Gábor Koncz

BCR mediated signal transduction in immature and mature B cells

Ligation of B cell receptors (BCR) on immature B cells may induce apoptosis, while in mature B cells it stimulates cell activation and growth. The signaling pathway regulating the differential functional response, death or survival of the B cell is not fully characterized. We have tested the intracellular signaling requirement of these processes using B cells isolated from the spleen of irradiated auto-reconstituted (transitional immature B cells) and untreated mice (mature B cells), respectively. We compared the BCR induced intracellular [Ca2+] transient, protein tyrosine phosphorylation and ERK phosphorylation, furthermore, the activation of Elk-1 and CREB transcription factors. The BCR induced rise of intracellular [Ca2+] did not significantly differ in the two populations, only a slight difference in the late phase of the response was observed. Immature B cells responded with a maximum tyrosine phosphorylation to a five times lower dose of anti-IgM compared to the mature population. Most importantly, we have found a significant difference in the tyrosine phosphorylation of the Gab family adaptor proteins, Gab1/2. In contrast to mature B cells, crosslinking of BCR on immature B cells did not induce tyrosine phosphorylation of Gab2, thus the Gab2-organized signal amplification complex could not be produced. Furthermore, we detected a significant difference in the kinetics of BCR induced ERK, Elk-1 and CREB phosphorylation. In immature B cells, ERK was transiently phosphorylated, ceasing after 120 min, while in mature cells, ERK phosphorylation was sustained. Elk-1 and CREB activation was also transient in immature B cells, followed the kinetics of ERK phosphorylation. The lack of sustained Erk1/2 activation suppresses the transcription factors necessary for the proliferation signal. Since ERK is regulated by the phosphorylated Gab1/2, these data demonstrate that BCR triggered phosphorylation and signal amplification of Gab1/2 is a critical step in a life or death decision of B cells.

Human Type II Fcγ Receptors Inhibit B Cell Activation by Interacting with the p21ras-dependent Pathway

Co-ligation of antigen receptors and type II Fcγ receptors (FcγRIIb) on B cells interrupts signal transduction and ultimately inhibits antibody production. We have identified p52 Shc in the FcγRIIb1-specific immunoprecipitates isolated from the membrane fraction of BL41 Burkitt lymphoma cells following B cell receptor-FcγRIIb1 co-ligation. The insolubilized synthetic peptide representing the phosphorylated form of the tyrosine-based inhibitory motif of FcγRIIb also binds Shc from the lysates of activated but not from resting BL41 cells. This suggests that the binding does not depend on the interaction of FcγRIIb1-phosphotyrosine with the SH2 domain of Shc. Tyr phosphorylation of FcγRIIb1-associated Shc is low, indicating an impaired function. Shc is implicated in regulating p21ras activation; thus, we have compared p21ras activities in BL41 cells treated in different ways. p21ras activity is reduced when B cell receptor and FcγRIIb1 are co-ligated. p21ras couples protein-tyrosine kinase-dependent events to the Ser/Thr kinase-mediated signaling pathway leading to the activation of mitogen-activated protein kinases (MAPK). Our results show that B cell receptor-FcγRIIb1 co-cross-linking partially inhibits mitogen-activated protein kinase activity. We conclude that FcγRIIb1-dependent inhibition of human B cell activation may be based on interrupting signal transduction between protein-tyrosine kinases and the p21ras/mitogen-activated protein kinase-dependent activation pathway.

Fcγ receptor type IIb induced recruitment of inositol and protein phosphatases to the signal transductory complex of human B-cell

Co-clustering of Fc gamma RIIb and B-cell receptor (BCR) inhibits cell activation by interrupting BCR stimulated signal transduction. The immunoreceptor tyrosine-based inhibitory motif (ITIM) of Fc gamma RIIb becomes tyrosyl phosphorylated (P-ITIM) upon co-clustering with BCR then P-ITIM interacts with several signalling molecules, some of which negatively regulate the cell activation process. The molecules recruited by the P-ITIM of human Fc gamma RIIb have not been characterised yet. In order to affinity isolate the potential functional partner molecules of human Fc gamma RIIb, synthetic peptides were designed to cover almost the entire intracellular Fc gamma RIIb domain, including Fc gamma RIIb2 specific sequences and stretches containing the phosphorylated and non-phosphorylated ITIM. We report here that several tyrosyl phosphorylated proteins bind to the P-ITIM peptide from both resting and activated B-cell lysates, the 53-56 kDa being the most prominent one. A fraction of the 53-56 kDa bands were identified as the protein tyrosine kinase (PTK), Lyn which also bound to ITIM peptide, pointing to its role in initiating Fc gamma RIIb-mediated negative regulation. Among the P-ITIM associated tyr phosphorylated components, the 145 kDa one was identified as the inositol polyphosphate 5-phosphatase, SHIP and the 72 kDa protein as the protein tyrosine phosphatase (PTP) SHP2, whereas SHP1 was not detected. Phosphatase activity assays showed that P-ITIM bound about five times higher SHIP and four times higher PTP activity than the ITIM containing peptide. Furthermore, we detected PKC and MAPK in both ITIM and P-ITIM peptides precipitated samples. Since human B-cells express both Fc gamma RIIb1 and Fc gamma RIIb2, differing in a 19 amino acid insert in the cytoplasmic tail of the former, we investigated the components binding to Fc gamma RIIb1 and Fc gamma RIIb2 specific sequences. Synthetic peptide representing Fc gamma RIIb1 and Fc gamma RIIb2 specific sequences weakly bound unidentified tyr phosphorylated proteins at 50-56 kDa, while the insert itself did not bind a detectable amount of protein. Neither of the ITIM or P-ITIM bound molecules were observed in samples precipitated with peptides corresponding to Fc gamma RIIb1 or Fc gamma RIIb2 specific sequences. These observations suggest that protein kinases associate with both ITIM and P-ITIM of human Fc gamma RIIb, Lyn being responsible for the tyrosyl phosphorylation of ITIM. SHIP and SHP2 phosphatases selectively bind to the phosphorylated ITIM. Based on these data we assume that SHIP and SHP2 recruited in vivo to the Fc gamma RIIb co-clustered BCR are responsible for the Fc gamma RIIb mediated negative regulation of human B-cell activation.

The Fas/CD95 Receptor Regulates the Death of Autoreactive B Cells and the Selection of Antigen-Specific B Cells.

Cell death receptors have crucial roles in the regulation of immune responses. Here we review recent in vivo data confirming that the Fas death receptor (TNFSR6) on B cells is important for the regulation of autoimmunity since the impairment of only Fas function on B cells results in uncontrolled autoantibody production and autoimmunity. Fas plays a role in the elimination of the non-specific and autoreactive B cells in germinal center, while during the selection of antigen-specific B cells different escape signals ensure the resistance to Fas-mediated apoptosis. Antigen-specific survival such as BCR or MHCII signal or coreceptors (CD19) cooperating with BCR inhibits the formation of death inducing signaling complex. Antigen-specific survival can be reinforced by antigen-independent signals of IL-4 or CD40 overproducing the anti-apoptotic members of the Bcl-2 family proteins.

Integration of activatory and inhibitory signals in human B-cells

Fc gamma receptors type IIb1 (Fc gammaRIIb1) inhibit B-cell activation when co-ligated with B-cell antigen receptors (BCR) by immune complexes. In murine B-cells the inhibition is mediated by the interaction of the phosphorylated immunoreceptor tyrosine-based inhibitory motif (P-ITIM) of Fc gammaRIIb1 with the SH2 domain containing protein tyrosine phosphatase. SHP1. To clarify the mechanism of Fc gammaRIIb mediated inhibition of human B-cells we have studied the association of signaling molecules with human Fc gammaRIIb1 after co-ligating with BCR. Fc gammaRIIb1 were affinity purified from the Burkitt lymphoma cell line, BL41. Several tyrosine phosphorylated proteins were co-isolated with Fc gammaRIIb1 at 145, 110, and 50 60 kDa, which were not present in Fc gammaRIIb1 free immune complexes. Among these molecules we have identified the p52 Shc adaptor protein. Furthermore, we have shown that the insolubilised synthetic peptide corresponding P-ITIM bound Shc, Lyn and the p75 and p 10 unidentified tyrosine phosphorylated proteins. Here we describe that the cell membrane associated Shc is partially dephosphorylated in BCR-Fc gammaRIIb1 co-ligated samples, suggesting that its function in regulating p21ras monomeric G protein is impaired. Indeed, we have detected a lower p21ras activity in BCR-Fc gammaRIIb1 co-crosslinked samples. These data indicate that co-ligation of BCR and Fc gammaRIIb1 interrupts signal transduction between protein tyrosine kinase activation and p21ras mediated activation pathway. Since in contrast to the mouse B-cells both Fc gammaRIIb1 and Fc gammaRIIb2 are expressed in human B-cells, we have investigated the inhibitory function of the two receptors in Fc gammaRIIb negative Burkitt lymphoma cell line ST486 transfected with Fc gammaRIIb1 and Fc gammaRIIb2, respectively. Both Fc gammaRIIb1 and Fc gammaRIIb2 inhibited the rise of intracellular Ca2+ induced by the crosslinking of BCR. The rate of the inhibition depended on the ratio of the co-crosslinked receptors (BCR-Fc gammaRIIb1) to the crosslinked BCR (BCR-BCR). Co-crosslinking of the two receptors inhibited not only the capacitive Ca2+ entry but rather the total Ca2+ response in both Fc gammaRIIb1 and Fc gammaRIIb2 transfected human B-cells. CD19 represents the signal transduction unit of complement receptor, CR2 (CD21), and is responsible for the complement activating IgM-immune complex induced enhancement of B-cell activation. Co-crosslinking of CD19 and BCR was shown to enhance B-cell activation due to the recruitment of further signaling molecules to the activator complex by the phosphorylated tyrosine residues of CD19. Here we show a novel finding that co-ligation of CD19 with Fc gammaRIIb1 inhibits the CD19-induced upregulation of Ca2+ response. The results indicate that IgG plus complement containing immune complexes may inhibit B-cell activation in vivo, due to the Fc gammaRIIb1-mediated interruption of signal transduction via both BCR and CD19.

Fcγ receptor-mediated inhibition of human B cell activation: The role of SHP-2 phosphatase

Co‐clustering of the type II receptors binding the Fc part of IgG (FcγRIIb) and B cell receptors results in the translocation of cytosolic, negative regulatory molecules to the phosphorylated immunoreceptor tyrosine‐based inhibitory motif (P‐ITIM) of the FcγRIIb. SH2 domain‐containing protein tyrosine phosphatases (SHP‐1 and SHP‐2), and the polyphosphoinositol 5′‐phosphatase (SHIP) have been reported earlier to bind to murine FcγRIIb P‐ITIM. However, neither the functional substrates of these enzymes, nor the mechanism of the inhibition are fully resolved. We show here that the human FcγRIIb binds SHP‐2 when co‐clustered with the B cell receptors, whereas its synthetic P‐ITIM peptide bindes SHP‐2 and SHIP in lysates of the Burkitts lymphoma cell line BL41. The P‐ITIM peptide binding enhances SHP‐2 activity, resulting in dephosphorylation and release of P‐ITIM‐bound SHIP and Shc. Moreover, P‐ITIM‐bound SHP‐2 dephosphorylates synthetic peptides corresponding to the sites of tyrosine phosphorylation on SHIP and Shc, indicating that these proteins are its potential substrates. Thus SHP‐2‐induced dephosphorylation may modulate the intracellular localization and/or activity of SHIP and Shc, thereby inhibiting further activation pathways which they mediate.

Cooperation between SHP-2, phosphatidyl inositol 3-kinase and phosphoinositol 5-phosphatase in the FcγRIIb mediated B cell regulation

Co-clustering B cell receptors (BCR) and type II receptors binding the Fc part of IgG (Fc gamma RIIb) inhibits B cell activation and antibody production. Tyrosine phosphorylation of an intracellular motif of Fc gamma RIIb has been shown to be a prerequisite of the inhibition. After being phosphorylated by BCR-activated tyrosine kinases, the immunoreceptor tyrosine-based inhibitory motif (P-ITIM) of Fc gamma RIIb recruits SH2 domain containing protein tyrosine phosphatase(s) (PTPs) and polyphosphoinositol 5-phosphatase (SHIP) to the vicinity of BCR, which in turn dephosphorylate their specific substrates. This leads to the interruption of signal transduction, consequently to the anergy and/or apoptosis of the cell. The downstream signaling pathways affected by Fc gamma RIIb-BCR co-clustering are not clarified yet, neither the substrates of PTPs are known. We have studied the Fc gamma RIIb mediated B cell inhibition on human Burkitt lymphoma cell line (BL41). From the lysates of BL41 cells SHP-2 and phosphatidylinositol 3-kinase (PI3-K), as well as the protein tyrosine kinase (PTK) Lyn bind both to the BCR-co-clustered Fc gamma RIIb and to its P-ITIM peptide. Lyn hyperphosphorylates the P-ITIM associated molecules, including SHIP in the in vitro protein tyrosine kinase activity assay. The P-ITIM-compelled multi-phosphoprotein complex binds to and activates SHP-2, which in turn dephosphorylates SHIP and Shc and probably other substrates. Subcellular localisation of these signaling molecules is regulated by the phosphotyrosine-SH2 domain interactions, thus dephosphorylation may result in the re-direction of Shc and SHIP within the cell, consequently, in the modulation of their activity. Finally, co-clustering Fc gamma RIIb and BCR or Fc gamma RIIb and CD19 on the intact cells inhibited PI3-K activity as detected in the anti-phosphotyrosine (anti-PY) precipitates. The results indicate that SHP-2 bound to and activated by the BCR co-clustered Fc gamma RIIb, may down-regulate PI3-K activity by dephosphorylating a yet unidentified regulatory molecule, which recruits PI3-K to the cell membrane.

Functional mapping of the FcγRII binding site on human IgG1 by synthetic peptides

Receptors specific for the Fc part of IgG (FcγR) are expressed by several cell types and play diverse roles in immune responses. Impaired function of the activating and inhibitory FcγRmay result in autoimmunity. Thus, the modulation of IgG‐FcγR interaction can be a target for the development of treatments for some autoimmune and inflammatory diseases. This study addresses the localization and functional characterization of linear sequences in human IgG1 which bind to FcγRII. Peptides with overlapping sequences derived from the CH2 domain of human IgG1 between P234 and S298 were synthesized and used in binding and functional experiments. Binding of the peptides to FcγR was assayed in vitro and ex vivo, and peptides foundto interact were functionally tested. The shortest effective peptide was T256–P271, which bound to soluble recombinant FcγRIIb with Kd=6×106 M–1. The biotinylated peptides R255–P271 and T256–P271 complexed by avidin exhibited functional activity; they induced FcγRIIb‐mediated inhibition of the BCR‐triggered Ca2+ response of human Burkitt lymphoma cells, and inflammatory cytokine production (TNF‐α and IL‐6) by the human monocyte cell line MonoMac. In conclusion, our results suggest that the selected peptides functionally represent the FcγRII‐binding part of IgG1.

Regulating Vav1 phosphorylation by the SHP-1 tyrosine phosphatase is a fine-tuning mechanism for the negative regulation of DISC formation and Fas-mediated cell death signaling.

The actin cytoskeleton association is required for caspase 8-independent Fas/CD95 receptor internalization, a critical step for an optimal death-inducing signaling complex formation along the endocytic pathway, leading to efficient activation of the caspase cascade and, ultimately, cell death. However, the way in which this initiation phase of Fas receptor signaling is regulated is still unknown. We report herein that, in B cells, upon Fas engagement, the tyrosine phosphatase SHP-1-regulated Vav dephosphorylation, by downmodulating the Fas–ezrin–actin linkage is a fine-tune switch-off mechanism that the cell uses as a way to terminate the receptor internalization, controlling therefore the time and extent of the DISC formation and cell death.

Grb2 associated binder 2 couples B-cell receptor to cell survival

B-cell fate during maturation and the germinal center reaction is regulated through the strength and the duration of the B-cell receptor signal. Signaling pathways discriminating between apoptosis and survival in B cells are keys in understanding adaptive immunity. Gab2 is a member of the Gab/Dos adaptor protein family. It has been shown in several model systems that Gab/Dos family members may regulate both the anti-apoptotic PI3-K/Akt and the mitogenic Ras/MAPK pathways, still their role in B-cells have not been investigated in detail. Here we studied the role of Gab2 in B-cell receptor mediated signaling. We have shown that BCR crosslinking induces the marked phosphorylation of Gab2 through both Lyn and Syk kinases. Subsequently Gab2 recruits p85 regulatory subunit of PI3-K, and SHP-2. Our results revealed that Ig-alpha/Ig-beta, signal transducing unit of the B-cell receptor, may function as scaffold recruiting Gab2 to the signalosome. Overexpression of Gab2 in A20 cells demonstrated that Gab2 is a regulator of the PI3-K/Akt but not that of the Ras/MAPK pathway in B cells. Accordingly to the elevated Akt phosphorylation, overexpression of wild-type Gab2 in A20 cells suppressed Fas-mediated apoptosis, and enhanced BCR-mediated rescue from Fas-induced cell death. Although PH-domain has only a stabilizing effect on membrane recruitment of Gab2, it is indispensable in mediating its anti-apoptotic effect.