Pertussis toxin-sensitive G-proteins are not involved in activation of T-lymphocytes.

Multiple effects of pertussis toxin (PT) on Jurkat T-cells can be distinguished on the basis of their dose-response and their kinetics. High concentrations of PT deliver to cells an activating signal resulting in a rapid rise in [Ca2+]i followed by IL-2 synthesis. This activation is accompanied (within 2 h) by a down-regulation of the CD3/TCR complex from the cell surface. Cells then become refractory towards stimulation by CD3 mAb or PHA. All these effects, referred to as 'mitogenic effects', present the same dose-response curves with an EC50 of 0.5 micrograms/ml. Short term effects (PT-induced Ca2+ movements, down-regulation of CD3/TCR complex and inhibition of PHA and CD3-induced Ca2+ signal) are observed under conditions where no PT-induced ADP-ribosylation can be detected. In contrast, ADP-ribosylation of the 40,000 alpha-subunit of G-proteins requires a sustained (18 h) incubation of intact cells in the presence of low concentration (EC50 = 0.3 ng/ml) of PT. Dose-response curves for PT-dependent ADP-ribosylation and mitogenic effects are separated by three orders of magnitude. Covalent modification of G-protein has no effect on CD3-induced increase in [Ca2+]i and IL-2 synthesis induced by a combination of phorbol ester and either CD3 mAb, PHA or calcium ionophore. These data indicate that transduction of the mitogenic signal does not involve a PT-sensitive G-protein. Furthermore, inhibition of mitogenic signals following PT treatment results from a PT-induced activation leading to a down-regulation of the CD3/T cell receptor complex.     

Pertussis toxin effects on T lymphocytes are mediated through CD3 and not by pertussis toxin catalyzed modification of a G protein

Pertussis toxin (PT) has been shown to have a variety of effects on T lymphocyte function, and its activity has been used to suggest the involvement of a G protein in the early events of T lymphocyte activation. In this report, the effects of PT on T lymphocytes have been investigated in detail. PT at a concentration of 10 micrograms/ml rapidly stimulated early events that are normally induced by occupancy of the TCR complex in Jurkat cells and cloned, murine CTL including increased intracellular Ca2+ concentration, serine esterase release, and induction of Ag non-specific target cell lysis. However, 1-h treatment with this concentration of PT induced a state that was refractory to further receptor stimulation in Jurkat cells but not cloned CTL although substrate membrane proteins were modified to a similar extent in both cell lines. The functional effects of PT were mimicked by the B oligomer of PT which did not, however, catalyze ADP-ribosylation of membrane proteins. In addition, overnight exposure of Jurkat cells to a lower concentration of PT also modified substrate membrane proteins but did not inhibit receptor stimulation. These findings indicate that PT catalyzed ADP-ribosylation of a G protein does not account for the actions of the toxin on T lymphocytes. Finally, direct stimulation of increased intracellular Ca2+ concentration by PT and the B oligomer only occurred in T lymphocytes expressing CD3. This suggests that the mitogenic effect of PT holotoxin is mediated by the interaction of the B oligomer with CD3 and that this may account for many of the effects of PT holotoxin both in vivo and in vitro.     

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.     

Difference in effects of sodium fluoride and cholecystokinin on diacylglycerol accumulation and calcium increase in guinea pig gastric chief cells

In isolated guinea pig gastric chief cells, sodium fluoride (NaF) stimulated a monophasic increase in diacylglycerol accumulation, while cholecystokinin (CCK) strongly stimulated its biphasic accumulation. NaF evoked an increase in initial Ca2+ influx rate with a slow increase in intracellular free Ca2+ concentration [( Ca2+]i), while CCK stimulated a rapid increase in [Ca2+]i followed by a late sustained phase of the [Ca2+]i increase. Lanthanum chloride (La3+) effectively blocked NaF-stimulated increase in [Ca2+]i, but it blocked only CCK-stimulated late sustained phase of [Ca2+]i increase. The effect of NaF on pepsinogen secretion was enhanced in the presence of 100 microM AlCl3. Furthermore, pertussis toxin did not affect NaF-evoked diacylglycerol accumulation at all. These results suggest that NaF may activate a pertussis-toxin insensitive guanine nucleotide regulatory protein (G protein) coupled to a signal transducing mechanism which seems to be distinct from that activated by CCK, thereby inducing increases in diacylglycerol accumulation, Ca2+ influx and pepsinogen secretion in guinea pig gastric chief cells.     

Effects of modulators of adenylyl cyclase on interleukin-2 production, cytosolic Ca2+ elevation, and K+ channel activity in Jurkat T cells

We have studied the effects of prostaglandin E2 (PGE2) and cholera toxin, two modulators of adenylyl cyclase, and 8-bromo cAMP (8-BrcAMP) on various parameters of lymphocyte activation using the human T cell line Jurkat. Our results show that PGE2 and cholera toxin inhibit, in a dose-related manner, the phytohemagglutinin (PHA)-dependent production of interleukin 2 by these cells. The data are consistent with the interpretation that the inhibition is due to an intracellular increase in cAMP, since the metabolically stable 8-BrcAMP analog produced the same inhibitory effect. However, PGE2 or 8-BrcAMP did not interfere with the PHA-induced elevation in the cytosolic concentration of Ca2+, suggesting that changes in the intracellular concentration of cAMP does not affect the internal release or the influx of Ca2+. In contrast, cholera toxin prevented the Ca2+ response of Jurkat cells to PHA. We studied the effects of PGE2, cholera toxin, and 8-BrcAMP on the amplitude of the K+ outward current using the patch clamp technique in the whole cell configuration. Results showed that PGE2, 8-BrcAMP, and cholera toxin inhibited K+ channel activity. For instance, the amplitude of the outward K+ current was reduced to 43 +/- 19%, 50 +/- 26%, and 46 +/- 16% of control values in the case of cells perfused in the presence of PGE2, 8-BrcAMP, and cholera toxin, respectively. Blocking K+ channels with tetraethylammonium ions did not prevent the characteristic Jurkat Ca2+ response to PHA. Our observations that cAMP inhibits K+ channel activity in a T cell line provide an additional explanation for its reported inhibition of lymphocyte activation. Increasing the intracellular concentration of cAMP may result in reduction of K+ movements and in negative modulation of signal transduction via G-proteins as previously suggested. These two effects could act in synergy to impair signal transduction.     

Regulation of the CD2 alternate pathway of T cell activation by CD3. Evidence for heterologous desensitization

Normal resting T cells were stimulated through the alternate CD2 pathway. A CD3 mAb VIT3 completely blocked their proliferative response. The time interval for 50% inhibition lasted for 24 h after the onset of CD2 stimulation. Mitogen-activated cloned long term cultured T cells could also be stimulated via CD2. This proliferative response was again inhibitable by VIT3, indicating that CD3 regulates the CD2 pathway not only in resting cells, but also in lymphocytes actively involved in an Ir. T cells were further loaded with Quin2 and their free cytoplasmic Ca2+ levels were monitored in response to CD3 and CD2 stimulation. Antibodies directed against both surface R triggered a rapid elevation of Ca2+ levels. Both responses were abrogated when the cells had been treated overnight with VIT3. The free cytoplasmic Ca2+ levels of VIT3-pretreated cells, however, were not higher than those of control cells. These results point to a functional interaction between CD3 and CD2 possibly at the level of signal transducing proteins. Finally, cholera toxin was found to inhibit the Ca2+ response in Jurkat T cells. Both the CD3 and CD2 stimulation were sensitive to cholera toxin, indicating that a GTP-binding protein may be involved in signal transduction for both surface structures.     

Two different signal transductions of neuropeptide Y1 receptor in SK-N-MC cells.

The signal transduction systems of the neuropeptide Y (NPY) Y1 receptor were studied in SK-N-MC human neuroblastoma cells. NPY induced an increase in intracellular calcium ion concentration ([Ca2+]i) and inhibition of forskolin-stimulated cyclic AMP accumulation, which were mediated through Y1 receptors. One-min preincubation of cells with phorbol 12-myristate 13-acetate (PMA) inhibited both signal transductions dose-dependently, but its effect on [Ca2+]i was about 100-fold more potent than that on cyclic AMP. PMA had no effect on [125I]BH-NPY binding in SK-N-MC cells and hardly inhibited the endothelin-1-induced increase in [Ca2+]i. Pertussis toxin also inhibited the NPY-induced [Ca2+]i increase 30-fold more effectively than the NPY-mediated inhibition of cyclic AMP accumulation. These results indicate that Y1 receptors in SK-N-MC cells couple to two signal transduction systems that have different sensitivities to phorbol ester and pertussis toxin treatments.     

The role of the L3T4 molecule in mitogen and antigen-activated signal transduction

We investigated the role of the L3T4 molecule in mitogen and antigen-initiated signal transduction in the L3T4(+) murine T cell hybridoma, 3DT52.5.9 and an L3T4(-) variant, 3DT52.5.24. Both Concanavalin A (Con A) and specific antigen stimulated increases in cytosolic-free calcium ([Ca2+]i), phosphatidylinositol turnover, and interleukin-2 (IL-2) production in both cell lines. About 85% of the stimulated rise in [Ca2+]i was from an extracellular source. Anti-L3T4 monoclonal antibody (MAb) inhibited 90% of antigen- and 50% of Con A-stimulated increases in [Ca2+]i and IL-2 production but had no effect on the ability of either activation signal to stimulate phosphatidylinositol turnover in the parent L3T4(+) cells. Stimulus-response coupling in the L3T4(-) cells was unaffected by the MAb. The anti-L3T4-insensitive increase in [Ca2+]i induced by Con A was inhibited by EGTA, suggesting that this mitogen also stimulated an influx of Ca2+ via an additional transport mechanism distinct from that stimulated by antigen. The fact that anti-L3T4 antibodies inhibit antigen and Con A-stimulated Ca2+ transport and IL-2 production without affecting phosphatidylinositol turnover suggests that L3T4 may play a critical role in modulating the activation of the T cell receptor-associated Ca2+ transporter in T cell stimulus-response coupling.     

Dissection of thymocyte signaling pathways by in vivo expression of pertussis toxin ADP-ribosyltransferase.

Stimulation of the T lymphocyte antigen receptor-CD3 complex (TCR-CD3) causes T cell activation by a process associated with increased phosphatidylinositol-specific phospholipase C (PI-PLC) activity. Evidence exists suggesting that GTP-binding (G) proteins, particularly the pertussis toxin (PT)-sensitive Gi proteins, participate in this signal transduction pathway. To clarify the role of Gi proteins in TCR-CD3 signaling, and to investigate other possible functions of Gi molecules in T cells, we expressed the S1 subunit of PT in the thymocytes of transgenic mice using the lymphocyte-specific lck promoter. Transgenic thymocytes contained S1 activity and exhibited profound depletion of Gi protein PT substrates in a manner suggesting their inactivation by S1 in vivo. Nevertheless, treatment of transgenic thymocytes with mitogenic stimuli provoked normal increases in intracellular free Ca2+ concentrations and IL-2 secretion, indicating that Gi proteins are not required for T cell activation. These normal signaling responses notwithstanding, mature thymocytes accumulated in lck-PT mice and did not appear in secondary lymphoid organs or in the circulation. Viewed in the context of the known features of Bordetella pertussis infection, our results suggest that a PT-sensitive signaling process, probably involving Gi proteins, regulates thymocyte emigration.     

Identification of a 43-kilodalton human T lymphocyte membrane protein as a receptor for pertussis toxin

Pertussis toxin (PTx), an exotoxin of Bordetella pertussis has been used as a molecular probe to study stimulus-response coupling in a wide variety of cells. We have previously shown that PTx activates the same signal transduction pathways as Ag or mAb directed against the CD3-T cell Ag receptor complex in human T cells. Because the EC50 for mitogenic stimulation by PTx was 1.7 nM, we suspected that the toxin was specifically interacting with a membrane protein or receptor. We have used both chemical cross-linking and Western blotting techniques to demonstrate that PTx shows specific binding to a 43 kDa-membrane protein on cells that respond to PTx by rapid second messenger production. The PTx receptor can be detected in both the E6-1 Jurkat cell line and a CD3-TCR-negative Jurkat line, demonstrating that it is not coordinately expressed with the Ag receptor complex. The 43 kDa-protein is also found in the HPB-ALL human T cell line and PBL, but not in a murine T cell hybridoma or human neutrophils, both of which are unresponsive to PTx activation. These data suggest that the biochemical basis for the mitogenic activity of PTx may lie in its binding to a specific membrane receptor that is capable of transmitting an activation signal.     

Studies of Signal Transduction of Salicylic Acid in Cucumber Cells

For the first time, the signal transduction pathway of salicylic acid (SA) was investigated by using 3H-labelling, thin-layer chromatography and anion exchange column chromatography. It was found that SA stimulated the activity of membrane bound phospholipase C (PLC), accelerated the bm&down of phosphatidylinositol-4-monophosphate (PIP) and phosphatidylinositol-4,5-bisphos- phate (PIP2) and increased the levels of inositol-1,4-bisphosphate (IP2), inositol-1, 4,5-trisphos- phate (IP3) and diacylglycerol (DAG). These indicated that signal transduction of SA was probably accomplished through the mediation of phosphatidylinositide signal transduction system in cucumber ( Cucumis sativa L. ).     

Cell shape and increased free cytosolic calcium [Ca2+]i induced by growth factors

Growth factors stimulate DNA synthesis of neoplastic cells but not of non-neoplastic cells in suspension cultures. Similarly, growth ceases in dense monolayers of non-neoplastic cells, while crowded neoplastic cells continue to grow. The mechanism of these important phenotypic changes is unknown; the block in growth stimulation could occur in early events of signal transduction at the plasma membrane or in a late step in the final steps of gene activation and induction of DNA synthesis. One particular early intracellular event, [Ca2+]i increases, is in fact necessary for the induction of DNA synthesis in attached non-neoplastic Balb/c 3T3 cells stimulated by platelet-derived growth factor (PDGF). We therefore used digital image analysis of intracellular Fura-2 fluorescence to determine whether PDGF can stimulate [Ca2+]i transients in suspension or in dense monolayer cultures of Balb/c 3T3 cells. In dense cells (greater than 8 x 10(4) cells/cm2) the basal [Ca2+]i and [Ca2+]i response to PDGF stimulation were both lower than those in sparser, more spread cells. PDGF also did not release internal stores of Ca2+ or produce Ca2+ influx in completely suspended cells. Remarkably, attachment alone, with minimal cell spreading, was enough to reinitiate the entire early signalling mechanism stimulated by PDGF. Thus, a block in PDGF-induced [Ca2+]i increases may contribute to the inability of PDGF to stimulate DNA synthesis in suspended non-neoplastic cells. This early block in signal transduction must be abrogated in neoplastic cells growing in suspension and dense monolayer cultures.     

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。     

Pertussis toxin abolishes angiotensin II-induced phosphoinositide hydrolysis and prostaglandin synthesis in rat renal mesangial cells.

Incubation of rat renal mesangial cells with angiotensin II (0.1 microM) resulted in transient breakdown of phosphatidylinositol 4,5-bisphosphate, rapid generation of diacylglycerol and phosphatidic acid, increased 45Ca2+ influx, increased intracellular [Ca2+] as measured by quin 2, and increased prostaglandin E2 synthesis. All of these processes were markedly inhibited time- and dose-dependently by prior exposure of cells to pertussis toxin. In contrast, the effects of the ionophore A23187 on 45Ca2+ influx and prostaglandin E2 synthesis were not altered by the exposure of the cells to pertussis toxin. The action of the toxin was not associated with alterations in cellular concentrations of cyclic AMP. Incubation of membrane fraction of mesangial cells with pertussis toxin resulted in ADP-ribosylation of Mr-42,000 protein. From all these results, it is likely that a G protein is involved in receptor-mediated signal transduction in renal mesangial cells.     

Cholera toxin inhibits the increase in cytoplasmic free calcium induced via the CD2 pathway of human T-lymphocyte activation

We investigated the action of cholera toxin on the intracellular ionized calcium [Ca2+]i increase induced by anti-CD2 and anti-CD3 monoclonal antibodies in the leukemic human T-cell line Jurkat. Cholera toxin inhibits in a dose-dependent manner these two pathways of human T-lymphocyte activation but with different half maximal inhibition doses (75 ng/ml for CD3, 30 ng/ml for CD2). This effect cannot be accounted for only by the increase in cAMP induced by cholera toxin because forskolin, which raises cellular cyclic adenosine monophosphate (cAMP) to the same levels, induced only a small inhibition of the [Ca2+]i increase in similar conditions. Cholera toxin induced a decrease in the surface expression of the CD3 molecule, suggesting a down-regulation of the CD3 molecules. On the other hand, the expression of CD2 remained unchanged. Cell surface disappearance of the CD3 molecule cannot account for all the inhibitory effects of cholera toxin because CD2 molecule expression was not affected (no modifications in the half maximal binding of anti-CD2 monoclonal antibodies). All together, these results suggest that cholera toxin acts on substrates, possibly G proteins, that could regulate the [Ca2+]i increase induced by anti-CD2 and anti-CD3 mAbs in Jurkat cells. In addition, the present study demonstrated that the rise in cellular cAMP partially inhibits the [Ca2+]i increase induced by anti-CD2 and anti-CD3 mAbs.     

Signaling from LFA-1 contributes signal transduction through CD2 alternative pathway in T cell activation.

LFA-1, a member of the integrin family of molecules, is involved in mediating cellular adhesion in all phases of the immune response, playing a role in the interaction of helper T cells as well as in killing of target cells by both cytotoxic T cells and natural killer cells. We have developed a monoclonal antibody, anti-HVS6B6, which recognizes a functionally unique epitope of the LFA-1 molecule. Although this mAb itself was not mitogenic against T cells, it induced a strong proliferative response when added to T cells with submitogenic concentrations of anti-CD2 (anti-T11(2) and anti-T11(3)) mAbs. In contrast, other anti-LFA-1 mAbs (CD11a and CD18) suppressed this anti-CD2 mAb-induced T cell proliferation. Kinetic studies showed that anti-HVS6B6 acts on an early event in CD2-mediated T cell activation. Although T11(3)-epitope expression induced by anti-T11(2) mAb was not affected by treatment of cells with anti-HVS6B6, both Ca2+ influx and phosphatidylinositol turnover induced by anti-CD2 mAbs were markedly enhanced by the pretreatment of T cells with anti-HVS6B6 mAb. These results indicate that the LFA-1 mediating signal contributes to a very early phase of signal transduction during CD2-mediated T cell activation.     

Synergy between the T3/antigen receptor complex and Tp44 in the activation of human T cells

In addition to the T3/antigen receptor complex (T3/Ti), other T cell surface molecules participate in early events involved in human T cell activation. In this report we document that monoclonal antibody 9.3, which recognizes a 90,000 dalton homodimer expressed on human T cells, synergizes with ligands reacting with T3/Ti to activate purified T cells and Jurkat, a human T cell leukemic line. Unlike phorbol myristate acetate (PMA), 9.3 was able to synergize only with anti-T3 or anti-Ti if these antibodies were immobilized. Moreover, 9.3 failed to synergize with the calcium ionophore ionomycin. At high concentrations only, 9.3 could synergize with PMA in the activation of Jurkat and a T3/Ti negative mutant of Jurkat. At such high concentrations of 9.3, small transient increases in cytoplasmic free calcium ((Ca++)i) were detected in quin 2-loaded Jurkat cells. This increase in (Ca++)i was the result of release of internal stores of calcium. 9.3 induced the hydrolysis of polyphosphoinositides, albeit the magnitude of inositol phosphates generated in response to 9.3 was substantially less than that observed with anti-Ti. No effect on pkC translocation was observed in Jurkat cells stimulated with 9.3. Although the small increase in (Ca++)i induced by 9.3 may account for its synergy with PMA, this effect is unlikely to account for the more potent synergistic effect observed with 9.3 and phytohemagglutinin or immobilized anti-T3 and anti-Ti antibodies.     

Pertussis toxin B-subunit-induced Ca2(+)-fluxes in Jurkat human lymphoma cells: the action of long-term pre-treatment with cholera and pertussis holotoxins

The exotoxins of Bordetella pertussis and Vibrio cholera have been used to investigate signal transduction in the human T-cell lymphoma Jurkat. Stimulation of the cells, leading to an increase in cytoplasmic free calcium, could be achieved by the anti-T-cell receptor complex antibody OKT3 and by pertussis holotoxin (PTHT), or its B-subunit (PTB), but not by cholera holotoxin (CTHT) or its B-subunit (CTB). Both holotoxins ADP-ribosylated specifically G-proteins in the plasma membrane of intact cells, while their B-subunits had no ADP-ribosyltransferase activity. Incubation of the cells with CTHT led to a state of unresponsiveness to all stimulants. CTB was without any effect, indicating that the ADP-ribosyltransferase activity of cholera toxin (located in the A-subunit of the holotoxin) was necessary for the inhibition of cellular signalling. The inhibitory effect of cholera toxin on the pertussis toxin action was not due to a blockade of pertussis toxin interaction with the cell surface, because pertussis toxin was still able to ADP-ribosylate membrane proteins in cholera toxin treated intact cells. In addition, the cholera toxin mediated inhibition was not due to elevated levels of cyclic-AMP, as forskolin (a direct activator of the adenylate cyclase) and no inhibitory effect. The stimulating effect of PTHT was independent of its ADP-ribosyltransferase activity, because it could also be obtained by the B-subunit alone. In addition, the increase of cytoplasmic free calcium after stimulation by PTHT clearly preceded the ADP-ribosylation. Pre-treatment with PTHT, PTB or OKT3, led to a long lasting increase in the level of intracellular Ca2+ in Jurkat cells, which could not, therefore, be stimulated further. Inhibition by cholera holotoxin of the stimulation by OKT3 and pertussis toxin (PTHT and PTB) imply that the mitogenic effect of pertussis toxin is perhaps mediated via the T-cell antigen receptor signalling cascade. The presented data do not support the idea that a pertussis toxin-sensitive G-protein is involved in coupling the T-cell antigen receptor to the phospholipase C.