B lymphocytes and their differentiated daughters are charged with responding to the myriad pathog... more B lymphocytes and their differentiated daughters are charged with responding to the myriad pathogens in our environment and production of protective antibodies. A sample of the protective antibody produced by each clone is utilized as a component of the cell's antigen receptor (BCR). Transmembrane signals generated upon antigen binding to this receptor provide the primary directive for the cell's subsequent response. In this report, we discuss recent progress and current controversy regarding B cell receptor signal initiation, transduction and regulation.
F1000 - Post-publication peer review of the biomedical literature, 2013
Genetic ablation of the Lyn tyrosine kinase has revealed unique inhibitory roles in B lymphocyte ... more Genetic ablation of the Lyn tyrosine kinase has revealed unique inhibitory roles in B lymphocyte signaling. We now report the consequences of sustained activation of Lyn in vivo using a targeted gain-of-function mutation (Lyn up/up mice). Lyn up/up mice have reduced numbers of conventional B lymphocytes, down-regulated surface immunoglobulin M and costimulatory molecules, and elevated numbers of B1a B cells. Lyn up/up B cells are characterized by the constitutive phosphorylation of negative regulators of B cell antigen receptor (BCR) signaling including CD22, SHP-1, and SHIP-1, and display attributes of lymphocytes rendered tolerant by constitutive engagement of the antigen receptor. However, exaggerated positive signaling is also apparent as evidenced by the constitutive phosphorylation of Syk and phospholipase C ␥ 2 in resting Lyn up/up B cells. Similarly, Lyn up/up B cells show a heightened calcium flux in response to BCR stimulation. Surprisingly, Lyn up/up mice develop circulating autoreactive antibodies and lethal autoimmune glomerulonephritis, suggesting that enhanced positive signaling eventually overrides constitutive negative signaling. These studies highlight the difficulty in maintaining tolerance in the face of chronic stimulation and emphasize the pivotal role of Lyn in B cell signaling.
Many self-reactive B cells exist in the periphery in a rapidly reversible state of unresponsivene... more Many self-reactive B cells exist in the periphery in a rapidly reversible state of unresponsiveness referred to as anergy. Reversibility of anergy indicates that chronically occupied BCR must transduce non-durable regulatory signals that maintain unresponsiveness. Consistent with such a mechanism, studies of immunoglobulin transgenic, as well as naturally occurring polyclonal autoreactive B cells demonstrate activation of the inositol 5-phosphatase SHIP-1 in anergic cells, and low affinity chromatin autoantigen-reactive B cells have been shown to require expression of this phosphatase to maintain anergy. However, it has been reported that anergy of B cells recognizing high affinity soluble antigen may not require SHIP-1, and is instead mediated by upregulation of the inositol 3-phosphatase PTEN. To further explore this apparent difference in mechanism we analyzed the effect of B cell-targeted SHIP-1 deletion on immune tolerance of high affinity anti-HEL B cells in mice expressing so...
Bruton’s tyrosine kinase (Btk) plays a critical role in B cell Ag receptor (BCR) signaling, as in... more Bruton’s tyrosine kinase (Btk) plays a critical role in B cell Ag receptor (BCR) signaling, as indicated by the X-linked immunodeficiency and X-linked agammaglobulinemia phenotypes of mice and men that express mutant forms of the kinase. Although Btk activity can be regulated by Src-family and Syk tyrosine kinases, and perhaps by phosphatidylinositol 3,4,5-trisphosphate, BCR-coupled signaling pathways leading to Btk activation are poorly understood. In view of previous findings that CD19 is involved in BCR-mediated phosphatidylinositol 3-kinase (PI3-K) activation, we assessed its role in Btk activation. Using a CD19 reconstituted myeloma model and CD19 gene-ablated animals we found that BCR-mediated Btk activation and phosphorylation are dependent on the expression of CD19, while BCR-mediated activation of Lyn and Syk is not. Wortmannin preincubation inhibited the BCR-mediated activation and phosphorylation of Btk. Btk activation was not rescued in the myeloma by expression of a CD1...
FcγRIIB are low-affinity receptors for IgG whose intracytoplasmic domain contains an immunorecept... more FcγRIIB are low-affinity receptors for IgG whose intracytoplasmic domain contains an immunoreceptor tyrosine-based inhibition motif (ITIM). FcγRIIB 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 FcγRIIB phosphorylation and its consequences in mast cells. We demonstrate that the phosphorylation of FcγRIIB requires coaggregation with FcεRI and that, once phosphorylated, FcγRIIB selectively recruit the inositol polyphosphate 5 phosphatase SHIP, in vivo. In vitro, however, the phosphorylated FcγRIIB ITIM binds not only SHIP, but also the two protein tyrosine phosphatases, SHP-1 and SHP-2. We show that the coaggregation of FcγRIIB with FcεRI does not prevent FcεRI-mediated activation of lyn and syk. Both ki...
Accumulating evidence indicates that the Src homology 2-containing tyrosine phosphatase 2 (SHP-2)... more 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 FcγRIIB1 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 FcγRIIB1 immunoreceptor tyrosine-based inhibitory motif. The role of SHP-2 in BCR-mediated cell activation and in FcγRIIB1-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. Co...
Antigen binding to the B cell receptor (BCR) induces receptor desensitization, a condition charac... more 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 activate...
B cell antigen receptor (BCR) signaling is a tightly regulated process governed by both positive ... more B cell antigen receptor (BCR) signaling is a tightly regulated process governed by both positive and negative mediators/regulators to ensure appropriate responses to exogenous and autologous antigens. Upon naïve B cell recognition of antigen CD79 [the immunoreceptor tyrosine-based activation motif (ITAM)-containing signaling subunit of the BCR] is phosphorylated and recruits Src and Syk family kinases that then phosphorylate proximal intermediaries linked to downstream activating signaling circuitry. This plasma membrane localized signalosome activates PI3K leading to generation of PIP3 critical for membrane localization and activation of plecktrin homology domain-containing effectors. Conversely, in anergic B cells, chronic antigen stimulation drives biased monophosphorylation of CD79 ITAMs leading to recruitment of Lyn, but not Syk, which docks only to bi-phosphorylated ITAMS. In this context, Lyn appears to function primarily as a driver of inhibitory signaling pathways promoting the inhibition of the PI3K pathway by inositol phosphatases, SHIP-1 and PTEN, which hydrolyze PIP3 to PIP2. Lyn may also exert negative regulation of signaling through recruitment of SHP-1, a tyrosine phosphatase that dephosphorylates activating signaling molecules. Alleles of genes that encode or regulate expression of components of this axis, including SHIP-1, SHP-1, Csk/PTPn22, and Lyn, have been shown to confer risk of autoimmunity. This review will discuss functional interplay of components of this pathway and the impact of risk alleles on its function.
Many autoreactive B cells persist in the periphery in a state of unresponsiveness called anergy. ... more Many autoreactive B cells persist in the periphery in a state of unresponsiveness called anergy. This unresponsiveness is rapidly reversible, requiring continuous BCR interaction with self-antigen and resultant regulatory signaling for its maintenance. Using adoptive transfer of anergic B cells with subsequent acute induction of gene deletion or expression, we demonstrate that the continuous activities of independent inhibitory signaling pathways involving the tyrosine phosphatase SHP-1 and the inositol phosphatase SHIP-1 are required to maintain anergy. Acute breach of anergy by compromise of either of these pathways leads to rapid cell activation, proliferation, and generation of short-lived plasma cells that reside in extrafollicular foci. Results are consistent with predicted/observed reduction in the Lyn-SHIP-1-PTEN-SHP-1 axis function in B cells from systemic lupus erythematosus patients.
Receptors for immunoglobulin Fc regions play multiple critical roles in the immune system, mediat... more Receptors for immunoglobulin Fc regions play multiple critical roles in the immune system, mediating functions as diverse as phagocytosis, triggering degranulation of basophils and mast cells, promoting immunoglobulin class switching, and preventing excessive activation. Transmembrane signaling associated with these functions is mediated primarily by two amino acid sequence motifs, ITAMs (immunoreceptor tyrosine-based activation motifs) and ITIMs (immunoreceptor tyrosine-based inhibition motifs) that act as the receptors' interface with activating and inhibitory signaling pathways, respectively. While ITAMs mobilize activating tyrosine kinases and their consorts, ITIMs mobilize opposing tyrosine and inositol-lipid phosphatases. In this review, we will discuss our current understanding of signaling by these receptors/motifs and their sometimes blurred lines of function.
So SelectiveThe key elements for long-lived antibody-mediated immunity—memory B cells and plasmab... more So SelectiveThe key elements for long-lived antibody-mediated immunity—memory B cells and plasmablasts—are generated in germinal centers, where B cells expressing high-affinity antigen receptors are selected for survival and proliferation in a process called affinity maturation. Unexpectedly,Khalilet al.(p.1178, published online 3 May; see the Perspective byBannard and Cyster) found that, in contrast to naïve B cells and B cells outside the germinal center, proximal signaling events are impaired downstream of the antigen receptor in mouse germinal center B cells.
C3dg is a cleavage product of the C3 component of complement that can facilitate the coligation o... more C3dg is a cleavage product of the C3 component of complement that can facilitate the coligation of the complement receptor 2 (CR2/CD21) with the BCR via C3dg/Ag complexes. This interaction can greatly amplify BCR-mediated signaling events and acts to lower the threshold for B cell activation. Although previous studies have used anti-CR2 Abs or used chimeric Ags in the context of BCR transgenic mice as surrogate C3d-containing ligands, we have used a physiological form of C3d to study signaling in B cells from wild-type C57BL/6 mice. We find that while CR2-enhanced BCR signaling causes intracellular Ca2+ mobilization and total pTyr phosphorylation of an intensity comparable to optimal BCR ligation using anti-IgM Abs, it does so with limited activation of inhibitory effectors (such as CD22, Src homology region 2 domain containing phosphatase 1, and SHIP-1) and without substantial receptor cross-linking. In summary, we demonstrate that CR2-enhanced BCR signaling may proceed not only th...
The low-affinity receptor for IgG, FcγRIIB, functions broadly in the immune system, blocking mast... more The low-affinity receptor for IgG, FcγRIIB, functions broadly in the immune system, blocking mast cell degranulation, dampening the humoral immune response, and reducing the risk of autoimmunity. Previous studies concluded that inhibitory signal transduction by FcγRIIB is mediated solely by its immunoreceptor tyrosine-based inhibition motif (ITIM) that, when phosphorylated, recruits the SH2-containing inositol 5′- phosphatase SHIP and the SH2-containing tyrosine phosphatases SHP-1 and SHP-2. The mutational analysis reported here reveals that the receptor’s C-terminal 16 residues are also required for detectable FcγRIIB association with SHIP in vivo and for FcγRIIB-mediated phosphatidylinositol 3-kinase hydrolysis by SHIP. Although the ITIM appears to contain all the structural information required for receptor-mediated tyrosine phosphorylation of SHIP, phosphorylation is enhanced when the C-terminal sequence is present. Additionally, FcγRIIB-mediated dephosphorylation of CD19 is ind...
FcγRIIB functions as an inhibitory receptor to dampen B cell Ag receptor signals and immune respo... more FcγRIIB functions as an inhibitory receptor to dampen B cell Ag receptor signals and immune responses. Accumulating evidence indicates that ex vivo B cells require the inositol 5-phosphatase, Src homology domain 2-containing inositol 5-phosphatase (SHIP), for FcγRIIB-mediated inhibitory signaling. However, we report here that LPS-activated primary B cells do not require SHIP and thus differ from resting B cells. SHIP-deficient B cell blasts display efficient FcγRIIB-dependent inhibition of calcium mobilization as well as Akt and extracellular signal-related protein kinase phosphorylation. Surprisingly, FcγRIIB-dependent degradation of phosphatidylinositol 3,4,5-trisphosphate and conversion into phosphatidylinositol 3,4-bisphosphate occur in SHIP-deficient B cell blasts, demonstrating the function of an additional inositol 5-phosphatase. Further analysis reveals that while resting cells express only SHIP, B cell blasts also express the recently described inositol 5-phosphatase, SHIP-...
Recent studies demonstrate that MHC class II molecules can signal via associated Ig-αβ dimers, si... more Recent studies demonstrate that MHC class II molecules can signal via associated Ig-αβ 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-αβ signaling. Interestingly, CD22 is actively excluded from cell surface MHC class II aggregates.
The low-affinity receptor for IgG, FcγRIIB, is expressed widely in the immune system and function... more The low-affinity receptor for IgG, FcγRIIB, is expressed widely in the immune system and functions to attenuate Ag-induced immune responses. In mast cells, coaggregation of FcγRIIB with the high-affinity IgE receptor, FcεRI, leads to inhibition of Ag-induced degranulation and cytokine production. FcγRIIB inhibitory activity requires a conserved motif within the FcγRIIB cytoplasmic domain termed the immunoreceptor tyrosine-based inhibition motif. When coaggregated with an activating receptor (e.g., FcεRI, B cell Ag receptor), FcγRIIB is rapidly phosphorylated on tyrosine and recruits the SH2 domain-containing inositol 5-phosphatase (SHIP). However, the mechanisms by which SHIP mediates FcγRIIB inhibitory function in mast cells remain poorly defined. In this report we demonstrate that FcγRIIB coaggregation with FcεRI stimulates enhanced SHIP tyrosine phosphorylation and association with Shc and p62dok. Concurrently, enhanced p62dok tyrosine phosphorylation and association with RasGAP ...
Coaggregation of FcγRIIB1 with B cell Ag receptors (BCR) leads to inhibition of BCR-mediated sign... more Coaggregation of FcγRIIB1 with B cell Ag receptors (BCR) leads to inhibition of BCR-mediated signaling via recruitment of Src homology domain 2 (SH2)-containing phosphatases. In vitro peptide binding experiments using phosphotyrosine-containing sequences derived from the immunoreceptor tyrosine-based inhibitory motif (ITIM) known to mediate FcγRIIB1 effects suggest that the receptor uses SH2-containing inositol phophatase (SHIP) and SH2-containing phophotyrosine phosphatase (SHP)-1, as well as SHP-2 as effectors. In contrast, coimmunoprecipitation studies of receptor-effector associations suggest that the predominant FcγRIIB1 effector protein is SHIP. However, biologically significant interactions may be lost in such studies if reactants’ dissociation rates (Kd) are high. Thus, it is unclear to what extent these assays reflect the relative recruitment of SHIP, SHP-1, and SHP-2 to the receptor in vivo. As an alternative approach to this question, we have studied the effects of ectopi...
B lymphocytes and their differentiated daughters are charged with responding to the myriad pathog... more B lymphocytes and their differentiated daughters are charged with responding to the myriad pathogens in our environment and production of protective antibodies. A sample of the protective antibody produced by each clone is utilized as a component of the cell's antigen receptor (BCR). Transmembrane signals generated upon antigen binding to this receptor provide the primary directive for the cell's subsequent response. In this report, we discuss recent progress and current controversy regarding B cell receptor signal initiation, transduction and regulation.
F1000 - Post-publication peer review of the biomedical literature, 2013
Genetic ablation of the Lyn tyrosine kinase has revealed unique inhibitory roles in B lymphocyte ... more Genetic ablation of the Lyn tyrosine kinase has revealed unique inhibitory roles in B lymphocyte signaling. We now report the consequences of sustained activation of Lyn in vivo using a targeted gain-of-function mutation (Lyn up/up mice). Lyn up/up mice have reduced numbers of conventional B lymphocytes, down-regulated surface immunoglobulin M and costimulatory molecules, and elevated numbers of B1a B cells. Lyn up/up B cells are characterized by the constitutive phosphorylation of negative regulators of B cell antigen receptor (BCR) signaling including CD22, SHP-1, and SHIP-1, and display attributes of lymphocytes rendered tolerant by constitutive engagement of the antigen receptor. However, exaggerated positive signaling is also apparent as evidenced by the constitutive phosphorylation of Syk and phospholipase C ␥ 2 in resting Lyn up/up B cells. Similarly, Lyn up/up B cells show a heightened calcium flux in response to BCR stimulation. Surprisingly, Lyn up/up mice develop circulating autoreactive antibodies and lethal autoimmune glomerulonephritis, suggesting that enhanced positive signaling eventually overrides constitutive negative signaling. These studies highlight the difficulty in maintaining tolerance in the face of chronic stimulation and emphasize the pivotal role of Lyn in B cell signaling.
Many self-reactive B cells exist in the periphery in a rapidly reversible state of unresponsivene... more Many self-reactive B cells exist in the periphery in a rapidly reversible state of unresponsiveness referred to as anergy. Reversibility of anergy indicates that chronically occupied BCR must transduce non-durable regulatory signals that maintain unresponsiveness. Consistent with such a mechanism, studies of immunoglobulin transgenic, as well as naturally occurring polyclonal autoreactive B cells demonstrate activation of the inositol 5-phosphatase SHIP-1 in anergic cells, and low affinity chromatin autoantigen-reactive B cells have been shown to require expression of this phosphatase to maintain anergy. However, it has been reported that anergy of B cells recognizing high affinity soluble antigen may not require SHIP-1, and is instead mediated by upregulation of the inositol 3-phosphatase PTEN. To further explore this apparent difference in mechanism we analyzed the effect of B cell-targeted SHIP-1 deletion on immune tolerance of high affinity anti-HEL B cells in mice expressing so...
Bruton’s tyrosine kinase (Btk) plays a critical role in B cell Ag receptor (BCR) signaling, as in... more Bruton’s tyrosine kinase (Btk) plays a critical role in B cell Ag receptor (BCR) signaling, as indicated by the X-linked immunodeficiency and X-linked agammaglobulinemia phenotypes of mice and men that express mutant forms of the kinase. Although Btk activity can be regulated by Src-family and Syk tyrosine kinases, and perhaps by phosphatidylinositol 3,4,5-trisphosphate, BCR-coupled signaling pathways leading to Btk activation are poorly understood. In view of previous findings that CD19 is involved in BCR-mediated phosphatidylinositol 3-kinase (PI3-K) activation, we assessed its role in Btk activation. Using a CD19 reconstituted myeloma model and CD19 gene-ablated animals we found that BCR-mediated Btk activation and phosphorylation are dependent on the expression of CD19, while BCR-mediated activation of Lyn and Syk is not. Wortmannin preincubation inhibited the BCR-mediated activation and phosphorylation of Btk. Btk activation was not rescued in the myeloma by expression of a CD1...
FcγRIIB are low-affinity receptors for IgG whose intracytoplasmic domain contains an immunorecept... more FcγRIIB are low-affinity receptors for IgG whose intracytoplasmic domain contains an immunoreceptor tyrosine-based inhibition motif (ITIM). FcγRIIB 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 FcγRIIB phosphorylation and its consequences in mast cells. We demonstrate that the phosphorylation of FcγRIIB requires coaggregation with FcεRI and that, once phosphorylated, FcγRIIB selectively recruit the inositol polyphosphate 5 phosphatase SHIP, in vivo. In vitro, however, the phosphorylated FcγRIIB ITIM binds not only SHIP, but also the two protein tyrosine phosphatases, SHP-1 and SHP-2. We show that the coaggregation of FcγRIIB with FcεRI does not prevent FcεRI-mediated activation of lyn and syk. Both ki...
Accumulating evidence indicates that the Src homology 2-containing tyrosine phosphatase 2 (SHP-2)... more 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 FcγRIIB1 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 FcγRIIB1 immunoreceptor tyrosine-based inhibitory motif. The role of SHP-2 in BCR-mediated cell activation and in FcγRIIB1-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. Co...
Antigen binding to the B cell receptor (BCR) induces receptor desensitization, a condition charac... more 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 activate...
B cell antigen receptor (BCR) signaling is a tightly regulated process governed by both positive ... more B cell antigen receptor (BCR) signaling is a tightly regulated process governed by both positive and negative mediators/regulators to ensure appropriate responses to exogenous and autologous antigens. Upon naïve B cell recognition of antigen CD79 [the immunoreceptor tyrosine-based activation motif (ITAM)-containing signaling subunit of the BCR] is phosphorylated and recruits Src and Syk family kinases that then phosphorylate proximal intermediaries linked to downstream activating signaling circuitry. This plasma membrane localized signalosome activates PI3K leading to generation of PIP3 critical for membrane localization and activation of plecktrin homology domain-containing effectors. Conversely, in anergic B cells, chronic antigen stimulation drives biased monophosphorylation of CD79 ITAMs leading to recruitment of Lyn, but not Syk, which docks only to bi-phosphorylated ITAMS. In this context, Lyn appears to function primarily as a driver of inhibitory signaling pathways promoting the inhibition of the PI3K pathway by inositol phosphatases, SHIP-1 and PTEN, which hydrolyze PIP3 to PIP2. Lyn may also exert negative regulation of signaling through recruitment of SHP-1, a tyrosine phosphatase that dephosphorylates activating signaling molecules. Alleles of genes that encode or regulate expression of components of this axis, including SHIP-1, SHP-1, Csk/PTPn22, and Lyn, have been shown to confer risk of autoimmunity. This review will discuss functional interplay of components of this pathway and the impact of risk alleles on its function.
Many autoreactive B cells persist in the periphery in a state of unresponsiveness called anergy. ... more Many autoreactive B cells persist in the periphery in a state of unresponsiveness called anergy. This unresponsiveness is rapidly reversible, requiring continuous BCR interaction with self-antigen and resultant regulatory signaling for its maintenance. Using adoptive transfer of anergic B cells with subsequent acute induction of gene deletion or expression, we demonstrate that the continuous activities of independent inhibitory signaling pathways involving the tyrosine phosphatase SHP-1 and the inositol phosphatase SHIP-1 are required to maintain anergy. Acute breach of anergy by compromise of either of these pathways leads to rapid cell activation, proliferation, and generation of short-lived plasma cells that reside in extrafollicular foci. Results are consistent with predicted/observed reduction in the Lyn-SHIP-1-PTEN-SHP-1 axis function in B cells from systemic lupus erythematosus patients.
Receptors for immunoglobulin Fc regions play multiple critical roles in the immune system, mediat... more Receptors for immunoglobulin Fc regions play multiple critical roles in the immune system, mediating functions as diverse as phagocytosis, triggering degranulation of basophils and mast cells, promoting immunoglobulin class switching, and preventing excessive activation. Transmembrane signaling associated with these functions is mediated primarily by two amino acid sequence motifs, ITAMs (immunoreceptor tyrosine-based activation motifs) and ITIMs (immunoreceptor tyrosine-based inhibition motifs) that act as the receptors' interface with activating and inhibitory signaling pathways, respectively. While ITAMs mobilize activating tyrosine kinases and their consorts, ITIMs mobilize opposing tyrosine and inositol-lipid phosphatases. In this review, we will discuss our current understanding of signaling by these receptors/motifs and their sometimes blurred lines of function.
So SelectiveThe key elements for long-lived antibody-mediated immunity—memory B cells and plasmab... more So SelectiveThe key elements for long-lived antibody-mediated immunity—memory B cells and plasmablasts—are generated in germinal centers, where B cells expressing high-affinity antigen receptors are selected for survival and proliferation in a process called affinity maturation. Unexpectedly,Khalilet al.(p.1178, published online 3 May; see the Perspective byBannard and Cyster) found that, in contrast to naïve B cells and B cells outside the germinal center, proximal signaling events are impaired downstream of the antigen receptor in mouse germinal center B cells.
C3dg is a cleavage product of the C3 component of complement that can facilitate the coligation o... more C3dg is a cleavage product of the C3 component of complement that can facilitate the coligation of the complement receptor 2 (CR2/CD21) with the BCR via C3dg/Ag complexes. This interaction can greatly amplify BCR-mediated signaling events and acts to lower the threshold for B cell activation. Although previous studies have used anti-CR2 Abs or used chimeric Ags in the context of BCR transgenic mice as surrogate C3d-containing ligands, we have used a physiological form of C3d to study signaling in B cells from wild-type C57BL/6 mice. We find that while CR2-enhanced BCR signaling causes intracellular Ca2+ mobilization and total pTyr phosphorylation of an intensity comparable to optimal BCR ligation using anti-IgM Abs, it does so with limited activation of inhibitory effectors (such as CD22, Src homology region 2 domain containing phosphatase 1, and SHIP-1) and without substantial receptor cross-linking. In summary, we demonstrate that CR2-enhanced BCR signaling may proceed not only th...
The low-affinity receptor for IgG, FcγRIIB, functions broadly in the immune system, blocking mast... more The low-affinity receptor for IgG, FcγRIIB, functions broadly in the immune system, blocking mast cell degranulation, dampening the humoral immune response, and reducing the risk of autoimmunity. Previous studies concluded that inhibitory signal transduction by FcγRIIB is mediated solely by its immunoreceptor tyrosine-based inhibition motif (ITIM) that, when phosphorylated, recruits the SH2-containing inositol 5′- phosphatase SHIP and the SH2-containing tyrosine phosphatases SHP-1 and SHP-2. The mutational analysis reported here reveals that the receptor’s C-terminal 16 residues are also required for detectable FcγRIIB association with SHIP in vivo and for FcγRIIB-mediated phosphatidylinositol 3-kinase hydrolysis by SHIP. Although the ITIM appears to contain all the structural information required for receptor-mediated tyrosine phosphorylation of SHIP, phosphorylation is enhanced when the C-terminal sequence is present. Additionally, FcγRIIB-mediated dephosphorylation of CD19 is ind...
FcγRIIB functions as an inhibitory receptor to dampen B cell Ag receptor signals and immune respo... more FcγRIIB functions as an inhibitory receptor to dampen B cell Ag receptor signals and immune responses. Accumulating evidence indicates that ex vivo B cells require the inositol 5-phosphatase, Src homology domain 2-containing inositol 5-phosphatase (SHIP), for FcγRIIB-mediated inhibitory signaling. However, we report here that LPS-activated primary B cells do not require SHIP and thus differ from resting B cells. SHIP-deficient B cell blasts display efficient FcγRIIB-dependent inhibition of calcium mobilization as well as Akt and extracellular signal-related protein kinase phosphorylation. Surprisingly, FcγRIIB-dependent degradation of phosphatidylinositol 3,4,5-trisphosphate and conversion into phosphatidylinositol 3,4-bisphosphate occur in SHIP-deficient B cell blasts, demonstrating the function of an additional inositol 5-phosphatase. Further analysis reveals that while resting cells express only SHIP, B cell blasts also express the recently described inositol 5-phosphatase, SHIP-...
Recent studies demonstrate that MHC class II molecules can signal via associated Ig-αβ dimers, si... more Recent studies demonstrate that MHC class II molecules can signal via associated Ig-αβ 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-αβ signaling. Interestingly, CD22 is actively excluded from cell surface MHC class II aggregates.
The low-affinity receptor for IgG, FcγRIIB, is expressed widely in the immune system and function... more The low-affinity receptor for IgG, FcγRIIB, is expressed widely in the immune system and functions to attenuate Ag-induced immune responses. In mast cells, coaggregation of FcγRIIB with the high-affinity IgE receptor, FcεRI, leads to inhibition of Ag-induced degranulation and cytokine production. FcγRIIB inhibitory activity requires a conserved motif within the FcγRIIB cytoplasmic domain termed the immunoreceptor tyrosine-based inhibition motif. When coaggregated with an activating receptor (e.g., FcεRI, B cell Ag receptor), FcγRIIB is rapidly phosphorylated on tyrosine and recruits the SH2 domain-containing inositol 5-phosphatase (SHIP). However, the mechanisms by which SHIP mediates FcγRIIB inhibitory function in mast cells remain poorly defined. In this report we demonstrate that FcγRIIB coaggregation with FcεRI stimulates enhanced SHIP tyrosine phosphorylation and association with Shc and p62dok. Concurrently, enhanced p62dok tyrosine phosphorylation and association with RasGAP ...
Coaggregation of FcγRIIB1 with B cell Ag receptors (BCR) leads to inhibition of BCR-mediated sign... more Coaggregation of FcγRIIB1 with B cell Ag receptors (BCR) leads to inhibition of BCR-mediated signaling via recruitment of Src homology domain 2 (SH2)-containing phosphatases. In vitro peptide binding experiments using phosphotyrosine-containing sequences derived from the immunoreceptor tyrosine-based inhibitory motif (ITIM) known to mediate FcγRIIB1 effects suggest that the receptor uses SH2-containing inositol phophatase (SHIP) and SH2-containing phophotyrosine phosphatase (SHP)-1, as well as SHP-2 as effectors. In contrast, coimmunoprecipitation studies of receptor-effector associations suggest that the predominant FcγRIIB1 effector protein is SHIP. However, biologically significant interactions may be lost in such studies if reactants’ dissociation rates (Kd) are high. Thus, it is unclear to what extent these assays reflect the relative recruitment of SHIP, SHP-1, and SHP-2 to the receptor in vivo. As an alternative approach to this question, we have studied the effects of ectopi...
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Papers by John Cambier