The tetraspanin CD81 regulates the expression of CD19 during B cell development in a postendoplasmic reticulum compartment

CD81 is a widely expressed tetraspanin that associates in B cells with CD19 in the CD19-CD21-CD81 signaling complex. CD81 is necessary for normal CD19 expression; cd81(-/-) B cells express lower levels of CD19, especially cd81(-/-) small pre-BII cells, which are almost devoid of surface CD19. The dependence of CD19 expression on CD81 is specific to this particular tetraspanin since cd9(-/-) B cells express normal levels of CD19. Furthermore, expression of human CD81 in mouse cd81(-/-) B cells restored surface CD19 to normal levels. Quantitative analysis of CD19 mRNA demonstrated normal levels, even in cd81(-/-) pre-BII cells. Analysis of CD19 at the protein level identified two CD19 glycoforms in both wild-type and cd81(-/-) B cells. The higher M(r) glycoform is significantly reduced in cd81(-/-) B cells and is endoglycosidase H (endo-H) resistant. In contrast, the low M(r) glycoform is comparably expressed in cd81(-/-) and in wild-type B cells and is endo-H sensitive. Because endo-H sensitivity is tightly correlated with endoplasmic reticulum localization, we suggest that the dependency of CD19 expression on CD81 occurs in a postendoplasmic reticulum compartment where CD81 is necessary for normal trafficking or for surface membrane stability of CD19.     

B cell antigen receptor and CD40 differentially regulate CD22 tyrosine phosphorylation

Cell surface molecules on lymphocytes positively or negatively modulate the Ag receptor signaling, and thus regulate the fate of the cell. CD22 is a B cell-specific cell surface protein that contains multiple ITIMs in the cytoplasmic tail, and critically regulates B cell activation and survival. CD22 regulation on B cell signaling is complex because CD22 can have both positive and negative roles in various contexts. We generated phosphospecific polyclonal Abs reacting four major CD22 tyrosine motifs (Y762, Y807, Y822, and Y842) and analyzed the pattern and intensity of phosphorylation of these tyrosine residues. The tyrosine motifs, Y762, Y822, and Y842, are considered as ITIM, whereas the other, Y807, is suggested to be important for Grb2 recruitment. Approximately 10% of the four tyrosine residues were constitutively phosphorylated. Upon anti-IgM ligation, CD22 Y762 underwent most rapid phosphorylation, whereas all four tyrosine residues were eventually phosphorylated equally at approximately 35% of all CD22 molecules in the cell. By contrast, anti-CD40 stimulation specifically up-regulated anti-IgM-induced phosphorylation of tyrosines within two ITIM motifs, Y762 and Y842, which was consistent with in vivo finding of the negative role of CD22 in CD40 signaling. Thus, CD22 phosphorylation is not only quantitatively but also qualitatively regulated by different stimulations, which may determine the outcome of B cell signaling.     

Human basophils express CD22 without expression of CD19.

BACKGROUND:Even modern automatic cell counters cannot count basophils precisely. Therefore, we need a rapid, accurate, precise, and easy method for counting basophils. METHODS:Using flow cytometry, basophils (CD22+/CD19-) and B cells (CD22+/CD19+) were counted. Within a large lymphocyte light scatter gate, % basophils (G%baso) and % B cells (G%B) were determined from the total count. Another method of analysis was to make two regions (R1 for basophils and R2 for B cells) and to determine in those the % basophils (R1%baso) and % B cells (R2%B) without gating. The flow cytometric basophil counts of the blood of 21 normal controls and 43 chronic myelogenous leukemia (CML) patients were compared with manual basophil count (Ma%baso) and basophil count by Coulter electronic cell counter (Hialeah, FL) (Auto%baso). CD22+/CD19- cells were sorted by a FACSCalibur (Becton Dickinson, San Jose, CA). RESULTS:The G%baso of all samples was 4.66 +/- 5.35%, and R1%baso was 4.23 +/- 4.88%, and they were well-correlated (r = 0.996, P < 0.001). The G%B of all samples was 1.55 +/- 1.68%, and R2%B was 1.59 +/- 1.67%, and they were also well-correlated (r = 0.993, P < 0.001). Their correlation was better in normal controls than in CML. G%baso was well-correlated to Ma%baso (r = 0.827) and Auto%baso (r = 0.806), and R1%baso was well-correlated to Ma%baso (r = 0.831) but showed poor correlation to Auto%baso (r = 0.734). Auto%baso revealed the poorest correlation to Ma%baso (r = 0.692). The sorted CD22+/CD19- cells were all basophils (99.48 +/- 0.30%), and they revealed CD13, CD33, and dim CD45 expression, whereas CD3, CD14, CD16, and HLA-DR were not detected on them. CONCLUSIONS: We discovered a specific marker combination to identify basophils (CD22+/CD19-), and we suggest that flow cytometric analysis using these markers is an easy, reliable, and accurate method of basophil counting.     

CD19 signaling pathways play a major role for murine AIDS induction and progression

Infection of genetically susceptible mice with the LP-BM5 mixture of murine leukemia viruses including an etiologic defective virus (BM5def) causes an immunodeficiency syndrome called murine AIDS (MAIDS). The disease is characterized by interactions between B cells and CD4(+) T cells resulting in polyclonal activation of both cell types. It is known that BM5def is expressed at highest levels in B cells and that B cells serve as viral APC. The CD19-CD21 complex and CD22 on the surface of B cells play critical roles as regulators of B cell responses to a variety of stimuli, influencing cell activation, differentiation, and survival. CD19 integrates positive signals induced by B cell receptor ligation by interacting with the protooncogene Vav, which leads to subsequent tyrosine phosphorylation of this molecule. In contrast, CD22 negatively regulates Vav phosphorylation. To analyze the role of CD19, CD21, Vav, and CD22 in MAIDS, we infected mice deficient in CD19, CD21 (CR2), Vav-1, or CD22 with LP-BM5 murine leukemia viruses. Infected CR2(-/-) mice developed MAIDS with a time course and severity indistinguishable from that of wild-type mice. In contrast, CD19 as well as Vav-1 deficiency restricted viral replication and suppressed the development of typical signs of MAIDS including splenomegaly, lymphadenopathy, and hypergammaglobulinemia. Finally, CD22 deficiency was found to accelerate MAIDS development. These results provide novel insights into the B cell signaling pathways required for normal induction and progression of MAIDS.     

Characterization of acetylcholine receptor subunits in developing and in denervated mammalian muscle

We have used subunit-specific antibodies to identify and to characterize partially the alpha, beta, gamma, and delta subunits of rat skeletal muscle acetylcholine receptor (AChR) on immunoblots. The alpha subunit of rat muscle is a single band of 42 kDa, whereas the beta subunit has an apparent molecular mass of 48 kDa. Both alpha and beta subunits are glycosylated and contain one or more N-linked oligosaccharide chains that are sensitive to endoglycosidase H digestion. The gamma and delta subunits, on the other hand, each appear as doublets on immunoblots, with apparent molecular masses of 52 kDa (gamma), 48 kDa (gamma') and 58 kDa (delta), 53 kDa (delta'), respectively. In each case, the two bands are structurally related and the lower band is probably the partial degradation product of the corresponding upper band. Each of the four gamma and delta polypeptides is N-glycosylated and contains both endoglycosidase H-sensitive and endoglycosidase H-resistant oligosaccharides. When the AChRs purified from embryonic, neonatal, adult, and denervated adult rat muscles were compared, no differences in the mobilities of alpha, beta, or delta subunits on sodium dodecyl sulfate gels were detected among them, either with or without endoglycosidase treatment. The gamma subunits, which were present in AChRs purified from neonatal, embryonic, or denervated rat muscles, were also identical; no gamma subunit was detected, however, in AChRs of normal adult rat muscle.     

Severely impaired B lymphocyte proliferation, survival, and induction of the c-Myc:Cullin 1 ubiquitin ligase pathway resulting from CD22 deficiency on the C57BL/6 genetic background

Understanding the molecular mechanisms through which CD22 regulates B lymphocyte homeostasis, signal transduction, and tolerance is critical to defining normal B cell function and understanding the role of CD22 in autoimmunity. Therefore, CD22 function was examined in vivo and in vitro using B cells from CD22-deficient (CD22(-/-)) mice. Backcrossing of founder CD22(-/-) mice onto the C57BL/6 (B6) genetic background from a B6/129 mixed background resulted in a dramatically reduced B cell proliferative response following IgM ligation, characterized by a paucity of lymphoblasts and augmented apoptosis. Also, the phenotype of splenic B6 CD22(-/-) B cells was uniquely HSA(high) and IgD(low)/CD21(low) with intermediate levels of CD5 expression, although the percentages of mature and transitional B cells were normal. That B6 CD22(-/-) B cells predominantly underwent apoptosis following IgM ligation correlated with this unique tolerant phenotype, as well as defective induction of the c-Myc:Cullin 1 (CUL1) ubiquitin ligase pathway that is necessary for progression to the S phase of cell cycle. CD40 ligation compensated for CD22 deficiency by restoring lymphoblast development, proliferation, c-Myc and CUL1 expression, and protein ubiquitination/degradation in IgM-stimulated B6 CD22(-/-) B cell cultures. Thereby, this study expands our current understanding of the complex role of CD22 during B cell homeostasis and Ag responsiveness, and reveals that the impact of CD22 deficiency is dictated by the genetic background on which it is rendered. Moreover, this study defines CD22 and CD40 as the first examples of lymphocyte coreceptors that influence induction of the c-Myc:CUL1 ubiquitin ligase pathway.     

Amplification of human B cell activation by a monoclonal antibody to the B cell-specific antigen CD22, Bp 130/140

The B cell-specific antigen CD22 is a 130/140-Kd complex and is unique among human B cell antigens, since its surface expression is restricted to a subpopulation of Ig+ B cells. Here the function of the CD22 antigen was evaluated by using the mAb HD6, directed against one of the epitopes on the molecule. The HD6 antibody was constimulatory with anti-Ig in inducing small, dense tonsillar cells to proliferate; however, the antibody by itself was devoid of stimulatory activity. Anti-CD22 antibody also induced more anti-Ig-treated B cells to leave G0 and enter the G1 phase of the cell cycle. It also was constimulatory with low-m.w. BCGF and with an antibody to a 50-Kd polypeptide, Bp50, which mediates a BCGF-like activity. Results of kinetic experiments and analysis of different B cell fractions suggested that anti-CD22 acts during an early phase of B cell activation, probably by amplifying the anti-Ig signal. F(ab')2 fragments of anti-CD22 HD6 were as effective as the whole antibody in inducing augmentation of B cell proliferation, showing that the Fc portion of the molecule was not required for the activity. The results of these experiments, together with the intriguing distribution of the Bp 130/140 antigen in B cell ontogeny, suggest that this molecule plays an important role in the process that leads to B cell activation and proliferation.     

Detection and enzymatic deglycosylation of a glycosylated variant of prolactin in human plasma

Immunoperoxidase electrophoresis was applied to the plasma of a patient showing a high level of prolactin (PRL) secreted by a pituitary adenoma. Two PRL monomers were detected with an anti-hPRL antiserum: a major 22 kDa form and a minor 25 kDa form. Concanavalin A-Sepharose 4B chromatography revealed that the 25 kDa form was a glycosylated variant of PRL. Incubation of this variant with endoglycosidase F led to its transformation into the 22 kDa form.     

Expansion of CD22lo B cells in the spleen of autoimmune-prone flaky skin mice

Similar to murine models with compromised CD22/SHP-1 function, flaky skin (fsn) mutant mice exhibit lymphocyte hyperactivation and an autoimmune phenotype characterized by circulating autoantibodies to dsDNA and glomerulonephritis. Immunophenotyping of fsn/fsn splenic B cells was performed to determine if abnormalities in CD22 expression contributed to the phenotype. We identified an expansion of an IgM(bright) CD22lo population consistent with immature B-lymphocytes. While normal B-lymphocytes require IL-4 to achieve down-modulation of CD22 expression in response to BCR cross-linking, culture with anti-IgM alone led to reduced CD22 expression in fsn/fsn mice. Furthermore, when IL-4 was added to fsn/fsn cultures, no further reduction in CD22 expression was observed. This suggested that fsn/fsn B cells were pre-activated in vivo by chronic IL-4 exposure. A portion of these CD22lo cells expressed the B-1 surface marker CD11b. We contend that decreased activation thresholds among CD22lo B-lymphocytes contributes to the expansion of immature and B-1 B cell populations and to the development of autoimmune pathology in fsn/fsn mice.     

Epitope specificity of the anti-(B cell lymphoma) monoclonal antibody,LL2

LL2 is a murine monoclonal antibody IgG2a reactive with B cells and non-Hodgkin's B-cell lymphoma, which, in a radioiodinated form, induces responses in lymphoma patients [Goldenberg et al. (1991) J Clin Oncol 9:548–564]. In this report we identify LL2 as a member of the CD22 cluster. The molecular size of the antigen, its expression profile, and competitive blocking studies were used to establish this identification. By Western blot analysis and immunoprecipitation studies using the Raji Burkitt's lymphoma cell line metabolically labelled with [³H]leucine, the LL2 antigen was determined to correspond to a molecular mass of 140 kDa. The molecular mass of the LL2 antigen, and the B-cell-restricted reactivity of the LL2 antibody, were consistent with both the CD21 and CD22 clusters. To assess additional similarities and differences between LL2 and anti-CD22 and anti-CD21, the binding of these mAb to cultured cell lines. Nalm-6 and Molt-4, was compared by flow cytometry. The binding profile of LL2 on these cell lines was consistent with anti-CD22, but not anti-CD21. Sequential immunoprecipitation and cross-blocking studies with anti-CD22 monoclonal antibodies recognizing established CD22 epitopes were performed to confirm that LL2 reacts with CD22 and to determine which epitope LL2 recognizes. Binding of¹³¹I-LL2 to Raji cells is inhibited over 90% by prior incubation of the target cells with unlabelled RFB4, indicating that LL2 belongs to the same epitope group as RFB4, i.e., epitope B.This work was supported in part by USPHS grant CA39841 from the NIH     

CD22 ligand binding regulates normal and malignant B lymphocyte survival in vivo

The CD22 extracellular domain regulates B lymphocyte function by interacting with alpha2,6-linked sialic acid-bearing ligands. To understand how CD22 ligand interactions affect B cell function in vivo, mouse anti-mouse CD22 mAbs were generated that inhibit CD22 ligand binding to varying degrees. Remarkably, mAbs which blocked CD22 ligand binding accelerated mature B cell turnover by 2- to 4-fold in blood, spleen, and lymph nodes. CD22 ligand-blocking mAbs also inhibited the survival of adoptively transferred normal (73-88%) and malignant (90%) B cells in vivo. Moreover, mAbs that bound CD22 ligand binding domains induced significant CD22 internalization, depleted marginal zone B cells (82-99%), and reduced mature recirculating B cell numbers by 75-85%. The CD22 mAb effects were independent of complement and FcRs, and the CD22 mAbs had minimal effects in CD22AA mice that express mutated CD22 that is not capable of ligand binding. These data demonstrate that inhibition of CD22 ligand binding can disrupt normal and malignant B cell survival in vivo and suggest a novel mechanism of action for therapeutics targeting CD22 ligand binding domains.     

Synthesis and biological evaluation of sialyl-oligonucleotide conjugates targeting leukocyte B trans-membranal receptor CD22 as delivery agents for nucleic acid drugs

Antisense oligonucleotides (ASOs) modified with ligands which target cell surface receptors have the potential to significantly improve potency in the target tissue. This has recently been demonstrated using triantennary N-acetyl d-galactosamine conjugated ASOs. CD22 is a cell surface receptor expressed exclusively on B cells thus presenting an attractive target for B cell specific delivery of drugs. Herein, we reported the synthesis of monovalent and trivalent ASO conjugates with biphenylcarbonyl (BPC) modified sialic acids and their study as ASO delivery agents into B cells. CD22 positive cells exhibited reduced potency when treated with ligand modified ASOs and mechanistic examination suggested reduced uptake into cells potentially as a result of sequestration of ASO by other cell-surface proteins.     

The activation and subsequent regulatory roles of Lyn and CD19 after B cell receptor ligation are independent

The cell surface glycoprotein CD19 and the Src-related protein tyrosine kinase Lyn are key mediators of, respectively, positive and negative signaling in B cells. Despite the apparent opposition of their regulatory functions, a recent model of the biochemical events after B cell receptor (BCR) ligation intimately links the activation of Lyn and CD19. We examined the biochemical consequences of BCR ligation in mouse B cells lacking either Lyn or CD19 for evidence of interaction or codependence. In contrast to published results, we found CD19 phosphorylation after BCR ligation to be unaffected by the absence of Lyn, yet dependent on Src family protein tyrosine kinases as it was inhibited fully by PP2, an Src family-specific inhibitor. Consistent with normal CD19 phosphorylation in lyn(-/-) B cells, the recruitment of phosphoinositide-3 kinase to CD19 and the ability of CD19 to enhance both intracellular calcium flux and extracellular signal-regulated kinase 1/2 activation after coligation with the BCRs were intact in the absence of Lyn. Similarly, unique functions of Lyn were found to be independent of CD19. CD19(-/-) B cells were normal for increased Lyn kinase activity after BCR ligation, inhibition of BCR-mediated calcium flux after CD22 coligation, and inhibition of extracellular signal-regulated kinase phosporylation after FcgammaRIIB coligation. Collectively, these data show that the unique functions of Lyn do not require CD19 and that the signal amplification mediated by CD19 is independent of Lyn. We conclude that the roles of Lyn and CD19 after BCR ligation are independent and opposing, one being primarily inhibitory and the other stimulatory.     

Infection of human B lymphoma cells by Mycoplasma fermentans induces interaction of its elongation factor with the intracytoplasmic domain of Epstein-Barr virus receptor (gp140, EBV/C3dR, CR2, CD21)

Human cell lines are often infected by mycoplama strains. We have demonstrated that when infected by Mycoplasma fermentans, human B lymphoma cell proliferation increased strongly. These infected B cells expressed a p45 kDa protein which interacted with the intracellular domain of CD21, the EBV/C3d receptor. p45 analysis demonstrated that this is a new gene which encodes an elongation factor originating from Mycoplasma fermentans. p45 interaction with CD21 was specific, there being no interaction with CD19. This is the first demonstration that Mycoplasma fermentans, in infecting human B cells, generates a p45 Mycoplasma component that interacts with CD21, which is involved in B cell proliferation.     

CD72 negatively regulates signaling through the antigen receptor of B cells

The immunoreceptor tyrosine-based inhibition motif (ITIM) is found in various membrane molecules such as CD22 and the low-affinity Fc receptor for IgG in B cells and the killer cell-inhibitory receptor and Ly-49 in NK cells. Upon tyrosine phosphorylation at the ITIMs, these molecules recruit SH2 domain-containing phosphatases such as SH2-containing tyrosine phosphatase-1 and negatively regulate cell activity. The B cell surface molecule CD72 carries an ITIM and an ITIM-like sequence. We have previously shown that CD72 is phosphorylated and recruits SH2-containing tyrosine phosphatase-1 upon cross-linking of the Ag receptor of B cells (BCR). However, whether CD72 modulates BCR signaling has not yet been elucidated. In this paper we demonstrate that expression of CD72 down-modulates both extracellular signal-related kinase (ERK) activation and Ca2+ mobilization induced by BCR ligation in the mouse B lymphoma line K46micromlambda, whereas BCR-mediated ERK activation was not reduced by the ITIM-mutated form of CD72. Moreover, coligation with CD72 with BCR reduces BCR-mediated ERK activation in spleen B cells of normal mice. These results indicate that CD72 negatively regulates BCR signaling. CD72 may play a regulatory role in B cell activation, probably by setting a threshold for BCR signaling.     

The B lymphocyte adhesion molecule CD22 interacts with leukocyte common antigen CD45RO on T cells and alpha 2-6 sialyltransferase, CD75, on B cells.

Functional maturation of B lymphocytes correlates with expression of the B lineage-specific cell surface glycoprotein CD22. Two CD22 polypeptides have been characterized and suggested to play a role in B cell-B cell interaction as well as in B cell adhesion to monocytes. In this work we provide evidence that CD22 is directly involved in the cognate interaction between B and T cells. One of the two CD22 polypeptides, CD22 beta, interacts with a specific ligand on a subpopulation of CD4+ T cells. Our results suggest that the T cell ligand of CD22 is CD45RO, an isoform of the leukocyte common antigen class of phosphotyrosine phosphatases associated with the helper T cell phenotype. We further demonstrate that CD22 recognizes a second ligand, CD75, expressed predominantly on activated B cells and shown to be a cell surface alpha 2-6 sialyltransferase.     

Engagement of CD22 on B cells with the monoclonal antibody epratuzumab stimulates the phosphorylation of upstream inhibitory signals of the B cell receptor

The binding of antigen to the B cell receptor (BCR) results in a cascade of signalling events that ultimately drive B cell activation. Uncontrolled B cell activation is regulated by negative feedback loops that involve inhibitory co-receptors such as CD22 and CD32B that exert their functions following phosphorylation of immunoreceptor tyrosine-based inhibition motifs (ITIMs). The CD22-targeted antibody epratuzumab has previously been shown to inhibit BCR-driven signalling events, but its effects on ITIM phosphorylation of CD22 and CD32B have not been properly evaluated. The present study therefore employed both immunoprecipitation and flow cytometry approaches to elucidate the effects of epratuzumab on direct phosphorylation of key tyrosine (Tyr) residues on both these proteins, using both transformed B cell lines and primary human B cells. Epratuzumab induced the phosphorylation of Tyr⁸²² on CD22 and enhanced its co-localisation with SHP-1. Additionally, in spite of high basal phosphorylation of other key ITIMs on CD22, in primary human B cells epratuzumab also enhanced phosphorylation of Tyr⁸⁰⁷, a residue involved in the recruitment of Grb2. Such initiation events could explain the effects of epratuzumab on downstream signalling in B cells. Finally, we were able to demonstrate that epratuzumab stimulated the phosphorylation of Tyr²⁹² on the low affinity inhibitory Fc receptor CD32B which would further attenuate BCR-induced signalling. Together, these data demonstrate that engagement of CD22 with epratuzumab leads to the direct phosphorylation of key upstream inhibitory receptors of BCR signalling and may help to explain how this antibody modulates B cell function.     

Suppression of IgE responses in CD23-transgenic animals is due to expression of CD23 on nonlymphoid cells

Serum IgE is suppressed in CD23-transgenic (Tg) mice where B cells and some T cells express high levels of CD23, suggesting that CD23 on B and T cells may cause this suppression. However, when Tg B lymphocytes were compared with controls in B cell proliferation and IgE synthesis assays, the two were indistinguishable. Similarly, studies of lymphokine production suggested that T cell function in the Tg animals was normal. However, adoptive transfer studies indicated that suppression was seen when normal lymphocytes were used to reconstitute Tg mice, whereas reconstitution of controls with Tg lymphocytes resulted in normal IgE responses, suggesting that critical CD23-bearing cells are irradiation-resistant, nonlymphoid cells. Follicular dendritic cells (FDC) are irradiation resistant, express surface CD23, and deliver iccosomal Ag to B cells, prompting us to reason that Tg FDC may be a critical cell. High levels of transgene expression were observed in germinal centers rich in FDC and B cells, and IgE production was inhibited when Tg FDCs were cultured with normal B cells. In short, suppressed IgE production in CD23-Tg mice appears to be associated with a population of radioresistant nonlymphoid cells. FDCs that interface with B cells in the germinal center are a candidate for explaining this CD23-mediated IgE suppression.     

Antigen receptor triggered upregulation of CD86 and CD80 in human B cells: augmenting role of the CD21/CD19 co-stimulatory complex and IL-4

The impact of BCR:CD21 co-engagement on B cell expression of molecules critical for T cell activation was investigated with receptor-specific mAbs conjugated to high MW dextran as stimulatory ligands. In the absence of IL-4, BCR:CD21 co-ligation augmented BCR-triggered CD86 only under conditions of very low BCR ligand dose or affinity, and CD80 was minimally induced by BCR and/or CD21 crosslinking. In the presence of IL-4, BCR:CD21 co-ligation augmented CD86 and CD80 expression under conditions of greater BCR engagement. However, with very high level BCR engagement, no bonus effect of BCR:CD21 crosslinking was observed. Co-ligation-promoted CD86 and CD80 expression was associated with heightened B cell activation of resting allogeneic T cells. The data suggest that co-clustering of BCR and the CD21/CD19 co-stimulatory complex following B cell engagement with C3d-bound microbial or self-antigens will enhance B cell recruitment of T cell help only when IL-4 is present and/or BCR engagement is very limiting.     

The tetraspanin CD81 is necessary for partitioning of coligated CD19/CD21-B cell antigen receptor complexes into signaling-active lipid rafts

Tetraspanins have been hypothesized to facilitate the organization of functional multimolecular membrane complexes. In B cells the tetraspanin CD81 is a component of the CD19/CD21 complex. When coligated to the B cell Ag receptor (BCR), the CD19/CD21 complex significantly enhances BCR signaling in part by prolonging the association of the BCR with signaling-active lipid rafts. In this study CD81 is shown to associate with lipid rafts upon coligation of the BCR and the CD19/CD21 complex. Using B cells from CD81-deficient mice we demonstrate that in the absence of CD81, coligated BCR and CD19/CD21 complexes fail to partition into lipid rafts and enhance BCR signaling from rafts. Furthermore, a chimeric CD19 protein that associates only weakly if at all with CD81 fails to promote the association of coligated BCR with lipid rafts. The requirement for CD81 to promote lipid raft association may define a novel mechanism by which tetraspanins function as molecular facilitators of signaling receptors.