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Results for “CD3 Complex”

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PubMed1989

Triggering T cells by otherwise inert hybrid anti-CD3/antitumor antibodies requires encounter with the specific target cell.

We used a purified bispecific antibody (Ab) against CD3 and an ovarian carcinoma (OVCA) antigen to ask whether the binding of a monovalent ligand to CD3 can induce triggering of T cells. In the presence of OVCA cells, this Ab bridges the CD3 complex to the target cell and triggers proliferation and cytotoxicity in T cells. In the absence of target cells, however, this monovalent Ab, even when bound to T cells at high levels, fails to induce any increase in cytosolic Ca2+, nor does it induce responsiveness to IL-2 or modulation of the CD3 complex. Because it is inert when bound monovalently, this hybrid Ab can be used to arm in vitro CTL clones, which then retain the capacity to kill the specific tumor for up to 2 d.

Antibodies, Monoclonal↗

PubMed1990

The G protein coupling T cell antigen receptor/CD3-complex and phospholipase C in the human T cell lymphoma Jurkat is not a target for cholera toxin.

Intact Jurkat cells could be stimulated by monoclonal antibodies against the Tcell antigen receptor complex (OKT3 directed against the CD3 complex, BMA031 directed against constant framework epitopes in the alpha/beta heterodimer). The accumulation of inositol phosphates was inhibited by prior incubation of the cells with cholera holotoxin. The inhibitory effect of cholera toxin (CT) was not cAMP mediated because forskolin (a direct activator of adenylate cyclase) did not mimic the inhibitory effect. When measuring phospholipase C (PLC) in a cell-free assay system by using [3H]inositol-labeled membranes, the enzyme could be stimulated by the poorly hydrolyzable GTP analogue guanosine 5'-O-(thiotriphosphate (GTP gamma S). Both anti-receptor antibodies augmented the GTP gamma S stimulatory effect, while the antibodies alone had no stimulatory capacity. In membranes from CT-pretreated cells, whereas the antibodies lost their stimulatory effect on PLC as in untreated cells, whereas the antibodies lost their stimulatory capacity in the presence of GTP gamma S. These data imply that CT exerts its inhibitory effect on signaling by acting at the receptor level while the PLC regulating G protein is not a target for CT-mediated alterations. This assumption is supported by the finding that in intact Jurkat cells CT, which ADP ribosylated only the alpha-subunit of the stimulatory G protein of the adenylate cyclase, led to a loss of the T cell antigen receptor complex from the cell surface as demonstrated by a decrease of receptor density using flow cytometry analysis. Receptor loss could not be achieved by forskolin treatment or incubation of the cells with the binding subunit of the toxin alone.

Adenosine Diphosphate Ribose↗

PubMed1992

Monoclonal antibodies anti-CD3, anti-TCR alpha beta and anti-CD2 act synergistically with tumor cells to stimulate lymphokine-activated killer cells and tumor-infiltrating lymphocytes to secrete interferon gamma.

Peripheral blood lymphocytes cultured in interleukin-2 IL-2 acquire the ability to recognize and kill a wide range of tumor cells. Such promiscuous killer cells are termed lymphokine-activated killer (LAK) cells. We recently reported that the interaction of LAK cells with tumor cells stimulated the LAK cells to release interferon (IFN) gamma. Here, we report that the release of IFN gamma by LAK cells can be further enhanced by addition of the monoclonal antibodies (mAbs), anti-CD3, anti-(T cell receptor alpha beta) (TCR alpha beta) and a mitogenic combination of anti-CD2 (T112 + T113). Other antibodies, including a non-mitogenic anti-CD2 mAb (Leu5b), that recognize T cell-associated antigens were not stimulatory. The same stimulatory mAbs also synergized with tumor cells to stimulate tumor-infiltrating lymphocytes (TIL) to secrete IFN gamma. Additional experiments indicated that it was the T cell subset of LAK cells (LAK-T cells) that was stimulated by tumor cells and mAbs to release IFN gamma. Inhibition studies with specific mAbs suggest that the stimulation of IFN gamma release by LAK-T cells was dependent both on the aggregation of TCR-CD3 complexes on the LAK-T cell, and on the interaction of accessory molecules with their ligands. The accessory molecules we have identified as critical are LFA 1 and CD2/LFA-2 on LAK-T cells interacting with their respective ligands ICAM-1 and LFA3. Thus our data suggest that cytokine production in LAK-T cells can be regulated by multiple molecular interactions, involving the TCR-CD3 complex and adhesion molecules.

Antibodies, Monoclonal↗

PubMed1990

Stimulation of T-cells with OKT3 antibodies increases forskolin binding and cyclic AMP accumulation.

It has recently been shown that elevation of cAMP by adenosine receptor stimulation may be potentiated by stimulation of the T-cell receptor/CD3 complex on human T-cells with the monoclonal antibody OKT3, and that this is mimicked by activation of protein kinase C [Kvanta, A. et al. (1989) Naunyn-Schmeideberg's Arch. Pharmac. 340, 715-717]. In this study the diterpene forskolin, which binds to and activates the adenylate cyclase, has been used to examine further how the CD3 complex may influence the adenylate cyclase pathway. Stimulation with OKT3 alone was found to cause a small dose-dependent increase in basal cAMP accumulation. When combining OKT3 with a concentration of forskolin (10 microM), which by itself had little effect on the cyclase activity, the cAMP accumulation was markedly potentiated. This potentiation was paralleled by an increase in [3H]forskolin binding to saponine permeabilized Jurkat cells from 24 to 41 fmol/10(6) cells. The OKT3 effect on cAMP was blocked by chelating extracellular Ca2+ with EGTA or intracellular Ca2+ with BAPTA and also by W-7, an inhibitor of calmodulin, but was unaffected by H-7, an inhibitor of protein kinase C. Even though OKT3 caused an increase in inositolphosphate turnover, and activated protein kinase C, neither phorbol 12,13 dibutyrate (PDBu) nor the Ca2(+)-ionophore A23187 could mimic the OKT3 effect, whereas a combination of PDBu and A23187 at high concentrations could potentiate forskolin stimulated cyclase activity. Together, these results indicated that stimulation of the CD3 complex could influence the adenylate cyclase by two different mechanisms, one involving activation of protein kinase C and another which does not.

Adenylyl Cyclases↗

PubMed1995

Physical interaction with monocytes rescues human mature CD4+ T-cell lines from anti-CD3-induced apoptosis.

Crosslinking of the TcR-CD3 complex with immobilized anti-CD3 antibodies without sufficient co-stimulation induced cell death in human mature CD4+ T-cell lines. In these T cells, DNA fragmentation and morphological characteristics of apoptosis were seen. The anti-CD3-induced apoptosis was inhibited by co-culture with monocytes. The rescue signal provided by monocytes does not need to be present simultaneously with signals mediated by anti-CD3. When T cells were precultured with monocytes for 24 h before anti-CD3 stimulation and then the monocytes were removed from the culture, anti-CD3-induced T-cell apoptosis was also inhibited. To determine whether the monocyte-derived rescue signals were transduced by soluble factors or by direct cell-to-cell interaction with monocytes, we precultured T cells with monocytes separated by a micropore membrane which prevented T cell-monocyte physical interaction but not the diffusion of secreted molecules. In this system, rescue signals could not reach the T cells. To further assess the importance of physical interaction, we precultured T cells with fixed monocytes. T cells could not be rescued from apoptosis under these experimental conditions, either. The results considered collectively suggest that sufficient physical interaction with viable monocytes is important for the rescue of anti-CD3-induced apoptosis of CD4+ T cells.

Antibodies, Monoclonal↗

PubMed1994

Prevention of TCR-mediated apoptosis by the elevation of cAMP.

The stimulation through TCR-CD3 complexes by immobilized anti-CD3 antibody induced the production of IL-2 and activation-induced cell death (ACD) in the majority of T cell hybridomas. However, some hybridomas produced IL-2 without showing any signs of ACD by the same stimulation, indicating that TCR-CD3-mediated signaling pathways of IL-2 production and of ACD are different. These pathways were discriminated from each other by protein kinase inhibitors and cAMP-elevating reagents such as forskolin. The pathway of IL-2 production but not of ACD was inhibited by protein kinase inhibitors. On the other hand, various cAMP-elevating reagents prevented the T cell hybridomas from TCR-mediated ACD with minimal inhibition of IL-2 production. The elevated cytoplasmic cAMP did not block dexamethasone-induced apoptosis. This indicates that apoptosis is regulated by multiple pathways. Furthermore, the inhibitory effect of cAMP is specific for the TCR-mediated signaling pathway of ACD. Messenger RNA for bcl-2 was detected after treatment with forskolin.

Animals↗

PubMed1994

Adenovirus infection induces loss of HLA class I and CD3 antigens, but does not induce cell surface presentation of the La (SS-B) autoantigen.

Antibodies to the RNA polymerase III transcription termination factor La are frequently found in the serum of patients with various autoimmune diseases. The mechanisms by which autoimmune responses are evoked remain largely obscure, but the presentation of autoantigens on the cell surface during stress conditions has been reported as a possible factor. In this study we analysed the effects of adenovirus infection on the binding of anti-La antibodies to the surface of several human cell lines and on the levels of the membrane-expressed glycoproteins HLA class I, CD44 and the CD3 complex. In addition, we studied the relative amount and the intracellular distribution of the La protein as well as its association with the major species of non-coding virus-associated (VAI) RNA. While immunofluorescence patterns revealed a redistribution and possibly cell surface expression of the La protein during infection, this could not be confirmed by other techniques. In contrast, surface levels of HLA class I proteins and CD3 complex were severely affected. The data suggest that the subcellular distribution of the La protein is not detectably influenced by adenovirus infection.

Adenovirus Infections, Human↗

PubMed1994

Enhancement of T cell receptor signaling of tumor-infiltrating lymphocytes by retrovirally mediated fyn gene transduction.

We conducted retrovirally mediated transduction of fyn gene into tumor-infiltrating lymphocytes (TILs) in an attempt to augment T cell receptor-CD3 complex signal transduction, using TILs obtained from six patients with epithelial ovarian cancer. The expression of the transduced gene was more than five times that of endogenous fyn gene. In all preparations, the cytolytic activity of the TILs against autologous tumor cells, but not allogeneic tumor cells, was significantly enhanced (P < 0.01) by transduction of fyn gene. In addition, when TILs were treated with anti-CD3 antibody to stimulate T cell receptor-CD3 complex, fyn-gene-transduced TILs showed about two times higher proliferation (P < 0.05) and secreted 2.2 to 11.7 times more tumor necrosis factor-beta (P < 0.05) than nontransduced TILs. These results could have important implications for TILs-mediated gene therapy for cancer.

Female↗

PubMed1993

Interleukin-7 specifically induces the B7/BB1 antigen on human cord blood and peripheral blood T cells and T cell clones.

B7/BB1 is a cell surface molecule and member of the Ig super family that is constitutively expressed on dendritic cells. In addition, B7 is expressed on B cells, macrophages, T cells, and T cell clones following activation. Interaction of B7 with its natural ligand CD28 is required for optimal stimulation of T cells, activated via the TCR-CD3 complex, which is thought to be due to stabilization of cytokine mRNA. Here we demonstrate that the expression of B7 on T cells can specifically be induced by IL-7. Induction of B7 expression on T cells and T cell clones requires at least 5-7 days of culture and represents a late activation event. Results of studies using T cell clones, as well as resting purified B7- T cells, demonstrate that B7 is induced on a substantial proportion of T cells after IL-7 activation and is not due to an outgrowth of pre-existing B7+ T cells. In addition, CD4+ as well as CD8+ T cells could be induced to express B7. Stimulation of purified cord blood T cells with cross-linked anti-CD3 mAb resulted in a relatively fast (48 h) induction of B7, which could not be inhibited by a neutralizing anti-IL-7 mAb, whereas no endogenous IL-7 production by activated T cells and T cell clones could be detected. Together, these results indicate that the B7 molecule can be induced on T cells by IL-7, but also by an IL-7 independent pathway involving triggering of the TCR-CD3 complex.

Antigens, Surface↗

PubMed1989

Inhibition of phosphatidylserine synthesis induced by triggering CD2 or the CD3-TCR complex in a human T cell line. Relationships with G proteins and receptors modulation.

Activation of Jurkat T cells with phytohemagglutinin, CD3 or CD2 mAbs results in a marked inhibition of phosphatidylserine (PS) synthesis. Monitoring PS synthesis in T cells shows that: (i) after modulation of CD3 molecules the cells become refractory to further treatment with CD3 mAbs as well as to a further challenge with CD2 mAbs; and (ii) treatment of T cells with fluoride ions and cholera toxin, two known effectors of guanosine triphosphate-binding proteins, also resulted in a strong inhibition of the synthesis of this phospholipid. The inhibition of PS synthesis thus appears to be regulated similarly to the other activation events, suggesting that transmembrane signalling mechanisms leading to PS inhibition are the same as those previously proposed for increasing phosphatidylinositides turnover and subsequent rise in the intracellular calcium concn in lymphocytes.

Antibodies, Monoclonal↗

PubMed1987

Structural and functional characteristics of the CD3 (T3) molecular complex on human thymocytes.

The CD3 (T3) molecular complex is noncovalently associated with the antigen receptor molecule on T cells. The mitogenic properties of anti-CD3 antibodies have suggested that this complex may be the transducer of the antigenic signal to the intracellular environment. In the present investigation, we studied some of the structural and functional characteristics of the CD3 complex on human thymocytes. In 11 specimens tested, we found that anti-CD3 antibodies react with 50 to 76% of the thymocytes. Two-color immunofluorescence analysis revealed that the majority (greater than 50%) of thymocytes express both CD3 and CD1 on their surfaces. The latter is a marker of immature thymocytes. However, a distinct subpopulation comprising 13 to 19% of the total cells displays only CD3, while an approximately equal percentage of cells expresses only CD1. The mitogenic potential of anti-CD3 antibodies on peripheral T cells is dependent on the presence of monocytes. Anti-CD3 antibodies by themselves cannot activate thymocytes, indicating that functionally active monocytes are absent from the thymocyte population. Even the addition of peripheral monocytes does not allow a response of thymocytes to anti-CD3 antibodies. However, when the anti-CD3 antibody 64.1 is added in the presence of exogenous rIL 2, a strong antibody and lymphokine dose-dependent response ensues. Only CD1- CD3+ thymocytes are stimulated by the addition of antibody and IL 2. The mere expression of CD3 on the CD1+ CD3+ subpopulation of thymocytes apparently is not sufficient to render the cells responsive to the signals of anti-CD3 and IL 2.

Antibodies, Monoclonal↗

PubMed1993

Efficient immunoselection of cytolytic effectors with a magnetic cell sorter.

This paper describes a rapid and efficient method for the sorting of in vitro activated cytolytic effectors cells. For cytotoxic assays, a large number of cells with conserved function must be rapidly obtained. Immunomagnetic sorting was chosen because it is faster than flow cytometry sorting. The MACS system requires the use of paramagnetic beads of small diameter (100-150 nm), reputed to interfere minimally with cell function. In order to generate the cytolytic effectors, peripheral blood lymphocytes were cultivated in the presence of interleukin-2 (50 U/ml) and anti-CD3 monoclonal antibody (BMA030, 100 ng/ml) for 4 days. Cell separation was based on the membrane expression of the CD3 complex. The purity obtained for positive (CD3+) cell sorting with the MACS was higher than 95%. The purity of negative (CD3-) cell fraction was more variable, but further purification by flow cytometry rapidly yielded purity higher than 95%. Cytotoxic assays were performed against four target cell lines (K562, Daudi, HL60 and U937) and proliferation assays showed that both negatively and positively selected populations had conserved their function acquired during culture in the presence of anti-CD3 mAb and IL2.

Antibodies, Monoclonal↗

PubMed1993

Analysis of T cell repertoire and function in mice transgenic for the human V beta 3 TCR.

We have constructed mice containing the human V beta 3 TCR gene from the influenza virus haemagglutinin specific human CD4+ T cell clone HA1.7. Similar cell yields were obtained from transgenic and non-transgenic lymphoid tissue, with normal levels of T cells and with no unusual bias of the CD4 or CD8 subpopulations. Immunostaining and FACS analysis of transgenic thymocytes, spleen, and mesenteric lymph nodes revealed that the majority of T cells expressed the human V beta 3 TCR on the cell surface. Small numbers of cells expressing murine TCR beta chain were also detected. Polymerase chain reaction analysis revealed that an extensive V alpha TCR repertoire was used in the human V beta 3 transgenic mice. Lymphocytes from the spleen and mesenteric lymph nodes of transgenic mice were assessed for functional activity in vitro. Isolated cells were stimulated with mitogen or superantigen, as well as directly through the TCR-CD3 complex, and their ability to proliferate and secrete lymphokines analysed. Cells from transgenic mice responded well after stimulation with phytohaemagglutinin, concanavalin A, anti-CD3 antibody, anti-CD3 antibody with phorbol ester, and Staphylococcus aureus enterotoxin B, and also showed alloreactivity in a mixed lymphocyte reaction. Minimal levels of response were detected after stimulation with murine TCR beta antibody. Together, these data suggest that a human TCR beta chain is able to associate with a murine TCR alpha chain, to form a fully functional surface TCR-CD3 complex.

Animals↗

PubMed1987

Independent association of T cell receptor beta and gamma chains with CD3 in the same cell.

We have demonstrated that the PEER cell line, which expresses a CD3-associated TCR gamma chain on the cell surface, synthesizes TCR beta chain intracellularly. A percentage of this TCR beta chain associates with the CD3 complex intracellularly. These results indicate that TCR beta and gamma chains can be synthesized by one cell line, and that these chains can independently associate with the CD3 complex. However, the results argue against the formation of TCR beta gamma chain complexes in this cell line.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1991

The effect of human T cell leukaemia virus type I infection on a herpes simplex virus-specific CD8+ cytotoxic T cell clone.

In an effort to clarify the effect of human T cell leukaemia virus type I (HTLV-I) infection on virus-specific CD8+ cytotoxic T cells, a herpes simplex virus-specific CD8+ cytotoxic T cell clone was infected with HTLV-I in vitro. The cytotoxic activity of the clone was found to have declined early after HTLV-I infection when the expression of T cell receptor-CD3 complex on the cell surface still showed no difference in comparison with that of uninfected parent cells. After 16 weeks of HTLV-I infection, expression of T cell receptor-CD3 complex on HTLV-I-infected clone cells became decreased. This phenomenon is similar to the effect of HTLV-I infection on CD4+ cytotoxic T cells as we previously reported, and suggests that there are common mechanisms of declined cytotoxic activity mediated by both CD4+ and CD8+ cytotoxic T cells following infection with HTLV-I. Such functional alterations of cytotoxic effector cells might be one of the mechanisms underlying immunodeficiency caused by HTLV-I infection.

Antigens, CD↗

PubMed1995

Developmental arrest of NK1.1+ T cell antigen receptor (TCR)-alpha/beta+ T cells and expansion of NK1.1+ TCR-gamma/delta+ T cell development in CD3 zeta-deficient mice.

The relationship between the structure of the T cell antigen receptor (TCR)-CD3 complex and development of NK1.1+ T cells was investigated. The TCR complex of freshly isolated NK1.1+ TCR-alpha/beta+ thymocytes contained CD3 zeta homodimers and CD zeta-FcR gamma heterodimers, whereas that of the majority of NK1.1- T cells did not contain FcR gamma. The function of CD3 zeta and FcR gamma in the development of NK1.1+ T cells was determined by analyzing CD3 zeta- and FcR gamma-deficient mice. The NK1.1+ T cells from wild-type and CD3 zeta-deficient mice had equal levels of CD3 expression. However, the development of NK1.1+ TCR-alpha/beta+ T cells was almost completely disrupted in thymus and spleen in CD3 zeta-deficient mice, whereas no alteration was observed in FcR gamma-deficient mice. In contrast, the number of novel NK1.1+ TCR-gamma/delta+ thymocytes expressing a surface phenotype similar to NK1.1+ TCR-alpha/beta+ thymocytes increased approximately six times in CD3 zeta-deficient mice. These findings establish the distinct roles of the CD3 zeta chain in the development of the following different thymic T cell compartments: NK1.1- TCR+, NK1.1+ TCR-alpha/beta+, and NK1.1+ TCR-gamma/delta+ thymocytes, which cannot be replaced by CD3 eta or FcR gamma chains.

Animals↗

PubMed1995

[Towards immuno-modulation through the molecular mechanism of lymphocyte activation].

Recognition of the antigen/MHC complex by the T cell receptor (TCR)-CD3 complex in T cells triggers early activation events such as tyrosine phosphorylation, phosphatidylinositol turnover, intracellular Ca2+ mobilization or activation of protein kinases, and finally exhibits effector functions such as lymphokine secretion by helper T cells or cytotoxicity by killer T cells as late activation events. Several key molecules have been shown to engage in these signaling cascades. In addition to the TCR-CD3 molecules, other surface molecules such as CD28 or LFA-1 contribute to the regulation of T cell activation as a co-stimulator. Growing knowledge about the downstream of antigen recognition is promoting the attempt to modulate the signal transduction by specific drugs, mAbs, altered peptides or cytokines. Further investigations on the molecular mechanism of T cell activation will provide clinical successes to control immune responses.

Animals↗

PubMed1991

Modification of membrane phospholipid fatty acyl composition in a leukemic T cell line: effects on receptor mediated intracellular Ca2+ increase.

The effect of modifying fatty acyl composition of cellular membrane phospholipids on receptor-mediated intracellular free Ca2+ concentration ([Ca2+]i) increase was investigated in a leukemic T cell line (JURKAT). After growing for 72 h in medium supplemented with unsaturated fatty acids (UFAs) and alpha-tocopherol, the fatty acyl composition of membrane phospholipids in JURKAT cells was extensively modified. Each respective fatty acid supplemented in the culture medium was readily incorporated into phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine in the JURKAT cells. The total n-6 fatty acyl content was markedly reduced in phosphatidylinositol and phosphatidylcholine of cells grown in the presence of n-3 fatty acids (alpha-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid). Conversely, in the presence of n-6 fatty acids (linoleic acid and arachidonic acid), the total n-3 fatty acyl content was reduced in all the phospholipids examined. In n-3 and n-6 polyunsaturated fatty acid (PUFA) modified JURKAT cells, the total n-9 monounsaturated fatty acyl content in the phospholipids were markedly reduced. Changing the fatty acyl composition of membrane phospholipids in the JURKAT cells appears to have no affect on the presentation of the T cell receptor/CD3 complex or the binding of anti-CD3 antibodies (OKT3) to the CD3 complex. However, the peak increase in [Ca2+]i and the prolonged sustained phase elicited by OKT3 activation were suppressed in n-3 and n-6 PUFA but not in n-9 monounsaturated fatty acid modified cells. In Ca2+ free medium, OKT3-induced transient increase in [Ca2+]i representing Ca2+ release from the inositol 1,4,5-trisphosphate-sensitive Ca2+ stores, were similar in control and UFA modified cells. Using Mn2+ entry as an index of plasma membrane Ca2+ permeability, the rate of fura-2 fluorescence quenching as a result of Mn2+ influx stimulated by OKT3 in n-9 monounsaturated fatty acid modified cells was similar to control cells, but the rates in n-3 and n-6 PUFA modified cells were significantly lower. These results suggest that receptor-mediated Ca2+ influx in JURKAT cells is sensitive to changes in the fatty acyl composition of membrane phospholipids and monounsaturated fatty acids appears to be important for the maintenance of a functional Ca2+ influx mechanism.

Antigens, Differentiation, T-Lymphocyte↗