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.     

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.     

B lymphocyte activation. Transmembrane signal transduction by membrane immunoglobulin in isolated cell membranes

We report the development of cell-free systems in which ligation of B cell membrane immunoglobulin leads to demonstrable mono- and polyphosphatidylinositol hydrolysis. Membranes were prepared by differential centrifugation of sonicates of normal murine B cells. Incubation of these membranes with 32P-adenosine triphosphate in the presence of Mg2+ effected the radiolabeling of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) and phosphatidylinositol 4-phosphate (PtdInsP). Alternately, membranes were labeled with exogenous 3H(inositol)-PtdInsP2 in sodium cholate. Stimulation of labeled membranes with anti-immunoglobulin, but not anti-Ia or anti-H2 antibodies, resulted in hydrolysis of phosphatidylinositol, PdtInsP, PtdInsP2 and generation of inositol phosphates indicative of activation of a phospholipase C. The response was rapid, being detectable within 30 sec of stimulation, and independent of Ca2+ and guanosine 5'-triphosphate. Optimal responses were dependent on the presence of a cytosolic factor presumed to be phospholipase C. Development of these systems represents an important step towards reconstitution of membrane immunoglobulin-mediated transmembrane signaling in artificial membranes.     

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.     

Block of T lymphocyte differentiation by activation of the cAMP-dependent signal transduction pathway.

Differentiation of T lymphocytes is a complex and finely tuned process. Here we show that treatment of mouse fetal thymus organ cultures with agents activating the cAMP-dependent signalling pathway results in the block of thymocyte differentiation. This is due to severe impairment of maturation beyond the CD4-/CD8- stage. In addition, rearrangements at the TCR alpha gene locus, but not at the TCR beta locus, are completely inhibited. The cAMP effect is reversible and is restricted to TCR alpha beta+ cells. cAMP acts both by triggering apoptosis and by inducing cell-cycle block in thymocytes. Thus, activation of the cAMP pathway provides a mechanism to modulate thymic function for hormones and ligands whose receptors are coupled to adenylate cyclase.     

Agrin‐independent activation of the agrin signal transduction pathway

The neural factor agrin induces the aggregation of acetylcholine receptors (AChRs) and other synaptic molecules on cultured myotubes. This aggregating activity can be mimicked by experimental manipulations that include treatment with neuraminidase or elevated calcium. We report evidence that neuraminidase and calcium act through the agrin signal transduction pathway. The effects of neuraminidase and calcium on AChR clustering are additive with that of agrin at low concentrations and cosaturating at high concentrations. In addition, like agrin, both neuraminidase and calcium cause rapid tyrosine phosphorylation of the muscle‐specific kinase (MuSK) and the AChR‐β subunit. Our results argue that all three agents act directly on components of the same signal transduction complex. We suggest that sialic acids on components of the complex inhibit interactions necessary for signal transduction and that disinhibition can result in activation. In such a model, agrin could activate signal transduction by disinhibition or by circumventing the inhibition. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 356–365, 1999     

B and T lymphocyte attenuator-mediated signal transduction provides a potent inhibitory signal to primary human CD4 T cells that can be initiated by multiple phosphotyrosine motifs

The B and T lymphocyte attenuator (BTLA) is a recently identified member of the CD28 family of cell receptors. Initial reports demonstrated that mice deficient in BTLA expression were more susceptible to experimental autoimmune encephalomyelitis, indicating that BTLA was likely to function as a negative regulator of T cell activation. However, cross-linking of BTLA only resulted in a 2-fold reduction of IL-2 production, questioning the potency with which BTLA engagement blocks T cell activation. We established a model in which BTLA signaling could be studied in primary human CD4 T cells. We observed that cross-linking of a chimeric receptor consisting of the murine CD28 extracellular domain and human BTLA cytoplasmic tail potently inhibits IL-2 production and completely suppresses T cell expansion. Mutation of any BTLA tyrosine motifs had no effect on the ability of BTLA to block T cell activation. Only mutation of all four tyrosines rendered the BTLA cytoplasmic tail nonfunctional. We performed structure-function studies to determine which factors recruited to the BTLA cytoplasmic tail correlated with BTLA function. Using pervanadate as a means to phosphorylate the BTLA cytoplasmic tail, we observed both Src homology protein (SHP)-1 and SHP-2 recruitment. However, upon receptor engagement, we observed only SHP-1 recruitment, and mutations that abrogated SHP-1 recruitment did not impair BTLA function. These studies question whether SHP-1 or SHP-2 have any role in BTLA function and caution against the use of pervanadate as means to initiate signal transduction cascades in primary cells.     

Role of cAMP-dependent signal transduction in the control of T lymphocyte activation

The role of cAMP in the regulation of antigen-dependent differentiation of T cells is discussed with consideration of the molecular mechanisms of cAMP effects. Characteristics of activation signal in various T lymphocyte subpopulations determining differential sensitivity to cAMP are reviewed. Specific attention is given to the involvement of the cAMP-dependent messenger system in the formation of the spectrum of secreted cytokines because their level and ratio determine the type of immune response.     

Suppression of lymphocyte signal transduction by murine mastocytoma ascites.

The lymphocyte signal transduction, as determined by intracellular free Ca2+ mobilization of concanavalin A-stimulated T lymphocytes and of anti-immunoglobulin mu chain antibody-stimulated B lymphocytes, was suppressed in spleen cells from mice injected with murine P1.HTR mastocytoma-induced ascites and in spleen cells treated with the ascites in vitro. The suppression was observed both at the peak level and in the reactive pattern of Ca2+ influx. In the suppression, the ascites were replaceable with tumor culture supernatants or tumor homogenates. Correspondingly, primary and secondary cytotoxic T lymphocyte (CTL) responses of DBA/2 mice to allogeneic antigen were also significantly suppressed by injection of the syngeneic P1.HTR tumor-derived ascites. This new finding suggested that the mechanism of the tumorous ascites or of the tumor-derived factor-mediated immunosuppression involves at least in part the suppression of the early event of the signal transduction for lymphocyte activation.     

Agrin-independent activation of the agrin signal transduction pathway.

The neural factor agrin induces the aggregation of acetylcholine receptors (AChRs) and other synaptic molecules on cultured myotubes. This aggregating activity can be mimicked by experimental manipulations that include treatment with neuraminidase or elevated calcium. We report evidence that neuraminidase and calcium act through the agrin signal transduction pathway. The effects of neuraminidase and calcium on AChR clustering are additive with that of agrin at low concentrations and cosaturating at high concentrations. In addition, like agrin, both neuraminidase and calcium cause rapid tyrosine phosphorylation of the muscle-specific kinase (MuSK) and the AChR-beta subunit. Our results argue that all three agents act directly on components of the same signal transduction complex. We suggest that sialic acids on components of the complex inhibit interactions necessary for signal transduction and that disinhibition can result in activation. In such a model, agrin could activate signal transduction by disinhibition or by circumventing the inhibition.     

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.     

Down-modulation of B cell signal transduction by ligation of mucins to CD22

Epithelial cancer cells secrete mucins carrying carbohydrate antigens such as a sialyl-Tn antigen into cancer tissues and/or the bloodstream, in which mucins may interact with CD22 (Siglec-2). Mucins isolated from colon cancer cells and bovine submaxillary mucins bound to CD22 cDNA transfectants and a human B cell line, Daudi cell, and the binding of soluble recombinant CD22 to the mucins was confirmed by means of a plate assay. The binding specificity was demonstrated by the fact that the mucins bound to the recombinant CD22 with an intact ectodomain but not to that with a mutated ectodomain. Daudi cells were stimulated with anti-IgM F(ab′)₂ in the presence or absence of mucins. Ligation of mucins to CD22 decreased the phosphorylation of CD22 and SHP-1 recruitment, and the phosphorylation of ERK-1/2 prominently. The in vivo effect of mucins on splenic B cells in the tumor-bearing state was investigated using mucin-producing (TA3-Ha) and non-producing (TA3-St) mammary adenocarcinoma-bearing mice. When fluorescence-labeled epiglycanins were administered to normal mice, a portion of them was taken up by the spleen and became associated with splenic B cells. We found that splenic B cells were reduced in TA3-Ha-bearing mice but not in TA3-St-bearing ones. These results suggest that in the tumor-bearing state a portion of the mucins in the bloodstream was taken up by the spleen and ligated to CD22 expressed on splenic B cells, which may have led to down-regulation of signal transduction.     

Complement component C3d-antigen complexes can either augment or inhibit B lymphocyte activation and humoral immunity in mice depending on the degree of CD21/CD19 complex engagement

C3d can function as a molecular adjuvant by binding CD21 and thereby enhancing B cell activation and humoral immune responses. However, recent studies suggest both positive and negative roles for C3d and the CD19/CD21 signaling complex in regulating humoral immunity. To address whether signaling through the CD19/CD21 complex can negatively regulate B cell function when engaged by physiological ligands, diphtheria toxin (DT)-C3d fusion protein and C3dg-streptavidin (SA) complexes were used to assess the role of CD21 during BCR-induced activation and in vivo immune responses. Immunization of mice with DT-C3d3 significantly reduced DT-specific Ab responses independently of CD21 expression or signaling. By contrast, SA-C3dg tetramers dramatically enhanced anti-SA responses when used at low doses, whereas 10-fold higher doses did not augment immune responses, except in CD21/35-deficient mice. Likewise, SA-C3dg (1 microg/ml) dramatically enhanced BCR-induced intracellular calcium concentration ([Ca2+]i) responses in vitro, but had no effect or inhibited [Ca2+]i responses when used at 10- to 50-fold higher concentrations. SA-C3dg enhancement of BCR-induced [Ca2+]i responses required CD21 and CD19 expression and resulted in significantly enhanced CD19 and Lyn phosphorylation, with enhanced Lyn/CD19 associations. BCR-induced CD22 phosphorylation and Src homology 2 domain-containing protein tyrosine phosphatase-1/CD22 associations were also reduced, suggesting abrogation of negative regulatory signaling. By contrast, CD19/CD21 ligation using higher concentrations of SA-C3dg significantly inhibited BCR-induced [Ca2+]i responses and inhibited CD19, Lyn, CD22, and Syk phosphorylation. Therefore, C3d may enhance or inhibit Ag-specific humoral immune responses through both CD21-dependent and -independent mechanisms depending on the concentration and nature of the Ag-C3d complexes.     

Stathmin phosphorylation patterns discriminate between distinct transduction pathways of human T lymphocyte activation through CD2 triggering.

CD2 triggering of human T lymphocyte activation has been associated with the activation of different interacting protein kinases, including protein kinase C (PKC). However the precise roles of its phosphorylated substrates are still unknown. We show here that PKC-dependent and -independent pathways are responsible for the CD2-induced phosphorylation of stathmin, a ubiquitous soluble phosphoprotein, most likely acting as a general intracellular relay integrating various second messenger pathways. The phosphorylated variants of stathmin provide a fingerprint reflecting the second messenger pathway(s) stimulated. The respective roles of both PKC and stathmin in the regulation of T lymphocyte proliferation are discussed.     

Signaling from LFA-1 contributes signal transduction through CD2 alternative pathway in T cell activation.

LFA-1, a member of the integrin family of molecules, is involved in mediating cellular adhesion in all phases of the immune response, playing a role in the interaction of helper T cells as well as in killing of target cells by both cytotoxic T cells and natural killer cells. We have developed a monoclonal antibody, anti-HVS6B6, which recognizes a functionally unique epitope of the LFA-1 molecule. Although this mAb itself was not mitogenic against T cells, it induced a strong proliferative response when added to T cells with submitogenic concentrations of anti-CD2 (anti-T11(2) and anti-T11(3)) mAbs. In contrast, other anti-LFA-1 mAbs (CD11a and CD18) suppressed this anti-CD2 mAb-induced T cell proliferation. Kinetic studies showed that anti-HVS6B6 acts on an early event in CD2-mediated T cell activation. Although T11(3)-epitope expression induced by anti-T11(2) mAb was not affected by treatment of cells with anti-HVS6B6, both Ca2+ influx and phosphatidylinositol turnover induced by anti-CD2 mAbs were markedly enhanced by the pretreatment of T cells with anti-HVS6B6 mAb. These results indicate that the LFA-1 mediating signal contributes to a very early phase of signal transduction during CD2-mediated T cell activation.