B-cell Antigen Receptor Complex Research Articles

A symmetric geometry of transmembrane domains inside the B cell antigen receptor complex

B lymphocytes have the ability to sense thousands of structurally different antigens and produce cognate antibodies against these molecules. For this they carry on their surface multiple copies of the B cell antigen receptor (BCR) comprising the membrane-bound Ig (mIg) molecule and the Igα/Igβ heterodimer functioning as antigen binding and signal transducing components, respectively. The mIg is a symmetric complex of 2 identical membrane-bound heavy chains (mHC) and 2 identical light chains. How the symmetric mIg molecule is asymmetrically associated with only one Igα/Igβ heterodimer has been a puzzle. Here we describe that Igα and Igβ both carry on one side of their α-helical transmembrane domain a conserved amino acid motif. By a mutational analysis in combination with a BCR rebuilding approach, we show that this motif is required for the retention of unassembled Igα or Igβ molecules inside the endoplasmic reticulum and the binding of the Igα/Igβ heterodimer to the mIg molecule. We suggest that the BCR forms within the lipid bilayer of the membrane a symmetric Igα-mHC:mHC-Igβ complex that is stabilized by an aromatic proline-tyrosine interaction. Outside the membrane this symmetry is broken by the disulfide-bridged dimerization of the extracellular Ig domains of Igα and Igβ. However, symmetry of the receptor can be regained by a dimerization of 2 BCR complexes as suggested by the dissociation activation model.

Tumor necrosis factor receptor associated factor 3 (TRAF3) regulates B cell antigen receptor (BCR) signaling

Abstract Tumor necrosis factor receptor associated factor 3 (TRAF3) is an intracellular adaptor protein that plays multiple cell type- and context-dependent roles. In B cells, TRAF3 negatively regulates signaling by several receptors important to survival and activation, including the B cell activating factor receptor (BAFF-R), CD40, toll-like receptors and the IL-6 receptor. Deletions or inactivating mutations of TRAF3 are common in the human B cell malignancies multiple myeloma and B cell lymphoma (BCL). Enhanced homeostatic B cell survival in B cell-specific TRAF3-deficient mice (B-Traf3−/−) causes enlarged secondary lymphoid organs, spontaneous germinal centers, and autoantibody production. Highlighting the role TRAF3 plays as a B cell tumor suppressor, B-Traf3−/− mice develop BCL at an increased rate as they age. The B cell antigen receptor (BCR) is crucial for B cell antigen recognition, survival, proliferation, and antibody production. Defects in BCR signaling can lead to malignancy, and many BCLs are dependent upon BCR signaling for survival. TRAF3 regulates the signaling of several pathways that interact with the BCR pathway, but the role of TRAF3 in regulating BCR signals is unknown, a knowledge gap we are currently addressing. Our initial studies show that TRAF3 associates with CD79b of the BCR complex. Phosphorylation of Syk, a proximal BCR signaling target, is increased after BCR stimulation in the absence of TRAF3, with increased downstream phosphorylation and activation of Btk, PLCγ2, Akt, Erk, and canonical NF-κB. This suggests that TRAF3 plays an important role as a regulator of BCR signaling. Ongoing studies are examining the mechanisms of this regulation.

Crystalline silica activates the T-cell and the B-cell antigen receptor complexes and induces T-cell and B-cell proliferation

Silicosis is an occupational fibrotic lung disease, which is associated with an increased incidence of autoimmune diseases. The effect of crystalline silica on the immune system is thought to be mediated by the antigen presenting cells. However, the direct effect of silica on T-cells and B-cells has not been evaluated adequately. For this purpose, CD4(+)T-cells and B-cells from 10 healthy individuals were isolated and cultured with or without Min-U-Sil 5. Cell proliferation was assessed with BrdU assay. In cell proliferation experiments, tacrolimus, an inhibitor of the signal transduction derived from the activation of the T-cell or the B-cell antigen receptor (BCR) complex, was also used. The levels of phosphorylated zeta and phosphorylated Igα, indicative of the T-cell and BCR complex activation respectively, and of the transcription factor c-Myc, required for cell proliferation, were assessed by Western blotting. Crystalline silica triggered CD4(+)T-cell and B-cell proliferation, while tacrolimus significantly decreased the silica-induced proliferation in both cell types. Crystalline silica enhanced the level of phosphorylated zeta and phosphorylated Igα in CD4(+)T-cells and B-cells, respectively. In both cell types, treatment with silica increased c-Myc expression. Thus, crystalline silica may induce T-cell and B-cell proliferation by activating T-cell and BCR complexes. It is likely that the direct activation of CD4(+)T-cells and B-cells by silica crystals detected in this study circumvents many self-tolerance check-points and offers a mechanistic explanation for the crystalline silica-induced autoimmune diseases.

Grouper (Epinephelus coioides) BCR signaling pathway was involved in response against Cryptocaryon irritans infection

B cell antigen receptor (BCR) plays a crucial role in B cell development and antibody production. It comprises membrane immunoglobulin non-covalently associated with CD79a/CD79b heterodimer. After B cell activation, initial extracellular signals are transduced by BCR complex and amplified by two protein tyrosine kinases, LYN and SYK, which then trigger various pathways. In the present study, we cloned grouper genes for BCR accessory molecules, EcCD79a (669 bp) and EcCD79b (639 bp), as well as two protein tyrosine kinases, EcLYN (1482 bp) and EcSYK (1854 bp). Homology analysis showed that all four molecules had a relatively high amino acid identity compared with those in other animals. Among them, they all shared the highest identity with Takifugu rubripes (EcCD79a 49%, EcCD79b 52%, EcLYN 82% and EcSYK 77%). The conserved features and important functional residues were analyzed. Together with IgM and IgT, tissue distribution analysis showed that all six molecules were mainly expressed in immune organs, particularly systematic immune organs. In groupers infected with Cryptocaryon irritans, up-regulation of EcCD79a and b, EcIgM and EcIgT were not seen in the early stage skin and gill until 14–21 days. Up-regulation of EcCD79a was seen in head kidney at most time points, while EcCD79a and b were only significantly up-regulated in day 14 spleen. Significant up-regulation of EcIgT were seen in day 21 head kidney and day 1, day14 spleen. Significant up-regulation of EcIgM were seen in day 1 head kidney and 12 h spleen. In addition, two protein kinase genes, EcLYN and EcSYK, were up-regulated in the skin at most time points, which suggested that B cells may be activated at the skin local infection site.

Receptor Dissociation and B-Cell Activation.

The B-cell antigen receptor (BCR) is one of the most abundant receptors on the surface of B cells with roughly 100,000-200,000 copies per cell. Signaling through the BCR is crucial for the activation and differentiation of B cells. Unlike other receptors, the BCR can be activated by a large set of structurally different ligands, but the molecular mechanism of BCR activation is still a matter of controversy. Although dominant for a long time, the cross-link model (CLM) of BCR activation is not supported by recent studies of the nanoscale organization of the BCR on the surface of resting B cells. In contrast to the prediction of CLM, the numerous BCR complexes on these cells are not randomly distributed monomers but rather form oligomers which reside within membrane confinements. This finding is more in line with the dissociation activation model (DAM), wherein B-cell activation is accompanied by an opening of the auto-inhibited BCR oligomers instead of a cross-linking of the BCR monomers. In this review, we discuss in detail the new findings and their implications for BCR signaling.

Probing of a human proteome microarray with a recombinant pathogen protein reveals a novel mechanism by which hookworms suppress B-cell receptor signaling.

Na-ASP-2 is an efficacious hookworm vaccine antigen. However, despite elucidation of its crystal structure and studies addressing its immunobiology, the function of Na-ASP-2 has remained elusive. We probed a 9000-protein human proteome microarray with Na-ASP-2 and showed binding to CD79A, a component of the B-cell antigen receptor complex. Na-ASP-2 bound to human B lymphocytes ex vivo and downregulated the transcription of approximately 1000 B-cell messenger RNAs (mRNAs), while only approximately 100 mRNAs were upregulated, compared with control-treated cells. The expression of a range of molecules was affected by Na-ASP-2, including factors involved in leukocyte transendothelial migration pathways and the B-cell signaling receptor pathway. Of note was the downregulated transcription of lyn and pi3k, molecules that are known to interact with CD79A and control B-cell receptor signaling processes. Together, these results highlight a previously unknown interaction between a hookworm-secreted protein and B cells, which has implications for helminth-driven immunomodulation and vaccine development. Further, the novel use of human protein microarrays to identify host-pathogen interactions, coupled with ex vivo binding studies and subsequent analyses of global gene expression in human host cells, demonstrates a new pipeline by which to explore the molecular basis of infectious diseases.

Suppression of rheumatoid arthritis B cells by XmAb5871, an anti-CD19 antibody that coengages B cell antigen receptor complex and Fcγ receptor IIb inhibitory receptor.

Engagement of Fcγ receptor IIb (FcγRIIb) suppresses B cell activation and represents a promising target for therapy in autoimmunity. The aim of this study was to characterize B cell immunosuppression mediated by the Fc-engineered antibody, XmAb5871, which coengages FcγRIIb with the B cell antigen receptor (BCR) complex and that is currently in clinical development for the treatment of rheumatoid arthritis (RA). Because rheumatoid factor (RF) might interfere with the binding of XmAb5871 to FcγRIIb, we correlated RF titers with the potency of XmAb5871. We analyzed the expression of CD19, FcγRIIb, and CD86 on naive and memory B cells from 50 patients with RA and 66 healthy donors, quantified XmAb5871-induced promotion of FcγRIIb phosphorylation and suppression of calcium flux in activated B cells, measured CD86 inhibition in whole blood, and correlated RF and anti-citrullinated protein antibody (ACPA) levels with drug potency. We engrafted RA peripheral blood mononuclear cells (PBMCs) into SCID mice, treated them with XmAb5871, and quantified human total IgG, total IgM, and anti-tetanus IgG antibody levels in vivo. B cells from all donors expressed CD19 and FcγRIIb, and the expression of FcγRIIb was higher on naive, but not memory, B cells from donors with RA compared with healthy donors. BCR-mediated calcium flux was suppressed by XmAb5871 and was associated with FcγRIIb phosphorylation. XmAb5871 inhibited CD86 induction, and the levels of RF and ACPAs did not affect efficacy. XmAb5871 suppressed B cell activation regardless of disease severity. In SCID mice engrafted with PBMCs from a patient with RA, XmAb5871 suppressed humoral responses. Coengagement of the BCR complex and FcγRIIb by XmAb5871 inhibits B cell activation and function. The similar potency in patients with RA and healthy donors and the absence of autoantibody interference suggest that XmAb5871 may represent a new therapeutic strategy to suppress autoreactive B cells in RA.

Synthetic Antigens Reveal Dynamics of BCR Endocytosis during Inhibitory Signaling

B cells detect foreign antigens through their B cell antigen receptor (BCR). The BCR, when engaged by antigen, initiates a signaling cascade. Concurrent with signaling is endocytosis of the BCR complex, which acts to downregulate signaling and facilitate uptake of antigen for processing and display on the cell surface. The relationship between signaling and BCR endocytosis is poorly defined. Here, we explore the interplay between BCR endocytosis and antigens that either promote or inhibit B cell activation. Specifically, synthetic antigens were generated that engage the BCR alone or both the BCR and the inhibitory co-receptor CD22. The lectin CD22, a member of the Siglec family, binds sialic acid-containing glycoconjugates found on host tissues, inhibiting BCR signaling to prevent erroneous B cell activation. At low concentrations, antigens that can cocluster the BCR and CD22 promote rapid BCR endocytosis; whereas, slower endocytosis occurs with antigens that bind only the BCR. At higher antigen concentrations, rapid BCR endocytosis occurs upon treatment with either stimulatory or inhibitory antigens. Endocytosis of the BCR, in response to synthetic antigens, results in its entry into early endocytic compartments. Although the CD22-binding antigens fail to activate key regulators of antigen presentation (e.g., Syk), they also promote BCR endocytosis, indicating that inhibitory antigens can be internalized. Together, our observations support a functional role for BCR endocytosis in downregulating BCR signaling. The reduction of cell surface BCR levels in the absence of B cell activation should raise the threshold for BCR subsequent activation. The ability of the activating synthetic antigens to trigger both signaling and entry of the BCR into early endosomes suggests strategies for targeted antigen delivery.

Oligomeric organization of the B-cell antigen receptor on resting cells

B lymphocytes are activated by many different antigens to produce specific antibodies protecting higher organisms from infection. To detect its cognate antigen, each B cell contains up to 120,000 B-cell antigen receptor (BCR) complexes on its cell surface. How these abundant receptors stay silent on resting B cells and how they can be activated by a molecularly diverse set of ligands is poorly understood. Here we show, with the use of a quantitative bifluorescence complementation assay (BiFC), that the BCR has an intrinsic ability to form oligomers on the surface of living cells. A BCR mutant that fails to form oligomers is more active and cannot be expressed stably on the B-cell surface, whereas BiFC-stabilized BCR oligomers are less active and more strongly expressed on the surface. We propose that oligomers are the autoinhibited form of the BCR and that it is the shift from closed BCR oligomers to clustered monomers that drives B-cell activation in a way that is independent of the structural input from the antigen.

Negative modulation of B-cell function via inhibitory receptor signaling by using novel diabody-based Dual-Affinity Re-Targeting (DART) molecules (96.14)

Abstract The Fc receptor, CD32B, functions as an inhibitory receptor and, when co-ligated with the B-cell antigen receptor (BCR) complex, dampens signaling and immune activation. Ligation of activating and inhibitory receptors is an obligatory event for CD32B inhibition coupling in B cells, which heightens the need for a bispecific molecule for interventional purposes. To exploit this mechanism, we have engineered a novel and stable bispecific diabody scaffold that facilitates heterodimeric assembly with high predictability. This Dual-Affinity Re-Targeting (DART) scaffold was used to couple an Fv specific for the inhibitory receptor, CD32B, with an Fv specific for the Ig-beta component (CD79B) of the BCR. The resulting CD32BxCD79B DART molecule was capable of co-ligating the activating and the inhibitory receptors on the B-cell surface, resulting in reduced BCR-induced calcium mobilization and phosphorylation of selected signaling molecules. These biochemical changes correlated with inhibition of B-cell proliferation and Ig secretion upon B-cell activation. In addition, a DART protein specific for the mouse orthologs of CD32B and CD79B reduced disease severity in a collagen-induced arthritis model. This novel approach to B-cell function modulation forms the basis for translating inhibitory signaling into therapeutic tools for managing autoimmune and inflammatory diseases.

Low-valency, but not monovalent, antigens trigger the B-cell antigen receptor (BCR)

Antigen binding to the B-cell antigen receptor (BCR) leads to receptor triggering and B-lymphocyte activation. Here, we have probed the molecular requirements for BCR triggering in primary murine B cells using a set of defined soluble haptenated peptides. Bi- and trivalent haptens activated the BCR, as measured by protein phosphorylation, Ca(2+) influx, BCR down-modulation and CD69, CD86 and MHC class II up-regulation. In contrast, four distinct monovalent haptens were ineffective. Next, we used two different anti-idiotypic antibodies, which bind to the antigen-combining site of the BCR. Again, monovalent Fab fragments were ineffective, whereas bivalent antibodies could stimulate the BCR. These findings are compatible with ligand-induced clustering of monomeric BCRs or re-organization of BCR complexes within pre-formed BCR oligomers. Lastly, an increase in the valency of the haptenated peptides improved the activation potential, whereas variations in the distance between two haptens had no effect. This finding contributes to understand how the immune system can efficiently recognize structurally diverse antigens but still discriminate between foreign and self.

HVCN1 modulates BCR signal strength via regulation of BCR-dependent generation of reactive oxygen species.

Voltage-gated proton currents regulate generation of reactive oxygen species (ROS) in phagocytic cells. In B cells, stimulation of the B cell antigen receptor (BCR) results in the production of ROS that participate in B cell activation, but the involvement of proton channels is unknown. We report here that the voltage-gated proton channel HVCN1 associated with the BCR complex and was internalized together with the BCR after activation. BCR-induced generation of ROS was lower in HVCN1-deficient B cells, which resulted in attenuated BCR signaling via impaired BCR-dependent oxidation of the tyrosine phosphatase SHP-1. This resulted in less activation of the kinases Syk and Akt, impaired mitochondrial respiration and glycolysis and diminished antibody responses in vivo. Our findings identify unanticipated functions for proton channels in B cells and demonstrate the importance of ROS in BCR signaling and downstream metabolism.

Resting and activated states of the B cell antigen receptor

Each B cell contains up to 100,000 B cell antigen receptor (BCR) complexes on its surface, which only become fully active on exposure of the B cell to its cognate antigen. Most studies on BCR function aims at a better understanding how the BCR becomes active and transmits its signal to the many signaling pathways inside the cell. Another, albeit related question, is how the B cells ensure that the many BCR complexes stay (in most cases) in a silent inert conformation. This problem of activation control the BCR shares with many of its signaling elements, be it kinases, phosphatases or adaptors, which all have to be regulated tightly in their localization and activity. For the later element autoinhibition plays an essential role in this regulation. In a biochemical study we previously have found that on resting B cells the BCR resides in oligomeric and monomeric forms. We now have conducted experiments that suggest that the BCR also can resume an autoinhibitory conformation and that BCR oligomerization plays an important role in this process. Specifically oligomerization of the BCR in conjunction with the kinase-phosphatase equilibrium at the BCR contributes to the tight control of BCR activation. Our studies resulted in a new model of the resting BCR and its activation. We also study how the cytosolic protein tyrosine kinase Syk is regulated and activated by the BCR. Syk is also regulated by auto-inhibition and only becomes fully active when recruited to the BCR. We now have identified Syk mutant that are altered in auto-inhibition and tested their activity in the S2 Schneider cell reconstitution system as well as in reconstituted Syk deficient B cell lines. Our data show that a tight Syk regulation is essential for normal B-lymphocytes development.

High CD79b Expression Predicts a Poor Outcome in B-Cell Chronic Lymphocytic Leukemia (B-CLL).

[Display omitted] The immunoglobulin (Ig) beta protein (CD79b) dimerizes with Ig alpha (CD79a) to form the signaling component of the B-cell antigen receptor complex (BCR). Given the critical role of Ig beta in BCR signaling, it has been suggested that its variable expression in CLL may induce either cell proliferation or apoptosis. High CD79b expression has been associated with atypical morphology, strong expression of surface Ig, advanced clinical stage and short overall survival in B-CLL (Garcia Vela, 1999; Zucchetto, 2006). Moreover, high CD38 expression was correlated with high CD40, CD69 and CD79b which are consistent with an activation phenotype (Damle, 2002). Furthermore, we have demonstrated that the ZAP-70+ CLL subgroup shows a rapid disease progression and an inferior overall survival (Del Principe, 2006). Since it has been described that BCR engagement has significant effects on B-CLL cell survival, activation and cell cycle progression (Deglesne, 2006), we decided to evaluate the real impact of CD79b expression on B-CLL prognosis. The primary aims of our research were: 1) to determine progression-free survival (PFS) and overall survival (OS) upon CD79b expression; 2) whether CD79b could predict varied outcome within ZAP-70+ and ZAP-70 negative subgroups; and finally 3) whether CD79b was an independent prognostic factor. Therefore, we investigated 401 patients (pts), median age 65 years (range 33–89), 213 males and 188 females. With regard to modified Rai stages, 123 pts had a low stage, 261 an intermediate stage and 17 a high stage. CD79b was determined by multicolor flow cytometry, using the monoclonal antibody anti-CD79 beta-FITC (clone SN8, Dako) and fixing a cut-off value of 30%. CD79b+ B-CLL pts were 207/401 (52%). CD79b>30% was significantly associated with the intermediate/high Rai stage (p=0.00001), beta-2 microglobulin >2.2 mg/dl (p<0.0001) and with multiple intrathoracic/abdominal lymphadenopathies and/or splenomegaly (p<0.0001). Low CD79b was significantly correlated either with IgVH mutated status (>2%) (86/105; p<0.0001) or lower soluble CD23 levels (125/161; p<0.00001). Significant associations were found either between CD38<30% and lower CD79b (172/194; p<0.0001) or ZAP- 70<20% and CD79b<30% (145/193; p<0.0001). With regard to cytogenetics, there were strict correlations either between high CD79b and trisomy 12 (25/33; p=0.001) or high CD79b and del17p (16/20; p=0.001). Median follow up duration from diagnosis was 68 months (range 6–230). Concerning clinical outcome, both a shorter PFS (Figure) and OS were observed in CD79b+ pts (12% vs 58% at 16 years; p<0.0001 and 49% vs 85% at 16 years; p<0.0001) as well as in ZAP-70+ pts (5% vs 52% at 11 years; p<0.0001 and 33% vs 87% at 18 years; p<0.0001). To further explore the prognostic value of CD79b, we investigated its expression within ZAP-70+ (159 pts) and ZAP-70 negative (242 pts) subsets. As a matter of fact, CD79b+ pts showed a shorter PFS and OS both within the ZAP-70+ subset (7% vs 19% at 10 years; p=0.00006 and 25% vs 65% at 16 years; p=0.006) and within the ZAP-70 negative subset (29% vs 67% at 14 years, p<0.00001 and 86% vs 100% at 14 years; p=0.01). In multivariate analysis of PFS and OS, in which age, Rai stages, CD38, CD69, CD79b and ZAP-70 entered, ZAP-70 (p=0.001 and p=0.001), CD69 (p=0.001 and p=0.01) and CD79b (p=0.0007 and p=0.03) resulted to be independent prognostic factors. Therefore CD79b, easily determined by flow cytometry, should be considered another important prognostic parameter and could be used to early identify and stratify B-CLL progressive pts.

The transmembrane tyrosine of -heavy chain is required for BCR destabilization and entry of antigen into clathrin-coated vesicles

The B cell antigen receptor (BCR) delivers antigen to the endocytic compartment and transduces signals that regulate the stability of the receptor complex. Previous studies showed that BCR-mediated signal transduction dissociates micro-heavy chain (microm) from Ig-alpha/Ig-beta, facilitating the delivery of antigen to clathrin-coated vesicles (CCVs). Herein, we demonstrate that the dissociation of Ig-alpha/Ig-beta from microm requires tyrosine-587 of the microm transmembrane domain. Receptors expressing a mutation at tyrosine-587 (Y587F) transduced signals that were comparable to wild type, yet they failed to dissociate microm from Ig-alpha/Ig-beta. Further, receptors harboring the Y587F mutation failed to associate with CCVs, resulting in diminished antigen in the lysosome-associated membrane protein-1 (LAMP-1(+)) compartment and severely impaired antigen presentation, indicating that endocytosis through CCVs is required for antigen presentation. Thus, the transmembrane tyrosine of mum mediates destabilization of the BCR complex, facilitating antigen processing by promoting the association of antigen with CCVs.

CD79a Is Heterogeneously Expressed in Neoplastic and Normal Myeloid Precursors and Megakaryocytes in an Antibody Clone–Dependent Manner

CD79a, a component of the B-cell antigen receptor complex, can also be expressed in certain non-B-cell malignancies. The reported frequency of CD79a expression in acute myeloid leukemias (AML) ranges from 0% to 90%. We evaluated 39 bone marrow biopsy specimens (29 AML and 10 normal cases) using 5 different commercially available anti-CD79a monoclonal antibody (MoAb) clones. Of 7 acute promyelocytic leukemia (APL) cases, 6 (86%) stained for CD79a with clones HM47/A9 (Novocastra, Newcastle Upon Tyne, England) and HM57 (DAKO, Carpinteria, CA) but were negative with clones 11E3 (Novocastra), and JCB117 (DAKO). Half of 6 acute megakaryoblastic leukemia (AMKL) cases and normal megakaryocytes in 14 (67%) of 21 cases were immunoreactive using clone 11D10 (Novocastra). Approximately one third of non-APL/non-AMKL AML and myeloid precursors in normal marrow specimens stained with clones HM57 and 11D10. This heterogeneity of CD79a expression in AML, megakaryocytes, and myeloid precursors is MoAb clone-dependent, likely owing to different epitope detection, and may be of diagnostic usefulness.

The Adaptor Protein HSH2 Attenuates Apoptosis in Response to Ligation of the B Cell Antigen Receptor Complex on the B Lymphoma Cell Line, WEHI-231

Signals transduced by the B cell antigen receptor (BCR) play a central role in regulating the functional response of the cell to antigen. Depending on the nature of the antigenic signal and the developmental or differentiation state of the B cell, antigen receptor signaling can promote either apoptosis or survival and activation. Understanding the molecular mechanisms underlying BCR-mediated apoptosis constitutes an important area of research because aberrations in programmed cell death can result in the development of autoimmunity or cancer. Expression of the adaptor protein hematopoietic Src homology 2 (HSH2) was found to significantly decrease BCR-mediated apoptosis in the murine WEHI-231 cell line. Analysis of signal transduction pathways activated in response to BCR ligation revealed that HSH2 does not significantly alter total protein tyrosine phosphorylation or Ca2+ mobilization. HSH2 does not potentiate the activation-dependent phosphorylation of AKT either. With respect to MAPK activation, HSH2 was not observed to alter the activation of ERK or p38 in response to BCR ligation, but it does significantly potentiate JNK activation. Analysis of processes directly associated with apoptosis revealed that HSH2 inhibits mitochondrial depolarization to a significant degree, whereas it has only a slight effect on caspase activation and poly ADP-ribose polymerase cleavage. BCR-induced apoptosis of WEHI-231 cells is associated with the loss of endogenous HSH2 expression within 12 h, whereas inhibition of apoptosis in response to CD40-mediated signaling leads to stabilization of HSH2 expression. Thus, endogenous HSH2 expression correlates directly with survival of WEHI-231 cells, which supports the hypothesis that HSH2 modulates the apoptotic response through its ability to directly or indirectly promote mitochondrial stability.

Genetic analysis of patients with defects in early B‐cell development

Approximately 85% of patients with defects in early B-cell development have X-linked agammaglobulinemia (XLA), a disorder caused by mutations in the cytoplasmic Bruton's tyrosine kinase (Btk). Although Btk is activated by cross-linking of a variety of cell-surface receptors, the most critical signal transduction pathway is the one initiated by the pre-B cell and B-cell antigen receptor complex. Mutations in Btk are highly diverse, and no single mutation accounts for more than 3% of patients. Although there is no strong genotype/phenotype correlation in XLA, the specific mutation in Btk is one of the factors that influences the severity of disease. Mutations in the components of the pre-B cell and B-cell antigen receptor complex account for an additional 5-7% of patients with defects in early B-cell development. Patients with defects in these proteins are clinically indistinguishable from those with XLA. However, they tend to be younger at the time of diagnosis, and whereas most patients with XLA have a small number of B cells in the peripheral circulation, these cells are not found in patients with defects in micro heavy chain or Igalpha. Polymorphic variants in the components of the pre-B cell and B-cell receptor complex, particularly micro heavy chain and lambda5, may contribute to the severity of XLA.

B cell antigen receptor assembly and Syk activation in the S2 cell reconstitution system

Signal transduction from the B cell antigen receptor (BCR) involves a multitude of signaling molecules often organized in dynamic protein complexes. The molecular mechanisms operating during signaling are difficult to study solely by loss-of-function analysis. For a better understanding of the transient interaction of signaling molecules and their regulation by feedback loops, as well as their dynamic behavior in living cells, new techniques are required. We have developed a method allowing the reconstitution of the BCR complex and several of its key signaling elements in the evolutionary distant environment of the Drosophila S2 Schneider cell line. With this gain-of-function approach, we study here the assembly of the BCR complex and the control of its transport to the cell surface of S2 cells. We find that without binding to a light chain, the membrane-bound μm heavy chain (μmHC) homodimer, together with the Ig-α/Ig-β heterodimer, can come to the cell surface where it is signaling competent. This finding could have implications for potential signaling functions of such a receptor molecule during pro-/pre-B cell development. We also studied the activation of the BCR-proximal kinase Syk. We found that a truncated Syk mutant lacking the first (N-terminal) SH2 domain and the linker regions, is still regulated by autoinhibition and can only become activated in the presence of the BCR. This indicates that the C-terminal SH2 domain of Syk is the dominant regulatory subunit of this kinase.

Modulation and function of caspase pathways in B lymphocytes

During their development, B-lineage cells are selected to mature, to die, to divide, or to survive and wait, ready to respond to external signals. The homeostatic balance between growth, death, and survival is mediated by signaling pathways through the B-cell antigen receptor (BCR) complex, cytokine and chemokine receptors or cell-cell coreceptor interactions. The BCR complex is a master regulator essential at key checkpoints during development. These checkpoints involve various processes, including negative selection (deletion), anergy, receptor editing, and positive selection. Without BCRs or downstream BCR-signaling components, B-lineage cells arrest during development. Removal of BCRs from mature B cells leads to their death. Here, we discuss signaling pathways in B cells that activate members of the caspase family of cysteine proteases. In some B-cell subsets, BCR signaling activates caspases, which in turn induce a program leading to cell death. However, in other contexts, caspases are involved in the proliferation of B cells. The outcome depends in part on the presence or absence of modifiers that affect signaling thresholds and on which caspases are activated. These mechanisms allow the coordinated regulation of proliferation and apoptosis that is essential for lymphoid homeostasis.