Tunicamycin modulates binding of 125I-erythropoietin to Friend erythroleukemia cells

The effects of tunicamycin and neuraminidase treatment on the specific binding of 125I-erythropoietin to a murine erythroleukemia cell clone, B8, were investigated. Neuraminidase treatment of B8 cells did not affect the specific binding of erythropoietin, but tunicamycin treatment caused a 2.5 to 4-fold increase in the amount of 125I-erythropoietin binding. Scatchard analysis of the binding data showed that the increase in the amount of binding resulted from increased affinity of the receptor. These results suggest that N-linked sugars of the erythropoietin receptor protein are involved in the interaction of erythropoietin with the cell-surface receptors on B8 cells.     

The role of accessory cells in polyclonal T cell activation II. Induction of interleukin 2 responsiveness requires cell-cell contact

It has previously been reported (Hünig, T. et al., Eur. J. Immunol. 1983. 13:1) that highly purified peripheral T cells do not respond to concanavalin A (Con A) even in the presence of Con A-induced spleen cell supernatant as a source of interleukin 2 (IL 2). In the present report, the hypothesis was tested whether this unresponsiveness correlates with the observed inability of Con A to mediate cell-cell contact between highly purified T cells. It was found that T cells, pretreated with neuraminidase to reduce their net negative charge, were both aggregated and rendered IL 2-reactive by Con A. In addition, leukoagglutinin (LA) was found to be able to both agglutinate untreated T cells and to make them IL 2-reactive. Neuraminidase treatment reduced the concentration of LA required to mediate both effects. Neuraminidase treatment did not alter the overall Con A-binding capacity of T cells, nor did it induce reactivity to IL 2 in the absence of lectin. Furthermore, irradiated, neuraminidase-treated T cells served as accessory cells (AC) in the induction of responsiveness to IL 2, but not for production of IL2, which depends on Ia+ AC. Finally, Lyt-2- neuraminidase-treated peripheral T cells responded in the same fashion as whole T cell populations, indicating that at least some T cells of the helper phenotype need not interact with Ia+AC for the induction of IL 2 responsiveness.     

Role of carbohydrate in binding of IgG to the Fc receptor of neonatal rat enterocytes

Monoclonal IgG1, IgG2a, IgG2b and IgG2c were prepared from rat hybridoma cells treated with tunicamycin in order to inhibit N-linked glycosylation. The IgG produced by these cells was about 70% lower in carbohydrate content compared to IgG from equivalent untreated cells, but was similar to the corresponding normal IgG in terms of antigen binding. However, the ability of carbohydrate deficient (CHO-) IgG to bind in vitro to Fc receptor extracted from jejunum of neonatal rats was impaired in most cases and, in all but one case, the amount of CHO- IgG transported from gut lumen to blood in vivo was markedly reduced. No reduction in binding of normal IgG to extracted receptor was observed in the presence of various sugars. It is postulated that N-linked carbohydrate acts to stabilize the structure within the IgG molecule which is responsible for binding to this Fc receptor, possibly in the CH2 domain.     

Synergy of tumor necrosis factor with protein kinase C activators on T cell activation

The ability of tumor necrosis factor (TNF)-alpha to activate T lymphocytes in combination with other stimuli has been studied. TNF was strongly co-mitogenic with low doses of anti-CD3 antibodies or phorbol esters (those which are strong activators of protein kinase C, PKC) but poorly with phytohemagglutinin or concanavalin A. No synergism was seen with the calcium ionophore A23187. TNF was co-mitogenic with several phorbol esters known to activate PKC but was uneffective with inactive phorbol esters such as methyl-phorbol 12-myristate 13-acetate. Furthermore, H-7 a known inhibitor of PKC, inhibited the proliferative response of T cells induced by esters plus TNF. This effect took place at low doses of TNF and was also observed with purified T lymphocytes indicating that the effect of TNF was not dependent on accessory cells. This proliferative effect of TNF was inhibited by an anti-interleukin 2 receptor (IL2R) antibody, MAR 108, which blocks IL2 binding to its receptor. Although PKC activation induced CD25 (IL2R) expression but very little IL2 synthesis, TNF did not synergize by augmenting the synthesis of this lymphokine in peripheral blood lymphocytes stimulated with phorbol esters. By contrast, TNF strongly increased the membrane level of CD25 and to a lesser extent that of the activation antigen, 4F2, over the levels already induced by phorbol esters on T cells. More interestingly, TNF significantly increased the number of high-affinity IL2R on purified T cells in the presence of phorbol 12,13-dibutyrate. Our results indicate that TNF is co-mitogenic with those stimuli which strongly activate PKC and suggest that TNF may play a role on T cell activation increasing the number of effective IL2/IL2R interactions when these are limiting.     

Modification of simian virus 40 large tumor antigen by glycosylation

The SV40-encoded transforming protein, large tumor antigen (T-ag), is multifunctional. Chemical modifications of the T-ag polypeptide may be important for its multifunctional capacity. T-ag is additionally modified by glycosylation. T-ag was metabolically labeled in SV40-infected cells with tritiated galactose or glucosamine, but not with mannose or fucose. The identity of glycosylated T-ag was established by immunoprecipitation with a variety of T-ag-specific antisera, including monoclonal antibodies. Incorporation of labeled sugar into T-ag was inhibited in the presence of excess unlabeled sugars, but not in the presence of excess unlabeled amino acids. Labeled monosaccharides could be preferentially removed from T-ag with a mixture of glycosidic enzymes. In addition, galactose was removed from purified T-ag by acid hydrolysis and identified as such by thin-layer chromatography. T-ag oligosaccharides were resistant to treatment with EndoH, and glycosylation was not inhibited by tunicamycin. Together, these data strongly suggest that T-ag is glycosylated. Several characteristics, including lack of mannose labeling, EndoH resistance, and tunicamycin resistance, suggest that T-ag is not an N-linked glycoprotein. Rather, these properties are more consistent with the identification of T-ag as an O-linked glycoprotein.     

Fingolimod targeting protein phosphatase 2A differently affects IL‐33 induced IL‐2 and IFN‐γ production in CD8+ lymphocytes

Multiple sclerosis patients are treated with fingolimod (FTY720), a prodrug that acts as an immune modulator. FTY720 is first phosphorylated to FTY720‐P and then internalizes sphingosine‐1‐phosphate receptors, preventing lymphocyte sequestration. IL‐33 is released from necrotic endothelial cells and contributes to MS severity by coactivating T cells. Herein we analyzed the influence of FTY720, FTY720‐P, and S1P on IL‐33 induced formation of IL‐2 and IFN‐ γ , by using IL‐33 receptor overexpressing EL4 cells, primary CD8 ⁺ T cells, and splenocytes. EL4‐ST2 cells released IL‐2 after IL‐33 stimulation that was inhibited dose‐dependently by FTY720‐P but not FTY720. In this system, S1P increased IL‐2, and accordingly, inhibition of S1P producing sphingosine kinases diminished IL‐2 release. In primary CD8 ⁺ T cells and splenocytes IL‐33/IL‐12 stimulation induced IFN‐ γ , which was prevented by FTY720 but not FTY720‐P, independently from intracellular phosphorylation. The inhibition of IFN‐ γ by nonphosphorylated FTY720 was mediated via the SET/protein phosphatase 2A (PP2A) pathway, since a SET peptide antagonist also prevented IFN‐ γ formation and the inhibition of IFN‐ γ by FTY720 was reversible by a PP2A inhibitor. While our findings directly improve the understanding of FTY720 therapy in MS, they could also contribute to side effects of FTY720 treatment, like progressive multifocal leukoencephalopathy, caused by an insufficient immune response to a viral infection.     

Interaction between prostaglandin D2 and chemoattractant receptor‐homologous molecule expressed on Th2 cells mediates cytokine production by Th2 lymphocytes in response to activated mast cells

The mechanisms by which immunologically activated mast cells stimulate the production of proinflammatory cytokines by T helper type 2 (Th2) lymphocytes were investigated in a human cell culture system. Supernatants collected from cord blood‐derived mast cells after treatment with immunoglobulin E (IgE)/anti‐IgE contained an activity that stimulated the production of interleukin (IL)‐4, IL‐5 and IL‐13 (both mRNA and protein) by Th2 lymphocytes. This activity was not detected in supernatants from unactivated mast cells and its production was inhibited by treatment of activated mast cells with the cyclo‐oxygenase inhibitor diclofenac. The concentration of diclofenac used inhibited completely the production of prostaglandin D₂ (PGD₂) but did not inhibit the release of histamine or leukotriene C₄. The effect of supernatants from activated mast cells was mimicked by exogenous PGD₂ at concentrations similar to those detected in the cultures of activated mast cells, and addition of exogenous PGD₂ to supernatants from diclofenac‐treated mast cells restored their ability to stimulate Th2 cytokine production. The ability of the mast cell supernatants to stimulate production of Th2 cytokines was not affected by addition of diclofenac to the Th2 cells directly, indicating that the production, but not the action, of the factor was sensitive to diclofenac treatment. Inhibition of chemoattractant receptor‐homologous molecule expressed on Th2 cells (CRTH2) abolished the effect of the mast cell supernatants on Th2 cytokine production. These data indicate that mast cells have the ability to stimulate Th2 cells to elaborate cytokines independently of T cell receptor activation or co‐stimulation and this response is mediated by PGD₂ acting upon CRTH2 expressed by Th2 cells.     

Effect of simple sugars on natural killing: evidence against the involvement of a lectin like mechanism in target recognition

The spontaneous lysis of target cells sensitive to natural killer (NK) activity is accomplished in two distinct phases: (i) binding between target and effector cells and (ii) post-binding events leading to target cell destruction. To test the hypothesis that cell surface carbohydrate(s) might be involved in recognitive and/or lytic events, the binding and cytotoxicity of peripheral blood lymphocytes (PBL) towards NK sensitive K-562 targets was studied in the presence of simple sugars and after treatment of the targets with the antibiotic, tunicamycin. Lysis by peripheral blood lymphocytes was found to be inhibited by N-acetyl glucosamine, N-acetyl galactosamine and alpha-methyl mannoside in a dose-dependent manner under conditions where neither these sugars nor those (fucose, galactose) which had little effect on lysis inhibited the binding of effector cells to targets. Further, growth of K-562 in tunicamycin (which inhibits N-linked glycosylations occurring through the lipid intermediate pathway) with or without subsequent treatment with the enzyme neuraminidase, markedly reduced cell surface expression of sugars monitored by lectin binding. Treated cells showed no loss of NK susceptibility and were frequently more sensitive to lysis. Sugar inhibition profiles were the same as for untreated cells. These data suggest that carbohydrates are not the target sites of NK recognition but that simple sugars may have an inhibitory action at a later stage of the lytic process.     

Characterization of a 105-kDa plasma membrane associated glycoprotein that is involved in West Nile virus binding and infection

This study attempts to isolate and characterize West Nile virus-binding molecules on the plasma membrane of Vero and murine neuroblastoma cells that is responsible for virus entry. Pretreatment of Vero cells with proteases, glycosidases (endoglycosidase H, alpha-mannosidase), and sodium periodate strongly inhibited West Nile virus infection, whereas treatments with phospholipases and heparinases had no effect. The virus overlay protein blot detected a 105-kDa molecule on the plasma membrane extract of Vero and murine neuroblastoma cells that bind to WN virus. Treatment of the 105-kDa molecules with beta-mercaptoethanol resulted in the virus binding to a series of lower molecular weight bands ranging from 30 to 40 kDa. The disruption of disulfide-linked subunits did not affect virus binding. N-linked sugars with mannose residues on the 105-kDa membrane proteins were found to be important in virus binding. Specific antibodies against the 105-kDa glycoprotein were highly effective in blocking virus entry. These results strongly supported the possibility that the 105-kDa protease-sensitive glycoprotein with complex N-linked sugars could be the putative receptor for WN virus.     

雷公藤对IL2的产生和IL2受体表达的抑制作用

观察雷公藤对T淋巴细胞免疫应答的抑制作用,发现雷公藤水煎液抑制小鼠脾细胞和胸腺细胞用ConA诱导的增殖反应,抑制作用与雷公藤剂量呈正比例。5mg/ml雷公藤完全抑制脾细胞的ConA增殖反应;该剂量雷公藤与脾细胞共育2小时不影响脾细胞的活率,细胞经洗涤后对CouA的增殖反应与正常细胞相同。进一步研究,在ConA诱导脾细胞72小时培养的早期加入雷公藤,完全抑制细胞的增殖反应;而在培养后期加入仅见部份抑制作用,推测雷公藤是抑制细胞的活化而不是对细胞的直接毒性作用和对DNA合成的抑制。ConA诱导脾细胞表达IL2受体和分泌IL2。雷公藤的加入完全抑制受体的表达,也抑制但不完全抑制细胞分泌IL2。外源性IL2的加入也不能逆转雷公藤对脾细胞ConA增殖反应的抑制作用,提示IL2分泌的减少与细胞DNA合成的抑制无直接联系。此外,IL2受体表达的受阻与DNA合成受抑制之间的联系还有待深入研究。     

Activated mast cells synthesize and release soluble ST2‐a decoy receptor for IL‐33

IL‐33 released from damaged cells plays a central role in allergic inflammation by acting through its membrane‐bound receptor, ST2 receptor (ST2L). IL‐33 activity can be neutralized by the soluble spliced variant of ST2 (sST2) that has been associated with allergic inflammation but its source is not well defined. We investigated whether mast cells (MCs) are a significant source of sST2 following activation through FcεRI or ST2. We find that antigen and IL‐33 induce substantial production and release of sST2 from human and mouse MCs in culture and do so synergistically when added together or in combination with stem cell factor. Moreover, increases in circulating sST2 during anaphylaxis in mice were dependent on the presence of MCs. Human MCs activated via FcεRI failed to generate IL‐33 and IL‐33 produced by mouse bone marrow‐derived MCs was retained within the cells. Therefore, FcεRI‐mediated sST2 production is independent of MC‐derived IL‐33 acting in an autocrine manner. These results are consistent with the conclusion that both mouse and human MCs when activated are a significant inducible source of sST2 but not IL‐33 and thus have the ability to modulate the biologic impact of IL‐33 produced locally by other cell types during allergic inflammation.     

Chromatofocusing as a tool for the characterization and partial purification of human interleukin-2

Human interleukin-2 (IL2) has been characterized and partially purified with a sequence of chromatofocusing, gel filtration and SDS-PAGE analysis. IL2 when tested in a [³H]thymidine incorporation assay by human IL2-dependent T cells, appeared to have a MW of 25,000 as determined by Ultrogel ACA 54 gel filtration. Chromatofocusing of an 80% ammonium sulfate precipitate from crude conditioned medium yielded 4 peaks of activity corresponding to fractions of pH 7.65, 7.28, 6.72 and 6.58. Neuraminidase treatment of IL2 prior to chromatofocusing reduced its charge heterogeneity to a single peak of activity at pH 7.63. IL2 which had been treated with neuraminidase, purified by chromatofocusing, radioiodinated and further separated by gel filtration was subjected to SDS gel electrophoresis. We observed a band, migrating in the 15,000 region which only occurred in the active fractions and which we tentatively identified as IL2. These findings indicate that the purification procedure described is appropriate to the characterization and preparation of quantities of human IL2.     

Induction of interleukin 2 (IL2) and interferon-γ and enhancement of IL2 receptor expression by a CD26 monoclonal antibody

The ability of the 134-2C2 monoclonal antibody (mAb; CD26) to transmit an activation signal and to affect T cell proliferation has been studied. The 134-2C2 mAb, although not being mitogenic by itself, is able to increase the proliferation of purified T cells in the presence of exogenous interleukin 2 (IL2) or phorbol 12-myristate 13-acetate (PMA). No effect of our mAb was observed on the proliferation of T cells induced by other stimuli such as Sepharose-bound CD3 mAb, phytohemagglutinin or calcium ionophore. Since the co-stimulatory effect of 134-2C2 mAb on PMA-induced T cell proliferation was strongly inhibited by an anti-Tac antibody, its involvement on the IL2/IL2 receptor pathway was investigated. An increased IL2 secretion in T cells cultured with PMA plus 134-2C2 mAb was observed and Northern blot analysis showed that the mAb 134-2C2 acts synergistically with PMA favoring the induction of both IL2 and interferon-γ mRNA synthesis, as well as the enhancement of IL2 receptor and transferrin receptor mRNA expression. Studies on mechanisms implicated in signal transduction showed that 134-2C2 mAb modifies neither intracellular calcium levels nor phosphoinositide breakdown. Additionally, no effect was exerted on protein kinase C translocation. These data suggest that the CD26 antigen is involved in T cell activation in an IL2/IL2 receptor-dependent pathway.     

T cell triggering by lectins. I. Requirements for interleukin 2 production; lectin concentration determines the accessory cell dependency

The requirements for lectin-induced interleukin 2 (IL2) production by human T cells have been investigated. With two different types of T cells, the Jurkat T cell lymphoma and highly purified HLA class II- peripheral T cells, the amount of IL2 produced was strongly dependent on the lectin concentration used. Addition of accessory cells caused a shift in the dose-response curve, resulting in strongly enhanced IL2 production at low concentrations. Thus, the (absolute) accessory cell dependency for T cells to produce IL2 is defined by experimental conditions. Only at lectin concentrations that were found to be optimal in the presence of accessory cells, removal of these cells abrogates IL2 production. Furthermore, after depletion of monocytes IL 2 production by peripheral T cells became almost completely dependent on the presence of thiols in the culture medium. In contrast, the IL2 production by the Jurkat line was not influenced by addition of thiols. The Jurkat model was used to study the nature of accessory cell because this cell line does not show any reactivity to allogeneic cells. Various myeloid and B lymphoid cell lines were tested as accessory cells. The capacity to function as accessory cell was not related to the monocytic origin of the cell. B cell lines were far more effective than monocytes, as two HLA class II- monocytic cell lines were not active. Even after HLA class II determinants were induced on these cells by incubation with an interferon-gamma-containing conditioned medium, they failed to act as as accessory cells. These experiments question the importance of HLA class II molecules and monokines, such as IL1, for lectin-induced IL2 production.     

Endogenous interleukin 6 plays an obligatory role in interleukin 4-dependent human IgE synthesis

The lymphokine interleukin (IL) 4 plays a crucial role in the regulation of IgE synthesis. In the present study, the cellular and cytokine requirements for the IL4-dependent induction of IgE synthesis in humans were analyzed. Recombinant IL4 could induce IgE synthesis by peripheral blood mononuclear cells and autologous T/B cell mixtures, but not by highly purified B cells. IgE induction by IL4 was strongly decreased in monocyte-depleted peripheral blood mononuclear cells. These results show that the induction of IgE synthesis by recombinant IL4 is T cell dependent and optimal in the presence of monocytes. IL5 and IL6, but not IL2, IL1 and tumor necrosis factor-alpha, strongly up-regulated the IL4-dependent synthesis of IgE, with modest effects on cell proliferation. An anti-IL6 polyclonal antibody strongly inhibited IL4-driven IgE production. Endogenous IL6 plays, therefore, an obligatory role in the IL4-dependent induction of IgE. However, a combination of IL4, IL5 and IL6 (with or without IL1) at optimal concentrations could not induce IgE synthesis by purified normal B cells, indicating that cytokine-mediated signals, although essential, are not sufficient for the IL4-dependent induction of IgE synthesis.     

Selective induction of growth factor production and growth factor receptor expression by different signals to a single T cell

Stimulation of lymphokine production and the expression of receptors for growth factors can be dissociated in AK-8, a line of CD4+, I-A-restricted, conalbumin-specific mouse T cells. When activated by antibodies specific for the T cell receptor (TcR; F23.1) or the CD3 complex (145-2C11) adsorbed to plastic culture wells, AK-8 cells produce lymphokines but are unable to proliferate. Proliferation takes place using the same stimuli upon addition of interleukin 1 (IL 1). Autocrine growth induced by anti-TcR, anti-CD3 or by antigen is dependent on IL 4 and not on IL 2 in this cell line, as shown by the effect of antibodies against IL 4 or the IL 2 receptor. Similarly to plastic-adsorbed antibodies, phorbol myristic acetate (PMA) or a combination of PMA and the calcium ionophore Ionomycin also induces secretion of growth factors without inducing proliferation, but in this case addition of IL 1 is ineffective in inducing AK-8 proliferation. When incubated with anti-TcR or anti-CD3 antibodies in soluble form these cells neither proliferate nor produce IL 4 even in the presence of IL 1. However, soluble antibodies in the presence of IL 1 induce enhanced expression of IL 2 receptors, as measured both by induction of responsiveness to exogenous IL 2 or flow cytometry analysis using anti-IL 2 receptor antibodies. These results show that the pathways for the activation of growth factor receptor expression and the induction of lymphokine secretion can be differentiated in this cell line using anti-TcR or anti-CD3 reagents in different physical forms. The transmembrane signals delivered by these different forms of anti-receptor antibody may allow an understanding of these distinct requirements for T cell growth.     

Experimental Trypanosoma brucei infections selectively suppress both interleukin 2 production and interleukin 2 receptor expression

Experimental infections with Trypanosoma brucei AnTat 1.1.E not only account for a suppression of interleukin 2 (IL 2) production, but also induce an impairment of IL 2 receptor expression. Indeed, lymph node cells derived from infected mice failed to express IL 2 receptors following mitogenic stimulation as compared to normal controls. This impairment was not attributed to a modulation of the number of T cells and was not caused by the presence of living parasites. Furthermore, the basal level of IL 2 receptor expression could not be re-established through the exogenous supply of recombinant IL 2. This impairment of receptor expression was found to be mediated by suppressive cells that affect the relative number of receptor-positive cells as well as the mean receptor density. On the other hand, the mitogen-induced secretion of other T cell-derived lymphokines was not inhibited during infection, indicating that the severe suppression of IL 2 regulation was not attributed to a total paralysis of the T cell responsiveness.     

High-affinity interleukin 2 receptors on B cell chronic lymphocytic leukemia cells are induced by phorbol myristate acetate but not by calcium ionophore

The role of phorbol myristate acetate (PMA: a protein kinase-C (PKC) activator) and calcium ionophore A23187 in the induction mechanism of the interleukin 2 receptor (IL2R) on B-cell chronic lymphocytic leukemia (B-CLL) cells was studied. B-CLL cells from five patients were cultured with PMA or A23187 for 72 h and used for the following experiments. Interleukin 2 (IL2) cross-linking assays showed that PMA induced the expression of IL2R subunits (p55 and p70/75) in all cases examined, but that A23187 induced neither subunit. Radiolabeled IL2 binding assays also demonstrated that PMA induced both high-affinity IL2R (HA-IL2R) and low-affinity IL2R (LA-IL2R) on B-CLL cells, but that A23187 did not. After treatment with PMA, three of five cases did not respond to IL2 even though they expressed HA-IL2R, suggesting impaired signal transduction. No cases responded to IL2 after treatment with A23187. In conclusion, PMA but not A23187 stimulates B-CLL cells to induce the expression of p55 and p70/75, indicating that the PKC pathway plays a more important role than the calcium pathway in the induction of IL2R subunits in B-CLL cells.     

Ag and IL‐2 immune complexes efficiently expand Ag‐specific Treg cells that migrate in response to chemokines and reduce localized immune responses

An intravenous administration of a high‐dose antigen (Ag) can induce immune tolerance and suppress the immune response, but the mechanism remains unclear. We recently proved that a combined i.v. administration of OVA and IL‐2‐anti‐IL‐2 Ab immune complexes (IL‐2 ICs) efficiently expands OVA‐specific Treg cells in the thymus and induces their migration into peripheral blood, by using OVA‐specific TCR Tg‐expressing DO11.10 mice. Here, we demonstrate that the expanded OVA‐specific Treg cells rapidly move into the air pouch after OVA injection in DO11.10 mice. The migration was inhibited by blocking the axis of a chemokine receptor, CCR2. Moreover, prior treatment with OVA and IL‐2 ICs enhanced OVA‐specific Treg‐cell migration and inhibited OVA‐induced delayed‐type hypersensitivity (DTH) reactions in the skin of BM chimeric mice with 15% of T cells expressing OVA‐specific TCR. Blocking the CCR2 axis reversed this suppression of DTH in these mice. Furthermore, prior treatment with OVA and IL‐2 ICs effectively reduced DTH reactions even in WT mice possessing only a very small population of OVA‐specific T cells. Thus, the treatment with Ag and IL‐2 ICs can efficiently expand Ag‐specific Treg cells with the capacity to migrate and reduce localized immune responses.     

Effect of cyclosporin A on the early activation of human T helper lymphocytes: inhibition of RNA-synthesis and modification of the expression of activation antigens

The ability of cyclosporin A (CS-A) to inhibit induced lymphocyte activation and to modify expression of membrane receptors was assessed on human T helper cells. Flow cytometric cell cycle analyses of acridine orange-stained cells showed that CS-A (0.5 micrograms/ml) inhibits the G0-G1 activation process of a substantial proportion of PHA- and Con A-stimulated lymphocytes. The expression of Tac, OKT9 and 4F2 antigens (previously shown to be expressed or increased on activated cells) was investigated by immunofluorescence. Fewer cells expressed the Tac and OKT9 antigens after activation in presence of CS-A, but the percentage of 4F2-positive cells remained unchanged. Analyses of receptor densities measured by fluorescence intensity revealed for all three investigated antigens a decreased receptor density on positive cells in presence of CS-A. Thus, CS-A not only inhibited cell activation (G0-G1 transition) and the expression of Tac, OKT9 and 4F2 antigens, but it also diminished the number of Tac, OKT9 and 4F2 antigens per cell. Assessing specifically the activation of OKT4 (helper) and OKT8 (cytotoxic) cells after 24 h, either by double-fluorescence or by cell fractionation with anti-OKT4 or anti-OKT8 antibodies plus complement, showed that preferentially OKT4 cell activation as well as expression of Tac and OKT9 antigens on those cells was inhibited in the presence of CS-A.