The B-cell antigen receptor complex.
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Amino Acid Sequence↗
Biomedical subjects
J C Cambier
Publications and source records attributed to J C Cambier.
T-cell development and transmembrane signaling: changing biological responses through an unchanging receptor.
The antigen receptor repertoire is conditioned by discriminating forces in the thymus. Cells that see antigen only in the context of self major histocompatibility gene products are positively selected and those that recognize self antigens are deleted. The molecular mechanisms by which this complex conditioning is achieved via a single antigen receptor is one of the most fascinating problems in immunology. Here Terri Helman Finkel and colleagues review the literature and present a unifying mechanistic model of this process.
Animals↗
Capturing antigen receptor components.
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Journal Article↗
Increase in the intracellular free calcium concentration is not an obligatory early event in lipopeptide-induced B-cell activation.
We recently showed that synthetic lipopeptides, analogues of the N-terminal region of bacterial lipoprotein, induce DNA synthesis in B lymphocytes in the absence of enhanced phosphatidylinositol 4,5-bisphosphate hydrolysis and protein kinase C translocation. Here we demonstrate that lipopeptides are capable of inducing enhanced expression of MHC class II molecules and early increases in the intracellular free calcium concentration ([Ca2+]i) in B cells. However, they do not effect T cells. The increase in [Ca2+]i seen in B cells is due primarily to Ca2+ release from intracellular stores. Since lipopeptides differ in their capability to induce early increases in [Ca2+]i and since the calcium response does not correlate with the ability of lipopeptides to induce proliferation and expression of MHC class II molecules, we suggest that this biochemical event may not be essential for lipopeptide-mediated B-cell activation.
Amino Acid Sequence↗
Dual molecular mechanisms mediate ligand-induced membrane Ig desensitization.
We have previously shown that ligation of murine B cell membrane IgM or IgD can lead to inactivation of the signal transducing ability of unligated Ag receptors. We describe further studies of the molecular basis of this desensitization. Consistent with the possibility that ligand induced desensitization is mediated by protein kinase C (PKC) are findings that demonstrate that both Ig binding ligands and PKC activators (DIC8 or PMA) induce desensitization in virtually all resting B cells. However, ligand-induced desensitization is longer lived than PMA- or DIC8-induced desensitization and insensitive to the PKC inhibitor staurosporine. Further, biochemical studies indicate that insufficient PKC activation is induced by ligation of membrane Ig to mediate the observed desensitization. Thus data indicate that PKC must play only a minor role in ligand-induced membrane Ig desensitization. Further studies explored the molecular source and target of effectors that mediate ligand-induced desensitization. Data indicate that phosphoinositide hydrolysis is neither necessary nor sufficient for ligand induction of desensitization. Finally, ligand-induced desensitization appears to be mediated by uncoupling of membrane Ig from G proteins that regulate phospholipase C because ligand desensitized cells are hyperresponsive to agents including ALF4- and mastoparan which activate G proteins leading to mobilization of Ca2+. Thus, the function of G proteins and further downstream elements that mediate Ca2+ mobilization is intact. Taken together, these data are most consistent with ligand-induced membrane Ig desensitization being mediated by a non-PKC, non phosphatidylinositol 4,5-bisphosphate hydrolysis involving mechanism that has as its target a structure that is very proximal to the receptor, such as the receptor itself or a transducer complex analogous to CD3.
Amino Acid Sequence↗
Divalent cation regulation of phosphoinositide metabolism. Naturally occurring B lymphoblasts contain a Mg2(+)-regulated phosphatidylinositol-specific phospholipase C.
Membranes isolated from normal murine B lymphocytes were found to contain a novel phosphatidylinositol (PtdIns)-specific phospholipase C (PLC) which becomes activated as the Mg2+ concentration is raised from 30 to 1000 microM. This activity, which has not been described previously in any tissue, is restricted to naturally occurring B cell blasts, i.e. it was not detected in quiescent B cells, B lymphomas, or plasmacytomas. As seen in other cell systems, B cell membranes were found to contain Mg2(+)-stimulated inositol 1,4,5-trisphosphate phosphatase activity. Although neither the inositol 1,4,5-trisphosphate phosphatase nor the PtdIns PLC activities were affected by Ca2+, B cell membranes were found to contain a Ca2(+)-stimulated phosphatidylinositol 4,5-bisphosphate (PtdInsP2) PLC activity which is activated by [Ca2+] greater than 100 nM. Based on several characteristics, it appears that the Mg2(+)- and Ca2(+)-regulated PLCs are distinct species. First, they have distinct specificity for PtdIns and PtdInsP2, respectively. Second, they have distinct tissue distribution while the Ca2(+)-regulated activity was detected in all B cells, the Mg2(+)-regulated activity is restricted to low density, natural B blasts. Third, the kinetics of activation of the enzymes is distinct; the Mg2(+)-regulated enzyme exhibits slower and less transient activation kinetics. Fourth, the activities exhibit absolute specificity in terms of activation by Mg2+ and Ca2+, i.e. the PtdIns PLC is activated only by Mg2+ and the PtdInsP2 PLC is activated only by Ca2+. Data are consistent with the possibility that Mg2+ mobilization which follows ligation of certain receptors, may play an important role in the regulation of levels of the second messenger diacylglycerol.
Animals↗
Membrane IgM and IgD molecules fail to transduce Ca2+ mobilizing signals when expressed on differentiated B lineage cells.
We have measured Ca2+ mobilization in a panel of B lineage cell lines after stimulation with anti-Ig to assess whether membrane Ig transduces a functional signal in cells that are representative of immature, mature, or terminally differentiated stages. For these studies, three transfected cell lines which express the same IgM molecule (300-19 microns lambda 36/8, K46-17 microns lambda, and J558L microns lambda 3) as well as two lines expressing an identical IgD molecule (K46 delta m2.6 and J558L delta m8.8) were used. Cross-linking of membrane Ig on IgM+ or IgD+ lymphomas (K46-17 microns lambda or K46 delta m2.6) resulted in a Ca2+ mobilization response that is similar to that seen in mature, resting B cells. Both intracellular release and extracellular influx of Ca2+ were observed. In contrast, ligation of membrane Ig on an IgM+ pre-B cell line (300 - 19 microns lambda 36/8) induced extracellular influx of Ca2+ but no detectable intracellular release. Finally, cross-linking of membrane Ig on IgM+ or IgD+ plasmacytomas (J558L microns 3 or J558L delta m8.8) or an IgD+ B cell hybridoma (B1.8.delta 1) expressing an endogenous Ig gene, did not result in a detectable Ca2+ mobilization response. Importantly, stimulation of cells with the GTP-binding protein activator, aluminum fluoride, resulted in a comparable Ca2+ mobilization response in all cell lines. In view of the fact that aluminum fluoride induced a Ca2+ response in the terminally differentiated B cell lines, J558L microns 3, J558L delta m8.8, and B1.8.delta 1, it is likely that there is an alteration in the signal transduction cascade at some point proximal to GTP binding protein activation. This finding suggests that differentiation of the B cell is accompanied by the loss or alteration of one or more components that couple membrane Ig to subsequent signal transduction elements. Finally, it has previously been demonstrated that the IgM+ cell lines described above, express the recently described membrane Ig-associated protein, B34. Thus, it is apparent based on the fact that the J558L microns 3 cell line does not mobilize Ca2+ after stimulation with anti-Ig, that coexpression of B34 in association with membrane Ig does not constitute a functional receptor complex capable of activating GTP-binding proteins that in turn regulate Ca2+ mobilization.
Aluminum↗
Transforming growth factor beta 1 has differential effects on B cell proliferation and activation antigen expression.
There is accumulating evidence that TGF beta 1 is an important immunoregulatory molecule. Here we report evidence that TGF beta 1 has potent effects on murine B cells. It is profoundly inhibitory to the proliferation of quiescent B cells activated in model systems using both thymus-independent and thymus-dependent stimuli and arrests cells predominantly at the G1 cell-cycle stage. It also blocks the proliferation of B cell blasts, with a similar accumulation at stage G1. In parallel with this antiproliferative effect, TGF beta 1 inhibits induction of the expression of a series of "activation Ag" including transferrin receptor, RL388, and Ly-6, in mitogen-stimulated B cells. It also inhibits the induction of Ly-6 expression by IL-4, a nonmitogenic stimulus. In contrast to these negative influences, TGF beta 1 induces modestly increased expression of MHC class II Ag in quiescent B cells, and more marked increases in both B cell blasts and mitogen-stimulated cells. We speculate that in the appropriate context TGF beta 1 may be a cytokine that promotes productive B cell-Th cell interaction.
Animals↗
Improved method for measuring intracellular Ca++ with fluo-3.
The accuracy of flow cytometric measurement of intracellular calcium with fluo-3 is compromised by variation in basal fluorescence intensity due to heterogeneity in dye uptake or compartmentalization. We have loaded cells simultaneously with fluo-3 and SNARF-1. When SNARF-1 fluorescence is collected at approximately 600 nm, its intensity does not change upon cell activation. Furthermore, fluo-3 and SNARF-1 fluorescence signals exhibit a linear relationship. The ratio of fluo-3 to SNARF-1 eliminates a significant proportion of variation in fluorescence intensity caused by variation in fluo-3 uptake and thus can be used as a sensitive parameter for measuring changes in [Ca2+]i.
Aniline Compounds↗
B lymphocyte antigen receptors (mIg) are non-covalently associated with a disulfide linked, inducibly phosphorylated glycoprotein complex.
T and B lymphocyte antigen receptors exhibit single transmembrane spanning regions and very short, three to five amino acid, C-terminal cytoplasmic tails. Ligation of these receptors leads, apparently through GTP binding protein activation, to rapid stimulation of a polyphosphoinositide specific phosphodiesterase (PPI-PDE). T lymphocyte antigen receptors (alpha beta) are coupled to PPI-PDE via a receptor associated complex of membrane proteins, designated CD3. Although an analogous transducer complex is presumed to exist in B cells, no such structure has been defined. We utilized in vitro [32P]phosphorylation to identify and characterize a membrane immunoglobulin (mIg) associated phosphoprotein complex which appears to represent a B cell analog of CD3. The phosphoprotein complex consists of three N-glycosylated polypeptides which occur as disulfide linked dimers, non-covalently associated with mIg. The complex associated with mIgM (pp32, pp34 and pp37 subunits) differs from that associated with mIgD (pp33, pp34 and pp37 subunits), and the isotype specific phosphoprotein (pp32 or pp33) appears to exist as a disulfide linked heterodimer with either pp34 or pp37. Aluminum fluoride stimulates phosphorylation of all of the subunits, and at least one of the proteins is phosphorylated on a tyrosine residue(s).
Animals↗
Signal transmission pathways and lymphocyte function.
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Animals↗
Ligation of membrane immunoglobulin leads to inactivation of the signal-transducing ability of membrane immunoglobulin, CD19, CD21, and B-cell gp95.
We have examined the ability of membrane immunoglobulin-binding ligands to desensitize several human B-cell surface molecules that normally transduce signals leading to Ca2+ mobilization. Ligation of membrane IgM or IgD leads to heterologous desensitization of the reciprocal receptor in Epstein-Barr virus-transformed B-cell lines and peripheral blood B cells, as evidenced by a failure of cells to mobilize in response to receptor ligation. Under these conditions CD19, CD21, and B-cell gp95 ligation also did not lead to normal Ca2+ mobilization, indicating that these transducers are also desensitized. The desensitization does not reflect receptor modulation from the cell surface or reduced accessibility to ligand and is long lived, lasting greater than 16 hr. Finally, data that indicate that desensitized cells remain responsive to the G protein activating agent AIF4-, as measured by Ca2+ mobilization, suggest that desensitization reflects uncoupling of these receptors from G proteins that are intermediaries in their transduction of signals. We hypothesize that the molecular target of desensitization may be a recently described membrane immunoglobulin-associated and inducibly tyrosine-phosphorylated protein complex that may function as a master transducer in B cells, analogous to CD3 in T cells.
Aluminum↗
Signal transduction by B lymphocyte receptors: structure-function relationships of membrane immunoglobulins and associated molecules.
Membrane immunoglobulins function in transmembrane transduction of information leading to gene activation and to receptor desensitization. These receptors also function in focusing and internalization of antigen for subsequent processing and presentation to T cells. These functions require that antigen receptors communicate physically with molecules in the cytoplasm. Recent evidence suggests that this is accomplished via an mIg-associated protein complex analogous to T cell CD3. Here we review evidence regarding the molecular basis of signal transduction by mIg and the structure of mIg-associated proteins which may be involved in signal transduction.
Amino Acid Sequence↗
The thymus has two functionally distinct populations of immature alpha beta + T cells: one population is deleted by ligation of alpha beta TCR.
During T cell development, the processes of selection and tolerance act on the universe of expressed T cell receptors in the thymic cortex to form the repertoire of mature T cells that will respond to foreign antigen in the context of self-MHC in that animal. We have subdivided the cortical thymocytes into three functionally distinct populations: one population which is antigen-receptor negative, a second population which is antigen-receptor positive and is resistant to deletion by signaling through the antigen receptor, and a third population which is also antigen-receptor positive but is sensitive to deletion. These results have implications for the cellular compartments in which positive and negative selection occur and for the biochemical mechanisms that mediate selection and tolerance.
Animals↗
Ia-mediated signal transduction leads to proliferation of primed B lymphocytes.
One of the most controversial questions in immunology is the molecular basis by which Th lymphocytes deliver activating signals to quiescent B lymphocytes during T cell-dependent immune responses. Recent studies suggest that T cell-dependent activation of quiescent B lymphocytes may involve signaling mediated by direct T helper cell-B cell contact. Since B cell membrane-associated MHC-encoded class II molecules (Ia) must be recognized by Th lymphocytes for generation of T cell-dependent humoral immune responses, they are obvious candidates for receptors of this signal. Here we report that stimulation of quiescent murine B cells with IL-4 and antibodies against the B cell antigen receptor for 12-16 h primes cells to proliferate in response to immobilized mIa binding ligands. In the presence of additional lymphokines, these B cells differentiate to secrete Ig of IgM and IgG classes. These results suggest that Ia molecules are receptors for direct, T helper cell-B cell contact mediated signaling that results in B cell proliferation.
Animals↗
Production of multiple lymphokines by the A20.1 B cell lymphoma after cross-linking of membrane Ig by immobilized anti-Ig.
Cross-linking of membrane IgG2a or IgD on the B cell lymphoma A20.1 resulted in the elaboration of lymphokines which were able to support the growth of HT-2 cells and to induce increased Ia expression on resting B cells. Unstimulated A20.1 cell did not produce detectable levels of lymphokine activity. Lymphokine secretion did not occur in response to cross-linking of MHC class II (Ia) or class I (H2K) molecules. The kinetics for secretion were rapid, with detectable levels of lymphokine arising within 3 to 4 h of stimulation. Maximal lymphokine production was reached by 8 to 10 h. Soluble intact anti-Ig antibodies failed to stimulate lymphokine production due to Fc-mediated effects. This was concluded based on the fact that soluble F(ab')2 fragments of anti-IgG, but not soluble intact antibody, stimulated the production of lymphokine by A20.1 cells. Based on serologic criteria, membrane Ig cross-linking by ligand induced secretion of IL-2 but not IL-4 by A20.1 cells. Induction of Ia expression by resting B cells in response to A20.1 supernatant was not mimicked by stimulation with IL-1, -3, -5, or -6 either singly or in combination. Furthermore, preliminary physicochemical characterization revealed that the Ia-inducing factor in A20.1 supernatant has a molecular weight greater than 50,000. These data suggest that the Ia-inducing activity is a novel lymphokine. Thus, this report describes the first evidence for the existence of a B cell tropic lymphokine produced by B cells in response to Ag receptor-mediated signal transduction.
Animals↗
Induction of c-fos and c-myc expression during B cell activation by IL-4 and immunoglobulin binding ligands.
The data presented here indicated that both membrane Ig and IL-4 receptors transduce signals across the plasma membrane of quiescent B cells, which results in the induction of c-fos and c-myc proto-oncogene mRNA expression. Monoclonal anti-Ig antibodies with specificity for mu, delta, or kappa chains, regardless of mitogenicity, induced increased c-fos and c-myc mRNA expression with kinetics and magnitude similar to that observed following stimulation of B cells with IL-4. Maximal levels of c-fos mRNA, approximately 30-fold over background, were observed 30 min after stimulation. Maximal levels of c-myc mRNA, approximately 10-fold over background, were observed 60 min after stimulation. Phorbol myristate acetate alone induced expression of these two oncogenes in a similar fashion, suggesting that protein kinase C may be involved in the regulation of their expression following anti-Ig crosslinking. Ionomycin induced only a small increase in c-myc and c-fos message (three- to four-fold), and did not synergize with phorbol myristate acetate, suggesting that the membrane Ig-mediated calcium mobilization may not play a major role in regulation of c-myc or c-fos expression in mouse B cells. In vitro nuclear run-on analyses indicate that c-myc expression is primarily regulated post-transcriptionally, whereas c-fos expression is regulated at the level of transcription. Anti-sense transcription was found to be constitutive for both the c-myc and C-fos loci and was further induced by anti-Ig and IL-4, suggesting an additional mechanism for regulation of these genes. The observation that both anti-Ig and IL-4 regulate the expression of c-fos and c-myc suggests that multiple second messenger generating systems regulate the expression of these oncogenes in normal B cells and that their expression may be necessary, but is not sufficient to drive quiescent B cells into cell cycle.
Animals↗
Alpha beta T cell receptor and CD3 transduce different signals in immature T cells. Implications for selection and tolerance.
Ligand binding to alpha beta TCR has different consequences in thymocytes at different developmental stages, causing alternatively positive selection, clonal deletion, or activation. These various functional consequences may be due to changes in the signaling properties of the receptor complex during development. In this report we show that alpha beta TCR engagement on immature thymocytes has different effects on intracellular free calcium concentrations than alpha beta TCR engagement on mature T cells. In contrast, CD3 engagement on immature thymocytes and mature T cells has the same effect on intracellular free calcium, suggesting that altered signal transduction in immature thymocytes may be due to inefficient alpha beta TCR-CD3 coupling. These studies also suggest that in certain T cell populations, activation events resulting from ligation of CD3 may not accurately reflect the activation events resulting from ligation of the physiologic receptor, alpha beta TCR.
Animals↗