MS4A4A: a novel cell surface marker for M2 macrophages and plasma cells.

MS4A4A is a member of the membrane-spanning, four domain family, subfamily A (MS4A) that includes CD20 (MS4A1), FcRβ (MS4A2) and Htm4 (MS4A3). Like the first three members of this family, transcription of MS4A4A appears to be limited to hematopoietic cells. To evaluate expression of the MS4A4A protein in hematopoietic cell lineages and subsets we generated monoclonal antibodies against extracellular epitopes for use in flow cytometry.

Reduced T-dependent humoral immunity in CD20-deficient mice.

CD20 is a tetraspanning membrane protein expressed on B lymphocytes. CD20 deficiency in both mice and humans has recently been shown to have deleterious effects on Ab responses to T-independent Ags; however, no effect on T-dependent immunity has been reported. In this study, we used a Cd20⁻/⁻ mouse line to evaluate Ab responses to adeno-associated virus and SRBCs.

Expression of MS4A and TMEM176 Genes in Human B Lymphocytes.

The MS4A gene family in humans includes CD20 and at least 15 other genes. CD20 exists as homo-oligomers in the plasma membrane, however different MS4A proteins expressed in the same cell may hetero-oligomerize. Given the importance of CD20 in B-cell function and as a therapeutic target, we sought to explore the potential for CD20 hetero-oligomerization with other MS4A proteins.

Quantification of CD20 mRNA and protein levels in chronic lymphocytic leukemia suggests a post-transcriptional defect.

Chronic lymphocytic leukemia is less effectively treated than other B cell malignancies with the anti-CD20 agent, rituximab, presumably due, at least in part, to low CD20 expression. CD20 expression is typically measured by flow cytometry, which may not be quantitative. This study was undertaken to measure total CD20 protein in CLL B cells using quantitative immunoblot analysis.

Phylogenetic analysis of the MS4A and TMEM176 gene families.

Background: The MS4A gene family in humans includes CD20 (MS4A1), FcRbeta (MS4A2), Htm4 (MS4A3), and at least 13 other syntenic genes encoding membrane proteins, most having characteristic tetraspanning topology. Expression of MS4A genes is variable in tissues throughout the body; however, several are limited to cells in the hematopoietic system where they have known roles in immune cell functions. Genes in the small TMEM176 group share significant sequence similarity with MS4A genes and there is evidence of immune function of at least one of the encoded proteins.

Therapeutic (high) doses of rituximab activate calcium mobilization and inhibit B-cell growth via an unusual mechanism triggered independently of both CD20 and Fcgamma receptors.

Rituximab is a CD20-specific monoclonal antibody that effectively targets and depletes B lymphocytes in vivo, primarily via indirect cytotoxic mechanisms. Direct effects on B cells may also contribute to B-cell depletion but are less clearly defined. In this report, we demonstrate that monomeric rituximab, at the high concentrations found in plasma following infusion of therapeutic doses, induces prolonged low-amplitude release of calcium from thapsigargin-sensitive intracellular stores and reduces the growth of Ramos B cells in culture.

Efficient isolation of highly purified tonsil B lymphocytes using RosetteSep with allogeneic human red blood cells.

Background: Human tonsils are a rich source of B lymphocytes exhibiting a variety of phenotypes and activation states. Existing methods of purification are time consuming or costly. The aim of the present study was to optimize conditions to isolate large numbers of highly purified primary B lymphocytes from tonsils in a short and cost-effective single step, using a commercially available reagent designed for purifying cells from whole blood (RosetteSep).

Determination of apparent calcium affinity for endogenously expressed human sarco(endo)plasmic reticulum calcium-ATPase isoform SERCA3.

The sarco(endo)plasmic reticulum Ca(2+)-ATPases (SERCAs) play a crucial role in regulating free cytosolic Ca(2+) concentration in diverse cell types. It has been shown that recombinant SERCA3, when measured in heterologous systems, exhibits low apparent affinity for Ca(2+); however, Ca(2+) affinity of native SERCA3 in an endogenous setting has not been examined. Such a measurement is complicated, because SERCA3 is always coexpressed with the housekeeping isoform SERCA2b.

CD20 homo-oligomers physically associate with the B cell antigen receptor. Dissociation upon receptor engagement and recruitment of phosphoproteins and calmodulin-binding proteins.

B cell antigen receptor (BCR) signaling initiates sustained cellular calcium influx necessary for the development, differentiation, and activation of B lymphocytes. CD20 is a B cell-restricted tetraspanning protein organized in the plasma membrane as multimeric molecular complexes involved in BCR-activated calcium entry. Using coprecipitation of native CD20 with tagged or truncated forms of the molecule, we provide here direct evidence of CD20 homo-oligomerization into tetramers.

Activation-induced endocytosis of the raft-associated transmembrane adaptor protein LAB/NTAL in B lymphocytes: evidence for a role in internalization of the B cell receptor.

Linker for activation of B cell (LAB)/non-T cell activation linker (NTAL) and phosphoprotein associated with glycophospholipid-enriched membrane microdomain (PAG)/Csk-binding protein (Cbp) are raft-associated transmembrane adaptor proteins with distinct functions in immediate/early phases of receptor signaling pathways. Heterogeneous rafts are thought to compartmentalize membrane-associated signaling events. In order to investigate the subcellular localization of LAB/NTAL and PAG/Cbp, they were expressed as fluorescent chimeric fusion proteins in a human B cell line and their distribution was examined, along with the corresponding endogenous proteins, before and after B cell receptor (BCR) stimulation.

Cholesterol depletion inhibits src family kinase-dependent calcium mobilization and apoptosis induced by rituximab crosslinking.

The monoclonal antibody (mAb) rituximab produces objective clinical responses in patients with B-cell non-Hodgkin's lymphoma and antibody-based autoimmune diseases. Mechanisms mediating B-cell depletion by rituximab are not completely understood and may include direct effects of signalling via the target antigen CD20. Like most but not all CD20 mAbs, rituximab induces a sharp change in the solubility of the CD20 protein in the non-ionic detergent Triton-X-100, reflecting a dramatic increase in the innate affinity of CD20 for membrane raft signalling domains.

Improved access to CD20 following B cell receptor cross-linking at Burkitt's lymphoma cell surfaces.

Here we report that B cell receptor (BCR) engagement rapidly improves the capacity of CD20 to be accessed by cognate antibody at model Burkitt's lymphoma cell surfaces. None of eight other surface molecules demonstrated such BCR-dependent enhancement of ligand binding while the quantity of accessible CD20 remained unchanged on either CD19 or CD40 engagement. Neither the actin-depolymerizing agent cytochalasin D nor inhibitors targeting signalling pathways associated with the BCR attenuated the CD20 increase that could be uncoupled from BCR endocytosis.

The CD20 calcium channel is localized to microvilli and constitutively associated with membrane rafts: antibody binding increases the affinity of the association through an epitope-dependent cross-linking-independent mechanism.

CD20 is a B cell-specific membrane protein that functions in store-operated calcium entry and serves as a useful target for antibody-mediated therapeutic depletion of B cells. Antibody binding to CD20 induces a diversity of biological effects, some of which are dependent on lipid rafts. Rafts are isolated as low density detergent-resistant membranes, initially characterized using Triton X-100.

Store-operated cation entry mediated by CD20 in membrane rafts.

B cell activation requires sustained elevation of cytoplasmic free calcium, achieved by influx through store-operated calcium (SOC) channels. The molecular identity of these channels is not known. Ectopic expression of the raft-associated tetraspan protein CD20 in Chinese hamster ovary cells introduced a novel SOC entry pathway that was permeable to strontium as well as to calcium.

CD20-mediated apoptosis: signalling through lipid rafts.

CD20 is an effective target for therapeutic B-cell depletion with monoclonal antibodies. One proposed mechanism of action is direct cytotoxicity mediated via tyrosine kinase-dependent signalling pathways activated upon CD20 cross-linking. The association of CD20 with membrane microdomains known as lipid rafts, enriched in src-family tyrosine kinases and other signalling effectors, suggests an indirect mechanism of anti-CD20-induced apoptosis in which activation of src-family kinases occurs as a consequence of lipid raft clustering.

Colocalization of the B cell receptor and CD20 followed by activation-dependent dissociation in distinct lipid rafts.

The B cell Ag receptor (BCR) and CD20, a putative calcium channel, inducibly associate with cholesterol-dependent membrane microdomains known as lipid rafts. A functional association between the BCR and CD20 is suggested by the effects of CD20-specific mAbs, which can modulate cell cycle transitions elicited by BCR signaling. Using immunofluorescence microscopy we show here that the BCR and CD20 colocalize after receptor ligation and then rapidly dissociate at the cell surface before endocytosis of the BCR.

Alanine-170 and proline-172 are critical determinants for extracellular CD20 epitopes; heterogeneity in the fine specificity of CD20 monoclonal antibodies is defined by additional requirements imposed by both amino acid sequence and quaternary structure.

In vivo ablation of malignant B cells can be achieved using antibodies directed against the CD20 antigen. Fine specificity differences among CD20 monoclonal antibodies (mAbs) are assumed not to be a factor in determining their efficacy because evidence from antibody-blocking studies indicates limited epitope diversity with only 2 overlapping extracellular CD20 epitopes. However, in this report a high degree of heterogeneity among antihuman CD20 mAbs is demonstrated.