Modulation of CD40L antigen expression in Jurkat cells: involvement of protein kinase C activity

The CD40L expressed on activated CD4+ T cells delivers contact-dependent proliferative and anti-apoptotic signals to B lymphocytes. Little is known about molecular mechanisms of constitutive expression of CD40L on some non-Hodgkin's lymphomas, especially about involvement of two signal pathways regulating its expression in normal cells; one involving calcineurin, and the other protein kinase C. We analyzed by flow cytometry the effects of 6-hour stimulation of both pathways (stimuli: PMA and ionomycin) and their inhibitors: cyclosporin A and chelerythrine, on CD40L expression. Two Jurkat clones differing in CD40L surface expression: clone 217.6 (CD40L-) and 217.7 (CD40L+) were studied. Our experiments showed that high level of CD40L expression on the surface of 217.7 cells was reduced after stimulation with PMA. The same effect was observed for combination of PMA and chelerythrine or for PKC inhibitor alone. In 217.6 cells, only chelerythrine used alone induced low level of CD40L expression, while PMA and ionomycin were without effect. These results suggest that CD40L surface expression is mainly dependent on protein kinase C activity. By using PepTag Assay we have confirmed that in both Jurkat clones PKC activity is higher than in normal blood lymphocytes.     

Activation of human T cell clones and Jurkat cells by cross-linking class I MHC molecules

Cross-linking class I MHC molecules on human T cell clones by reacting them with various mAb directed at either monomorphic or polymorphic determinants on class I MHC molecules followed by cross-linking with GaMIg stimulated a rise in intracellular free calcium concentration ([Ca2+]i), and induced proliferation and IL-2 production. T cell clones varied in the mean density of class I MHC molecules and the capacity to respond to mAb to class I MHC molecules. However, the functional responses of the clones did not correlate with class I MHC density or the CD4/CD8 phenotype. mAb to polymorphic class I MHC determinants were less able to induce an increase in [Ca2+]i and a functional response in the T cell clones. Additive stimulatory effects were noted when mAb against both HLA-A and HLA-B determinants were employed. Cross-linking class I MHC molecules on Jurkat cells induced a rise by [Ca2+]i and induced IL-2 production upon co-stimulation with PMA. Cross-linking class I MHC molecules on mutant Jurkat cells that expressed diminished levels of CD3 and were unable to produce IL-2 in response to anti-CD3 stimulation triggered both a rise in [Ca2+]i and IL-2 production with PMA co-stimulation. In contrast, cross-linking class I MHC molecules on mutant Jurkat cells that were CD3- stimulated neither a rise in [Ca2+]i nor IL-2 production. The combination of mAb to CD28 or ionomycin and PMA, however, was able to induce IL-2 production by CD3- Jurkat cells. The data demonstrate that cross-linking class I MHC molecules delivers a functionally important signal to T cell clones and Jurkat cells and indicate that class I MHC molecules may function to transduce activation signals to T cells. In addition, the data demonstrate that transmission of an activation signal via class I MHC molecules requires CD3 expression. The data, therefore, support a central role for CD3 in the transduction of activation signals to T cells via class I MHC molecules.     

Heterogeneity of the regulation of phospholipase C by phorbol esters in T lymphocytes

In many cells, protein kinase C (PKC) activation inhibits cellular phospholipase C thereby preventing receptor-mediated phosphatidylinositol (PI) metabolism. In T lymphocytes, the T cell antigen receptor (Ti)/CD3 complex regulates PI hydrolysis and we have examined the consequences of PKC activation on Ti/CD3-mediated PI metabolism in human peripheral blood-derived T lymphocytes (T lymphoblasts) and the leukemic T cell line Jurkat. In Jurkat cells, PI metabolism after Ti/CD3 stimulation, is inhibited by PKC activation. PKC activation also inhibits calcium-induced PI metabolism in permeabilized Jurkat cells. In marked contrast, PI metabolism after Ti/CD3 stimulation in T lymphoblasts, is not inhibited by PKC activation. Moreover, in permeabilized T lymphoblasts PI metabolism can be induced by calcium in synergy with guanine 5'-O-(3-thiotrisphosphate) via a PKC-insensitive mechanism. The different effect of PKC stimulation on PI metabolism in Jurkat cells and T lymphoblasts reveals heterogeneity of PLC regulation in T lymphocytes. The data also indicate that the role of PKC as a regulator of Ti/CD3 signal transduction can differ depending on cell type.     

T cell activation via Leu-23 (CD69)

The CD69 (Leu-23) activation Ag is a phosphorylated 28 to 32-kDa disulfide-linked homodimer that is rapidly induced after lymphocyte activation. CD69 is not present on the surface of peripheral blood resting T cells, but is constitutively expressed by CD3bright thymocytes. Activation of protein kinase C (PKC) by stimulation of the TCR/CD3 or by phorbol esters directly induces CD69 expression on T cells. In the attempt to elucidate the function of CD69 we investigated the ability of the CD69 glycoprotein to transmit an activation signal. Cross-linking of CD69 by mAb induced a prolonged elevation of intracellular [Ca2+], mostly due to an influx of extracellular Ca2+. This signal alone was unable to effectively activate PKC. When PKC was simultaneously activated by PMA, stimulation of CD69 induced IL-2 and IFN-gamma gene expression, enhancement of CD25 expression, and ultimately IL-2-dependent T cell proliferation. Both CD4+ and CD8+ peripheral T cells responded to CD69-mediated activation. Stimulation of CD69 induced proliferation of thymocytes as well as peripheral T cells, but both required independent PKC activation by PMA. Cyclosporin A, which does not prevent PKC-induced CD69 expression, completely suppressed CD69-induced IL-2 and IFN-gamma gene expression. Although the signal delivered by the CD69 initiates T cell proliferation, it is unable to trigger cytotoxicity programs in CD69+-activated T cells or T cell clones.     

Bcl－2 over－expression and activation of protein kinase C suppresss the Trail－induced apoptosis in Jurkat T cells

Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role, like FasL in activation-induced cell death (AICD), has been demonstrated in immune system. However the mechanism of Trail induced apoptosis remains unclear. In this report, the recombinant Trail protein was expressed and purified. The apoptosis-inducing activity and the regulation mechanism of recombinant Trail on Jurkat T cells were explored in vitro. Trypan blue exclusion assay demonstrated that the recombinant Trail protein actively killed Jurkat T cells in a dose-dependent manner. Trail-induced apoptosis in Jurkat T cells were remarkably reduced by Bcl-2 over expression in Bcl-2 gene transfected cells. Treatment with PMA (phorbol 12-myristate 13-acetate), a PKC activator, suppressed Trail-induced apoptosis in Jurkat T cells. The inhibition of apoptosis by PMA was abolished by pretreatment with Bis, a PKC inhibitor. Taken together, it was suggested that Bcl-2 over-expression and PMA activated PKC actively down-regulated the Trail-mediated apoptosis in Jurkat T cell.     

Bcl-2 over-expression and activation of protein kinase C suppress the Trail-induced apoptosis in Jurkat T cells

INTRODUCTIONThaillll (TNF-related apoptosis inducing ligand)is a recently described member of the TNF family.Like other members of the TNF ligand family) availcould induce apoptosis of neoplastically transformedcells by priVating cell surface death receptors ThailRI and ThaiLR212].Trail has been demonstrated to play an important role in homeostasis of immune system includ.lug eradication of the old lymphocytel3], actiVationinduced T cell deathI41, regulation Of T cen eXPansion by…     

Protein kinase C plays a role in the induction of tyrosine phosphorylation of lymphoid microtubule-associated protein-2 kinase. Evidence for a CD3-associated cascade that includes pp56lck and that is defective in HPB-ALL.

Ligation of the CD3 receptor induces multiple signal transduction events that modify the activation state of the T cell. We have compared two lines that express biologically active CD3 receptors but differ in their biochemical activation pathways during ligation of this receptor. Jurkat cells respond to anti-CD3 with Ca2+ mobilization, PKC activation, induction of protein tyrosine phosphorylation, and activation of newly characterized lymphoid microtubule associated protein-2 kinase (MAP-2K). MAP-2K itself is a 43-kDa phosphoprotein that requires tyrosine phosphorylation for activation. Although ligation of the CD3 receptor in HPB-ALL could stimulate tyrosine phosphorylation of a 59- kDa substrate, there was no associated induction of [Ca2+]i flux, PKC, or MAP-2K activation. A specific PKC agonist, PMA, which bypasses the CD3 receptor, could, however, activate MAP-2K in HPB-ALL cells. This implies that defective stimulation of PKC by the CD3 receptor is responsible for its failure to activate MAP-2K in HPB-ALL. The defect in PKC activation is likely distal to the CD3 receptor as A1F14- failed to activate MAP-2K in HPB-ALL but was effective in Jurkat cells. The stimulatory effect of PMA on MAP-2K activity in HPB-ALL was accompanied by tyrosine phosphorylation of this kinase which implies that PKC may, in some way, regulate tyrosine phosphorylation of MAP-2K. A candidate for this role is pp56lck which underwent posttranslational modification (seen as mobility change on SDS-PAGE) during anti-CD3 and PMA stimulation in Jurkat or PMA treatment in HPB-ALL. There was, in fact, exact coincidence between induction of PKC activity, posttranslational modification of lck and tyrosine phosphorylation/activation of MAP-2K. Lck kinase activity in an immune complex kinase assay was unchanged during PMA treatment. An alternative explanation is that modification of lck may alter its substrate profile. We therefore looked at the previously documented ability of PKC to dissociate lck from the CD4 receptor and found that PMA could reduce the stoichiometry of the lck interaction with CD4 in HPB-ALL and to a lesser extent in Jurkat cells. These results imply the existence of a kinase cascade that is initiated by PKC and, in the course of which, lck and MAP-2K may interact.     

Signaling through T lymphocyte surface proteins, TCR/CD3 and CD28, activates the HIV-1 long terminal repeat

The state of T cell activation and proliferation controls HIV-1 replication and gene expression. Previously, we demonstrated that the administration of PHA and PMA to the human T cell line Jurkat activates the HIV-1 enhancer, which is composed of two nuclear factor kappa B (NF kappa B) binding sites. Here, we show that PMA alone is sufficient for this effect. In addition, activation of T cells through the surface proteins TCR/CD3 and CD28 increased gene expression directed by the HIV-1 long terminal repeat (LTR) to the same extent as PMA. Analysis of 5' deletions in the LTR revealed that the NF kappa B binding sites and sequences in the upstream U3 region are required for this response. Whereas cyclosporin A did not inhibit the effect of PMA, it reduced the effects of agonists to TCR/CD3 and CD28 on the LTR. H7, an inhibitor of protein kinase C (PKC), blocked the effects of all stimuli. Thus, PMA activates the NF kappa B sites through a PKC-dependent pathway while ligands to TCR/CD3 and CD28 activate the LTR through a cyclosporin A-sensitive, PKC-dependent pathway of T cell activation. We conclude that mechanisms involved in the expression of IL-2 and the alpha-chain of the IL-2R alpha genes also play a role in the regulation of HIV-1. Physiologic stimuli can activate HIV-1 gene expression; agents that block T cell activation also inhibit activation of the LTR. These observations might serve as a model for the regulation of HIV-1 gene expression in peripheral blood T cells.     

Cytotoxic T lymphocyte unresponsiveness induced by prolonged treatment with immobilized anti-CD3 antibody. Association of impairment of cytolytic activity with temporary depletion of intracellular protein kinase C

In our study we investigated the effect of pretreatment of bulk CTL and CTL clones with immobilized anti-CD3 antibody (Ab) or PMA. Primary CTL and CTL clones were cultured in dishes coated with anti-CD3 Ab or in medium containing PMA (5 nM) and assayed for Ag-specific or Ag-nonspecific "redirected" cytolysis using FcR+ P815 cells as targets. Cytotoxic activity of bulk CTL and five of six CTL clones tested in this study were inhibited by prolonged (longer than 6 h) pretreatment with immobilized anti-CD3 Ab or PMA, whereas proliferation of CTL clones or expression of surface CD3 molecules were not. The intracellular granule enzyme (N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester esterase) activity of CTL clones was not reduced under these suppressive conditions, indicating that the incompetence of CTL is not merely due to depletion of cytolytic granules by chronic stimulation. The suppressed cytotoxicity could be recovered by culturing CTL without perturbation of CD3 molecules for 24 h. In one exceptional clone, BM10-37, pretreatment with immobilized anti-CD3 Ab or PMA did not suppress the cytotoxic activity. Immunostaining of intracellular protein kinase C (PKC) revealed that PKC was depleted after prolonged treatment with immobilized anti-CD3 Ab or PMA in those susceptible CTL clones but not in the resistant BM10-37. These findings lead us to conclude that prolonged stimulation of CD3 of CTL results in depletion of PKC and that PKC may be essential for signal transduction to deliver a lethal hit to the target cells.     

Regulation of T cell receptor- and CD28-induced tyrosine phosphorylation of the focal adhesion tyrosine kinases Pyk2 and Fak by protein kinase C. A role for protein tyrosine phosphatases

The T cell receptor (TCR)-CD3 complex and the costimulatory molecule CD28 are critical for T cell function. Both receptors utilize protein tyrosine kinases (PTKs) for the phosphorylation of various signaling molecules, a process that is critical for the function of both receptors. The PTKs of the focal adhesion family, Pyk2 and Fak, have been implicated in the signaling of TCR and CD28. We show here evidence for the regulation of TCR- and CD28-induced tyrosine phosphorylation of the focal adhesion PTKs by protein kinase C (PKC). Thus, treating Jurkat T cells with the PKC activator phorbol 12-myristate 13-acetate (PMA) rapidly and strongly reversed receptor-induced tyrosine phosphorylation of the focal adhesion PTKs. In contrast, PMA did not affect TCR-induced tyrosine phosphorylation of CD3zeta or the PTKs Fyn and Zap-70. However, PMA induced a strong and rapid dephosphorylation of the linker molecule for activation of T cells. PMA failed to induce the dephosphorylation of proteins in PKC-depleted cells or in cells pretreated with the PKC inhibitor Ro-31-8220, confirming the role of PKC in mediating the PMA effect on receptor-induced protein tyrosine phosphorylation. The involvement of protein tyrosine phosphatases (PTPases) in mediating the dephosphorylation of the focal adhesion PTKs was confirmed by the failure of PMA to dephosphorylate Pyk2 in cells pretreated with the PTPase inhibitor orthovanadate. These results implicate PKC in the regulation of receptor-induced tyrosine phosphorylation of the focal adhesion PTKs in T cells. The data also suggest a role for PTPases in the PKC action.     

Quantitative analysis of CD5 antigen modulation by 12-O-tetradecanoylphorbol-13-acetate in T-lymphoblastic leukemia cells: individual response patterns and their relationships with both maturation and protein kinase C content.

Modulation of CD5 expression by TPA was investigated on T-leukemic cell lines corresponding to different stages of ontogeny. These CD5 changes have been analyzed simultaneously with modifications of cell growth, cell cycle, cell surface phenotype, and PKC content. CD5 expression was found 6- to 17-fold increased by TPA in a dose-dependent manner on phenotypically mature T-cells (Jurkat, JM, and T-CLL) while T-cells from earlier stages of differentiation (CEM III, CEM 95, and CEM 44) were found unresponsive. CD5 upregulation on TPA-sensitive JM cells appears correlated with inhibition of cell growth, blockage in G1 phase, and phenotypic maturation (downregulation of CD7 and CD1 antigens) and seemed to be related to PKC activation since DiC8 (a PKC activator) mimicked this TPA effect and H7 (a PKC inhibitor) partially reduced it. On the other hand, on CEM III cells TPA induced no modulation of CD5 antigen, a less dramatic effect on cell growth and cell cycle, but a CD7 downregulation. TPA appeared fully effective in binding and translocating PKC in both CEM III and JM cells, although the PKC activity level was three times higher in the latter. Finally, our study suggests that CD5 expression is at least partially under control of PKC in phenotypically mature neoplastic T-cells while PKC could not be directly involved in the regulation of CD5 antigen in leukemic cells arrested at earlier stages of differentiation.     

Characterization of functional GTP binding proteins in Jurkat T cell mutants lacking either CD3-Ti or CD2 surface receptors

G proteins are membrane-bound molecules involved in coupling of surface receptors with signal transduction effector systems in multiple cell types including T lymphocytes. Given that mature T cells which lack antigen receptors (CDl-Ti) are refractory to stimulation through CD2 or other accessory molecules, T cell receptor components likely play a critical role in coupling surface receptors with signal transduction effectors. It has recently been proposed that modulation of T cell receptor components with MAbs results in a physical loss or functional inactivation of G protein(s). In view of the importance of the T cell activation process, we herein examined G proteins in untreated or antibody-modulated Jurkat T cells as well as in genetic variants lacking either CD3-Ti or CD2 surface receptors. 43- and 41-kDa G protein alpha chains are ADP ribosylated with cholera (CTX) and pertussis (PTX) toxins, respectively, in wild type and receptor minus cell populations. In the wild type Jurkat cell line as well as in CD3- and CD2- variants, AlF4- can activate the G protein(s) presumably associated with phospholipase C to generate polyphosphoinositide turnover as well as an increase in cytoplasmic free calcium ions. Furthermore, G protein(s) linked to adenylylcyclase, a pathway which inhibits T lymphocyte activation, can be directly activated with CTX in the absence of CD3-Ti or CD2 on the membrane. Importantly, AlF4- can also induce polyphosphoinositide turnover in Jurkat cells whose T cell receptor proteins have been modulated with anti-CD3 MAb. These data provide functional and biochemical evidence that at least certain G proteins are intact in the absence of surface expression of CD3-Ti or CD2 molecules and imply that CD3-Ti desensitization is not singularly due to G protein loss.     

Regulation of galectin-9 expression and release in Jurkat T cell line cells

Ecalectin/galectin-9 was recently described as a novel eosinophil chemoattractant highly expressed in immune tissues. We investigated the regulation of galectin-9 expression and release in Jurkat (a T cell line) cells. We demonstrated that medium and long-sized galectin-9 isoforms were constitutively expressed, and phorbol 12-myriastate 13-acetate (PMA) upregulated the level of galectin-9 mRNA in Jurkat cells. Western blotting and flow cytometry analyses revealed that PMA stimulation resulted in the upregulation of both intracellular and surface galectin-9 protein. The stimulated Jurkat cells simultaneously released evident eosinophil chemoattractant activity (ECA). Main ECA was adsorbed by both lactose and anti-galectin-9 antibody affinity column, suggesting that the ECA was ascribed to galectin-9. When Jurkat cells were stimulated with PMA in the presence of a BB94, a matrix metalloproteinase (MMP) inhibitor, but not tissue inhibitor of metalloproteinase-1 (TIMP-1), the release of galectin-9 was suppressed in a dose-dependent manner. We further found that calphostin c, a protein kinase c (PKC) inhibitor, weakly but significantly suppressed the release of galectin-9. The present data suggested that galectin-9 production in Jurkat cells is provoked by the stimulation with PMA and that some MMP and PKC is, at least, partly involved in the release of galectin-9 from Jurkat cells.     

CD59、CD55在T细胞活化信号转导中的协同作用

目的:研究糖基磷脂酰肌醇(GPI)锚固蛋白CD59、CD55在脂筏介导T细胞信号转导通路中的协同效应。方法:应用siRNA技术,构建特异性针对CD55与CD59基因的重组载体pSUPER-siCD55,pSUPER-siCD59。实验分为未转染的Jurkat细胞组(Ⅰ组)、转染pSUPER空质粒的Jurkat细胞组(Ⅱ组)、转染pSUPER-siCD59重组质粒的Jurkat细胞组(Ⅲ组)及转染pSUPER-siCD55重组质粒的Jurkat细胞组(Ⅳ组)。RT-PCR检测转染细胞中CD55和CD59基因的表达噻唑蓝(MTT)比色法和激光共聚焦扫描显微镜分别检测CD55与CD59联合作用对4组Jurkat细胞的增殖效应以及细胞内钙离子的变化、结果:稳定转染后,Ⅲ组细胞CD59分子的表达和Ⅳ组细胞CD55分子的表达被成功抑制。Ⅰ组和Ⅱ组细胞CD55与CD59联合作用后增殖能力和钙离子浓度均明显高于Ⅲ组、Ⅳ组(P<0.05),Ⅰ组和Ⅱ组之间无差异结论:CD59和CD55在T细胞活化信号转导通路中存在协同效应。     

Stimulation of MAP-2 kinase activity in T lymphocytes by anti-CD3 or anti-Ti monoclonal antibody is partially dependent on protein kinase C

Signaling via the alpha-beta T cell Ag receptor (Ti)-CD3 complex is a complicated event that implicates several protein kinases, most notably protein kinase C (PKC). We have recently identified a serine kinase in T lymphocytes with the following characteristics: molecular mass 43 kDa, in vitro substrate affinity for microtubule associated protein 2 (MAP-2) with a preference for Mn2+ during the catalytic reaction, and elution from DEAE resin over a salt range 100 to 200 mM NaCl. This kinase is activated in a rapidly reversible fashion during ligation of CD3/Ti by a process which involves prior phosphorylation; in vitro exposure of activated 43-kDa MAP-2 kinase (MAP-K) to an immobilized phosphatase abrogated its kinase activity. We now show that a MAP-2K response could also be obtained during treatment with mAb to Ti and the specific PKC agonist, PMA. Although the kinetics of the former response was rapidly reversible, PMA elicited a more prolonged response. The dose responsiveness for PMA was similar to the requirements for PKC activation in intact lymphocytes. Moreover, as with PKC, we found that the CD3-induced MAP-2K response could be further enhanced by using a second layer cross-linking antibody. The specificity of CD3/Ti in the Jurkat cell response is demonstrated by the fact that OKT-11(CD2) and anti-CD4 mAb did not stimulate a MAP-2K response. It was also not possible to elicit a response in a Jurkat cell mutant that lacks surface expression of CD3 and Ti. The specificity of PKC in these events was further explored with the cell permeant diacylglycerol, 1-oleoyl-2-acetylglycerol, and the nonagonist phorbol ester, 4 alpha-phorbol 12,13-didecanoate: whereas the former was an effective inducer of the MAP-2K response, the latter failed to yield any stimulation. Prior exposure of Jurkat cells to 100 mM PMA for 24 h eliminated greater than 60% of the MAP-2K response during anti-CD3 treatment. This response could also be inhibited in dose-dependent fashion by prior treatment of Jurkat cells with the potent PKC inhibitor 1-(5-isoquinolinesulfonyl) 2-methylpiperazine dihydrochloride. Although a Ca2(+)-ionophore failed to synergize with PMA at inducing a MAP-2K response, depletion of extracellular Ca2+ by EGTA abrogated anti-CD3 responsiveness. The events culminating in MAP-2K activation were slightly inhibited in the presence of cholera toxin but not pertussis toxin.(ABSTRACT TRUNCATED AT 400 WORDS)