The role of the CD19/CD21 complex in B cell processing and presentation of complement-tagged antigens.

The CD19/CD21 complex is an essential B cell coreceptor that functions synergistically to enhance signaling through the B cell Ag receptor in response to T cell-dependent, complement-tagged Ags. In this study, we use a recombinant protein containing three tandemly arranged copies of C3d and the Ag hen egg lysozyme, shown to be a highly effective immunogen in vivo, to evaluate the role of the CD19/CD21 complex in Ag processing in B cells. Evidence is provided that coengagement of the CD19/CD21 complex results in more rapid and efficient production of antigenic peptide/class II complexes as compared with B cell Ag receptor-mediated processing alone. The CD19/CD21 complex does not itself target complement-tagged Ags for processing, but rather appears to influence B cell Ag processing through its signaling function. The ability of the CD19/CD21 complex to augment processing may be an important element of the mechanism by which the CD19/CD21 complex functions to promote B cell responses to T cell-dependent complement-tagged Ags in vivo.     

Regulation of MHC class II signal transduction by the B cell coreceptors CD19 and CD22

The major histocompatability class II heterodimer (class II) is expressed on the surface of both resting and activated B cells. Although it is clear that class II expression is required for Ag presentation to CD4(+) T cells, substantial evidence suggests that class II serves as a signal transducing receptor that regulates B cell function. In ex vivo B cells primed by Ag receptor (BCR) cross-linking and incubation with IL-4, or B cell lines such as K46-17 micromlambda, class II ligation leads to the activation of protein tyrosine kinases, including Lyn and Syk and subsequent phospholipase Cgamma-dependent mobilization of Ca(2+). In this study, experiments demonstrated reciprocal desensitization of class II and BCR signaling upon cross-linking of either receptor, suggesting that the two receptors transduce signals via common processes and/or effector proteins. Because class II and BCR signal transduction pathways exhibit functional similarities, additional studies were conducted to evaluate whether class II signaling is regulated by BCR coreceptors. Upon cross-linking of class II, the BCR coreceptors CD19 and CD22 were inducibly phosphorylated on tyrosine residues. Phosphorylation of CD22 was associated with increased recruitment and binding of the protein tyrosine phosphatase SHP-1. Similarly, tyrosine phosphorylation of CD19 resulted in recruitment and binding of Vav and phosphatidylinositol 3-kinase. Finally, co-cross-linking studies demonstrated that signaling via class II was either attenuated (CD22/SHP-1) or enhanced (CD19/Vav and phosphatidylinositol 3-kinase), depending on the coreceptor that was brought into close proximity. Collectively, these results suggest that CD19 and CD22 modulate class II signaling in a manner similar to that for the BCR.     

CD19 function in early and late B cell development: I. Maintenance of follicular and marginal zone B cells requires CD19-dependent survival signals

Loss of membrane-bound Ig results in the rapid onset of apoptosis in recirculating B cells. This observation implies that a competent B cell receptor (BCR) is not only required for Ag-dependent differentiation, but also for continued survival in the peripheral immune system. Expression of the B cell coreceptor, CD19, is likewise essential for key B cell differentiative events including the formation of B-1, germinal center, and marginal zone (MZ) B cells. In this study, we report that CD19 also exerts a role before Ag encounter by promoting the survival of naive recirculating B cells. This aspect of CD19 signaling was first suggested by the analysis of mixed bone marrow chimeras, wherein CD19-/- B cells fail to effectively compete with wild-type B cells to reconstitute the peripheral B cell compartment. Consistent with this observation, Bromodeoxyuridine- and CFSE-labeling studies reveal a shorter in vivo life span for CD19-/- B cells vs their wild-type counterparts. Moreover, we find that CD19 is necessary for propagation of BCR-induced survival signals and thus may contribute to homeostatic mechanisms of tonic signaling. To determine whether provision of a constitutive survival signal could compensate for the loss of CD19 in vivo, Bcl-2-transgenic mice were bred onto the CD19-/- background. Here, we observe an increase in follicular B cell numbers and selective recovery of the MZ B cell compartment. Together these findings suggest that maintenance of the follicular and MZ B cell compartments require CD19-dependent survival signals.     

Cognate B cell signaling via MHC class II: differential regulation of B cell antigen receptor and MHC class II/Ig-alpha beta signaling by CD22

Recent studies demonstrate that MHC class II molecules can signal via associated Ig-alphabeta dimers, signal transducers previously thought to function only in B cell Ag receptor (BCR) signaling. Surprisingly, the biologic outputs of MHC class II and BCR ligation (by thymus-dependent Ags) differ, e.g., MHC class II signaling leads to robust proliferation and extension of pseudopods. It seemed possible that these differences might be due, at least in part, to differential use of inhibitory coreceptors thought to modulate membrane Ig signals. In this study, we demonstrate that CD22, an inhibitory BCR coreceptor, neither associates with nor functions in MHC class II/Ig-alphabeta signaling. Interestingly, CD22 is actively excluded from cell surface MHC class II aggregates.     

Systematic analysis of the role of CD19 cytoplasmic tyrosines in enhancement of activation in Daudi human B cells: clustering of phospholipase C and Vav and of Grb2 and Sos with different CD19 tyrosines

CD19 is a coreceptor on B cells that enhances the increase in cytoplasmic calcium and ERK2 activation when coligated with the B cell Ag receptor. Constructs containing point mutations and truncations were expressed in Daudi human B lymphoblastoid cells to systematically determine the requirement for individual CD19 cytoplasmic tyrosines in these responses. Evidence for activity was found for Y330, Y360, and Y421 as well as that previously published for Y391. Precipitates formed with phosphopeptides consisting of CD19 sequences flanking these residues were used to screen for cytoplasmic proteins that mediate signaling. Phosphopeptide Y330 precipitated Grb2 and Sos, whereas phosphopeptides Y391 and Y421 both precipitated Vav and phospholipase C-gamma2. These molecules also were found associated with native CD19. In mapping studies with altered constructs, CD19 Y330 and/or Y360 were necessary for binding Grb2 and Sos. Vav associated with CD19 constitutively in unstimulated cells by a tyrosine-independent mechanism requiring the portion of CD19 encoded by exons 9-12. After B cell Ag receptor stimulation, Vav association was tyrosine-dependent, but binding was influenced by multiple residues. However, when maximally phosphorylated by pervanadate, Y391 and, to a lesser extent, Y421 were sufficient. CD19 Y391 was also both necessary and sufficient for binding phospholipase C-gamma2. Thus, different tyrosines along the CD19 cytoplasmic domain provide scaffolding for the formation of complexes of different signaling molecules.     

AP2 adaptor complex-dependent internalization of CD5: differential regulation in T and B cells

CD5 is a key regulator of Ag receptor-mediated activation, selection, and differentiation in both T and B cells. Accumulating evidence indicates that lymphocyte activation and selection are sensitive to variations in levels of CD5 on the cell surface. We now show that CD5 expression on the surface of B and T cells is regulated posttranslationally by direct interaction with the mu(2) subunit of the AP2 adaptor complex that links transmembrane proteins to clathrin-coated pits. CD5 is rapidly internalized from the cell surface in lymphoid cell lines, mature splenic T and B cells, and peritoneal CD5(+) B cells following monovalent or bivalent ligation of the receptor. We mapped the mu(2) subunit binding site on CD5 to Y(429) and determined that the integrity of this site was necessary for CD5 internalization. Cross-linking of the Ag receptor with intact Abs inhibited CD5 internalization in B cells, but had the opposite effect in T cells. However, if F(ab')(2) Abs were used to stimulate the Ag receptor in B cells, the effect on CD5 internalization was now similar to that observed in T cells, indicating that signals through the Ag receptor and FcR regulate CD5 endocytosis in B cells. This was confirmed using an FcgammaRIIB1-deficient B cell line. The ability to differentially alter posttranslational CD5 expression in T and B cells is likely to be key in regulation of Ag receptor signaling and generation of tolerance in T and B lymphocytes.     

The c-Abl tyrosine kinase is regulated downstream of the B cell antigen receptor and interacts with CD19

c-Abl is a nonreceptor tyrosine kinase that we have recently linked to growth factor receptor signaling. The c-Abl kinase is ubiquitously expressed and localizes to the cytoplasm, plasma membrane, cytoskeleton, and nucleus. Thus, c-Abl may regulate signaling processes in multiple subcellular compartments. Targeted deletion or mutation of c-Abl in mice results in a variety of phenotypes, including splenic and thymic atrophy and lymphopenia. Additionally, lymphocytes isolated from specific compartments of c-Abl mutant mice have reduced responses to a variety of stimuli and an increased susceptibility to apoptosis following growth factor deprivation. Despite these observations, little is known regarding the signaling mechanisms responsible for these phenotypes. We report here that splenic B cells from c-Abl-deficient mice are hyporesponsive to the proliferative effects of B cell Ag receptor (BCR) stimulation. The c-Abl kinase activity and protein levels are elevated in the cytosol following activation of the BCR in B cell lines. We show that c-Abl associates with and phosphorylates the BCR coreceptor CD19, and that c-Abl and CD19 colocalize in lipid membrane rafts. These data suggest a role for c-Abl in the regulation of B cell proliferation downstream of the BCR, possibly through interactions with CD19.     

CD38 signaling in B lymphocytes is controlled by its ectodomain but occurs independently of enzymatically generated ADP-ribose or cyclic ADP-ribose

CD38 is a type II transmembrane glycoprotein that is expressed by many cell types including lymphocytes. Signaling through CD38 on B lymphocytes can mediate B cell activation, proliferation, and cytokine secretion. Additionally, coligation of CD38 and the B cell Ag receptor can greatly augment B cell Ag receptor responses. Interestingly, the extracellular domain of CD38 catalyzes the conversion of NAD+ into nicotinamide, ADP-ribose (ADPR), and cyclic ADPR (cADPR). cADPR can induce intracellular calcium release in an inositol trisphosphate-independent manner and has been hypothesized to regulate CD38-mediated signaling. We demonstrate that replacement of the cytoplasmic tail and the transmembrane domains of CD38 did not impair CD38 signaling, coreceptor activity, or enzyme activity. In contrast, independent point mutations in the extracellular domain of CD38 dramatically impaired signal transduction. However, no correlation could be found between CD38-mediated signaling and the capacity of CD38 to catalyze an enzyme reaction and produce cADPR, ADPR, and/or nicotinamide. Instead, we propose that CD38 signaling and coreceptor activity in vitro are regulated by conformational changes induced in the extracellular domain upon ligand/substrate binding, rather than on actual turnover or generation of products.     

CD19 amplifies B lymphocyte signal transduction by regulating Src-family protein tyrosine kinase activation.

Ligation of the B cell Ag receptor (BCR) induces cellular activation by stimulating Src-family protein tyrosine kinases (PTKs) to phosphorylate members of the BCR complex. Subsequently, Src-family PTKs, particularly Lyn, are proposed to phosphorylate and bind CD19, a cell-surface costimulatory molecule that regulates mature B cell activation. Herein, we show that B cells from CD19-deficient mice have diminished Lyn kinase activity and BCR phosphorylation following BCR ligation. Tyrosine phosphorylation of other Src-family PTKs was also decreased in CD19-deficient B cells. In wild-type B cells, CD19 was constitutively complexed with Vav, Lyn, and other Src-family PTKs, with CD19 phosphorylation and its associations with Lyn and Vav increased after BCR ligation. Constitutive CD19/Lyn/Vav complex signaling may therefore be responsible for the establishment of baseline signaling thresholds in B cells before Ag receptor ligation, in addition to accelerating signaling following BCR engagement or other transmembrane signals. In vitro kinase assays using purified CD19 and purified Lyn revealed that the kinase activity of Lyn was significantly increased when coincubated with CD19. Thus, constitutive and induced CD19/Lyn complexes are likely to regulate basal signaling thresholds and BCR signaling by amplifying the kinase activity of Lyn and other Src-family PTKs. These in vivo and in vitro findings demonstrate a novel mechanism by which CD19 regulates signal transduction in B lymphocytes. The absence of this CD19/Src-family kinase amplification loop may account for the hyporesponsive phenotype of CD19-deficient B cells.     

CD23-bound IgE augments and dominates recall responses through human naive B cells

Human peripheral blood BCRμ(+) B cells express high levels of CD23 and circulate preloaded with IgE. The Ag specificity of CD23-bound IgE presumably differs from the BCR and likely reflects the Ag-specific mix of free serum IgE. CD23-bound IgE is thought to enhance B cell Ag presentation to T cells raising the question of how a B cell might respond when presented with a broad mix of Ags and CD23-bound IgE specificities. We recently reported that an increase in CD23(+) B cells is associated with the development of resistance to schistosomiasis, highlighting the potential importance of CD23-bound IgE in mediating immunity. We sought to determine the relationship between BCR and CD23-bound IgE-mediated B cell activation in the context of schistosomiasis. We found that crude schistosome Ags downregulate basal B cell activation levels in individuals hyperexposed to infectious worms. Schistosome-specific IgE from resistant, occupationally exposed Kenyans recovered responses of B cells to schistosome Ag. Furthermore, cross-linking of CD23 overrode intracellular signals mediated via the BCR, illustrating its critical and dominating role in B cell activation. These results suggest that CD23-bound IgE augments and dominates recall responses through naive B cells.     

B cell signaling is regulated by induced palmitoylation of CD81

Signaling through the B cell antigen receptor (BCR) is amplified and prolonged by coligation of the BCR to the CD19/CD21/CD81 coreceptor complex. Coligation is induced during immune responses by the simultaneous binding of complement-tagged antigens to the complement receptor, CD21, and to the BCR. Enhanced signaling is due in part to the ability of the CD19/CD21/CD81 complex to stabilize the BCR in sphingolipid- and cholesterol-rich membrane microdomains termed lipid rafts. The tetraspanin CD81 is essential for the raft-stabilizing function of the coreceptor. Here we show that coligation of the BCR and the CD19/CD21/CD81 complex leads to selective, rapid, and reversible palmitoylation of CD81 and that palmitoylation is necessary for the raft stabilizing function of the CD19/CD21/CD81 complex. Inducible palmitoylation may represent a novel mechanism by which tetraspanins function to facilitate lipid raft-dependent receptor signaling.     

Inhibitory coreceptors activated by antigens but not by anti-Ig heavy chain antibodies install requirement of costimulation through CD40 for survival and proliferation of B cells

Ag-induced B cell proliferation in vivo requires a costimulatory signal through CD40, whereas B cell Ag receptor (BCR) ligation by anti-Ig H chain Abs, such as anti-Ig micro H chain Ab and anti-Ig delta H chain Ab, alone induces proliferation of B cells in vitro, even in the absence of CD40 ligation. In this study, we demonstrate that CD40 signaling is required for survival and proliferation of B cells stimulated by protein Ags in vitro as well as in vivo. This indicates that the in vitro system represents B cell activation in vivo, and that protein Ags generate BCR signaling distinct from that by anti-Ig H chain Abs. Indeed, BCR ligation by Ags, but not by anti-Ig H chain Abs, efficiently phosphorylates the inhibitory coreceptors CD22 and CD72. When these coreceptors are activated, anti-Ig H chain Ab-stimulated B cells can survive and proliferate only in the presence of CD40 signaling. Conversely, treatment of Ag-stimulated B cells with anti-CD72 mAb blocks CD72 phosphorylation and induces proliferation, even in the absence of CD40 signaling. These results strongly suggest that activation of B cells by anti-Ig H chain Abs involves their ability to silence the inhibitory coreceptors, and that the inhibitory coreceptors install requirement of CD40 signaling for survival and proliferation of Ag-stimulated B cells.     

CD21/CD19 coreceptor signaling promotes B cell survival during primary immune responses

The adaptive immune response is tightly regulated to limit responding cells in an Ag-specific manner. On B cells, coreceptors CD21/CD19 modulate the strength of BCR signals, potentially influencing cell fate. The importance of the CD95 pathway was examined in response of B cells to moderate affinity Ag using an adoptive transfer model of lysozyme-specific Ig transgenic (HEL immunoglobulin transgene (MD4) strain) B cells. Although adoptively transferred Cr2+/+ MD4 B cells are activated and persist within splenic follicles of duck egg lysozyme-immunized mice, Cr2-/- MD4 B cells do not. In contrast, Cr2-/- MD4 lpr B cells persist after transfer, suggesting that lack of CD21/CD35 signaling results in CD95-mediated elimination. Cr2 deficiency did not affect CD95 levels, but cellular FLIP (c-FLIP) protein and mRNA levels were reduced 2-fold compared with levels in Cr2+/+ MD4 B cells. In vitro culture with Cr2+/+ MD4 B cells demonstrated that equimolar amounts of rHEL-C3d3 were more effective than hen egg lysozyme alone in up-regulating c-FLIP levels and for protection against CD95-mediated apoptosis. Collectively, this study implies a mechanism for regulating B cell survival in vivo whereby the strength of BCR signaling (including coreceptor) determines c-FLIP levels and protection from CD95-induced death.     

Receptor-facilitated antigen presentation requires the recruitment of B cell linker protein to Igalpha

Ags that cross-link the B cell Ag receptor are preferentially and rapidly delivered to the MHC class II-enriched compartment for processing into peptides and subsequent loading onto MHC class II. Proper sorting of Ag/receptor complexes requires the recruitment of Syk to the phosphorylated immunoreceptor tyrosine-based activation motif tyrosines of the B cell Ag receptor constituent Igalpha. We postulated that the Igalpha nonimmunoreceptor tyrosine-based activation motif tyrosines, Y(176) and Y(204), contributed to receptor trafficking. Igalpha(YDeltaF(176,204))/Igbeta receptors were targeted to late endosomes, but were excluded from the vesicle lumen and could not facilitate the presentation of Ag to T cells. Subsequent analysis demonstrated that phosphorylation of Y(176)/Y(204) recruited the B cell linker protein, Vav, and Grb2. Reconstitution of Igalpha(YDeltaF(176,204))/Igbeta with the B cell linker protein rescued both receptor-facilitated Ag presentation and entry into the MHC class II-enriched compartment. Thus, aggregation accelerates receptor trafficking by recruiting two separate signaling modules required for transit through sequential checkpoints.     

A critical role for complement C3d and the B cell coreceptor (CD19/CD21) complex in the initiation of inflammatory arthritis

Complement C3 cleavage products mediate the recognition and clearance of toxic or infectious agents. In addition, binding of the C3d fragment to Ag promotes B lymphocyte activation through coengagment of the BCR and complement receptor 2 (CD21). Signal augmentation is thought to be achieved through enhanced recruitment and activation of CD21-associated CD19. In this study we show, using the DBA/1 collagen-induced arthritis (CIA) model, that conjugation of C3d to heterologous type II collagen is sufficient to cause disease in the absence of the mycobacterial components of CFA. Transient depletion of C3 during the inductive phase of CIA delays and lessens the severity of disease, and DBA/1 mice deficient for coreceptor components CD19 or CD21 are not susceptible to CIA. Adoptive transfer experiments revealed that CD21 expression on either B cells or follicular dendritic cells is sufficient to acquire disease susceptibility. Although CD19(-/-) and CD21(-/-) mice produce primary Ab responses to heterologous and autologous type II collagen, they are impaired in the ability to activate T cells, form germinal centers, and produce secondary autoantibody responses. These findings indicate that binding of C3d to self-Ags can promote autoimmunity through enhanced Ag retention and presentation by follicular dendritic cells and B cells, respectively.     

CD1d and CD1c expression in human B cells is regulated by activation and retinoic acid receptor signaling

B cell activation and Ab production in response to protein Ags requires presentation of peptides for recruitment of T cell help. We and others have recently demonstrated that B cells can also acquire innate help by presenting lipid Ags via CD1d to NKT cells. Given the newfound contribution of NKT cells to humoral immunity, we sought to identify the pathways that regulate CD1 molecule expression in human B cells. We show that ex vivo, activated and memory B cells expressed lower levels of CD1d compared with resting, naive, and marginal zone-like B cells. In vitro, CD1d was downregulated by all forms of B cell activation, leaving a narrow temporal window in which B cells could activate NKT cells. CD1c expression and function also decreased following activation by CD40L alone, whereas activation via the BCR significantly upregulated CD1c, particularly on marginal zone-like B cells. We found that the CD40L-induced downregulation of CD1d and CD1c correlated with diminished expression of retinoic acid receptor α (RARα) response genes, an effect that was reversed by RARα agonists. However, BCR-induced upregulation of CD1c was independent of the RAR pathway. Our findings that both CD1d and CD1c are upregulated by RARα signaling in human B cells is distinct from effects reported in dendritic cells, in which CD1c is inversely downregulated. One functional consequence of CD1d upregulation by retinoic acid was NKT cell cytotoxicity toward B cells. These results are central to our understanding of how CD1-restricted T cells may control humoral immunity.     

Impaired light chain allelic exclusion and lack of positive selection in immature B cells expressing incompetent receptor deficient of CD19

Positive signaling is now thought to be important for B cell maturation, although the nature of such signals has not yet been defined. We are studying the regulatory role of B cell Ag receptor (BCR) signaling in mediating positive selection of immature B cells. To do so, we use Ig transgenic mice (3-83Tg) that are deficient in CD19, thus generating a monoclonal immature B cell population expressing signaling-incompetent BCR. Immature 3-83Tg CD19(-/-) B cells undergo developmental arrest in the bone marrow, allowing maturation only to cells that effectively compensate for the compromised receptor by elevated levels of BCR. We find that developmentally arrested 3-83Tg CD19(-/-) B cells fail to impose L chain allelic exclusion and undergo intensive V(D)J recombination to edit their BCR. Furthermore, immature 3-83Tg CD19(-/-) B cells, which were grown in vitro, failed to undergo positive selection and to survive when adoptively transferred into normal recipients. However, elevation of BCR expression levels, obtained by transgene homozygosity, effectively compensated for the compromised BCR and completely restored BCR-mediated Ca(2+) influx, allelic exclusion, and positive selection. Our results suggest that the BCR signaling threshold mediates positive selection of developing B cells, and that a receptor-editing mechanism has an important role in rescuing cells that fail positive selection because of incompetent receptors.     

Complementary roles for CD19 and Bruton's tyrosine kinase in B lymphocyte signal transduction

CD19 and Bruton's tyrosine kinase (Btk) may function along common signaling pathways in regulating intrinsic and B cell Ag receptor (BCR)-induced signals. To identify physical and functional interactions between CD19 and Btk, a CD19-negative variant of the A20 B cell line was isolated, and CD19-deficient (CD19(-/-)) and CD19-overexpressing mice with the X-linked immunodeficient (Xid; Btk) mutation were generated. In A20 cells, Btk physically associated with CD19 following BCR engagement. CD19 and Btk interactions were not required for initial Btk phosphorylation, but CD19 expression maintained Btk in an activated state following BCR engagement. In primary B cells, CD19 signaling also required downstream Btk function since CD19-induced intracellular Ca(2+) ([Ca(2+)](i)) responses were modest in Xid B cells. In addition, CD19 overexpression did not normalize the Xid phenotype and most phenotypic and functional hallmarks of CD19 overexpression were not evident in these mice. However, CD19 and Btk also regulate independent signaling pathways since their combined loss had additive inhibitory effects on BCR-induced [Ca(2+)](i) responses and CD19 deficiency induced a severe immunodeficiency in Xid mice. Thus, CD19 expression amplifies or prolongs Btk-mediated signaling, rather than serving as a required agent for Btk activation. Consistent with this, phosphatidylinositol 3-monophosphate kinase and Akt activation were normal in CD19(-/-) B cells following IgM engagement, although their kinetics of activation was altered. Thus, these biochemical and compound gene dosage studies indicate that Btk activation and [Ca(2+)](i) responses following BCR engagement are regulated through multiple pathways, including a CD19/Src family kinase-dependent pathway that promotes the longevity of Btk signaling.     

Continuous signaling of CD79b and CD19 is required for the fitness of Burkitt lymphoma B cells

Expression of the B‐cell antigen receptor (BCR) is essential not only for the development but also for the maintenance of mature B cells. Similarly, many B‐cell lymphomas, including Burkitt lymphoma (BL), require continuous BCR signaling for their tumor growth. This growth is driven by immunoreceptor tyrosine‐based activation motif (ITAM) and PI3 kinase (PI3K) signaling. Here, we employ CRISPR/Cas9 to delete BCR and B‐cell co‐receptor genes in the human BL cell line Ramos. We find that Ramos B cells require the expression of the BCR signaling component Igβ (CD79b), and the co‐receptor CD19, for their fitness and competitive growth in culture. Furthermore, we show that in the absence of any other BCR component, Igβ can be expressed on the B‐cell surface, where it is found in close proximity to CD19 and signals in an ITAM‐dependent manner. These data suggest that Igβ and CD19 are part of an alternative B‐cell signaling module that use continuous ITAM/PI3K signaling to promote the survival of B lymphoma and normal B cells.     

CD24 induces apoptosis in human B cells via the glycolipid-enriched membrane domains/rafts-mediated signaling system

The glycosylphosphatidylinositol-anchored CD24 protein is a B cell differentiation Ag that is expressed on mature resting B cells but disappears upon Ag stimulation. We used Burkitt's lymphoma (BL) cells, which are thought to be related to germinal center B cells, to examine the biological effect of Ab-mediated CD24 cross-linking on human B cells and observed 1) induction of apoptosis in BL cells mediated by cross-linking of CD24; and 2) synergism between the cross-linking of CD24 and that of the B cell receptor for Ag in the effect on apoptosis induction. We also observed activation of mitogen-activated protein kinases following CD24 cross-linking, suggesting that CD24 mediates the intracellular signaling that leads to apoptosis in BL cells. Although CD24 has no cytoplasmic portion to transduce signals intracellularly, analysis of biochemically separated glycolipid-enriched membrane (GEM) fractions indicated enhanced association of CD24 and Lyn protein tyrosine kinase in GEM as well as increased Lyn kinase activity after CD24 cross-linking, suggesting that CD24 mediates intracellular signaling via a GEM-dependent mechanism. Specific microscopic cocapping of CD24 and Lyn, but not of other kinases, following CD24 cross-linking supported this idea. We further observed that apoptosis induction by cross-linking is a common feature shared by GEM-associated molecules expressed on BL cells, including GPI-anchored proteins and glycosphingolipids. CD24-mediated apoptosis in BL cells may provide a model for the cell death mechanism initiated by GEM-associated molecules, which is closely related to B cell receptor for Ag-mediated apoptosis.