Immunoglobulin mu heavy chains do not mediate tyrosine phosphorylation of Ig alpha from the ER-cis-Golgi

Signals delivered by Ig receptors guide the development of functional B lymphocytes. For example, clonal expansion of early mu heavy chain ( mu HC)-positive pre-B cells requires the assembly of a signal-competent pre-B cell receptor complex (pre-BCR) consisting of a mu HC, a surrogate L chain, and the signal dimer Ig alpha beta. However, only a small fraction of the pre-BCR is transported to the cell surface, suggesting that pre-BCR signaling initiates already from an intracellular compartment, e.g., the endoplasmic reticulum (ER). The finding that differentiation of pre-B cells and allelic exclusion at the IgH locus take place in surrogate L chain-deficient mice further supports the presence of a mu HC-mediated intracellular signal pathway. To determine whether a signal-competent Ig complex can already be assembled in the ER, we analyzed the consequence of pervanadate on tyrosine phosphorylation of Ig alpha in J558L plasmacytoma and 38B9 pre-B cells transfected with either a transport-competent IgL chain-pairing or an ER-retained nonpairing micro HC. Flow cytometry, combined Western blot-immunoprecipitation-kinase assays, and confocal microscopy revealed that both the nonpairing and pairing mu HC assembled with the Ig alpha beta dimer; however, in contrast to a pairing mu HC, the nonpairing mu HC was retained in the ER-cis-Golgi compartment, and neither colocalized with the src kinase lyn nor induced tyrosine phosphorylation of Ig alpha after pervanadate treatment of cells. On the basis of these findings, we propose that a signal-competent Ig complex consisting of mu HC, Ig alpha beta, and associated kinases is assembled in a post-ER compartment, thereby supporting the idea that a pre-BCR must be transported to the cell surface to initiate pre-BCR signaling.     

Pre-B cell receptor assesses the quality of IgH chains and tunes the pre-B cell repertoire by delivering differential signals

It is well understood how a variety of Ig H and L chains, components of BCR, are generated in the DNA level during B cell development. However, it has remained largely unknown whether and how each component is monitored for its quality and selected before the assembly into the BCR. Here we show that muH chains produced by pre-B cells display a wide spectrum of ability to form the pre-BCR, which is composed of muH and surrogate light (SL) chains and is crucial for B cell development. The level of surface pre-BCR expression varies among pre-B cells, depending on the ability of their muH chains to pair with SL chains. The higher the level of pre-BCR expression by pre-B cells, the stronger their pre-BCR signaling, and the better they proliferate and differentiate. Thus, the extent of survival, proliferation, and differentiation of individual pre-B cells is primarily determined by the SL-pairing ability of their muH chains. Furthermore, IgH chains with higher potential to assemble with IgL chains appear to be positively selected and amplified through the assessment of their ability to pair with SL chains at the pre-BCR checkpoint before the assembly into the BCR. These results indicate that the pre-BCR assesses the quality of muH chains and tunes the pre-B cell repertoire by driving the preferential expansion and differentiation of cells with the higher quality of muH chains.     

Interaction of murine precursor B cell receptor with stroma cells is controlled by the unique tail of lambda 5 and stroma cell-associated heparan sulfate

Efficient clonal expansion of early precursor B (pre-B) cells requires signals delivered by an Ig-like integral membrane complex, the so-called pre-B cell receptor (pre-BCR). A pre-BCR consists of two membrane micro H chains, two covalently associated surrogate L chains, and the heterodimeric signaling transducer Igalphabeta. In contrast to a conventional Ig L chain, the surrogate L chain is a heterodimer composed of the invariant polypeptides VpreB and lambda5. Although it is still unclear how pre-BCR signals are initiated, two recent findings support a ligand-dependent initiation of pre-BCR signals: 1) a pre-BCR/galectin-1 interaction is required to induce phosphorylation of Igalphabeta in a human precursor B line, and 2) soluble murine as well as human pre-BCR molecules bind to stroma and other adherent cells. In this study, we show that efficient binding of a soluble murine pre-BCR to stroma cells requires the non-Ig-like unique tail of lambda5. Surprisingly however, a murine pre-BCR, in contrast to its human counterpart, does not interact with galectin-1, as revealed by lactose blocking, RNA interference, and immunoprecipitation assays. Finally, the binding of a murine pre-BCR to stroma cells can be blocked either with heparin or by pretreatment of stroma cells with heparitinase or a sulfation inhibitor. Hence, efficient binding of a murine pre-BCR to stroma cells requires the unique tail of lambda5 and stroma cell-associated heparan sulfate. These findings not only identified heparan sulfate as potential pre-BCR ligands, but will also facilitate the development of appropriate animal models to determine whether a pre-BCR/heparan sulfate interaction is involved in early B cell maturation.     

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.     

CD19 function in early and late B cell development. II. CD19 facilitates the pro-B/pre-B transition

Proliferative expansion of pro-B cells is an IL-7-dependent process that allows for the rearrangement of H chain genes and the expression of the pre-B cell receptor (pre-BCR). Further B cell differentiation is dependent upon signals elicited through the pre-BCR, which are thought to be responsible for allelic exclusion, induced L chain gene rearrangement, and continued proliferation. CD19 promotes the proliferation and survival of mature B cells, but its role in early B cell development is less well understood. Here we identify and characterize impairments in early B cell development in CD19(-/-) mice. Following sublethal irradiation, we found decreased numbers of autoreconstituted early B cells, which was first evident in the large cycling pre-B cell fraction. Reduced cell progression due to a defect in proliferation was made evident from cell cycle analysis and bromodeoxyuridine labeling of bone marrow cells from CD19(-/-) and wild-type mice. Studies of IL-7-dependent pre-B cell cultures derived from wild-type and CD19(-/-) mouse bone marrow suggested that CD19 has little affect on IL-7 signaling. By contrast, signaling through the pre-BCR was impaired in the absence of CD19, as demonstrated by reduced activation of Bruton's tyrosine kinase and extracellular signal-regulated kinase/mitogen-activated protein kinase. Thus, in addition to promoting mature B cell homeostasis and Ag-induced responses, the early onset of CD19 expression acts to enhance B cell generation.     

A unique role for the lambda5 nonimmunoglobulin tail in early B lymphocyte development

Precursor BCR (pre-BCR) signaling governs proliferation and differentiation of pre-B cells during B lymphocyte development. However, it is controversial as to which parts of the pre-BCR, which is composed of Igmu H chain, surrogate L chain (SLC), and Igalpha-Igbeta, are important for signal initiation. Here, we show in transgenic mice that the N-terminal non-Ig-like (unique) tail of the surrogate L chain component lambda5 is critical for enhancing pre-BCR-induced proliferation signals. Pre-BCRs with a mutated lambda5 unique tail are still transported to the cell surface, but they deliver only basal signals that trigger survival and differentiation of pre-B cells. Further, we demonstrate that the positively charged residues of the lambda5 unique tail, which are required for pre-BCR self-oligomerization, can also mediate binding to stroma cell-associated self-Ags, such as heparan sulfate. These findings establish the lambda5 unique tail as a pre-BCR-specific autoreactive signaling motif that could increase the size of the primary Ab repertoire by selectively expanding pre-B cells with functional Igmu H chains.     

A VH12 transgenic mouse exhibits defects in pre-B cell development and is unable to make IgM+ B cells

V(H)12 B cells undergo stringent selection at multiple checkpoints to favor development of B-1 cells that bind phosphatidylcholine. Selection begins with the V(H) third complementarity-determining region (CDR3) at the pre-B cell stage, in which most V(H)12 pre-B cells are selectively eliminated, enriching for those with V(H)CDR3s of 10 aa and a fourth position Gly (designated 10/G4). To understand this selection, we compared B cell differentiation in mice of two V(H)12 transgenic lines, one with the favored 10/G4 V(H)CDR3 and one with a non-10/G4 V(H)CDR3 of 8 aa and no Gly (8/G0). Both H chains drive B cell differentiation to the small pre-BII cell stage, and induce allelic exclusion and L chain gene rearrangement. However, unlike 10/G4 pre-B cells, 8/G0 pre-B cells are deficient in cell division and unable to differentiate to B cells. We suggest that this is due to poor 8/G0 pre-B cell receptor expression and to an inability to form an 8/G0 B cell receptor. Our findings also suggest that V(H)12 H chains have evolved such that association with surrogate and conventional L chains is most efficient with a 10/G4 CDR3. Thus, selection for phosphatidylcholine-binding B-1 cells is most likely the underlying evolutionary basis for the loss of non-10/G4 pre-B cells.     

Clustering of pre-B cell integrins induces galectin-1-dependent pre-B cell receptor relocalization and activation

Interactions between B cell progenitors and bone marrow stromal cells are essential for normal B cell differentiation. We have previously shown that an immune developmental synapse is formed between human pre-B and stromal cells in vitro, leading to the initiation of signal transduction from the pre-BCR. This process relies on the direct interaction between the pre-BCR and the stromal cell-derived galectin-1 (GAL1) and is dependent on GAL1 anchoring to cell surface glycosylated counterreceptors, present on stromal and pre-B cells. In this study, we identify alpha(4)beta(1) (VLA-4), alpha(5)beta(1) (VLA-5), and alpha(4)beta(7) integrins as major GAL1-glycosylated counterreceptors involved in synapse formation. Pre-B cell integrins and their stromal cell ligands (ADAM15/fibronectin), together with the pre-BCR and GAL1, form a homogeneous lattice at the contact area between pre-B and stromal cells. Moreover, integrin and pre-BCR relocalizations into the synapse are synchronized and require actin polymerization. Finally, cross-linking of pre-B cell integrins in the presence of GAL1 is sufficient for driving pre-BCR recruitment into the synapse, leading to the initiation of pre-BCR signaling. These results suggest that during pre-B/stromal cell synapse formation, relocalization of pre-B cell integrins mediated by their stromal cell ligands drives pre-BCR clustering and activation, in a GAL1-dependent manner.     

A Novel Mechanism for the Autonomous Termination of Pre-B Cell Receptor Expression via Induction of Lysosome-Associated Protein Transmembrane 5

The expression of the pre-B cell receptor (BCR) is confined to the early stage of B cell development, and its dysregulation is associated with anomalies of B-lineage cells, including leukemogenesis. Previous studies suggested that the pre-BCR signal might trigger the autonomous termination of pre-BCR expression even before the silencing of pre-BCR gene expression to prevent sustained pre-BCR expression. However, the underlying mechanism remains ill defined. Here we demonstrate that the pre-BCR signal induces the expression of lysosome-associated protein transmembrane 5 (LAPTM5), which leads to the prompt downmodulation of the pre-BCR. While LAPTM5 induction had no significant impact on the internalization of cell surface pre-BCR, it elicited the translocation of a large pool of intracellular pre-BCR from the endoplasmic reticulum to the lysosomal compartment concomitantly with a drastic reduction of the level of intracellular pre-BCR proteins. This reduction was inhibited by lysosomal inhibitors, indicating the lysosomal degradation of the pre-BCR. Notably, the LAPTM5 deficiency in pre-B cells led to the augmented expression level of surface pre-BCR. Collectively, the pre-BCR induces the prompt downmodulation of its own expression through the induction of LAPTM5, which promotes the lysosomal transport and degradation of the intracellular pre-BCR pool and, hence, limits the supply of pre-BCR to the cell surface.     

Core fucosylation of μ heavy chains regulates assembly and intracellular signaling of precursor B cell receptors

α1,6-Fucosyltransferase (Fut8) knock-out (Fut8(-/-)) mice showed an abnormality in pre-B cell generation. Membrane assembly of pre-BCR is a crucial checkpoint for pre-B cell differentiation and proliferation in both humans and mice. The assembly of pre-BCR on the cell surface was substantially blocked in the Fut8-knockdown pre-B cell line, 70Z/3-KD cells, and then completely restored by re-introduction of the Fut8 gene to 70Z/3-KD (70Z/3-KD-re) cells. Moreover, loss of α1,6-fucosylation (also called core fucosylation) of μHC was associated with the suppression of the interaction between μHC and λ5. In contrast to Fut8(+/+) CD19(+)CD43(-) cells, the subpopulation expressing the μHC·λ5 complex in the Fut8(-/-) CD19(+)CD43(-) cell fraction was decreased. The pre-BCR-mediated tyrosine phosphorylation of CD79a and activation of Btk were attenuated in Fut8-KD cells, and restored in 70Z/3-KD-re cells. The frequency of CD19(low)CD43(-) cells (pre-B cell enriched fraction) was also reduced in Fut8(-/-) bone marrow cells, and then the levels of IgM, IgG, and IgA of 12-week-old Fut8(-/-) mice sera were significantly lower than those of Fut8(+/+) mice. Our results suggest that the core fucosylation of μHC mediates the assembly of pre-BCR to regulate pre-BCR intracellular signaling and pre-B cell proliferation.     

Conventional and surrogate light chains differentially regulate Ig mu and Dmu heavy chain maturation and surface expression

Positive selection of precursor (pre-) B cells by Ig membrane mu H chains (mum HC) and counterselection mediated by the truncated HC Dmu depend on the ability of each HC to form a pre-B cell receptor (pre-BCR) signaling complex with the surrogate L chain (SLC) components lambda5 and Vpre-B. To better understand how pre-BCR signaling output is determined by its Ig components and the SLC, we investigated the regulation of pre-BCR surface expression and HC secretory maturation in a new nonlymphoid system. We took this approach as a means to distinguish B-lineage-specific effects from pre-BCR-intrinsic properties that may influence these aspects of pre-BCR homeostasis necessary for signaling. As in pre-B cells, the SLC in nonlymphoid cells supported only a limited degree of mum HC maturation and low pre-BCR surface expression levels compared with conventional LCs, indicating that this was due to an intrinsic property of the SLC. We identified the non-Ig region of lambda5 as harboring the restrictive activity responsible for this phenotype. This property of lambda5 was also evident with Dmu, but the overall SLC- and L chain-dependent requirements for Dmu maturation and surface expression were markedly different from those for mum. Surprisingly, Dmu was modified in an unusual manner that was only dependent on Vpre-B. These results establish a novel function of lambda5 in limiting surface pre-BCR levels and reveal biochemical properties of Ig molecules that may underlie the diverse consequences of pre-BCR signaling in vivo by different HCs.     

Cutting edge: signaling and cell surface expression of a mu H chain in the absence of lambda 5: a paradigm revisited

Pre-B cell receptor (pre-BCR) signals are essential for pro-B cells to mature efficiently into pre-B cells. The pre-BCR is an Ig-like transmembrane complex that is assembled from two mu H chains (mu HC) and two surrogate L chains consisting of the non-covalently associated polypeptides VpreB and lambda5. In lambda5(-/-) mice, pro-B cell maturation is impaired, but not completely blocked, implying that a mu HC induces differentiation signals in the absence of lambda5. Using a mouse model, in which transgenic mu HC expression can be controlled by tetracycline, we show that in the absence of lambda5, the transgenic mu HC promotes in vivo differentiation of pro-B cells, induces IL-7-dependent cell growth, and is expressed on the surface of pre-B cells. Our findings not only show that an incomplete pre-BCR can initiate signals, but also challenge the paradigm that an IgHC must associate with an IgLC or a SLC to gain transport and signaling competency.     

Cutting edge: B cell linker protein is dispensable for the allelic exclusion of immunoglobulin heavy chain locus but required for the persistence of CD5+ B cells

The pre-B cell receptor (pre-BCR) and the BCR are required for B lymphopoiesis and for the allelic exclusion of Ig genes. Mice lacking B cell linker (BLNK) protein that is a component of the BCR signaling pathway have impaired B cell development. In this report, we show that allelic exclusion is intact in BLNK(-/-) mice harboring a V(H)12 transgene. This differs from mice lacking the tyrosine kinase Syk that is upstream of BLNK in BCR signaling and contrasts with mice lacking SLP-76 that is the equivalent adaptor molecule in TCR-signal transduction. We also show that, whereas most wild-type V(H)12-expressing B cells are CD5(+), the majority of the splenic V(H)12-expressing BLNK(-/-) B cells are CD5(-). A small population of V(H)12-expressing, BLNK(-/-) CD5(+) B cells is detectable in the peritoneal cavity of younger but not older mice. This suggests that BLNK deficiency affects not only the generation but also the persistence of B-1 cells.     

The N terminus of the non-T cell activation linker (NTAL) confers inhibitory effects on pre-B cell differentiation

SLP-65 and the linker for activation of T cells (LAT) are central adaptor proteins that link the activated pre-BCR to downstream events in pre-B cells. Recently, a new transmembrane adaptor called NTAL/LAB/LAT2 (hereafter called NTAL for non-T cell activation linker) with striking functional and structural similarity to LAT has been identified in B cells. In this study, we compare the function of NTAL and LAT in pre-BCR signaling and show that, in contrast to LAT, NTAL does not induce pre-BCR down-regulation, calcium flux, or pre-B cell differentiation. To test whether differences between NTAL-mediated and LAT-mediated signaling are caused by the missing phospholipase C (PLC)-gamma binding motif in NTAL, we inserted the PLC-gamma1/2 binding motif of LAT into NTAL. This insertion rendered NTAL capable of activating pre-BCR down-regulation and calcium flux. Unexpectedly however, the ability of NTAL to induce calcium flux was not sufficient to promote pre-B cell differentiation, suggesting that the PLC-gamma binding motif has only partial effects on NTAL-mediated pre-BCR signaling. By generating chimeric swap mutants, we identified the N terminus of NTAL as an inhibitory domain that prevents pre-B cell differentiation while allowing pre-BCR down-regulation and receptor-mediated calcium flux. Our data suggest that, in addition to the missing PLC-gamma1/2 binding motif, the N terminus is responsible for the functional differences between NTAL and LAT in pre-B cells.     

VPREB3: cDNA characterization and expression in human and chromosome mapping in human and mouse

The pre-B cell receptor (pre-BCR) regulates pre-B cell expansion and allelic exclusion at the immunoglobulin (Ig) heavy chain locus and mediates the selection of Ig heavy chain variable gene segments. During the early phase of pre-BCR assembly in the mouse, the membrane Ig mu heavy chain transiently associates with the VPREB3 protein in the endoplasmic reticulum. Here, we present the human VPREB3 cDNA sequence and its B cell-specific expression in hematopoietic cell lines. We have localized this gene to chromosome 22q11 close to IGLL genes in human and to chromosome 10C in mouse.     

Developmental differences in B cell receptor-induced signal transduction

We have compared early signaling events at various stages of B cell differentiation using established mouse cell lines. Clustering of pre-B cell antigen receptor (BCR) or BCR induced the tyrosine phosphorylation of various proteins in all cells, although the phosphorylation pattern differed. In spite of the pre-BCR-induced tyrosine phosphorylation, we could not detect an intracellular Ca(2+) signal in pre-B cells. However, co-clustering of the pre-BCR with CD19 did induce Ca(2+) mobilization. In contrast to the immature and mature B cells, where the B cell linker protein (BLNK) went through inducible tyrosine phosphorylation upon BCR clustering, we observed a constitutive tyrosine phosphorylation of BLNK in pre-B cell lines. Both BLNK and phospholipase C (PLC)gamma were raft associated in unstimulated pre-B cells, and this could not be enhanced by pre-BCR engagement, suggesting a ligand-independent PLC gamma-mediated signaling. Further results indicate that the cell lines representing the immature stage are more sensitive to BCR-, CD19- and type II receptors binding the Fc part of IgG (Fc gamma RIIb)-mediated signals than mature B cells.     

Redundancy in B cell developmental pathways: c-Cbl inactivation rescues early B cell development through a B cell linker protein-independent pathway

Deficiency in the adaptor protein B cell linker protein (BLNK) results in a substantial but incomplete block in B cell development, suggesting that alternative pathways exist for B lineage differentiation. Another adaptor protein, c-Cbl, plays a negative regulatory role in several BCR-signaling pathways. We therefore investigated the role of c-Cbl during B cell development and addressed the possibility that redundancies in pathways for B cell differentiation could be further revealed by eliminating negative effects mediated by c-Cbl. Strikingly, c-Cbl inactivation reversed a number of the critical defects in early B cell differentiation that are seen in BLNK-deficient mice. c-Cbl(-/-)BLNK(-/-) mice exhibited normalized down-regulation of pre-BCR and CD43, up-regulation of MHC class II, and augmented L chain rearrangement, resulting in a successful transition from pre-B cells to immature B cells. c-Cbl inactivation also reversed the potentially tumor-predisposing hyperproliferative response of BLNK(-/-) pre-B cells to IL-7. Pre-BCR cross-linking induced enhanced and prolonged tyrosine phosphorylation in c-Cbl(-/-)BLNK(-/-) pre-BCR(+) pre-B cells compared with c-Cbl(+/-)BLNK(-/-) cells, including elevated phosphorylation of Lyn, Syk, Btk, and phospholipase C-gamma2. Our studies suggest that some, but not all, pre-BCR-triggered developmental events can be mediated by BLNK-independent pathways that are negatively regulated by c-Cbl, and further suggest that different events during early B cell development require different strength or duration of pre-BCR signaling.     

Multiple levels of selection responsive to immunoglobulin light chain and heavy chain structures impede the development of Dmu-expressing B cells

The truncated/V(H)-less mouse H chain Dmu forms precursor B cell receptors with the surrogate L chain complex that promotes allelic exclusion but not other aspects of pre-B cell development, causing most progenitor B cells expressing this H chain to be eliminated at the pre-B cell checkpoint. However, there is evidence that Dmu-lambda1 complexes can be made and are positively selected during fetal life but cannot sustain adult B lymphopoiesis. How surrogate and conventional L chains interpret Dmu's unusual structure and how that affects signaling outcome are unclear. Using nonlymphoid and primary mouse B cells, we show that secretion-competent lambda1 L chains could associate with both full-length H chains and Dmu, whereas secretion-incompetent lambda1 L chains could only do so with full-length H chains. In contrast, Dmu could not form receptors with a panel of kappa L chains irrespective of their secretion properties. This was due to an incompatibility of Dmu with the kappa-joining and constant regions. Finally, the Dmu-lambda1 receptor was less active than the full-length mouse mu-lambda1 receptor in promoting growth under conditions of limiting IL-7. Thus, multiple receptor-dependent mechanisms operating at all stages of B cell development limit the contribution of B cells with Dmu H chain alleles to the repertoire.