PubMed Health⌕ Search

Biomedical subjects

J C Cambier

Publications and source records attributed to J C Cambier.

At least 19 recordsLinked to original sources
PubMed1998

Antigens varying in affinity for the B cell receptor induce differential B lymphocyte responses.

The B cell receptor (BCR) triggers a variety of biological responses that differ depending upon the properties of the antigen. A panel of M13 phage-displayed peptide ligands with varying affinity for the 3-83 antibody was generated to explore the role of antigen-BCR affinity in cell activation studies using primary 3-83 transgenic mouse B cells. Multiple parameters of activation were measured. T cell-independent B cell proliferation, antibody secretion, induction of germline immunoglobulin gamma1 transcripts, and B cell production of interleukin (IL) 2 and interferon gamma responses were better correlated with antigen-BCR affinity than with receptor occupancy. In contrast, other responses, such as upregulation of major histocompatibility complex class II and B7.2 (CD86), secretion of IL-6, and B cell proliferation in the context of CD40 signaling were only weakly dependent on antigen affinity. Biochemical analysis revealed that at saturating ligand concentrations the ability of phage to stimulate some early signaling responses, such as Ca++ mobilization and tyrosine phosphorylation of syk or Igalpha, was highly affinity dependent, whereas the ability to stimulate Lyn phosphorylation was less so. These data suggest that the BCR is capable of differential signaling. The possibility that differential BCR signaling by antigen determines whether an antibody response will be T independent or dependent is discussed.

Amino Acid Sequence↗
PubMed1998

Antigen receptor signaling: integration of protein tyrosine kinase functions.

Antigen receptors on T and B cells function to transduce signals leading to a variety of biologic responses minimally including antigen receptor editing, apoptotic death, developmental progression, cell activation, proliferation and survival. The response to antigen depends upon antigen affinity and valence, involvement of coreceptors in signaling and differentiative stage of the responding cell. The requirement that these receptors integrate signals that drive an array of responses may explain their evolved structural complexity. Antigen receptors are composed of multiple subunits compartmentalized to provide antigen recognition and signal transduction function. In lieu of on-board enzymatic activity these receptors rely on associated Protein Tyrosine Kinases (PTKs) for their signaling function. By aggregating the receptors, and hence their appended PTKs, antigens induce PTK transphosphorylation, activating them to phosphorylate the receptor within conserved motifs termed Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) found in transducer subunits. The tyrosyl phosphorylated ITAMs then interact with Src Homology 2 (SH2) domains within the PTKs leading to their further activation. As receptor phosphorylation is amplified, other effectors, such as Shc, dock by virtue of SH2 binding, and serve, in-turn, as substrates for these PTKs. This sequence of events not only provides a signal amplification mechanism by combining multiple consecutive steps with positive feedback, but also allows for signal diversification by differential recruitment of effectors that provide access to distinct parallel downstream signaling pathways. The subject of antigen receptor signaling has been recently reviewed in depth (DeFranco, 1997; Kurosaki, 1997). Here we discuss the biochemical basis of antigen receptor signal transduction, using the B cell receptor (BCR) as a paradigm, with specific emphasis on the involved PTKs. We review several specific mechanisms by which responses through these receptors are propagated and modified by accessory molecules, and discuss how signal amplification and diversification are achieved.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1998

B cell antigen receptor (BCR)-mediated formation of a SHP-2-pp120 complex and its inhibition by Fc gamma RIIB1-BCR coligation.

Accumulating evidence indicates that the Src homology 2-containing tyrosine phosphatase 2 (SHP-2) plays an important role in signal transduction through receptor tyrosine kinase and cytokine receptors. In most models, SHP-2 appears to be a positive mediator of signaling. However, coligation of Fc gamma RIIB1 with B cell Ag receptors (BCR) inhibits BCR-mediated signaling by a mechanism that may involve recruitment of phosphatases SHP-1, SHP-2, and the SH2 containing inositol 5'phosphatase (SHIP) to the phosphorylated Fc gamma RIIB1 immunoreceptor tyrosine-based inhibitory motif. The role of SHP-2 in BCR-mediated cell activation and in Fc gamma RIIB1-mediated inhibitory signaling is unclear. In this study we assessed the association of SHP-2 with phosphotyrosine-containing cellular protein(s) before and after stimulation through these receptors. BCR stimulation induced the association of SHP-2 with a single major tyrosyl-phosphorylated molecule (pp120) that had an apparent molecular mass of 120 kDa. Coligation of Fc gamma RIIB1 with BCR led to a rapid decrease in SHP-2 association with pp120. Analysis of the subcellular localization of pp120 showed that the complex of SHP-2 and tyrosyl-phosphorylated p120 occurs predominantly in the cytosol. Furthermore, the binding of the two molecules was mediated by the interaction of tyrosyl-phosphorylated p120 with the SHP-2 N-terminal SH2 domain. These findings indicate that SHP-2 and pp120 function in BCR signaling, and this function may be inhibited by Fc gamma RIIB1 signaling.

Animals↗
PubMed1998

Asymmetrical phosphorylation and function of immunoreceptor tyrosine-based activation motif tyrosines in B cell antigen receptor signal transduction.

CD79a and CD79b function as transducers of B cell antigen receptor signals via a cytoplasmic sequence, termed the immunoreceptor tyrosine-based activation motif (ITAM). ITAMs contain two conserved tyrosines that may become phosphorylated upon receptor aggregation and bind distinct effectors by virtue of the distinct preference of phosphotyrosyl-containing sequences for SH2 domains. To explore the function of CD79a and CD79b ITAM tyrosines, we created membrane molecules composed of MHC class II I-Ak extracellular and transmembrane domains, and CD79a or CD79b cytoplasmic domains in which one or both of the ITAM tyrosines were mutated to phenylalanine. Functional analysis revealed that both ITAM tyrosines are required for ligand-induced Syk phosphorylation. However CD79a-ITAM and CD79b-ITAM tyrosine phosphorylations were asymmetrical, with >80% of phosphorylation occurring on the N-terminal tyrosine (Y-E-G-L). Thus, these findings suggest that following receptor ligation, only a minor proportion of phosphorylated ITAMs are doubly phosphorylated and thus can engage Syk. Only the N-terminal ITAM tyrosine of CD79a was required for ligand-mediated phosphorylation of the receptor and a subset of downstream substrates, including p62, p110, and Shc, and for Ca2+ mobilization. However, responses mediated through CD79b exhibited a greater dependence on the presence of both tyrosines. Neither tyrosine in CD79a or CD79b appeared absolutely essential for Src family kinase phosphorylation. These results indicate that phosphorylations of the tyrosines in CD79a and CD79b occur with very different stoichiometry, and the respective tyrosyl residues have distinct functions.

Antigens, CD↗
PubMed1998

Fc epsilon receptor I-associated lyn-dependent phosphorylation of Fc gamma receptor IIB during negative regulation of mast cell activation.

Fc gamma RIIB are low-affinity receptors for IgG whose intracytoplasmic domain contains an immunoreceptor tyrosine-based inhibition motif (ITIM). Fc gamma RIIB inhibit cell activation triggered by receptors that signal via immunoreceptor tyrosine-based activation motifs. This inhibition requires ITIM tyrosyl phosphorylation and is correlated with the binding of SH2 domain-containing phosphatases that may mediate the inhibitory signal. In the present work, we investigated the mechanism of Fc gamma RIIB phosphorylation and its consequences in mast cells. We demonstrate that the phosphorylation of Fc gamma RIIB requires coaggregation with Fc epsilon RI and that, once phosphorylated, Fc gamma RIIB selectively recruit the inositol polyphosphate 5 phosphatase SHIP, in vivo. In vitro, however, the phosphorylated Fc gamma RIIB ITIM binds not only SHIP, but also the two protein tyrosine phosphatases, SHP-1 and SHP-2. We show that the coaggregation of Fc gamma RIIB with Fc epsilon RI does not prevent Fc epsilon RI-mediated activation of lyn and syk. Both kinases can phosphorylate Fc gamma RIIB in vitro. However, when coaggregated with Fc epsilon RI, Fc gamma RIIB was in vivo phosphorylated in syk-deficient mast cells, but not in lyn-deficient mast cells. When Fc epsilon RI are coaggregated with Fc gamma RIIB by immune complexes, Fc epsilon RI-associated lyn may thus phosphorylate Fc gamma RIIB. By this mechanism, Fc epsilon RI initiate ITIM-dependent inhibition of intracellular propagation of their own signals.

Animals↗
PubMed1998

Developmental regulation of B lymphocyte immune tolerance compartmentalizes clonal selection from receptor selection.

B lymphocyte development is a highly ordered process that involves immunoglobulin gene rearrangements, antigen receptor expression, and a learning process that minimizes the development of cells with reactivity to self tissue. Two distinct mechanisms for immune tolerance have been defined that operate during early bone marrow stages of B cell development: apoptosis, which eliminates clones of cells, and receptor editing, which spares the cells but genetically reprograms their autoreactive antigen receptors through nested immunoglobulin L chain gene rearrangements. We show here that sensitivity to antigen-induced apoptosis arises relatively late in B cell development and is preceded by a functionally distinct developmental stage capable of receptor editing. This regulation compartmentalizes clonal selection from receptor selection.

Amino Acid Sequence↗
PubMed1998

CD72-mediated B cell activation involves recruitment of CD19 and activation of phosphatidylinositol 3-kinase.

Occupancy of the B cell glycoprotein, CD72 results in syk-independent activation of phospholipase-C gamma and calcium mobilization. The cytoplasmic tail of CD72 does not contain an immunoreceptor tyrosine-based activation motif to directly transduce signals into the B lymphocyte. Hence, we investigated whether other coreceptors such as CD19 and its associated phosphatidylinositol 3-kinase (PI 3-K) were involved in CD72 signaling. Two specific inhibitors of PI 3-K inhibited CD72-stimulated B cell proliferation in a dose-dependent manner. Activation of B lymphocytes via CD72 resulted in recruitment and activation of PI 3-K, which was mediated by CD19. Accordingly, CD72 ligation induced CD19 tyrosine phosphorylation. Thus, lipid products generated as a result of PI 3-K activation may have an important function in CD72-mediated B lymphocyte activation. The kinetics of CD19 tyrosine phosphorylation induced by CD72 ligation were strikingly different from those seen following B cell antigen receptor (BCR) stimulation. A transient increase in the tyrosine phosphorylation of the complement receptors, CD21 and CD35 was observed in BCR- but not CD72-stimulated cells. Co-cross-linking of CD72 and CD19 failed to induce syk tyrosine phosphorylation suggesting that even under these conditions, CD72 signaling was independent of syk activation. A transient and stimulation-dependent physical association between CD19 and CD72 was observed in CD72-ligated cells. These observations suggest a mechanism by which CD72 can recruit CD19 and influence activation of CD19-associated PI 3-K, which appears to be critical for CD72-mediated B cell activation.

Androstadienes↗
PubMed1997

Qualitative regulation of B cell antigen receptor signaling by CD19: selective requirement for PI3-kinase activation, inositol-1,4,5-trisphosphate production and Ca2+ mobilization.

Genetic ablation of the B cell surface glycoprotein CD19 severely impairs the humoral immune response. This requirement is thought to reflect a critical role of CD19 in signal transduction that occurs upon antigen C3dg coligation of antigen receptors with CD19 containing type 2 complement receptors (CR2). Here we show that CD19 plays a key accessory role in B cell antigen receptor signaling independent of CR2 coligation and define molecular circuitry by which this function is mediated. While CD19 is not required for antigen-mediated activation of receptor proximal tyrosines kinases, it is critical for activation of phosphatidylinositol 3-kinase (PI3-kinase). PI3-Kinase activation is dependent on phosphorylation of CD19 Y484 and Y515. Antigen-induced CD19-dependent PI3-kinase activation is required for normal phosphoinositide hydrolysis and Ca2+ mobilization responses. Thus, CD19 functions as a B cell antigen receptor accessory molecule that modifies antigen receptor signaling in a qualitative manner.

Androstadienes↗
PubMed1997

Delivery of B cell receptor-internalized antigen to endosomes and class II vesicles.

B cell receptor (BCR)-mediated antigen processing is a mechanism that allows class II-restricted presentation of specific antigen by B cells at relatively low antigen concentrations. Although BCR-mediated antigen processing and class II peptide loading may occur within one or more endocytic compartments, the functions of these compartments and their relationships to endosomes and lysosomes remain uncertain. In murine B cells, at least one population of class II- containing endocytic vesicles (i.e., CIIV) has been identified and demonstrated to be distinct both physically and functionally from endosomes and lysosomes. We now demonstrate the delivery of BCR-internalized antigen to CIIV within the time frame during which BCR-mediated antigen processing and formation of peptide-class II complexes occurs. Only a fraction of the BCR-internalized antigen was delivered to CIIV, with the majority of internalized antigen being delivered to lysosomes that are largely class II negative. The extensive colocalization of BCR-internalized antigen and newly synthesized class II molecules in CIIV suggests that CIIV may represent a specialized subcellular compartment for BCR-mediated antigen processing. Additionally, we have identified a putative CIIV-marker protein, immunologically related to the Igalpha subunit of the BCR, which further illustrates the unique nature of these endocytic vesicles.

Animals↗
PubMed1997

B cell antigen receptor desensitization: disruption of receptor coupling to tyrosine kinase activation.

Antigen binding to the B cell receptor (BCR) induces receptor desensitization, a condition characterized by cellular unresponsiveness to subsequent Ag stimulation despite the continued ability to bind Ag. To better understand the molecular mechanism of this unresponsiveness, we have used complementary lymphoma (K46 mu) and Ig transgenic (3-83 mu delta) mouse models to study regulation of BCR signaling. Our findings in the lymphoma model show that an initial Ag encounter renders receptors unresponsive to subsequent Ag challenge, as measured by their inability to mobilize Ca2+ and to mediate phosphorylation of receptor-proximal kinases, including Lyn, Blk, and Syk. Most importantly, the Ig alpha and Ig beta components of desensitized receptors are not phosphorylated, and receptor-associated kinases are not activated upon Ag challenge. The molecular defect does not appear to result from Lyn inactivation, sequestration, or repression, since Lyn from desensitized cell lysates is activated in vitro by synthetic doubly phosphorylated immunoreceptor tyrosine-based activation motif peptides. A similar deficit in Ag-induced receptor phosphorylation was observed in desensitized B cells from 3-83 mu delta transgenic mice. These studies indicate that Ag receptor desensitization reflects an inability to initiate activation of receptor-associated kinases that normally phosphorylate receptor Ig alphabeta subunits, leading to signal propagation.

Animals↗
PubMed1997

Syk, but not Lyn, recruitment to B cell antigen receptor and activation following stimulation of CD45- B cells.

B cell Ag receptor (BCR) signaling occurs via tyrosine phosphorylation of CD79a and CD79b ITAMs, leading to recruitment and activation of Lyn and Syk tyrosine kinases and subsequent downstream events. CD45 expression is required for BCR triggering of certain of these downstream events, such as calcium mobilization and p21ras activation. However, the site in the BCR signaling cascade at which CD45 impinges is poorly defined. To address this question, we have studied CD45 function in the CD45-deficient (CD45-) and CD45-reconstituted (CD45+) J558L mu m3 plasmacytoma. In both CD45+ and CD45- cells, Ag stimulation led to CD79a and CD79b tyrosine phosphorylation as well as Syk tyrosine phosphorylation, recruitment to the receptors, and activation. In contrast to CD45+ cells, Lyn exhibited high basal tyrosine phosphorylation in the CD45- cells and was not further phosphorylated upon Ag stimulation. Mapping studies indicated that the observed constitutive phosphorylation of Lyn reflects phosphorylation of its C-terminal tyrosine, Y508, at high stoichiometry. Constitutively Y508-phosphorylated Lyn was neither recruited to the BCR nor activated upon Ag stimulation. Moreover, CD79a-ITAM phosphopeptides failed to bind Lyn from the CD45- cells. Thus, Y508 phosphorylation of Lyn occurs in the absence of cellular CD45 expression and appears to render the kinase unable to associate with the phosphorylated receptor complex via its Src homology 2 domain and to participate in signal propagation. Surprisingly, in view of previous findings implicating Src family kinases in ITAM phosphorylation, the data indicate that Ag-induced CD79a and CD79b tyrosine phosphorylation and Syk recruitment and activation can occur in the absence of CD45 expression and, hence, Src-family kinase activation.

Animals↗
PubMed1997

Molecular targets of CD45 in B cell antigen receptor signal transduction.

Expression of the phosphotyrosine phosphatase CD45 is essential for B cell Ag receptor (BCR)-mediated p21ras activation and calcium mobilization. To examine the molecular basis of this requirement, we analyzed signaling events following BCR ligation in CD45-deficient (CD45-) and CD45-reconstituted (CD45+) variants of J558Lmicrom3 cells. Ag stimulation resulted in tyrosine phosphorylation of cellular proteins in both cells. However, the spectrum of proteins phosphorylated in the CD45+ cells was qualitatively and/or quantitatively distinct from that in the CD45- cells. Among the protein tyrosine kinases examined, the Src family kinases Fyn and Blk were inducibly tyrosine phosphorylated and activated by receptor ligation only in CD45+ cells. While Ag-induced Btk tyrosine phosphorylation occurred in both cells, its activation was greatly diminished in the CD45- cells. Analysis of specific effector molecules revealed that tyrosine phosphorylation of Shc, but not rasGAP or Vav, correlated with the unique ability of BCR ligation to trigger p21ras activation in CD45+ cells. BCR-mediated Shc phosphorylation and recruitment of Grb2 depended on CD45 expression. Thus, Shc tyrosine phosphorylation may be the primary CD45-dependent mechanism by which Ag receptors are coupled to the p21ras pathway in J558Lmicrom3. In addition, phospholipase Cgamma1 (PLCgamma1) and PLCgamma2 were tyrosine phosphorylated upon Ag stimulation in CD45- cells, despite much reduced inositol trisphosphate production and lack of calcium mobilization. These findings suggest that CD45 may modulate events other than PLCgamma phosphorylation, which regulate phosphoinositide hydrolysis and the calcium mobilization response following BCR ligation.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Qualitatively distinct signaling through T cell antigen receptor subunits.

T cell antigen receptors (TCR) contain several subunits including CD3gamma, delta, and epsilon, and TCRzeta and eta which are capable of mediating signal transduction. It is unclear whether the signaling function of these subunits is completely redundant. To assess the relative signaling capabilities of TCR subunits, we compared proximal events in signal transduction by wild-type TCR complexes and TCR devoid of functional zeta subunits, as well as chimeric receptors containing the cytoplasmic domains of TCRzeta or CD3epsilon. Results demonstrate that in BW5147 wild-type TCR, tail-less zeta TCR, CD3epsilon, and TCRzeta transduce signals leading to tyrosine phosphorylation of similar sets of cellular substrates, including the receptor subunits, Fyn, ZAP-70, and phospholipase Cgamma1 (PLCgamma1). Surprisingly, unlike wild-type TCR, tail-less zeta TCR, and CD3epsilon, TCRzeta was incapable of transducing signals resulting in inositol triphosphate (IP3) generation or intracellular free calcium ([Ca2+]i) mobilization. These data indicate that tyrosine phosphorylation of PLCgamma1 is not sufficient to drive IP3 production and [Ca2+]i mobilization. Most importantly, data presented indicate that TCRzeta and CD3epsilon engage partially distinct signaling pathways.

Amino Acid Sequence↗
PubMed1997

Differential association of phosphatases with hematopoietic co-receptors bearing immunoreceptor tyrosine-based inhibition motifs.

A novel family of inhibitory co-receptors has been recently defined according to the presence in their intracytoplasmic domain of immunoreceptor tyrosine-based inhibition motifs (ITIM). In particular, this family includes a low-affinity receptor for IgG, Fc gammaRIIB, which is widely expressed on hematopoietic cells, as well as killer cell inhibitory receptors (KIR) for major histocompatibility complex (MHC) class I proteins, expressed on both T and natural killer (NK) lymphocytes. Fc gammaRIIB and KIR inhibitory function depends upon the tyrosine phosphorylation of their respective ITIM. Phosphorylated Fc gammaRIIB and KIR ITIM bind the tandem SH2 tyrosine phosphatases, SHP-1 and SHP-2. Recently, Fc gammaRIIB has been shown to associate with a polyphosphate inositol 5-phosphatase, SHIP, which appears to be involved in its inhibitory function. Using cell lysate adsorption to phosphorylated ITIM peptides and surface plasmon resonance, we demonstrate here that, in contrast to Fc gammaRIIB, KIR (CD158b: p58.2) do not bind to SHIP, and only recruit SHP-1 and SHP-2. In addition, we show that point mutation of the amino acid residue in position tyrosine-2 of Fc gammaRIIB and KIR ITIM abolihes their binding to SHP-1 and SHP-2, but leaves intact the association of SHIP with Fc gammaRIIB ITIM. These data contribute to the structural definition of ITIM and document a differential recruitment of phosphatases by distinct ITIM. These findings also reveal that diverse strategies of inhibition are used by distinct members of the ITIM-bearing co-receptor family.

Amino Acid Sequence↗
PubMed1997

Fc gammaRIIB1 inhibition of BCR-mediated phosphoinositide hydrolysis and Ca2+ mobilization is integrated by CD19 dephosphorylation.

The B cell receptor for immunoglobulin G, Fc gammaRIIB1, is a potent transducer of signals that block antigen-induced B cell activation. Coligation of Fc gammaRIIB1 with B lymphocyte antigen receptors (BCR) causes premature termination of phosphoinositide hydrolysis and Ca2+ mobilization and inhibits proliferation. This inhibitory signal is mediated in part by phosphorylation of Fc gammaRIIB1 and recruitment of phosphatases; however, the molecular target(s) of effectors is unknown. Here we report that Fc gammaRIIB1 inhibition of BCR signaling is mediated in part by selective dephosphorylation of CD19, a BCR accessory molecule and coreceptor. CD19 dephosphorylation leads to failed CD19 association with phosphatidylinositol 3-kinase, and this in turn leads to termination of inositol-1,4,5-trisphosphate production, intracellular Ca2+ release, and Ca2+ influx. The results define a molecular circuit by which Fc gammaRIIB signals block phosphoinositide hydrolysis.

Animals↗
PubMed1997

Co-receptor and accessory regulation of B-cell antigen receptor signal transduction.

The development and function of the immune system is precisely regulated to assure the generation of protective immune responses while avoiding autoimmunity. This regulation is accomplished by the engagement of a multitude of cell-surface receptors which transduce signals that activate or regulate cell differentiative and proliferative pathways. In some cases biologic responses reflect the integration of signals generated by co-aggregation of multiple receptors by complex ligands. For example, B-cell responses to antigen receptor aggregation can be modulated by co-aggregation of receptors for immunoglobulin G (Fc gamma RIIB1), complement components (CR2), and alpha 2, 6-sialoglycoproteins (CD22). Here we review our recent studies of molecular mechanisms underlying co-receptor modulation of B-cell antigen receptor signaling. Our results define interesting circuitry involving interactions among the B-cell antigen receptor, CD19 and Fc gamma RIIB1. CD19 may function as an important integrator of positive and negative signals that regulate B-cell antigen receptor signal output.

Animals↗