Surface BCR Research Articles

Plasmablast response during acute SARS-CoV-2 infection

Plasmablasts are a population of short-lived B cells that appear in the circulation shortly after vaccination and during acute infection. Plasmablasts are formed from resting B lymphocytes, from which they differ in their ability to secrete antibodies, making them similar to plasma cells. Plasmablasts are terminally differentiated cells that can form at various nodes and branches of the B cell response. The plasmablast response is an indicator of the success of vaccination and also helps in predicting antibody levels after recovery or vaccination. However, the definition and classification of plasmablasts faces great experimental and theoretical difficulties. The aim of the work was to determine the characteristics of the plasmablast response during acute SARS-CoV-2 infection. The study included patients (n = 28) with a severe form of COVID-19. Blood sampling was carried out once on the 10–18th day from the moment of hospitalization. B cells were isolated by immunomagnetic separation. Cells were phenotyped using flow cytometry. Secretion of IgM and IgG was determined by ELISpot method. B cell subsets were isolated using a cell sorter. Patients with COVID-19 had an approximately fourfold increase in total plasmablast levels compared to healthy donors. An even more pronounced excess over the negative control was observed for RBD-specific plasmablasts. In terms of their composition, plasmablasts were one third IgM⁺ cells. This distribution between B-cell BCR receptor isotypes was consistent with the primary nature of the immune response in COVID-19. Approximately a third of plasmablasts carried the CD138 antigen. CD138 marker is characteristic of the late stage of plasmablast maturation and is also found on plasma cells. The CD27+CD38⁺ population was divided according to the expression of the CD138 antigen. Using the ELISpot method, we have shown that a significant portion of circulating plasmablasts are antibody-secreting cells. Among circulating plasmablasts, both early and late plasmablasts can be distinguished, which are characterized by the absence of a surface BCR, but which carry the CD138 antigen. Determining how plasmablasts relate to other B cell populations is of paramount importance for the development of new treatments for COVID-19 and for the creation of promising vaccines against SARS-CoV-2 infection.

Exit from germinal center to become quiescent memory B cells depends on metabolic reprograming and provision of a survival signal.

A still unanswered question is what drives the small fraction of activated germinal center (GC) B cells to become long-lived quiescent memory B cells. We found here that a small population of GC-derived CD38intBcl6hi/intEfnb1+ cells with lower mTORC1 activity favored the memory B cell fate. Constitutively high mTORC1 activity led to defects in formation of the CD38intBcl6hi/intEfnb1+ cells; conversely, decreasing mTORC1 activity resulted in relative enrichment of this memory-prone population over the recycling-prone one. Furthermore, the CD38intBcl6hi/intEfnb1+ cells had higher levels of Bcl2 and surface BCR that, in turn, contributed to their survival and development. We also found that downregulation of Bcl6 resulted in increased expression of both Bcl2 and BCR. Given the positive correlation between the strength of T cell help and mTORC1 activity, our data suggest a model in which weak help from T cells together with provision of an increased survival signal are key for GC B cells to adopt a memory B cell fate.

Multiple tolerance checkpoints suppress low-affinity germline precursor B cells of high affinity anti-dsDNA antibody-producing cells in BCR knock-in mice

Abstract Previously, we revealed that disease-associated dsDNA-reactive high-affinity antibodies evolve from ssDNA-reactive low-affinity germline precursors through clonal expansion and affinity maturation in acute systemic lupus erythematosus (SLE) patients. Our findings raise questions whether low-affinity precursors of anti-dsDNA antibody producing cells are subjected to immune tolerance, and under what conditions they expand and undergo affinity maturation in vivo. To address these questions, we generated G9gl, a site-directed knock-in (KI) mouse line carrying reverse mutated forms of VH-DH-JH and Vk-Jk regions of an acute SLE patient-derived high-affinity anti-dsDNA antibody. G9gl heterozygous mice had reduced numbers of B cells in their periphery, and half of peripheral B cells expressed endogenous IgH from non-KI allele due to receptor editing. G9gl-expressing B cells exhibited several anergic phenotypes, including reduced surface BCR and shorter life span. Yet, they underwent class-switching and produced antibodies following CD40 and IL-4 in vitro stimulation. G9gl B cells responded to ssDNA, but not dsDNA in vitro, and were selectively excluded from germinal center reaction in several conditions such as Treg depletion and DNA-conjugated mBSA immunization in vivo. However, some of G9gl-expressing B cells differentiated to somatically mutated IgG+ memory B cells when immunized with bacterial DNA-conjugated mBSA. Therefore, multiple tolerance checkpoints prevent low-affinity anti-DNA precursor B cells from undergoing clonal expansion and affinity maturation in germinal centers. However, G9gl-expressing B cell might undergo class-switching and somatic hypermutation through the extra-follicular pathway.

IgM, IgG, and IgA Influenza-Specific Plasma Cells Express Divergent Transcriptomes.

Ab-secreting cells (ASC) or plasma cells are essential components of the humoral immune system. Although Abs of different isotypes have distinct functions, it is not known if the ASC that secrete each isotype are also distinct. ASC downregulate their surface BCR upon differentiation, hindering analyses that couple BCR information to other molecular characteristics. In this study, we developed a methodology using fixation, permeabilization, and intracellular staining coupled with cell sorting and reversal of the cross-links to allow RNA sequencing of isolated cell subsets. Using hemagglutinin and nucleoprotein Ag-specific B cell tetramers and intracellular staining for IgM, IgG, and IgA isotypes, we were able to derive and compare the gene expression programs of ASC subsets that were responding to the same Ags following influenza infection in mice. Intriguingly, whereas a shared ASC signature was identified, each ASC isotype-specific population expressed distinct transcriptional programs controlling cellular homing, metabolism, and potential effector functions. Additionally, we extracted and compared BCR clonotypes and found that each ASC isotype contained a unique, clonally related CDR3 repertoire. In summary, these data reveal specific complexities in the transcriptional programming of Ag-specific ASC populations.

Influence of potassium hydroxide activation on characteristics and environmental risk of heavy metals in chars derived from municipal sewage sludge

To investigate the influence of KOH activation on characteristics and environmental risk of heavy metals in chars, sludge was pyrolyzed with varying amount of KOH. The analyzation of characteristics and potential ecological risk evaluation of heavy metals were conducted by surface area analyzer, FTIR, XRD and BCR sequential extraction. The activated chars have higher surface area and lower content of silica compared to those without being activated. The activation of KOH promoted residual fraction of Cd, meanwhile, Zinc, Cr, Ni and Mn were converted to relatively unstable fractions (F2 and F3). The results of risk assessment indicated that the potential ecological risk level of Cd was reduced in activated chars, while risk level of Zn, Cr, Ni and Mn were increased after pyrolysis with KOH activation. The potential ecological risk of heavy metals in activated chars was further declined, and the risk level transformed from moderate to low.

A model integrating tonic and antigen-triggered BCR signals to predict the survival of primary B cells

The BCR constitutively transmits a “tonic” survival signal in the absence of exogenous antigen-binding. However, the strength of tonic BCR signal and its relationship with antigen-triggered survival signal are poorly understood. We found that primary B cells expressing high levels of BCR had elevated BCR tonic signal and increased survival compared with those expressing low levels of BCR. In addition, we found that crosslinking BCR with low doses of F(ab′)2 α-IgM antibodies did not enhance, but rather decreased, B cell survival and that only when most of the BCR were occupied by F(ab′)2 α-IgM antibodies was B cell survival enhanced. Based on these experimental results, we present a mathematical model integrating tonic and antigen-triggered BCR signals. Our model indicates that the signal generated from crosslinked BCR is 4.3 times as strong as the tonic signal generated from free BCR and that the threshold of B cell activation corresponds to the signal generated by crosslinking 61% of the surface BCR. This model also allows the prediction of the survival probability of a B cell based on its initial BCR level and the strength and duration of antigen stimulation, and fits with the mechanism of B cell tolerance.

Surface characteristics and potential ecological risk evaluation of heavy metals in the bio-char produced by co-pyrolysis from municipal sewage sludge and hazelnut shell with zinc chloride

Municipal sewage sludge (MSS) and hazelnut shell were used for co-pyrolysis by chemical activation with ZnCl2. The surface characteristics and potential ecological risk evaluation of heavy metals in the bio-char produced by co-pyrolysis were analyzed by surface analyzer and BCR sequential extraction. When raw materials with ZnCl2 (3mol/L) were co-pyrolyzed at 500°C for 90min, specific surface area of the bio-char is 598.73m2/g, and iodine absorption number is 607.85mg/g. For microcosmic surface of the bio-char, the ratio of micropore area is stabilized from 0.74 to 0.80 of the total specific surface area, and hazelnut shell is effective to generate microporous construction. For the migration and transformation behavior of heavy metals, pyrolysis promoted mobile fraction (F1 and F2) to stable fraction (F3 and F4) with increasing pyrolysis temperature. The potential ecological risk of heavy metals transforms from considerable risk to low risk after pyrolysis at 500°C.

B-Cell Receptor Signaling in Diffuse Large B-Cell Lymphoma: Tonic Alone in the Germinal Center B-Cell Subtype, Plus Self Antigen-Induced in the Activated B-Cell Subtype

Introduction. Targeting BCR signaling with the BTK inhibitor ibrutinib is clinically effective against most B-cell lymphomas, including the activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL), but not the germinal center B-cell (GCB) subtype. Active BCR signaling in GCB-DLBCL was suggested by studies with a Syk inhibitor and our previous studies using BCR knockout (KO). We addressed these questions: why is the BCR active in DLBCL, and how does it signal in GCB-DLBCL?Methods. We used CRISPR/Cas9 technology to modify selected genes by KO or homologous recombination-mediated knock-in (KI). For some genes KI was used to express a fluorescent protein (FP; e.g., GFP) instead of the targeted gene (KI/KO), or to modify the targeted gene together with KI of an FP, for detection of modified cells.Results. In GCB lines (OCI-Ly7 and OCI-Ly19) and ABC lines (U2932 and HBL-1), we simultaneously replaced the hypervariable region (HVR) exons of both immunoglobulin heavy (IgH) and light chains (IgL) with HVR sequences from normal B cells recognizing tetanus toxoid (TT). GFP and CFP respectively marked KI of IgH and IgL HVRs, and KI of the endogenous HVR sequences in each line served as controls. In CFP+/GFP+ cells, the TT specific BCR (TT-BCR) was expressed at similar or higher levels than the endogenous BCR (endo-BCR) and was functional, as shown by calcium flux in response to TT. The TT-BCR maintained growth of GCB lines (Fig. 1), indicating that they use "tonic", antigen-independent BCR signaling. Other features of tonic signaling were confirmed in more GCB lines: 1) the toxicity of BCR KO, which eliminates AKT S473 phosphorylation, was rescued by PTEN KO or expression of constitutively active AKT (mAKT), showing that BCR signaling serves principally to activate PI3K/AKT; and 2) KO of SYK or CD19, or truncation or ITAM mutation of the cytoplasmic tail of CD79A, none of which affect surface BCR levels, were as toxic as BCR KO but were non-toxic in BCR/PTEN double-KO cells.In contrast, the TT-BCR was as growth-slowing as BCR KO to the ABC line U2932 (Fig. 1), and substantially toxic to HBL-1, indicating that BCR signaling is self antigen-dependent in ABC-DLBCL. Reversion of somatic hypermutations in the U2932 HVRs was also as growth-slowing as BCR KO (Fig. 1), suggesting that self-antigen reactivity developed during BCR affinity maturation. Tonic signaling by the TT-BCR provided a detectable benefit (as compared to BCR KO) in PTEN-expressing HBL-1, whereas there was no difference between TT-HVR BCR and BCR KO in PTEN-deficient U2932. The surface TT-BCR level was higher than the endo-BCR level in ABC lines, and dropped with TT stimulation, suggesting that endo-BCRs in ABC lines undergo constant antigen stimulation with BCR internalization. The presumed self-antigen in ABC lines seems to be cell line-specific, since HVRs from ABC lines TMD8 and HBL-1 did not rescue growth of U2932. BCR KO in ABC lines was also not rescued by PTEN KO or mAKT. In cells whose BCRs were labeled by KI to fuse GFP to CD79A, super-resolution microscopy showed macro-clustering of BCR complexes at the surface of ABC line HBL-1, not seen in GCB lines (Fig. 2).Several findings suggested the clinical potential of targeting tonic BCR signaling in DLBCL: 1) clinical trial-stage inhibitors of SYK (P505-15) and PI3K (idelalisib) were toxic to GCB lines (less so with PTEN KO); 2) GCB lines (6/8) were sensitized by BCR KO to an in vitro CHOP-like regimen; 3) P505-15 or idelalisib sensitized GCB lines (3/3) to CHOP in vitro; and 4) evidence of tonic signaling in ABC line HBL-1 after removing antigen-driven signaling by HVR replacement.Conclusion. The BCR provides antigen-independent tonic signals to activate PI3K/AKT in GCB-DLBCL and antigen-dependent signaling in ABC-DLBCL. Targeting of B-cell specific tonic signling alone or in combination could be clinically effective in both types of DLBCL. [Display omitted] [Display omitted] DisclosuresWestin:Spectrum: Research Funding.

The top ten clues to understand the origin of chronic lymphocytic leukemia (CLL)

The fundamental task of the immune system is to protect the individual from infectious organisms without serious injury to self. The essence of acquired immunity is molecular self/non self discrimination. Chronic lymphocytic leukemia is characterized by a global failure of immune system that begins with the failure of immunological tolerance mechanisms (autoimmunity) and finish with the incapacity to response to non-self antigens (immunodeficiency). Immunological tolerance mechanisms are involved in chronic lymphocytic leukemia (CLL) development. During B cell development some self-reactive B cells acquire a special BCR that recognize their own BCR. This self-autoantibody-self BCR interaction promotes survival, differentiation and proliferation of self-reactive B cells. Continuous self-autoantibody-self BCR interaction cross-linking induces an increased rate of surface BCR elimination, CD5+ expression, receptor editing and anergy. Unfortunately, some times this mechanisms increase genomic instability and promote additional genetic damage that immortalize self-reactive B cells and convert them into CLL like clones with the capability of clonal evolution and transformed CLL B cells. This review summarizes the immunological effects of continuous self-autoantibody-self BCR interaction cross-linking in the surface of self-reactive B cells and their role in CLL development.

Microenvironmental Interactions in Chronic Lymphocytic Leukemia: Hints for Pathogenesis and Identification of Targets for Rational Therapy

Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease characterized by the accumulation/expansion of a clonal population of small mature B lymphocytes in blood, bone marrow, and lymphoid organs. Although initial genetic events are considered primarily responsible for the first step(s) of neoplastic transformation, the development and progression of the CLL clone are thought to be affected by various micro-environmental signals that regulate proliferation and survival of malignant B cells. In the present review, we focus on specific interactions of CLL cells with the microenvironmental component, as they occur through the usage by CLL cells of specific molecular structures whose expression has been associated with prognosis, including: i) interactions of CLL cells via the surface BCR and dependent on specific molecular features of the BCR itself and/or the presence of the BCR-associated molecule ZAP- 70; ii) non-BCR-dependent proliferative and/or pro-survival interactions of CLL cells by CD49d and CD38. An overview of the putative drugs that could be employed to target specific molecules involved in CLL cells/tumor microenvironment interactions is also proposed in the closing chapter of the review.

A Role for Lysosomal-Associated Protein Transmembrane 5 in the Negative Regulation of Surface B Cell Receptor Levels and B Cell Activation

Mechanisms by which cell surface levels of the BCR are regulated remain largely unknown. We found that B cells lacking the lysosomal-associated protein transmembrane 5 (LAPTM5) expressed higher levels of cell surface BCR than did wild-type (WT) B cells after Ag stimulation in vitro and in vivo. In addition, LAPTM5-deficient mice contained an increased frequency of Ag-specific B cells and produced greater amounts of Abs than did WT mice after immunization with a T-dependent Ag. Adoptive transfer of LAPTM5-deficient B cells with WT T cells into RAG1-deficient mice revealed that the increased surface BCR levels and the enhanced B cell activation and Ab production were due to a B cell intrinsic defect. As they aged, the LAPTM5-deficient mice had increased titers of serum IgM and autoantibodies and immune complex deposition in the kidney. Immunofluorescent and biochemical analysis revealed that LAPTM5 physically interacted with the BCR complex and promoted its degradation in the lysosomal compartment in mouse B cells. These results demonstrate a role for LAPTM5 in the negative regulation of cell surface BCR levels and B cell activation.

BCR-induced actin rearrangement provides a driving force for the formation of surface BCR signalosomes (84.1)

Abstract In response to antigens, the BCR forms surface signalosomes where it initiates signaling cascades and antigen internalization. This study examines the role of the actin cytoskeleton in BCR signalosome formation. Both multi-valent soluble and membrane-associated antigens induce BCR clustering and actin reorganization. Actin is actively polymerized at BCR microclusters upon their formation and surrounding BCR clusters as they are merged into central clusters. Actin regulators, WASP, cofilin and gelsolin, are activated and recruited to BCR clusters. Latrunculin or jasplakinolide treatment, which blocks BCR-induced actin reorganization by depolymerizing or stabilizing F-actin, inhibits surface BCR clustering and B cell spreading. Jasplakinolide blocks antigen-triggered tyrosine phosphorylation. In contract, latrunculin alone, in the absence of antigenic stimulation, induces BCR microclustering and tyrosine phosphorylation, however, in a much slower kinetics than those induced by antigens. Btk deficiency inhibits not only antigen-induced actin polymerization, but also antigen-induced B cell spreading and BCR clustering. These results indicate that BCR-induced, Btk-dependent actin reorganization, consisting of temporospatially regulated actin polymerization and depolymerization, promotes the formation of surface BCR signalosomes by increasing the speed and the extend of BCR engagement and clustering. Our study reveals an actin-mediated positive feedback mechanism for BCR signaling.

Src-Like Adaptor Protein Regulates B Cell Development and Function

The avidity of BCRs and TCRs influences signal strength during processes of lymphocyte development. Avidity is determined by both the intrinsic affinity for Ag and surface levels of the Ag receptor. The Src-like adaptor protein (SLAP) is a regulator of TCR levels on thymocytes, and its deficiency alters thymocyte development. We hypothesized that SLAP, which is expressed in B cells, also is important in regulating BCR levels, signal strength, and B cell development. To test this hypothesis, we analyzed the B cell compartment in SLAP-deficient mice. We found increased splenic B cell numbers and decreased surface IgM levels on mature, splenic B cells deficient in SLAP. Immature bone marrow and splenic B cells from BCR-transgenic, SLAP-deficient mice were found to express higher surface levels of IgM. In contrast, mature splenic B cells from BCR-transgenic mice expressed decreased levels of surface BCR associated with decreased calcium flux and activation-induced markers, compared with controls. These data suggest that SLAP regulates BCR levels and signal strength during lymphocyte development.

Antinuclear Antigen B Cells That Down-Regulate Surface B Cell Receptor during Development to Mature, Follicular Phenotype Do Not Display Features of Anergy In Vitro

We previously demonstrated that B cells expressing a transgenic BCR with "dual reactivity" for the hapten arsonate and nuclear autoantigens efficiently complete development to follicular phenotype and stably reside in follicles in vivo. These B cells express very low levels of surface IgM and IgD, suggesting that they avoid central deletion and peripheral anergy by reducing their avidity for autoantigen via surface BCR (sBCR) down-regulation. Since a variety of states of B cell anergy have been previously described, a thorough examination of the functional capabilities of these B cells was required to test this hypothesis. In this study, we show that surface Ig cross-linking induces amounts of proximal BCR signaling in these B cells commensurate with their reduced sBCR levels. Functionally, however, they are comparable to nonautoreactive B cells in cell cycle progression, up-regulation of activation and costimulatory molecules, and Ab-forming cell differentiation when treated with a variety of stimuli in vitro. In addition, these B cells can efficiently process and present Ag and are capable of undergoing cognate interaction with naive TCR-transgenic T cells, resulting in robust IL-2 production. Together, these data reveal a lack of intrinsic anergy involving any known mechanism, supporting the idea that this type of antinuclear Ag B cell becomes indifferent to cognate autoantigen by down-regulating sBCR.

Survival of Resting Mature B Lymphocytes Depends on BCR Signaling via the Igα/β Heterodimer

We previously showed that type I interferon-induced, Cre-mediated ablation of surface BCR expression in mature B cells through Ig-heavy chain deletion results in apoptosis of these cells. This led to the hypothesis that survival signals from the BCR are vital for mature B cells. Here, we test two critical assumptions of this model. First, we demonstrate loss of mature B cells upon induced mutation of a signaling module of the BCR, not precluding BCR surface expression. Second, we show that the cells are also lost upon BCR inactivation in the absence of an exogenous inducer like interferon, excluding that cell death depends on previous cellular activation by the latter. Kinetic data demonstrate that BCR-less mature B cells have a severely reduced lifespan, with a half-life of 3–6 days. Together these results establish that BCR signaling is required to keep resting mature B cells alive in vivo.

Characterization of B lymphopoiesis in mouse bone marrow and spleen.

This chapter provides information on the application of flow cytometry for analysis of B-cell development, describing in detail the particular surface proteins that can serve as markers for recognizing distinct stages in this process. These cell fractions range from just prior to initial heavy chain rearrangement, the germline pro-B stage, through D-J rearranged pro-B and heavy chain expressing pre-B stages, to the maturing surface BCR positive B-cell stages. It also outlines assays for the characterization of these cells, including procedures for testing functional lineage restriction, determination of rearrangement status, analyses of gene expression at the ribonucleic acid (RNA) and protein level, and assessment of cell cycle state.

Review article: role of the surrogate light chain and the pre-B-cell receptor in mouse B-cell development.

The generation of B lymphocytes from committed progenitor cells is a complex process involving the transit of cells through several critical stages of development. Throughout their transit, developing B cells are subject to choices between survival, proliferation or death; fates that are dictated by combinations of intrinsic and extrinsic signals. Thus, failure to express certain signalling molecules on their surface at particular stages of development results in the cell failing to receive a viability and/or proliferative signal. Likewise, failure to link surface receptor expression to survival machinery will result in cell death. During the early stages of B-cell development, one such receptor molecule is the pre-B-cell receptor (pre-BCR). The pre-BCR is a heterodimer composed of an immunoglobulin (Ig) heavy chain molecule (IgH) covalently associated with an immunoglobulin light chain-like molecule called the surrogate light chain (SL).1–3 The SL itself is made up of two non-covalently associated proteins called lambda-5 (λ5) and VpreB, which together form a molecule having structural homology with conventional light (IgL) chains.4,5 Thus, in the SL, λ5 replaces a light chain constant region and VpreB the variable part. It is likely that the membrane pre-BCR complex is composed of two IgH molecules associated with two SLs.1 However, SL can reach the surface in the absence of IgH but in association with other molecules, for example with a glycoprotein of approximately 130 000 MW, so-called gp130.6,7 In this case, the surface SL-containing complex is referred to as a pro-BCR.8 The detailed description of SL structure must await crystallographic analysis, but recently Melchers has proposed a model of SL chain assembly.9 In this review we will discuss the molecular structure and gene organization of pre-BCR components and attempt to outline how surface expression of the pre-BCR impacts on cell survival, differentiation, proliferation and repertoire selection of developing B cells. General scheme of B lymphocyte development Different stages of B-cell development can be defined by the use of a combination of cell-surface markers and the status of immunglobulin gene rearrangements10,11 (Fig. 1, B-lymphocyte development in adult bone marrow). Following commitment to the B-cell lineage, an event which is still poorly understood, the first stage of the B-cell lineage is called a pro-B cell and is defined phenotypically as a B220+CD19− c-kit+CD43+CD25−IgM−IgD− cell in which there does not have to be any immunoglobulin gene rearrangement.12,13 It should be noted though that some pro-B cells have DJH rearrangements but only on one allele.13 Thus pro-B cells are found in mice in which the recombinase-activating genes (RAG) have been deleted,14,15 and in such mice, they are CD19+. However, pro-B cells do contain RNA transcripts for components of the SL but it is unclear at the present time whether they contain SL protein either in their cytoplasm or at the cell surface.13,16 It is possible that SL might reach the cell surface in association with molecules other than IgH, for example gp130.6,7 Open in a separate window Figure 1 B-lymphocyte development in the adult mouse bone marrow.

Altered Gene Expression upon BCR Cross-Linking in Burkitt's Lymphoma B Cell Line

Burkitt's lymphoma cell line, BL2 was stimulated by surface BCR cross-linking and altered gene expression was analyzed by RDA methodology. Consistent with previous reports, we detected up-regulated MDC, IL6R and adhesion molecule LFA1. We also detected gene expression of SIRPα, anti-apoptotic A-20, signal regulatory SLP76 and BCAR3, DNA binding proteins EGR2 and DEC1 in addition to some new genes.

A Tyrosine-Based Signal Present in Ig α Mediates B Cell Receptor Constitutive Internalization

AbstractB lymphocytes express Ag receptors (BCR) that are composed of ligand binding subunits, the membrane Igs, associated with Ig α/Ig β heterodimers. One main BCR function is to bind and to internalize Ags. Peptides generated from these internalized Ags may be presented to T lymphocytes. Here, we have analyzed the involvement of BCR Ig α/Ig β components in BCR constitutive endocytosis. The role of Ig α subunit in BCR constitutive endocytosis was first determined in the context of an IgM-based BCR. In contrast with BCR that contain wild-type Ig α, surface BCR lacking Ig α cytoplasmic domain were not constitutively internalized. The respective roles of Ig α and Ig β subunits were then analyzed by expressing chimeric molecules containing the cytoplasmic domains of either subunits in a B cell line. Only the Ig α cytoplasmic domain contained an internalization signal that allowed constitutive endocytosis of Ig α chimeras via coated pits and accumulation in sorting-recycling endosomes. This internalization signal is contained in its immunoreceptor tyrosine-based activation motif. These results indicate that Ig α, through its immunoreceptor tyrosine-based activation motif, may account for the ability of IgM/IgD BCR to constitutively internalize monovalent Ags.

Aberrations of the B-Cell Receptor B29 (CD79b) Gene in Chronic Lymphocytic Leukemia

AbstractLeukemic B cells in chronic lymphocytic leukemia (B-CLL) typically exhibit low or undetectable surface Ig. Because the B29 (CD79b and Igβ) and mb-1 (CD79a and Igα) gene products are required for surface Ig display in the B-cell receptor complex (BCR), we analyzed the expression of these genes in B-CLL cells. The majority (83%) of the randomly selected B-CLL patient samples analyzed exhibited low or undetectable surface BCR measured by μ heavy chain and B29 expression. Levels of mb-1 mRNA in these B-CLL samples with low surface BCR were similar to those in normal B cells. Among those with decreased surface expression, B29 mRNA was not detected in half of these B-CLL samples. The remaining B-CLL samples with diminished surface BCR contained normal levels of B29 mRNA. Further analysis of cDNA clones from the majority of these latter samples contained point mutations, insertions, or deletions that were largely located in the B29 transmembrane and cytoplasmic domains. These results indicate the occurrence of somatic mutations predicted to affect B29 expression and/or function in the majority of B-CLL and suggest that these aberrations underlie the diminished surface BCR display and loss of BCR signaling characteristic of this leukemia.