Ras regulation and function in lymphocytes.

The GTPase, Ras, is rapidly activated in antigen receptor stimulated T. cells, B cells and mast cells. Ras can bind to diverse effector molecules when activated and thereby switch on multiple downstream effector pathways. In lymphocytes Ras plays an important role in the signalling pathways that activate transcription factors involved in cytokine gene induction. Ras is also a key component of the complex regulatory networks that control T and B cell development.

The T-cell receptor regulates Akt (protein kinase B) via a pathway involving Rac1 and phosphatidylinositide 3-kinase.

The serine/threonine kinase Akt (also known as protein kinase B) (Akt/PKB) is activated upon T-cell antigen receptor (TCR) engagement or upon expression of an active form of phosphatidylinositide (PI) 3-kinase in T lymphocytes. Here we report that the small GTPase Rac1 is implicated in this pathway, connecting the receptor with the lipid kinase. We show that in Jurkat cells, activated forms of Rac1 or Cdc42, but not Rho, stimulate an increase in Akt/PKB activity. TCR-induced Akt/PKB activation is inhibited either by PI 3-kinase inhibitors (LY294002 and wortmannin) or by overexpression of a dominant negative mutant of Rac1 but not Cdc42. Accordingly, triggering of the TCR rapidly stimulates a transient increase in GTP-Rac content in these cells. Similar to TCR stimulation, L61Rac-induced Akt/PKB kinase activity is also LY294002 and wortmannin sensitive. However, induction of Akt/PKB activity by constitutive active PI 3-kinase is unaffected when dominant negative Rac1 is coexpressed, placing Rac1 upstream of PI 3-kinase in the signaling pathway. When analyzing the signaling hierarchy in the pathway leading to cytoskeleton rearrangements, we found that Rac1 acts downstream of PI 3-kinase, a finding that is in accordance with numerous studies in fibroblasts. Our results reveal a previously unrecognized role of the GTPase Rac1, acting upstream of PI 3-kinase in linking the TCR to Akt/PKB. This is the first report of a membrane receptor employing Rac1 as a downstream transducer for Akt/PKB activation.

p21ras initiates Rac-1 but not phosphatidyl inositol 3 kinase/PKB, mediated signaling pathways in T lymphocytes.

p21ras is activated by the T cell antigen receptor (TCR) and then co-ordinates important signaling pathways for T lymphocyte activation. Effector pathways for this guanine nucleotide binding protein in T cells are mediated by the serine/threonine kinase Raf-1 and the Ras-related GTPase Rac-1. In fibroblasts, an important effector for the Ras oncogene is Phosphatidylinositol 3-kinase (PtdIns 3-kinase). Activation of this lipid kinase is able to induce critical Rac-1 signaling pathways and can couple p21ras to cell survival mechanisms via the serine/threonine kinase Akt/PKB. The role of PtdIns 3-kinase in Ras signaling in T cells has not been explored. In the present study, we examined the ability of PtdIns 3-kinase to initiate the Rac-1 signaling pathways important for T cell activation. We also examined the possibility that Akt/PKB is regulated by Ras signaling pathways in T lymphocytes. The results show that Ras can initiate a Rac-1 mediated pathway that regulates the transcriptional function of AP-1 complexes. PtdIns 3-kinase signals cannot mimic p21ras and induce the Rac mediated responses of AP-1 transcriptional activation. Moreover, neither TCR or Ras activation of AP-1 is dependent on PtdIns 3-kinase. PKB is activated in response to triggering of the T cell antigen receptor; PtdIns 3-kinase activity is both required and sufficient for this TCR response. In contrast, p21ras signals are unable to induce Akt/PKB activity in T cell nor is Ras function required for Akt/PKB activation in response to the TCR. The present data thus highlight that PtdIns 3-kinase and Akt/PKB are not universal Ras effector molecules. Ras can initiate Rac-1 regulated signaling pathways in the context of T cell antigen receptor function independently of PtdIns 3-kinase activity.

Proliferative response of hairy cells to B cell growth factor (BCGF): in vivo inhibition by interferon-alpha and in vitro effects of interferon-alpha, -beta, and -gamma.

Hairy cell leukemia (HCL) is a pre-plasma B cell tumor which responds to interferon (IFN)-alpha therapy. In vitro, B cell growth factor (BCGF) can induce proliferation of hairy cells. We have investigated the effect of in vitro and in vivo treatments with different recombinant IFN on the capacity of hairy cells to proliferate in response to human BCGF. In vitro treatment of leukemic cells from HCL patients with recombinant IFN-alpha-2 (5/5 cases) or IFN-beta (4/5 cases) resulted in a marked inhibition of the BCGF-dependent response. This suppressive effect was obtained with IFN concentrations of 1000, 100 IU/ml, and even occasionally 10 IU/ml. In contrast, no such inhibition was observed with IFN-gamma, despite the presence of specific IFN-gamma receptors on hairy cells at densities similar to receptors for IFN-alpha/beta. The IFN-alpha-induced suppression of the proliferative response of hairy cells to BCGF was also observed in vivo in two patients within 6-12 hr after administration of single doses of IFN-alpha. When hairy cells were maintained in culture for 1 week, they recovered their capacity to be stimulated by BCGF. This reversion was also shown in vivo in hairy cells isolated 1 week after IFN administration. Since in vivo growth of hairy cells could possibly result from the autocrine secretion of BCGF, we propose that the therapeutic effect of IFN-alpha on HCL may be due in part to an inhibition of such autocrine loop.

Expression of the B8.7 antigen on hairy cells and relation with the LMW-BCGF response.

Hairy cells are classified as B cell tumors at a preplasma cell stage of differentiation and are believed to represent cells undergoing a switch process. These cells are stimulated in vitro to DNA synthesis and multiplication in the presence of the lymphokine LMW-BCGF. We have tested the level of expression on these cells of the newly described B8.7 activation marker which has been reported to be associated with the capacity of various B cells to respond to LMW-BCGF. The presence of this marker has been readily detected on the hairy cells of 10 of the 12 patients tested in this study; interestingly, for one of the negative cases, the tumor cells were unable to proliferate in response to LMW-BCGF. As on normal B cells, a marked inhibition of the LMW-BCGF dependent response could be achieved in the presence of a monoclonal anti-B8.7 antibody, sustaining the proposal that the B8.7 molecule is involved in the signaling pathway of this growth factor. IFN-alpha is highly efficient in the therapy of hairy cell leukemia (HCL), and we confirm in the present study that IFN-alpha also inhibits the LMW-BCGF dependent proliferation of hairy cells in vitro. In addition, we show that this inhibition is independent of a significant modulation of the B8.7 antigen, a molecule putatively associated with the LMW-BCGF receptor.

Interferon alpha and intracytoplasmic free calcium in hairy cell leukemia cells.

Hairy cell leukemia (HCL) is a B-cell tumor affecting the pre-plasma stage of B cell differentiation. One of the most striking characteristics of this disease is its remarkable responsiveness to alpha-interferon (IFN-alpha) therapy. Interferons constitute a heterologous family of multifunctional cytokines displaying anti-viral, anti-proliferative and immunoregulatory properties. These activities have been extensively studied in hairy cells, but the mechanism of action of IFN-alpha in hairy cell leukemia remains unknown. Our approach to investigate the mode action of IFN-alpha in HCL has been to identify abnormalities which occur in these tumor cells and then to ascertain whether these abnormalities can be rectified by IFN-alpha treatment. A high level of free Ca2+ in the cytoplasm of hairy cells was identified. Increases in cytosolic Ca2+ are believed to be a pivotal signal in regulating cell proliferation, cell differentiation and cell death. These high Ca2+ levels in hairy cells could be reduced upon treatment with IFN-alpha either in vitro or in vivo, probably acting by reducing Ca2+ influx into the leukemic cells. Moreover, the effect of IFN-alpha on [Ca2+]i seems to be correlated with down-regulation of CD20 phosphorylation, a B cell specific phosphoprotein involved in Ca2+ influx across the plasma membrane. The possible origins and implications of Ca2+ deregulation and the possible mechanisms or sites of action of IFN-alpha in tumor cells from HCL are explored in this review.

Investigating hairy cell leukemia dysregulations. Looking for interferon alpha site of action in hairy cells.

One of the most striking characteristics of hairy cell leukemia (HCL) is its remarkable responsiveness to alpha-interferon (IFN-alpha) therapy. IFN-alpha is a multifunctional cytokine able to regulate the growth, differentiation and recirculation of normal and malignant B lymphocytes. These activities have been extensively studied in hairy cells, but none of these properties has been shown to fully account for IFN-alpha responsiveness. Numerous data now support the idea that IFN-alpha exerts its therapeutic effect both indirectly, through stimulation of immunocompetent cells, and directly on the hairy cells. Our approach to investigate the mode action of IFN-alpha in HCL has been to identify abnormalities which occur in these tumor cells and then to ascertain whether these abnormalities can be rectified by IFN-alpha treatment. A high level of free Ca2+ in the cytoplasm of hairy cells was identified. Increases in cytosolic Ca2+ are believed to be a pivotal signal in regulating cell proliferation, cell differentiation and cell death. These high Ca2+ levels in hairy cells could be reduced upon treatment with IFN-alpha either in vitro or in vivo, probably acting by reducing Ca2+ influx into the leukemic cells. Moreover, the effect of IFN-alpha on [Ca2+]i seems to be correlated with alteration in the pattern of kinase/phosphatase activities and with down-regulation of CD20 phosphorylation, a B cell specific phosphoprotein involved in Ca2+ influx across the plasma membrane.

A method for the determination of the affinity constant of antibodies to tissue plasminogen activator.

A simple method for the determination of the affinity constant of antibodies to fibrin-bound tissue-type plasminogen activator (t-PA) is described. The method is based on the high-affinity binding of t-PA to a solid-phase fibrin support which allows the specific separation of t-PA present in any biological fluid. This provides as in the physiological fibrin-t-PA interaction, a molecular structure conserving its antigenic and functional properties which allows for the binding of specific anti-t-PA antibodies and hence facilitates the separation of free from bound antibody. Then, antibodies bound to t-PA are revealed with a labelled, second antibody and data relating Bound v Total antibody input can be analyzed. Affinities were calculated by least square nonlinear regression analysis using a computerized approach. The method allows the direct use of plasma or other crude sources where t-PA is present in its most native form.

Nonadherent cells switch to a Rac-mediated, SHIP regulated, Akt activation mode for survival.

Constitutively active Rac stimulates Akt activity in T lymphocytes cultured in suspension. This regulation contrasts with findings obtained in fibroblasts, endothelial or neuronal cells grown on substrate, where Akt stimulation occurs independently of Rac. We now show that V12Rac-mediated stimulation of Akt is not restricted to the hematopoietic lineage but is dependent on the adherence status of the cell. V12Rac-mediated stimulation of Akt as well as molecular association between Rac and Akt occurred exclusively in cells kept in suspension. Stimulation and complex formation are dependent on SHIP but in a manner that differs from its role in dephosphorylation of phosphoinositide lipids. Adherent cells lacking SHIP, but not those lacking PTEN, are able to activate Akt through the Rac pathway. Our data reveal the existence of a bona fide Rac to Akt signaling pathway, tightly regulated by SHIP and operational in suspended cells only. This pathway may point to an alternative survival signal that is called into action when cells lose contact with the substrate and/or with other cells.

A B cell growth factor-dependent cell line derived from a human lymphocytic nodular lymphoma.

A B cell line derived from a human nodular lymphocytic lymphoma (Brill-Symmers) was shown to be dependent on the presence of a low molecular weight B cell growth factor (BCGF) for its growth in vitro. The caryotype was normal and no contamination with Epstein-Barr virus (EBV) could be detected. These cells did not respond to recombinant gamma interferon or to recombinant human interleukin 2 (IL-2), although they displayed a weak density of IL-2 receptor sites. They were both responsive to and dependent on BCGF for their multiplication in vitro. Furthermore, the putative receptor for this growth factor (CD23) was detected on these cells and the BCGF-dependent proliferation could be blocked by a monoclonal anti-CD23 antibody. A tumour-derived cell line like this provides an interesting model for studying the mechanisms regulating B cell growth and the early events leading to the process of B cell immortalization.

Analysis of the role of protein kinase C-alpha, -epsilon, and -zeta in T cell activation.

T cells express multiple isotypes of protein kinase C (PKC) and although it is well accepted that PKCs have an important role in T cell activation, little is known about the function of individual PKC isotypes. To address this issue, mutationally active PKC-alpha, -epsilon, or -zeta have been transfected into T cells and the consequences for T cell activation determined. p21ras plays an essential role in T cell activation. Accordingly, the effects of the constitutively active PKCs were compared to the effects of mutationally activated p21ras. The data indicate that PKC-epsilon and, to a lesser extent PKC-alpha but not -zeta, can regulate the transcription factors AP-1 and nuclear factor of activated T cells (NF-AT-1). The ability of PKC-epsilon to induce transactivation of NF-AT-1 and AP-1 was similar to the stimulatory effect of a constitutively activated p21ras. PKC-epsilon, but not PKC-alpha nor activated p21ras, was able to induce NF-KB activity. Phorbol esters induce expression of CD69 whereas none of the activated PKC isotypes tested were able to have this effect. Activated Src and p21ras were able to induce CD69 expression. These results indicate selective functions for different PKC isotypes in T cells. Moreover, the data comparing the effects of activated Ras and PKC mutants suggest that PKC-alpha, p21ras, and PKC-epsilon are not positioned linearly on a single signal transduction pathway.

Multiple p21ras effector pathways regulate nuclear factor of activated T cells.

The transcription factor, Nuclear Factor of Activated T cells (NFAT) is a major target for p21ras and calcium signalling pathways in the IL-2 gene and is induced by p21ras signals acting in synergy with calcium/calcineurin signals. One p21ras effector pathway involves the MAP kinase ERK-2, and we have examined its role in NFAT regulation. Expression of dominant negative MAPKK-1 prevents NFAT induction. Constitutively active MAPKK-1 fully activates ERK-2 and the transcription factor Elk-1, but does not substitute for activated p21ras and synergize with calcium/calcineurin signals to induce NFAT. Expression of dominant negative N17Rac also prevents TCR and p21ras activation of NFAT, but without interfering with the ERK-2 pathway. The transcriptional activity of the NFAT binding site is mediated by a complex comprising a member of the NFAT group and AP-1 family proteins. The induction of AP-1 by p21ras also requires Rac-1 function. Activated Rac-1 could mimic activated p21ras to induce AP-1 but not to induce NFAT. Moreover, the combination of activated MAPKK-1 and Rac-1 could not substitute for activated p21ras and synergize with calcium signals to induce NFAT. Thus, p21ras regulation of NFAT in T cells requires the activity of multiple effector pathways including those regulated by MAPKK-1/ERK-2 and Rac-1.

Potentiation of the proliferative response of human B lymphocytes to low molecular weight B cell growth factor (LMW-BCGF) by fibroblast growth factors (FGFs).

Both acidic and basic fibroblast growth factor (FGF), although devoid alone of growth-promoting ability on resting or activated human lymphoid B cells, were found to markedly increase the proliferative response of anti-mu-chain or SAC preactivated B cell blasts to the low molecular weight B cell growth factor (LMW-BCGF) and to enhance the costimulatory response of resting B cells to anti-mu-chain and LMW-BCGF. This potentiating effect was also observed for a LMW-BCGF-dependent B cell tumor derived from a lymphocytic nodular lymphoma. Other growth factors acting on fibroblasts, such as epidermal growth factor, alpha-thrombin, platelet-derived growth factor, and insulin-like growth factor-I did not display such enhancing effect on LMW-BCGF-driven proliferation. Activated, but not resting B cells were found to bear receptor sites for FGFs and from kinetics experiments, it is suggested that LMW-BCGF induces competence expression for FGFs in those cells. Moreover, the LMW-BCGF-elicited generation of inositoltrisphosphate resulting from polyphosphoinositides hydrolysis was increased in the presence of FGF.

Activation of the phosphatidylinositol metabolic pathway by low molecular weight B cell growth factor.

The possible role of phosphatidylinositol breakdown in the induction of proliferation of human activated B cells by low molecular weight B cell growth factor (LMW-BCGF) was examined. LMW-BCGF was found to induce a rapid rise in the concentration of inositol trisphosphate (InsP3) in [3H]inositol-loaded B cell blasts, obtained by prior anti-mu antibody activation. A concomitant decrease in the concentration of phosphatidylinositol 4,5-bisphosphate could be detected at the same time. Maximum generation of InsP3 occurred within 15-30 s after the addition of the LMW-BCGF ligand to the activated B cells, then was followed by a slow decrease and return to control values. The amount of InsP3 generated by phosphatidylinositol hydrolysis was dependent on the concentration of LMW-BCGF. This effect was only detected in B cells already preactivated by a first signal such as anti-mu antibody and not in resting unstimulated B cells. In contrast, under similar conditions, interleukin 2, another B cell growth-promoting lymphokine, did not alter the rate of formation of the various phosphatidylinositol breakdown products. An augmentation of the [Ca2+]i concentration was also detected in activated B cells upon addition of LMW-BCGF and this increase could be blocked by TMB-8, a specific inhibitor of endoplasmic reticulum calcium release. Hydrolysis of phosphoinositides thus represents an essential component in the mechanism of transduction of the signal provided by LMW-BCGF.

Production of monoclonal antibodies to the high fibrin-affinity, tissue-type plasminogen activator of human plasma. Demonstration of its endothelial origin by immunolocalization.

Monoclonal antibodies (MAbs) to vascular plasminogen activator (vPA), the tissue-type plasminogen activator (tPA) in human plasma, were produced to be used as probes for immunochemical analysis. Human tissue sections and one of these MAbs were used to demonstrate the endothelial origin of plasma-tPA by immunohistochemistry. To produce MAbs, mice were immunized with semipurified vPA isolated from postocclusion human venous blood. Primed spleen cells were fused with the mouse myeloma cell line NS-1. Screening for MAb-producing hybridomas was performed with postocclusion euglobulins as a source of antigen by means of a solid-phase fibrin-vPA immunoassay. The selective and high-affinity binding of vPA for fibrin ensures the specificity and sensitivity of this test. Thus, eight hybridomas secreting MAbs to vPA were selected, cloned, and established as permanent hybridoma cell lines. Immunohistochemical analysis of cryostat sections of human tissues was performed with EA-delta 12D, a MAb having no inhibitory effect against vPA activity but binding to vPA with a high affinity. Thus, the only structures immunostained were endothelial cells of venules, capillaries, and arterioles. The EA-delta 12D monoclonal localization of plasma vPA in the endothelial lining of blood vessels provides evidence that tPA in plasma originates from the vascular wall and validates its designation as vascular plasminogen activator, ie, vPA. Also, our results are consistent with the fact that vPA in blood and tPA in tissues are immunologically identical and have a common endothelial origin.

Interferon-alpha downregulates the abnormal intracytoplasmic free calcium concentration of tumor cells in hairy cell leukemia.

Hairy cell leukemia (HCL) is a B-cell tumor affecting the preplasma stage of B-cell differentiation. One important feature of the disease is its exquisite sensitivity to interferon-alpha (IFN-alpha) therapy. Because we showed earlier that the CD20 molecule is consistently hyperphosphorylated in hairy cells and because previous studies showed that CD20 is involved in regulating intracytoplasmic free calcium concentrations ([Ca2+]i) in normal B lymphocytes, we measured [Ca2+]i in tumor cell samples from patients with HCL and studied the effect of IFN-alpha on this parameter. Using the Ca(2+)-sensitive fluorophore fura-2, we observed that hairy cells display a slightly but consistently higher [Ca2+]i than normal 48-hour-activated B cells or other leukemic cells. Furthermore, both in vitro preincubation of cell samples with IFN-alpha and in vivo administration of this cytokine reduced the [Ca2+]i in hairy cells. This effect was observed together with a decrease in transmembrane Ca2+ influx. However, preincubation with IFN-gamma had no effect. The in vivo correlation between the diminution of CD20 phosphorylation and [Ca2+]i in tumor cell samples from patients at the beginning of IFN-alpha therapy suggests that these two parameters are connected.

Hyperphosphorylation of CD20 in hairy cells. Alteration by low molecular weight B cell growth factor and IFN-alpha.

Hairy cell leukemia (HCL) is a B cell tumor affecting the pre-plasma stage of B cell differentiation. Hairy cells produce B cell growth factor (BCGF)-related growth factor(s) and we have previously shown that low mol wt (LMW)-BCGF-induced proliferation of hairy cells is inhibited in vitro and in vivo by IFN-alpha. We therefore suggested that this effect might contribute to the exquisite sensitivity of HCL to IFN-alpha therapy. To elucidate the mechanism involved in the therapeutic effect of IFN-alpha, we have analyzed the pattern of phosphorylated proteins in hairy cells. We detected the presence of a hyperphosphorylated protein with a molecular mass of about 35 kDa. This protein was identified as the CD20 molecule (B1), which is a structurally unique phosphoprotein exclusively detected on B cells and expressed during most stages of B cell development. Incubation of hairy cells with mitogenic concentrations of LMW-BCGF induces an additional increase in CD20 protein phosphorylation. In contrast, preincubation of cells with IFN-alpha, but not IFN-gamma, decreases both basal and LMW-BCGF-induced CD20 phosphorylation. CD20 phosphorylation in hairy cells is also reduced after in vivo IFN-alpha administration. In contrast, in one case of a patient unresponsive to IFN-alpha therapy, CD20 phosphorylation is not altered by in vitro IFN-alpha treatment, whereas LMW-BCGF still elicits CD20 phosphorylation stimulation. Our results suggest that IFN-alpha may act in HCL, at least in part, by inhibiting leukemic cell proliferation via regulation of phosphorylation, since CD20 phosphorylation is thought to be associated with cellular proliferation. A model involving dysregulation of CD20 is discussed.

The CD44v7/8 epitope as a target to restrain proliferation of fibroblast-like synoviocytes in rheumatoid arthritis.

CD44 is a receptor for the glycosaminoglycan hyaluronan. It exists in a large range of isoforms because of variability in the pattern of glycosylation (both N- and O-linked) and of multiple splice variants. Human fibroblast-like synoviocytes derived from patients with rheumatoid arthritis express certain CD44 splice variants and we have investigated the functional implications of their expression. We found that the rate of proliferation of fibroblast-like synoviocytes expressing the CD44v7/8 epitope (average doubling time 55 hours) exceeds those obtained from the same synovial specimen but lacking this particular epitope (69 hours). Antibodies against CD44v7/8, but not against other exons, inhibit cell proliferation with concomitant induction of the cell cycle inhibitors GADD45, GADD153 and the cyclin-dependent protein-kinase inhibitors p21Waf/Cip. These data show that expression of CD44v7/8 contributes to the transformed phenotype of fibroblast-like synoviocytes. More importantly, they reveal the presence of a target that might be amenable to pharmacological intervention in the treatment of rheumatoid arthritis.

Effect of bacterial toxins on human B cell activation. I. Mitogenic activity of pertussis toxin.

Pertussis toxin (PT) was found to elicit an increased thymidine uptake in resting B lymphocytes purified from human peripheral blood. A significant mitogenic effect was detected for toxin concentrations greater than 100 ng/ml (1nM) and a plateau of stimulation was reached at 1000 ng/ml (10 nM). B cell blasts, activated by a first signal such as Staphylococcus aureus Cowan I or insolubilized anti-mu chain antibody, were also stimulated to DNA synthesis by PT in the same range of concentrations. At lower sub-mitogenic concentrations, the toxin potentiated the response to the low-molecular weight B cell growth factor (LMW-BCGF or 12-kDa BCGF), a progression factor for activated B cells. The "A" or catalytic subunit was devoid of any activity on B cells, suggesting the stimulatory effect of the toxin might be associated with the binding or "B" subunit, as it has been shown for T cells. This hypothesis was strengthened by the observation that, as in T cell, the whole toxin but not the "A" promoter, was able to induce calcium influx in these cells. In addition, the purified "B" oligomer alone was found to promote DNA synthesis in B cells. Finally, a fragment of the soluble cleaved form of the CD23 molecule (Fc epsilon RII) could be involved in the process of PT mitogenicity for B cells.

Inhibition of the proliferative response of human B lymphocytes to B cell growth factor by transforming growth factor-beta.

The effects of transforming growth factor-beta (TGF-beta) on the proliferative response of human B cells to the low molecular weight B cell growth factor (BCGF) have been investigated in this study. It was found that TGF-beta, at picomolar concentrations, strongly inhibited the BCGF-induced proliferation of anti-mu chain or Staphylococcus aureus Cowan I-activated human B cells and also of a BCGF-dependent cell line derived from a human lymphocytic nodular lymphoma. This inhibitory effect was detected in normal and serum-free culture conditions. The suppression was greatly reduced when TGF-beta was added to the culture one day after BCGF and could be reverted by removing TGF-beta from the culture medium. Since TGF-beta has been detected in supernatants from activated T cells, this factor may represent an important regulatory molecule in the feedback control of B cell activation.