Induction of CD3δɛω) by phorbol 12-myristate 13-acetate (original) (raw)
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Activation of CD3/TCR negative human thymocytes via CD28 molecule
Cellular Immunology, 1991
We have observed that the CD28 molecule was present on the cell surface of a large fraction of resting CD3-thymocytes (40 to 100%). Interestingly, the majority (190%) of surface CD3-CD28cells reacted in the cytoplasm with anti-CD28 (CK248, 9.3) and anti-CD3c chain mAbs (Leu4, OKT3). Along this line, we found that CD28 surface expression could be induced within 18 hr on CD3-CD28-thymocytes using very low doses of phorbol-13-myristate-12-acetate (PMA). This event was accompanied by the appearance of CD25 and CD69 activation antigens but not of CD3/TCR complex. These results were further confirmed by immunoprecipitation studies. It is noteworthy that the T-cell activation pathway initiated via the CD28 molecule is functional in resting CD3-thymocytes in the presence of PMA and/or IL2. Finally, stimulation of CD3immature thymocytes via CD28 gave rise to a large fraction (about one-third) of CD3-CD8+ Cells.
Dephosphorylation of the human T lymphocyte CD3 antigen
1989
Previous studies demonstrated that activation of T lymphocytes by phorbol ester or mitogenic lectin leads to phosphorylation of Ser 126 of the CD3 antigen y chain, whereas treatment with ionomycin results in phosphorylation of both Ser 123 and 126 [Davies, A. A. et al. (1987) J . Biol. Clzem. 262, 10918-109211. In the present study, the dephosphorylation of Ser 123 and Ser 126 of the y chain was investigated. Phorbol-ester-induced phosphorylation of the y-chain Ser 126 in vivo was reversed following removal of phorbol ester. Dephosphorylation of both Ser 123 and 126 was also observed in vitro using the microsome fraction of T lymphocytes. In order to identify the phosphatases acting at these two sites, the immunoprecipitated y chain was used as substrate either following treatment with protein kinase C in vitro, in which case phosphorylation occurs mainly at Ser 123, or following in vivo phosphorylation of Ser 126. Purified oligomeric forms of the polycation-stimulated phosphatases were more effective in dephosphorylating both phosphorylated forms of the y chain compared with equivalent amounts of ATP,Mg2 +-dependent phosphatases or calcineurin. By using phosphopeptide analogues of the CD3 y chain containing Ser 123 or Ser 126 as substrates (A3 and A6), it was shown that polycation-stimulated phosphatases selectively dephosphorylated Ser 123 compared to Ser 126.
In Vitro Production and Characterization of Partly Assembled Human CD3 Complexes
Scandinavian Journal of Immunology, 2002
Pairwise assembly of human CD3 chains takes place in the endoplasmic reticulum of T cells. Subsequently, the CD3 heterodimers form complexes with Tia and Tiû chains forming hexameric TiabCD3gede complexes. Finally, association with the z 2 homodimer occurs in Golgi apparatus before the fully assembled T-cell receptor is transported to the cell surface. To study the structural properties of the human CD3 chains, we have developed new methods to produce and fold the extracellular domains of CD3g, CD3d and CD3e. Proteins were expressed in Escherichia coli as denatured chains and de novo folded in vitro. CD3g and CD3e folded as soluble monomers, whereas CD3d did not yield any soluble proteins. When folding the chains pairwise, soluble CD3ge and CD3de heterodimers could be isolated, whereas CD3gd heterodimers were not produced. Using antibodies as structural probes, we identified two different types of antigenic epitopes that were dependent on heterodimerization. Our data indicate that CD3e undergoes a conformational change after dimerization with CD3g or CD3d. Furthermore, we demonstrated that the CD3ge heterodimer could be purified using immunoaffinity chromatography.
Engagement of CD30 shapes the secretion of cytokines by human ??d T cells
Eur J Immunol, 2000
CD30 is a member of the TNF receptor superfamily, previously shown to be expressed on Hodgkin's lymphoma cells and on normal activated lymphocytes. We here show that CD30 is highly expressed on recently activated human +ˇT cells. Elevated surface levels of this molecule persisted in long-term cultures of +ˇcells, without further cell stimulation. CD30 acted as a co-stimulus in +ˇT cells by potentiating the intracellular Ca 2+ fluxes induced by CD3 cross-linking. The engagement of CD30 enhanced the expression of several cytokines induced upon CD3 stimulation such as IL-4 and IFN-+ but not IL-10. The CC chemokines RANTES and macrophage inflammatory protein-1 g were constitutively expressed and not affected by stimulation. The inducible expression of the neutrophil chemoattractant IL-8 was enhanced by CD30 co-stimulation, as well as that of the CC chemokines I-309 and MDC, whereas the secretion of the monocyte chemotactic protein-1 was not detected. Triggering of CD30 may therefore modulate the expression of several cytokines released by +ˇcells; the expression of its physiologic ligand by APC and neutrophils at the site of infection may contribute to determine the outcome of an immune response.
Construction and expression of a soluble form of human CD30 ligand with functional activity
Journal of leukocyte biology, 1998
CD30 engagement in human lymphoid cells induces pleiotropic cellular responses that affect cellular viability and proliferation, cytokine production, and nuclear factor kappaB (NF-kappaB) nuclear translocation. Studies examining the molecular basis for this pleiotropism thus far have relied on the use of antibodies and cells transfected with CD30L to trigger CD30, two methods of receptor induction that present important limitations: antibodies are not physiological receptor-triggering molecules and CD30L transfectants induce high background intracellular signaling in the cells under study. We have generated and expressed a functional soluble human CD30L molecule (sCD30L/CD8alpha) comprised of the extracellular domain of human CD30L fused to the extracellular domain of the human CD8alpha chain. Immunoprecipitation and Western blot analysis of sCD30L/CD8alpha revealed the existence of at least two forms of sCD30L/CD8alpha, which exhibited molecular sizes consistent with the existence ...
Journal of Leukocyte Biology
We have treated Jurkat T lymphocytes with a concentration (160 nM) of phorbol myristyl acetate (PMA) that down-regulates conventional and novel protein kinase C (PKC) isozymes and we have investigated the effects on Ca2+ signaling and protein tyrosine phosphorylation using mAb (C305) directed against the beta-subunit of the Ti heterodimer or the epsilon/delta-component of the CD3 complex (mAb Leu 4 or OKT 3). The levels of expression of PKC alpha, betaI, betaII, and delta were reduced by 90% or more in PMA-treated cells, whereas the expression of PKCtheta decreased by approximately 30%. In contrast, the chronic treatment with PMA increased the expression of PKCepsilon and PKCzeta. There was a lack of Ca2+ response and myo-inositol trisphosphate (IP3) production in PMA-treated cells when they were exposed to mAb Leu 4 but the cells responded to mAb C305. The treatment with PMA did not affect the surface expression of Ti or CD3. The overall levels of tyrosine-phosphorylated proteins w...
European Journal of Immunology, 1992
In this work we report that CDS, a T cell accessory activation antigen and receptor for the B cell surface protein CD72, is associated with theT cell antigen receptor (TcR)/CD3 complex in human T lymphocytes. In vitro phosphorylation of either CD3 or CDS immunoprecipitates prepared from CD3-stimulated Jurkat and peripheral blood T cells in the presence of the detergent polyoxyethylene 10 oleyl ether (Brij96) showed, unexpectedly, an identical pattern of five phosphopolypeptides of 70, 59, 56, 21 and 18 kDa, respectively. Peptide mapping of the five bands demonstrated that the same protein kinase substrates co-precipitated with both CD3 and CDS and that the majority of the protein phosphorylation occurred on tyrosine residues. These data suggested that the TcR/CD3 complex and and the CDS antigen might be associated inT cells. Evidence to support this hypothesis was obtained from analysis of immunoprecipitates prepared from surface-iodinated T cells. Bands characteristic of the TcR and CD3 antigens were identified in CDS immunoprecipitates and conversely, CDS was identified in CD3 immunoprecipitates. Conformation that CD3 and CDS co-precipitated in the presence of Brij96 was obtained by Western blotting. Quantitative immunodepletion demonstrated that between 10 % -20 % of cell surface CD5 was associated with the TcR/CD3 complex in Brij96 detergent lysates of human T cells and, furthermore, that this association was independent of T cell activation. The association of these two receptors provides a possible physical basis for the accessory role of the CDS antigen in T cell activation.
Cellular Immunology, 1987
Monoclonal antibody 3A35 (MA 3A35) has previously been shown to be an activation marker of macrophages and T lymphocytes. It immunoprecipitated from macrophages a 200-kDa molecule belonging to the T200 family and from T cells a 85kDa antigen. In the present work, the factors controlling the expression of the epitope identified by MA 3A35 on polyclonal activated T cells and T-cell clones, as well as the ability of 3A35 alone or together with complement to interfere with T-cell functions, were investigated. Corticoresistant thymocytes unreactive with MA 3A35 became fully reactive after 2 days of in vitro stimulation by PMA and IL-2 and the level of reactivity per cell declined to a low level thereafter, In helper and cytolytic T-cell clones, the expression ofthe epitope defined by MA 3A35 was also maximal soon after antigenic stimulation then declined. In helper-T-cell clones, the epitope remained detectable during the entire culture period, whereas in cytolytic clones its expression was markedly reduced at the end of the culture. The lineage of cytotoxic T lymphocytes (CTL) as studied in a bulk culture of spleen cells primed in vivo against a syngeneic tumor exhibited similar regulation by antigenic stimulation. The CTL precursors were resistant to lysis by MA 3A35 plus complement; after 3 days of culture with the stimulatory antigen, they became highly sensitive but their sensitivity then diminished and mature CTL were completely resistant. MA 3A35 plus complement also killed the activated T cells which responded to macrophage-presented antigens and were thought to be mainly Lyt-l+. Therefore, the epitope identified by MA 3A35 was expressed predominantly at an early stage of T-cell activation. At a late stage, it persisted almost exclusively on helper and Lyt-1' cells. In addition, MA 3A35 plus complement Iysed NK cells, AK cells, and their precursors present in normal spleen. In the absence ofcomplement, MA 3A35 had no detectable effect on T-cell functions. 0 1987 Academic PXSS, IX. ' This work was supported by the F&&ration Nationale des Centres de Lutte contre le Cancer.
Cellular Immunology, 1990
The signal requirements for activation and proliferation of CD1+ thymocytes have been studied in order to define whether this immature cell population could function as mature T cells do. We found that CD1+ cells expressed high levels of CD25 antigen upon triggering with specific monoclonal antibodies (mAbs) (anti-CD3, anti-CD2, anti-CD28) in association with low doses of Phorbol-13-myristate-12-acetate (PMA). More interestingly, we described that in the presence of PMA CD1+ thymocytes proliferate upon stimulation with anti-CD28 mAb as well as with a pair of anti-CD2 mAbs, without the need of exogenous interleukin-2 (IL2), whereas they respond to anti-CD3 mAb only if exogenous IL2 was provided. Furthermore, CD1+ cells stimulated under optimal proliferative conditions, gave rise to cell populations capable of lysing natural killer (NK)-sensitive (K562) and NK-resistant (MEL 10, Daudi, EPA1) tumor target cells. These data strongly support the idea that CD1+ thymocytes, under appropriate stimulations, display some of the functional capabilities of mature T cells.