B-cell Antigen Receptor Expression Research Articles

The CH1 domain influences the expression and antigen sensing of the HIV-specific CH31 IgM-BCR and IgG-BCR

How different classes of the B cell antigen receptor (BCR) sense viral antigens used in vaccination protocols is poorly understood. Here, we study antigen binding and sensing of human Ramos B cells expressing a BCR of either the IgM or IgG1 class with specificity for the CD4-binding-site of the envelope (Env) protein of the HIV-1. Both BCRs carry an identical antigen binding site derived from the broad neutralizing antibody (bnAb) CH31. We find a five times higher expression of the IgG1-BCR in comparison to the IgM-BCR on the surface of transfected Ramos B cells. The two BCR classes also differ from each other in their interaction with cognate HIV Env antigens in that the IgG1-BCR and IgM-BCR bind preferentially to polyvalent and monovalent antigens, respectively. By generating an IgM/IgG1 chimeric BCR, we found that the class-specific BCR expression and antigen-sensing behavior can be transferred with the CH1γ domain from the IgG1-BCR to the IgM-BCR. Thus, the class of CH1 domain has an impact on BCR assembly and expression as well as on antigen sensing.

Abstract B079: Molecular determinants of sensitivity to Polatuzumab-Vedotin

Abstract Purpose: Polatuzumab Vedotin (Pola-V) is an antibody-drug conjugate directed to the B cell surface antigen CD79B. When combined with conventional immunochemotherapy, Pola-V improves outcomes in DLBCL overall; however, there is noted heterogeneity in response to Pola-V, with germinal center b-cell (GCB) DLBCL showing no added benefit to the addition of Pola-V compared to standard immunochemotherapy. We aimed to identify molecular determinants of sensitivity or resistance to Pola-V. We hypothesized that these might lead us to innovative strategies to improve on-target tumor killing by Pola-V, or to find novel biomarkers that predict drug resistance. Methods: We employed combined drug-sensitization and CD79B-sorted CRISPR-Cas9 screening to identify molecular determinants of sensitivity to CD79B-directed, tumor killing by Pola-V in 9 cell lines representing different molecular subtypes of DLBCL. Results: Our results reveal the striking impact of epitope glycosylation, specifically a2,6 sialylation, on the binding of Pola-V to CD79B and thereby its ability to kill tumor cells. Specifically, we identify the exact glycosylated residues on CD79A and CD79B which create a sialylated glycan shield around the Pola-V binding site, precluding binding to its target. We show how genetic, pharmacological and enzymatic approaches that remove terminal sialic acid residues from these N-linked glycans lead to enhanced tumor killing by Pola-V. We hypothesize and test multiple methods of targeting this pathway in order to enhance Pola-V killing both in vitro and in vivo. Finally, we reveal a previously unappreciated role for the ubiquitin ligase KLHL6 in regulating CD79B protein abundance and surface expression of the B cell antigen receptor (BCR), including how this pathway is used by physiological germinal center B cells and how it is corrupted to enhance BCR expression in GCB DLBCL. Conclusions: These findings unravel the molecular basis of response heterogeneity to Pola-V and identify approaches that might be deployed therapeutically to enhance the efficacy of CD79B-specific tumor killing. In addition, we identify how KLHL6 determines expression of the BCR in both physiological and malignant germinal center B cells, and how KLHL6 mutation may modulate sensitivity of GCB DLBCL tumors to Pola-V. Citation Format: Sean R Corcoran, Jaewoo Choi, Rachel E Fenner, Xin Yu, Sebastian Scheich, Galina Schevchenko, Evangelia K Papachristou, Vivian M Morris, Kamal Kishore, Clive S D'Santos, Stefania Pittaluga, George Wright, Jagan Muppidi, Daniel J Hodson, Louis M Staudt. Molecular determinants of sensitivity to Polatuzumab-Vedotin [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B079.

Molecular Determinants of Sensitivity to Polatuzumab-Vedotin in Diffuse Large B-Cell Lymphoma

Purpose: Polatuzumab Vedotin (Pola-V) is an antibody-drug conjugate directed to the B cell surface antigen CD79B. When combined with conventional immunochemotherapy, Pola-V improves outcomes in DLBCL overall; however, there is noted heterogeneity in response to Pola-V, with germinal center b-cell (GCB) DLBCL showing no added benefit to the addition of Pola-V compared to standard immunochemotherapy. We aimed to identify molecular determinants of sensitivity or resistance to Pola-V. We hypothesized that these might lead us to innovative strategies to improve on-target tumor killing by Pola-V, or to find novel biomarkers that predict drug resistance. Methods: We employed combined drug-sensitization and CD79B-sorted CRISPR-Cas9 screening to identify molecular determinants of sensitivity to CD79B-directed, tumor killing by Pola-V in 9 cell lines representing different molecular subtypes of DLBCL. Results: Our results reveal the striking impact of epitope glycosylation, specifically a2,6 sialylation, on the binding of Pola-V to CD79B and thereby its ability to kill tumor cells. Specifically, we identify the exact glycosylated residues on CD79A and CD79B which create a glycan shield around the Pola-V binding site, precluding binding to its target. We show how genetic, pharmacological and enzymatic approaches that remove terminal sialic acid residues from these N-linked glycans lead to enhanced tumor killing by Pola-V. We hypothesize and test multiple methods of targeting this pathway in order to enhance Pola-V killing both in vitro and in vivo. Finally, we reveal a previously unappreciated role for the ubiquitin ligase KLHL6 in regulating CD79B protein abundance and surface expression of the B cell antigen receptor (BCR), including how this pathway is used by physiological germinal center B cells and how it is corrupted to enhance BCR expression in GCB DLBCL. Conclusions: These findings unravel the molecular basis of response heterogeneity to Pola-V and identify approaches that might be deployed therapeutically to enhance the efficacy of CD79B-specific tumor killing. In addition, we identify how KLHL6 determines expression of the BCR in both physiological and malignant germinal center B cells, and how KLHL6 mutation may modulate sensitivity of GCB DLBCL tumors to Pola-V.

Single-cell transcriptome landscape and antigen receptor dynamic during SARS-CoV-2 vaccination

Vaccination by inactivated vaccine is an effective strategy to prevent the COVID-19 pandemic. However, the detailed molecular immune response at single-cell level is poorly understood. In this study, we systematically delineated the landscape of the pre- and post-vaccination single-cell transcriptome, TCR (T cell antigen receptor) and BCR (B cell antigen receptor) expression profile of vaccinated candidates. The bulk TCR sequencing analysis of COVID-19 patients was also performed. Enrichment of a clonal CD8+ T cell cluster expressing specific TCR was identified in both vaccination candidates and COVID-19 patients. These clonal CD8+ T cells showed high expression of cytotoxicity, phagosome and antigen presentation related genes. The cell–cell interaction analysis revealed that monocytes and dendritic cells could interact with these cells and initiate phagocytosis via ICAM1-ITGAM and ITGB2 signaling. Together, our study systematically deciphered the detailed immunological response during SARS-CoV-2 vaccination and infection. It may facilitate understanding the immune response and the T-cell therapy against COVID-19.

B-cell antigen receptor expression and phosphatidylinositol 3-kinase signaling regulate genesis and maintenance of mouse chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a frequent lymphoproliferative disorder of B cells. Although inhibitors targeting signal proteins involved in B-cell antigen receptor (BCR) signaling constitute an important part of the current therapeutic protocols for CLL patients, the exact role of BCR signaling, as compared to genetic aberration, in the development and progression of CLL is controversial. In order to investigate whether BCR expression per se is pivotal for the development and maintenance of CLL B cells, we used the TCL1 mouse model. By ablating the BCR in CLL cells from TCL1 transgenic mice, we show that CLL cells cannot survive without BCR signaling and are lost within 8 weeks in diseased mice. Furthermore, we tested whether mutations augmenting B-cell signaling influence the course of CLL development and its severity. The phosphatidylinositol-3-kinase (PI3K) signaling pathway is an integral part of the BCR signaling machinery and its activity is indispensable for B-cell survival. It is negatively regulated by the lipid phosphatase PTEN, whose loss mimics PI3K pathway activation. Herein, we show that PTEN has a key regulatory function in the development of CLL, as deletion of the Pten gene resulted in greatly accelerated onset of the disease. By contrast, deletion of the gene TP53, which encodes the tumor suppressor p53 and is highly mutated in CLL, did not accelerate disease development, confirming that development of CLL was specifically triggered by augmented PI3K activity through loss of PTEN and suggesting that CLL driver consequences most likely affect BCR signaling. Moreover, we could show that in human CLL patient samples, 64% and 81% of CLL patients with a mutated and unmutated IgH VH, respectively, show downregulated PTEN protein expression in CLL B cells if compared to healthy donor B cells. Importantly, we found that B cells derived from CLL patients had higher expression levels of the miRNA-21 and miRNA-29, which suppresses PTEN translation, compared to healthy donors. The high levels of miRNA-29 might be induced by increased PAX5 expression of the B-CLL cells. We hypothesize that downregulation of PTEN by increased expression levels of miR-21, PAX5 and miR-29 could be a novel mechanism of CLL tumorigenesis that is not established yet. Together, our study demonstrates the pivotal role for BCR signaling in CLL development and deepens our understanding of the molecular mechanisms underlying the genesis of CLL and for the development of new treatment strategies.

Immunoglobulin expression in the endoplasmic reticulum shapes the metabolic fitness of B lymphocytes.

The major function of B lymphocytes is to sense antigens and to produce protective antibodies after activation. This function requires the expression of a B-cell antigen receptor (BCR), and evolutionary conserved mechanisms seem to exist that ensure that B cells without a BCR do not develop nor survive in the periphery. Here, we show that the loss of BCR expression on Burkitt lymphoma cells leads to decreased mitochondrial function and impaired metabolic flexibility. Strikingly, this phenotype does not result from the absence of a classical Syk-dependent BCR signal but rather from compromised ER expansion. We show that the reexpression of immunoglobulins (Ig) in the absence of the BCR signaling subunits Igα and Igβ rescues the observed metabolic defects. We demonstrate that immunoglobulin expression is needed to maintain ER homeostasis not only in lymphoma cells but also in resting B cells. Our study provides evidence that the expression of BCR components, which is sensed in the ER and shapes mitochondrial function, represents a novel mechanism of metabolic control in B cells.

Pten controls B‐cell responsiveness and germinal center reaction by regulating the expression of IgD BCR

In contrast to other B‐cell antigen receptor (BCR) classes, the function of IgD BCR on mature B cells remains largely elusive as mature B cells co‐express IgM, which is sufficient for development, survival, and activation of B cells. Here, we show that IgD expression is regulated by the forkhead box transcription factor FoxO1, thereby shifting the responsiveness of mature B cells towards recognition of multivalent antigen. FoxO1 is repressed by phosphoinositide 3‐kinase (PI3K) signaling and requires the lipid phosphatase Pten for its activation. Consequently, Pten‐deficient B cells expressing knock‐ins for BCR heavy and light chain genes are unable to upregulate IgD. Furthermore, in the presence of autoantigen, Pten‐deficient B cells cannot eliminate the autoreactive BCR specificity by secondary light chain gene recombination. Instead, Pten‐deficient B cells downregulate BCR expression and become unresponsive to further BCR‐mediated stimulation. Notably, we observed a delayed germinal center (GC) reaction by IgD‐deficient B cells after immunization with trinitrophenyl‐ovalbumin (TNP‐Ova), a commonly used antigen for T‐cell‐dependent antibody responses. Together, our data suggest that the activation of IgD expression by Pten/FoxO1 results in mature B cells that are selectively responsive to multivalent antigen and are capable of initiating rapid GC reactions and T‐cell‐dependent antibody responses.

The B-cell receptor in control of tumor B-cell fitness: Biology and clinical relevance.

Surface expression of a functional B cell antigen receptor (BCR) is essential for the survival and proliferation of mature B cells. Most types of B-cell lymphoproliferative disorders retain surface BCR expression, including B-cell non-Hodgkin lymphomas (B-NHL) and chronic lymphocytic leukemia (CLL). Targeting BCR effectors in B-NHL cell lines in vitro has indicated that this signaling axis is crucial for malignant B cell growth. This has led to the development of inhibitors of BCR signaling, which are currently used for the treatment of CLL and several B-NHL subtypes. Recent studies based on conditional BCR inactivation in a MYC-driven mouse B-cell lymphoma model have revisited the role of the BCR in MYC-expressing tumor B cells. Indeed, lymphoma cells losing BCR expression continue to grow unless subjected to competition with their BCR-expressing counterparts, which causes their elimination. Here, we discuss the molecular nature of the fitness signal delivered by the BCR to MYC-expressing malignant B cells, ensuring their preferential persistence within a rapidly expanding tumor population. We also review growing evidence of Ig-negative cases belonging to several B-NHL subtypes and CLL, and discuss the clinical implications of these findings in relation to an emerging picture of clinical resistances to anti-BCR therapies.

Myc and the Warburg Effect

Mature B cells recognize and respond in a highly-specific fashion to a multitude of environmental antigens through membrane-bound immunoglobulins forming together with the Igα and Igβ proteins a functional unit called the B cell antigen receptor (BCR). Through a complex network of effector molecules, the BCR transforms environmental signals into biochemical reactions which are responsible for highly codified cellular responses affecting survival, proliferation, migration and terminal differentiation of B cells. Surface BCR expression is conserved in most types of B cell malignancies arising from mature B cells. This observation, together with genetic and biochemical evidence pointing to sustained BCR signaling in different types of B cell neoplasms represents the rationale for the current use of pharmacological inhibitors of BCR signaling to treat several forms of B lymphoproliferative disorders. Nevertheless, our understanding of how the BCR influences malignant B cell behavior remains poorly understood. In an attempt to fill this knowledge gap, we engineered a mouse model to monitor the effects of acute ablation of the BCR in highly-aggressive MYC-driven lymphomas. Inducible BCR ablation did not, per se, prevent the outgrowth of receptor-less MYC lymphoma cells both in vitro and in vivo. Instead, BCR loss weakened the fitness of the malignant B cells leading to the rapid elimination of BCR-less tumor cells in the presence of their BCR-expressing counterparts (Varano et al., 2017). Through the integration of data generated from genomics, metabolomics and bulk/single cell transcriptomics analyses, comparing BCR-deficient lymphoma cells to their proficient counterparts, we have started to elucidate the gene networks and metabolic pathways influenced by BCR expression that sustain competitive fitness of MYC-transformed lymphoma B cells. Data from CRISPR/Cas9-mediated disruption of candidate fitness genes in primary malignant B cells will be presented. In support of the findings in the mouse model, we will provide evidence that BCR-less malignant B cells are spontaneously generated during tumor progression in several forms of human B cell lymphoproliferative disorders, establishing a possible Achilles heel of anti-BCR therapies. Finally, we will report possible strategies enabling the clearance of BCR-less lymphoma cells, taking advantage of their acquired addiction to specific signaling and metabolic pathways. Our results shed light on the coordinated regulation of signaling and metabolism imposed on malignant B cells by BCR expression/signaling and provide indications for improved treatment options to fight several forms of mature B cell malignancies.Reference:Varano G, Raffel S, Sormani M, Zanardi F, Lonardi S, Zasada C, Perucho L, Petrocelli V, Haake A, Lee AK, Bugatti M, Paul U, Van Anken E, Pasqualucci L, Rabadan R, Siebert R, Kempa S, Ponzoni M, Facchetti F, Rajewsky K, Casola S. Nature. 2017; 546:302-306. DisclosuresNo relevant conflicts of interest to declare.

The B-cell receptor controls fitness of MYC-driven lymphoma cells via GSK3β inhibition.

Similar to resting mature B cells, where the B-cell antigen receptor (BCR) controls cellular survival, surface BCR expression is conserved in most mature B-cell lymphomas. The identification of activating BCR mutations and the growth disadvantage upon BCR knockdown of cells of certain lymphoma entities has led to the view that BCR signalling is required for tumour cell survival. Consequently, the BCR signalling machinery has become an established target in the therapy of B-cell malignancies. Here we study the effects of BCR ablation on MYC-driven mouse B-cell lymphomas and compare them with observations in human Burkitt lymphoma. Whereas BCR ablation does not, per se, significantly affect lymphoma growth, BCR-negative (BCR-) tumour cells rapidly disappear in the presence of their BCR-expressing (BCR+) counterparts in vitro and in vivo. This requires neither cellular contact nor factors released by BCR+ tumour cells. Instead, BCR loss induces the rewiring of central carbon metabolism, increasing the sensitivity of receptor-less lymphoma cells to nutrient restriction. The BCR attenuates glycogen synthase kinase 3 beta (GSK3β) activity to support MYC-controlled gene expression. BCR- tumour cells exhibit increased GSK3β activity and are rescued from their competitive growth disadvantage by GSK3β inhibition. BCR- lymphoma variants that restore competitive fitness normalize GSK3β activity after constitutive activation of the MAPK pathway, commonly through Ras mutations. Similarly, in Burkitt lymphoma, activating RAS mutations may propagate immunoglobulin-crippled tumour cells, which usually represent a minority of the tumour bulk. Thus, while BCR expression enhances lymphoma cell fitness, BCR-targeted therapies may profit from combinations with drugs targeting BCR- tumour cells.

51 - The BCR Redox Machinery

Through the B cell antigen receptor (BCR), mature B cells recognize the presence of antigens in the external world and adapt their responses. The signaling output from this receptor is tightly controlled so as to prevent its hypo- or hyper-stimulation. Reactive oxygen species (ROS) can amplify BCR signaling, by inhibiting its intracellular negative regulators, i.e. protein tyrosine phosphatases. However, ROS fluxes and signals - most likely dependent on close proximity - have never been systematically studied. We recently showed that the NADPH oxidase complex 2 (NOX2) produces ROS extracellularly in B lymphocytes and that the water channel aquaporin 8 (AQP8) is consequently needed for their passage across the membrane for BCR signal amplification. On aquaporin 8 silencing, indeed, inducible B lymphoma cells respond poorly to BCR stimulation. Their differentiation is severely impaired, as demonstrated by retarded onset of IgM polymerization, low amounts of IgM secretion, and prolonged BCR expression on the cell surface. Thus, efficient induction of B cell activation and differentiation requires intact H 2 O 2 fluxes across the plasma membrane for signal amplification. The following step would be to understand whether there are physical connections between BCR, NOXes and AQPs. Are these connections induced or reinforced by antigen binding? To answer these questions, we are currently characterizing by PLA techniques the spatial organization and interaction of redox-regulators on the plasma membrane of resting and activated B cells.

Chronic lymphocytic leukaemia is driven by antigen-independent cell-autonomous signalling

B-cell antigen receptor (BCR) expression is an important feature of chronic lymphocytic leukaemia (CLL), one of the most prevalent B-cell neoplasias in Western countries. The presence of stereotyped and quasi-identical BCRs in different CLL patients suggests that recognition of specific antigens might drive CLL pathogenesis. Here we show that, in contrast to other B-cell neoplasias, CLL-derived BCRs induce antigen-independent cell-autonomous signalling, which is dependent on the heavy-chain complementarity-determining region (HCDR3) and an internal epitope of the BCR. Indeed, transferring the HCDR3 of a CLL-derived BCR provides autonomous signalling capacity to a non-autonomously active BCR, whereas mutations in the internal epitope abolish this capacity. Because BCR expression was required for the binding of secreted CLL-derived BCRs to target cells, and mutations in the internal epitope reduced this binding, our results indicate a new model for CLL pathogenesis, with cell-autonomous antigen-independent signalling as a crucial pathogenic mechanism.

Role of PI3K in the generation and survival of B cells

Engagement of the B-cell antigen receptor (BCR) or its precursor, the pre-BCR, induces a cascade of biochemical reactions that regulate the differentiation, selection, survival, and activation of B cells. This cascade is initiated by receptor-associated tyrosine kinases that activate multiple downstream signaling pathways. Since it is required for metabolism, cell growth, development, and survival, the activation of phosphoinositide 3-kinase (PI3K)-dependent pathways represents a crucial event of BCR/pre-BCR signaling. The phosphorylated substrates of the PI3K promote specific recruitment of selected signaling proteins to the plasma membrane, where important signaling complexes are formed to mediate the above-mentioned biological processes. Here, we review the principles of PI3K signaling and highlight the role of an important PI3K-driven module in VDJ recombination of immunoglobulin (Ig) genes during early B-cell development as compared with class switch recombination of Ig genes in mature B cells after activation by specific antigens. Furthermore, we discuss the role of PI3K in the survival of mature B cells, which is strictly dependent on BCR expression and basal BCR signaling.

Induction of Cytosolic Calcium Flux by CD20 Is Dependent upon B Cell Antigen Receptor Signaling

The anti-CD20 monoclonal antibody (mAb) rituximab is now routinely used for the treatment of non-Hodgkins lymphoma and is being examined in a wide range of other B-cell disorders, such as rheumatoid arthritis. Despite intensive study, the mechanism of action still remains uncertain. In the current study, anti-CD20 mAb-induced calcium signaling was investigated. Previously, we grouped anti-CD20 mAbs into Type I (rituximab-like) and Type II (B1-like) based upon various characteristics such as their ability to induce complement activation and redistribute CD20 into detergent-insoluble membrane domains. Here we show that only Type I mAbs are capable of inducing a calcium flux in B cells and that this is tightly correlated with the expression of the B-cell antigen receptor (BCR). Inhibitor analysis revealed that the signaling cascade employed by CD20 was strikingly similar to that utilized by the BCR, with inhibitors of Syk, Src, and PI3K, but not EGTA, p38, or ERK1/2, completely ablating calcium flux. Furthermore, binding of Type I but not Type II mAbs caused direct association of CD20 with the BCR as measured by FRET and resulted in the phosphorylation of BCR-specific adaptor proteins BLNK and SLP-76. Crucially, variant Ramos cells lacking BCR expression but with unchanged CD20 expression were completely unable to induce calcium flux following ligation of CD20. Collectively, these data indicate that CD20 induces cytosolic calcium flux through its ability to associate with and "hijack" the signaling potential of the BCR.

Analysis of B-cell signaling using DT40 B-cell line.

A chicken DT40 B-cell line provides an excellent model system for the analysis of the function of genes related to B-cell antigen receptor (BCR) signaling. Crosslinking of BCR by micro-chain-specific antibody stimulates DT40 cells to undergo apoptosis. Activation of protein- tyrosine kinases (PTKs) and intracellular calcium signaling are essential for BCR-induced apoptosis. Because DT40 B cells are highly proliferative and readily integrate exogenous DNA by homologous recombination, gene targeting is conveniently employed to inactivate genes of interest. This chapter describes procedures for (1) generation of knockout DT40 cell line, (2) analysis of surface BCR expression, (3) detection of tyrosine-phosphorylated proteins, (4) detection of intracellular calcium mobilization, and (5) reconstitution of depleted gene by transfection.

Alternatively spliced forms of Igα and Igβ prevent B cell receptor expression on the cell surface

The B cell antigen receptor (BCR) includes an Igalpha/Igbeta heterodimer non-covalently associated with surface immunoglobulin. Recently, variant Igalpha and Igbeta transcripts, arising from alternative mRNA splicing, have been reported. The present study examined the function of the potential products of these transcripts, by utilizing cDNA expression plasmids to reconstitute human BCR expression in transfected 293T cells. Spliced transcripts produced truncated proteins (deltaIgalpha and deltaIgbeta), that failed to form heterodimers with their full-length counterparts, and did not mediate transport of IgM to the cell surface. When overexpressed, both deltaIgalpha and deltaIgbeta acted as competitors of Igalpha and Igbeta, leading to down-modulated surface IgM expression, and retention of IgM in the endoplasmic reticulum. These findings document a possible novel mechanism for controlling BCR expression in B cells, based on up-regulated synthesis of components devoid of transport function.

Alternatively spliced forms of Igalpha and Igbeta prevent B cell receptor expression on the cell surface.

The B cell antigen receptor (BCR) includes an Igalpha/Igbeta heterodimer non-covalently associated with surface immunoglobulin. Recently, variant Igalpha and Igbeta transcripts, arising from alternative mRNA splicing, have been reported. The present study examined the function of the potential products of these transcripts, by utilizing cDNA expression plasmids to reconstitute human BCR expression in transfected 293T cells. Spliced transcripts produced truncated proteins (deltaIgalpha and deltaIgbeta), that failed to form heterodimers with their full-length counterparts, and did not mediate transport of IgM to the cell surface. When overexpressed, both deltaIgalpha and deltaIgbeta acted as competitors of Igalpha and Igbeta, leading to down-modulated surface IgM expression, and retention of IgM in the endoplasmic reticulum. These findings document a possible novel mechanism for controlling BCR expression in B cells, based on up-regulated synthesis of components devoid of transport function.

The tyrosine kinase Lyn is required for B cell development beyond the T1 stage in the spleen: rescue by over-expression of Bcl-2.

We have analyzed the effects of deficiency in the tyrosine kinase Lyn on B cell development using transgenic mice that express a B cell antigen receptor (BCR) of defined specificity (3-83,anti-H-2K(k or b)). In the absence of Lyn, immature B cells are abundant in the bone marrow and spleen up until the T1 stage (IgM(hi) IgD(-) CD21(-)CD23(-)), after which B cell development is severely impaired. The small number of mature B cells that do develop in Lyn-deficient mice express normal levels of the transgenic BCR and lack expression of CD80 and CD86, suggesting they are not activated. In Lyn-deficient animals the presence of a Bcl-2 transgene leads to a dramatic increase in B cell numbers and restores T2 stage (IgM(hi) IgD(hi) CD21(hi) CD23(int)) and mature populations. In 3-83 lyn-/- Bcl-2 Tg mice, a population of lambda-positive cells that also express the 383 idiotype is evident, suggesting that in the absence of lyn isotype exclusion by the transgenic BCR is less efficient. The results indicate that Lyn plays a positive role in the selection and survival of mature B cells in addition to its previously documented negative role in tolerance and B cell activation.

In Vivo Ablation of Surface Immunoglobulin on Mature B Cells by Inducible Gene Targeting Results in Rapid Cell Death

Gene targeting experiments have demonstrated that the expression of immunoglobulin heavy chain in the pre-B cell receptor (pBCR) and of heavy and light chains in the B cell antigen receptor (BCR) marks checkpoints in early B cell development that the cells have to pass to survive. To investigate whether the persistence of mature B cells in the peripheral immune system also depends on BCR expression, we have generated a transgenic mouse in which the BCR can be inducibly ablated through V region gene deletion. Ablation leads to rapid death of mature B lymphocytes, which is preceded by down-regulation of MHC antigens and up-regulation of CD95 (Fas) and can be delayed by constitutive bcl-2 expression.

Antigen endocytosis and presentation mediated by human membrane IgG1 in the absence of the Ig(alpha)/Ig(beta) dimer.

Membrane immunoglobulin (mIg) M and D heavy chains possess minimal (KVK) cytoplasmic tails and associate with the Ig alpha/Ig beta (CD79) dimer to achieve surface expression and antigen presentation function. In contrast, the cytoplasmic tail of mIgG is extended by 25 residues (gamma ct). We have tested the possibility that mIgG can perform antigen capture and presentation functions independently of the Ig(alpha)/beta dimer. We show that CD4/(gamma)ct chimeras are efficiently endocytosed partially dependent on a tyrosine residue in (gamma)ct. In addition, human mIgG was expressed on the surface of Ig(alpha)/Ig(beta)-negative non-lymphoid cells and mediated antigen capture and endocytosis. Antigen-specific human mIgG targeted antigen to MIIC-type vesicles in the Ig(alpha)/beta negative melanoma Mel JuSo and augmented antigen presentation 1000-fold, identical to the augmentation seen in Ig(alpha)/beta-positive B-cells expressing the same transfected mIgG. Thus, unlike mIgM, mIgG has autonomous antigen capture and presentation capacity, which may have evolved to reduce or eliminate the BCR's dependence on additional accessory molecules.