraction (2%) of newly synthesized pre-BCR is transported to the cell surface within 4-6 h after synthesis, where it is constitutively re-internalized. Membrane Ig-heavy chain cross-linking induced internalization of surface pre-BCR within a few minutes, and the mechanisms underlying endocytosis were analyzed by immunofluorescence and confocal microscopy. Prein-cubation of the cells with either genistein or orthovanadate, which inhibit, respectively, tyrosine kinases and tyrosine phosphatases, blocked pre-BCR internalization in a dose-dependent manner, indicating that both activities are required for endocytosis. BCR internalization was also inhibited in a reversible manner by the drugs. In contrast, neither drug affected the size of the steadystate pool of internalized transferrin receptors. Thus, our data show that tyrosine phosphorylation and dephosphorylation are both required for cross-linkinginduced pre-BCR and BCR internalization.     

Btk and phospholipase C gamma 2 can function independently during B cell development

The pre-BCR and the BCR regulate B cell development via a signalosome nucleated by the adaptor protein B cell linker protein (BLNK). Formation of this complex facilitates activation of phospholipase C (PLC) gamma2 by Bruton's tyrosine kinase (Btk). To determine whether Btk and PLCgamma2 also have separate functions, we generated Btk(-/-)PLCgamma2(-/-) mice. They demonstrated a block in development at the pre-B stage and increased pre-BCR surface expression. This phenotype was more severe than that of Btk(-/-) or PLCgamma2(-/-) mice. Although both Btk and PLCgamma2 were required for proliferation of splenic B cells in response to BCR cross-linking, they contributed differently to anti-IgM-induced phosphorylation of ERK. Btk(-/-) and PLCgamma2(-/-) mice each had a reduced frequency of Iglambda-expressing B cells and impaired migration of pre-B cells towards stromal cell-derived factor 1. However, the increase in pre-B cell malignancy that occurs in BLNK(-/-) mice in the absence of Btk was not observed in the absence of PLCgamma2. Thus, Btk and PLCgamma2 act both in concert and independently throughout B cell development.     

The molecular requirements for LAT-mediated differentiation and the role of LAT in limiting pre-B cell expansion

Successful recombination of the heavy-chain locus in developing B cells results in the expression of the pre-BCR, which induces the proliferation and expansion of pre-B cells. To avoid uncontrolled proliferation, pre-BCR signals transmitted via the adaptor protein SLP-65 (SH2-domain-containing leukocyte protein of 65 kDa) lead to the down-regulation of pre-BCR expression and to pre-B cell differentiation. Here, we show that, similarly to SLP-65, the adaptor protein LAT (linker for activation of T cells) limits pre-B cell proliferation and reduces the potential of a tumorgenic pre-B cell line to develop leukemia in immune-deficient mice. We further show that the four distal tyrosines are required for LAT activity in pre-B cells. Mutation at Y136 completely abolishes LAT activity, whereas single point-mutations at Y175, Y195 or Y235 impair, but do not block, LAT-induced pre-B cell differentiation. As LAT is also expressed in human pre-B cells, our results suggest that LAT cooperates with SLP-65 to promote the differentiation and control the proliferation of both murine and human pre-B cells.     

Cross-linking of B cell antigen receptor-related structure of pre-B cell lines induces tyrosine phosphorylation of p85 and p110 subunits and activation of phosphatidylinositol 3-kinase

To understand the function of B cell antigen receptor (BCR)-relatedcomplex on pre-B cells (pre-BCR, V_preB5/µ heavy chain/lg-/lg-ß),we examined pre-BCR- and BCR-mediated signaling events in humanand mouse pre-B (Nalm-6, 697, NFS-5), Immature B (lgM⁺ Daudi,WEH1–231) and mature B (lgM⁺;lgD⁺ BALL1) cell lines. Antl-µcross-linking induced tyrosine phosphorylation of the cytoplasmicproteins In each cell type, but did not induce a detectableCa²⁺ mobilization response in pre-B cells. While the pre-B cellsexpressed Syk protein at levels similar to those found In Bcell lines, pre-BCR cross-linkage did not Induce phosphorylationof Syk tyrosine residues. Different protein kinase C Isozymeswere expressed by pre-B (PKC-), Immature B (PKC- and -ß)and mature B (PKC-ß) cell lines. Anti-µ cross-linkingInduced PKC translocatlon from the cytosolic to the membranecompartment In Immature and mature B cells, but did not havethis effect In a pre-B cell line. Antl-µ cross-linkinginduced tyrosine phosphorytation of the p85 and p110 subunitsof phophatldylinositol 3-kinase (PI3-kinase) In both pre-B andB cell lines, but the pre-BCR induced PI3-kinase activationwas Syk independent. Ligation of the pre-BCR complex thus triggersa characteristic signaling pattern In pre-B cells.     

LAT links the pre-BCR to calcium signaling

SLP-65(-/-) mice display a partial block at the pre-B cell stage of development. Here, we show that LAT is required for the differentiation of SLP-65(-/-) pre-B cells. We show that LAT and SLP-76 are recruited to the pre-BCR and associated with Ig-alpha upon pre-BCR engagement, whereas LAT interaction with SLP-76 is already detected in untreated pre-B cells. Reconstitution of LAT or SLP-65 expression in SLP-65/LAT(-/-) pre-B cells restored their calcium (Ca2+) mobilization capacity, led to downregulation of surface pre-BCR, and induced differentiation to BCR+ cells. Together, our results suggest that the adaptor proteins LAT and SLP-76 are involved in pre-BCR signaling, thereby rescuing arrested murine SLP-65(-/-) pre-B cells.     

Deficiency of BLNK hampers PLC-gamma2 phosphorylation and Ca2+ influx induced by the pre-B-cell receptor in human pre-B cells

B-cell linker protein (BLNK) is a component of the B-cell receptor (BCR) as well as of the pre-BCR signalling pathway, and BLNK(-/-) mice have a block in B lymphopoiesis at the pro-B/pre-B cell stage. A recent report described the complete loss or drastic reduction of BLNK expression in approximately 50% of human childhood pre-B acute lymphoblastic leukaemias (ALL), therefore we investigated BLNK expression in human pre-B ALL cell lines. One of the four cell lines tested, HPB-NULL cells, was found to lack BLNK expression, and we used these human pre-B ALL cell lines that express and do not express BLNK to investigate the intracellular signalling events following pre-BCR cross-linking. When pre-BCR was cross-linked with anti-micro heavy-chain antibodies, significant phosphorylation of intracellular molecules, including Syk, Shc, ERK MAP kinase, and AKT, and an activation of Ras were observed without regard to deficiency of BLNK expression, suggesting that BLNK is not required for pre-BCR-mediated activation of MAP kinase and phosphatidyl-inositol 3 (PI3) kinase signalling. By contrast, phospholipase C-gamma2 (PLC-gamma2) phosphorylation and an increase in intracellular Ca(2+) level mediated by pre-BCR cross-linking were observed only in the BLNK-expressing cells, indicating that BLNK is essential for PLC-gamma2-induced Ca(2+) influx. Human pre-B cell lines expressing and not expressing BLNK should provide an in vitro model for investigation of the role of BLNK in the pre-BCR-mediated signalling mechanism.     

The skewed heavy-chain repertoire in peritoneal B-1 cells is predetermined by the selection via pre-B cell receptor during B cell ontogeny in the fetal liver

As many as 5–15% of B-1 cells in the peritoneal cavityof adult mice produce antibodies reactive to phosphatidylcholine(PtC) and the vast majority of them express B cell receptors(BCRs) composed of V_H11-µH chains utilizing the J_H1 segmentand V9-L chains. This extremely skewed repertoire of PtC-reactiveB-1 cells is traditionally attributed to the expansion of particularclones in response to self or exogenous antigens. Here, we showthat the strong bias toward the J_H1 usage among V_H11-µHchains is already established prior to the BCR assembly, namelyat the transition from the large to the small pre-B cell stageduring B cell ontogeny in the fetal liver. Among V_H11-µHclones isolated from large pre-B cells where the J_H1 skewingwas not established yet, the J_H1 users showed the highest abilityto form pre-B cell receptor (pre-BCR) and to induce cellularproliferation and differentiation when expressed in fetal liverpro-B cells. Thus, the J_H1 users were positively selected andamplified at the pre-BCR checkpoint. When co-expressed withV9-L chains to form BCR, the J_H1 users almost exclusively conferredthe PtC reactivity on BCR even though other J_H users could alsoform BCR on the cell surface. Therefore, the pre-BCR-mediatedpositive selection of the J_H1 users among V_H11-µH chainsappears to be beneficial to the efficient generation of ‘innate-type’PtC-reactive B cells during the fetal B cell development, evenbefore the self-renewal or the antigen-driven clonal expansionof B-1 cells takes place in the peritoneal cavity.     

The Ig-alpha ITAM is required for efficient differentiation but not proliferation of pre-B cells

Signals from the pre-B cell receptor (pre-BCR) mediated by the cytoplasmic tails of Ig-alpha/Ig-beta are essential for developing B cells. To analyze the role of Ig-alpha ITAM and non-ITAM tyrosines in pre-BCR signaling, we reconstituted individual tyrosine mutants of Ig-alpha in src homology 2 domain-containing leukocyte protein of 65 kDa (SLP-65)/Ig-alpha double-deficient pre-B cells. We show that the Ig-alpha mutants led to comparable pre-BCR expression on the cell surface, while the pre-BCR-induced tyrosine phosphorylation was different. We further show that the reconstitution of Ig-alpha and the resulting pre-BCR expression led to enrichment of the pre-BCR-expressing cells in vitro irrespective of the introduced Ig-alpha mutation. We show that, even though the enrichment rate increased by lowering the IL-7 concentration, residual amounts of IL-7 were required for optimal enrichment. Our results indicate that surface IL-7 receptor expression is modulated by the pre-BCR, thereby increasing the IL-7 sensitivity of the respective cells. In contrast to the comparable pre-B cell proliferation, however, the Ig-alpha mutants differed in their capacity to induce calcium flux and activate efficient pre-B cell differentiation. Together, our data suggest that ITAM tyrosines and Y204 are required for efficient pre-B cell differentiation but not proliferation.     

Role of the BCR complex in B cell development,activation, and leukemic transformation

A primary focus of signal transduction in B cells, from the pre-B cell to the mature B cell, is the B cell receptor complex. Here we describe work demonstrating the importance of signaling via the pre-B cell receptor complex (pre-BCR) to the pre-B cell transition, the central checkpoint in B-cell development. We have shown tht pre-BCR complex components Igα and Igβ are critical to allowing the pre-B cell to move through thistransition, but may not be required for allelic exclusion. Pre-BCR expression also directly affects the response of leukemic cells to steroid treatment, suggesting that signals initiated by the pre-BCR complex may present therapeutic targetsin acute leukemia. Additionally, interleukin-7 may also modulate the response of leukemic cells arising from early B-cell stages to treatment. This observation has lead directly to proposals to test drugs which may antagonize early B-cell growth signals, such as rapamycin, in acute lymphoid leukemia.     

The adaptor protein SLP-65 acts as a tumor suppressor that limits pre-B cell expansion

Mice deficient in the adaptor protein SLP-65 (also known as BLNK) have reduced numbers of mature B cells, but an increased pre-B cell compartment. We show here that compared to wild-type cells, SLP-65(-/-) pre-B cells show an enhanced ex vivo proliferative capacity. This proliferation requires interleukin 7 and expression of the pre-B cell receptor (pre-BCR). In addition, SLP-65(-/-) mice have a high incidence of pre-B cell lymphoma. Reintroduction of SLP-65 into SLP-65(-/-) pre-B cells led to pre-BCR down-regulation and enhanced differentiation. Our results indicate that SLP-65 regulates a developmental program that promotes differentiation and limits pre-B cell expansion, thereby acting as a tumor suppressor.