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PubMed2001

Evolution of T cell receptor (TCR) alpha beta heterodimer assembly with the CD3 complex.

T cell antigen receptors (TCR) are composed of an antigen-recognizing unit, the TCRalpha beta heterodimer, and a signal transduction ensemble, the CD3 complex. Whereas mammals possess three CD3 dimers (delta epsilon, gamma epsilon, and zeta2), birds and amphibians have only two (delta/gamma-epsilon and zeta2). To understand evolutionary changes in TCR/CD3 assembly,a phylogenetic approach was employed to dissect the interaction of TCRalpha beta heterodimers with the CD3 components. While sheep and mouse TCRalpha and TCRbeta chains could replace the corresponding human chains in mutant human T cells to restore surface TCR/CD3 expression and function, chicken TCRalpha, TCRbeta and CD3delta/gamma chains were unable to replace the corresponding human chains in forming a chimeric TCR/CD3 complex. The inability of chicken TCR/CD3 components to replace the human molecules in T cells was found to result from the lack of interaction between chicken TCRalpha beta heterodimers and the human CD3 complex. In contrast, if no CD3 molecules are present (non-T cells), TCRalpha -TCRbeta chain pairing can take place in an apparently non-controlled way. Thus, the TCR-CD3 interactions have changed with the evolutionary divergence of two mammalian CD3gamma and CD3delta genes from a single prototypic chicken delta/gamma gene. Our data suggest that the structures in mammalian TCR.C regions, which distinguish between CD3delta and CD3gamma chains, have evolved with the appearance of two separate CD3delta and CD3gamma functions.

Animals↗

PubMed1990

CD4, CD8 and the TCR-CD3 complex: a novel class of protein-tyrosine kinase receptor.

A novel form of receptor-kinase interaction was first described in the interaction between the CD4 and CD8 antigens and the protein-tyrosine kinase p56lck. This linkage, between a regulatory antigen on T cells and a member of a family of intracellular molecules with an established ability to activate and transform cells, is likely to be of great importance in the regulation of T-cell growth. Recently, data have been obtained on the molecular basis of regulation of the CD4/CD8-p56lck interaction and an interaction between the T-cell receptor complex (TCR-CD3) and another src-kinase p59fyn has been described. Here, Christopher Rudd examines these interactions and outlines their potential roles in normal and malignant T-cell growth.

Amino Acid Sequence↗

PubMed2002

In vitro production and characterization of partly assembled human CD3 complexes.

Pairwise assembly of human CD3 chains takes place in the endoplasmic reticulum of T cells. Subsequently, the CD3 heterodimers form complexes with Ti alpha and Tiss chains forming hexameric Ti alpha beta CD3 gamma epsilon delta epsilon complexes. Finally, association with the zeta 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 CD3 gamma, CD3 delta and CD3 epsilon. Proteins were expressed in Escherichia coli as denatured chains and de novo folded in vitro. CD3 gamma and CD3 epsilon folded as soluble monomers, whereas CD3 delta did not yield any soluble proteins. When folding the chains pairwise, soluble CD3 gamma epsilon and CD3 delta epsilon heterodimers could be isolated, whereas CD3 gamma delta 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 CD3 epsilon undergoes a conformational change after dimerization with CD3 gamma or CD3 delta. Furthermore, we demonstrated that the CD3 gamma epsilon heterodimer could be purified using immunoaffinity chromatography.

Antibodies, Monoclonal↗

PubMed2003

Pharmacological disintegration of lipid rafts decreases specific tetramer binding and disrupts the CD3 complex and CD8 heterodimer in human cytotoxic T lymphocytes.

Accumulating evidence strongly supports the role of lipid rafts in the regulation of T-lymphocyte activation, but the organization and molecular composition of these cholesterol- and sphingolipid-rich membrane microdomains in different subsets of T cells remain poorly investigated. Here, we show that pharmacological disruption of lipid rafts in human CD8+ cytotoxic T-lymphocyte (CTL) clones disturbs the integrity of CD3 complex and CD8 heterodimer, without affecting the reactivity with T-cell receptor (TCR)-specific antibodies. This demonstrates that interaction with completely assembled CD3 complex is not required for the stable expression of TCR at the cell surface. The effect of raft disruption on CD3 and CD8 expression correlates with failure to bind specific tetrameric complexes by a proportion of surface TCR molecules. However, the interaction of specific tetramer with the rest of surface TCR pools appears to be unaffected, demonstrating that TCR-signalling complexes may differ in their requirement for cholesterol to stably maintain their composition and to rearrange for efficient tetramer binding. Together with previously published data, our results support the existence of molecular and/or structural heterogeneity of lipid rafts that may play an important role in controlling distinct functional properties of T-cell subsets.

Antifungal Agents↗

PubMed1998

Expression of Tcf-1 mRNA and surface TCR-CD3 complexes are reduced during apoptosis of T cells.

When a T cell hybridoma, 70.7, was treated with a Ca2+ ionophore (A23187), apoptotic cell death was induced. Interestingly, we observed that the expression of Tcf-1, a T cell-specific transcription factor, mRNA was reduced by approximately 5-fold in the A23187-treated apoptotic cells compared to an ethanol-treated control. The hybridoma cells, however, did not display such a reduced expression of Tcf-1 mRNA upon treatment with buthionine sulfoxide, which is known to induce a necrosis-like cell death. When another T cell hybridoma, KMIs-8.3.5, was treated with A23187 and phorbol myristate acetate, which leads to activation-induced apoptosis, Tcf-1 expression was again greatly reduced. However, a mutant line (KCIT1-8.5) derived from KMIs-8.3.5, which produces IL-2 upon activation and is resistant to apoptosis, did not show such reduction in Tcf-1 expression. We also showed that the reduced expression level of CD3epsilon mRNA and surface TCR-CD3 complex in apoptotic T cells is caused by the reduced expression of Tcf-1. When 70.7 cells were transfected with a plasmid DNA pSVtcf-1, in which Tcf-1 gene expression is driven by the SV40 promoter, such reduction of the Tcf-1 mRNA and the surface expression of the TCR-CD3 complex were not observed upon apoptosis induction. Our results suggest that the reduced expression of Tcf-1 is specific for the apoptotic, but not for the activating, process of T cells and is also responsible for the reduced surface expression of the TCR-CD3 in apoptotic T cells.

Animals↗

PubMed1993

Transmembrane signalling through the T-cell-receptor-CD3 complex.

Recent data support the existence of activation motifs within different subunits of the T-cell-receptor-CD3 complex. This architecture generates a receptor composed of discrete modules, each capable of being coupled to an effector pathway. Although new T-cell specific protein tyrosine kinases have recently been identified, the nature of the proximal non-receptor protein tyrosine kinase linking the T-cell receptor complex to essential signalling effectors remains unknown. Developmentally regulated differences in T-cell-receptor-CD3 assembly or stability may lead to the expression of isoforms displaying different sets of activation motifs. Whether this may be the basis of differential signalling during T-cell development is still a matter of speculation.

Amino Acid Sequence↗

PubMed1995

Activated macrophages induce structural abnormalities of the T cell receptor-CD3 complex.

The mechanism of the structural alterations of the T cell receptor (TCR)-CD3 complex, which appear to be greatly responsible for immunosuppression in the tumor-bearing status, was investigated in tumor-bearing mice. Splenic T cells from tumor-bearing hosts lost the expression of the CD3 zeta chain without being replaced by FcR gamma, despite the normal expression of other components of the TCR complex. Tumor growth induced the accumulation of non-T, non-B cells in the spleen in correlation with the loss of zeta. Those cells were found to be macrophages that were able to induce the loss of zeta, as well as structural changes of CD3 gamma delta epsilon, even in freshly isolated normal T cells by cell contact-dependent interaction. More importantly, macrophages activated with zymosan A+LPS but not residential macrophages were able to induce the similar abnormality of the TCR complex. These results indicate that macrophages in certain activation stages play a crucial role in causing an abnormal TCR complex in tumor-bearing conditions, as well as in regulating the structure of the TCR complex in immune responses.

Animals↗

PubMed2005

Plasticity and rigidity in adaptor protein-2-mediated internalization of the TCR:CD3 complex.

Many cell surface proteins are internalized via dileucine- or tyrosine-based motifs within their cytoplasmic domains by the heterotetrameric adaptor protein complex, AP-2. In this study we have examined how AP-2 mediates internalization of large cell surface receptors, such as the eight-chain TCR:CD3 complex. Although most receptors have a single signal that drives internalization, the TCR complex has two (D/E)xxxL(L/I) motifs and 20 YxxØ motifs. Using 293T cells, we show that AP-2 is completely dependent on both signals to mediate TCR internalization, because deletion of either completely blocks this process. Significant plasticity and redundancy were observed in the use of the YxxØ motifs, with a clear hierarchy in their use (CD3delta > CD3gamma >or= CD3zeta >> CD3epsilon). Remarkably, a single, membrane-distal YxxØ motif in CD3delta could mediate approximately 75% of receptor internalization, whereas its removal only reduced internalization by approximately 20%. In contrast, significant rigidity was observed in use of the (D/E)xxxL(L/I) motif in CD3gamma. This was due to an absolute requirement for the position of this signal in the context of the TCR complex and for a highly conserved lysine residue, K128, which is not present in CD3delta. These contrasting requirements suggest a general principle by which AP-2 may mediate the internalization of large, multichain complexes.

Adaptor Protein Complex 2↗

PubMed1988

Molecular cloning and chromosomal localization of the human T-cell receptor zeta chain: distinction from the molecular CD3 complex.

The T-cell antigen receptor (TCR) is a multisubunit receptor complex specific to T cells subserving both antigen recognition and signal transduction functions. The zeta chain of the TCR is a component of all surface receptor complexes. This chain was first identified in murine T cells by virtue of the fact that it coimmunoprecipitates with the TCR complex using antibodies directed against either the clone-specific subunits or invariant CD3 subunits of the receptor. Recently, we have isolated a cDNA encoding the murine zeta. Using this as a probe, we have now isolated cDNAs encoding the human zeta. Sequence analysis of cDNAs encoding human and murine zeta reveals that it is a highly conserved protein. In addition to amino acid homology, there is remarkable interspecies conservation in the nucleotide sequence of the 5' and 3' untranslated regions of the zeta mRNA. The previously characterized invariant delta, epsilon, and gamma chains of the TCR, referred to as the CD3 complex, share significant sequence and structural homology with each other and are all located within 300 kilobases of each other on human chromosome 11 (11q23). zeta has no sequence similarity to the CD3 chains and the localization of the human zeta gene to the centromeric region of chromosome 1 underscores the fact that it is a distinct genetic component of the TCR.

Amino Acid Sequence↗

PubMed1987

T cell receptor-CD3 complex during early T cell differentiation. Analysis of immature T cell acute lymphoblastic leukemias (T-ALL) at DNA, RNA, and cell membrane level.

T cell acute lymphoblastic leukemias (T-ALL) can be regarded as the malignant counterparts of cells in various T cell differentiation stages. To study the expression of the human T cell receptor (TcR)-CD3 complex during the early stages of T cell differentiation, we have analyzed 22 T-ALL at the cell membrane level and the DNA level and 12 of them at the RNA level. According to their immunologic phenotype, the T-ALL could be divided into three main groups: 10 immature T-ALL (CD1-/CD3-), seven common thymocytic T-ALL (CD1+/CD3-or+), and five mature T-ALL (CD1-/CD3+). Among the 10 immature T-ALL three appeared to express the immunologic phenotype of the putative prothymocyte (TdT+/HLA-DR+/CD7+/CD2+/CD5-/CD1-/CD3-), whereas the other seven T-ALL appeared to be immature thymocytic (TdT+/HLA-DR-/CD7+/CD2+/CD5+/CD1-/CD3-). Transcripts of the CD3-delta and CD3-epsilon genes were present in all CD3- and CD3+ T-ALL tested, including prothymocytic T-ALL. However, prothymocytic T-ALL had germline TcR-beta genes and were not rearranged to the characterized TcR-gamma joining regions. The presence of CD3 transcripts and absence of TcR gene rearrangements in prothymocytic T-ALL supports their immature T cell character. Two immature thymocytic T-ALL also had germline TcR-gamma genes and one of them had germline TcR-beta genes. In all other T-ALL the TcR-gamma and TcR-beta genes were rearranged. The presumptive functional 1.3-kilobase TcR-beta transcripts were detected in the majority of T-ALL with rearranged TcR-beta genes. Distinct levels of TcR-gamma transcripts appeared to be present only in some thymocytic T-ALL, i.e., some immature thymocytic T-ALL and common thymocytic T-ALL. TcR-alpha mRNA could only be detected in CD3+ mature T-ALL, but was absent in all CD3+ common thymocytic T-ALL tested. Our data indicate that CD3 gene transcription is one of the earliest events during T cell differentiation and already occurs in prothymocytes. The TcR-gamma and TcR-beta genes rearrange early during thymocytic differentiation and can subsequently be transcribed. High levels of TcR-gamma gene transcription may only occur in a part of the T cells during thymic differentiation, while TcR-beta gene transcription continues during further differentiation. TcR-alpha gene transcription may be the final step in the production of the complete set of TcR and CD3 proteins, resulting in the expression of the TcR alpha beta-CD3 complex at the cell surface of mature T cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗

PubMed2000

On the dynamics of TCR:CD3 complex cell surface expression and downmodulation.

TCR downmodulation following ligation by MHC:peptide complexes is considered to be a pivotal event in T cell activation. Here, we analyzed the dynamics of TCR:CD3 cell surface expression on resting and antigen-activated T cells. We show that the TCR:CD3 complex is very stable and is rapidly internalized and recycled in resting T cells. Surprisingly, the internalization rate is not increased following TCR ligation by MHC:peptide complexes, despite significant TCR downmodulation, suggesting that constitutive internalization rather than ligation-induced downmodulation serves as the force that drives serial ligation. Furthermore, TCR downmodulation is mediated by the intracellular retention of ligated complexes and degradation by lysosomes and proteasomes. Thus, our data demonstrate that ligation induces TCR downmodulation by preventing recycling rather than inducing internalization.

Cell Line↗

PubMed1990

Lysine 271 in the transmembrane domain of the T-cell antigen receptor beta chain is necessary for its assembly with the CD3 complex but not for alpha/beta dimerization.

The T-cell antigen receptor (TcR) complex present on most T-cells is formed by a clone-specific disulfide-linked alpha/beta heterodimer noncovalently associated to the CD3 complex, the latter composed of five invariant polypeptides: gamma, delta, epsilon, zeta/zeta, or zeta/eta. The presence of conserved, oppositely charged, amino acids in the predicted transmembrane domains of all the subunits of the TcR.CD3 complex suggests that these residues may have a critical function in the assembly and/or stabilization of the complex. In order to analyze the role of the transmembrane-charged amino acids in the association and cell surface expression of the TcR.CD3 complex, we have carried out site-directed mutagenesis of Lys271 in the transmembrane domain of the TcR beta chain and analyzed the capacity of the altered chain to assemble in a TcR beta-negative T-cell line. Here we show that substitution of this positively charged residue by alanine or glutamine does not prevent cytoplasmic association of alpha and beta chains to form disulfide-linked heterodimers, but does abolish formation of an alpha/beta.CD3 complex and, consequently, its expression on the cell surface.

Antigens, CD↗

PubMed2006

A conformation- and avidity-based proofreading mechanism for the TCR-CD3 complex.

During antigen recognition, T cells show high sensitivity and specificity, and a wide dynamic range. Paradoxically, these characteristics are based on low-affinity receptor-ligand interactions [between the T-cell antigen receptor (TCR-CD3) complex and the antigen peptide bound to MHC]. Recent evidence indicates that the TCR-CD3 is expressed as multivalent complexes in the membrane of non-stimulated T cells and that conformational changes in the TCR-CD3 can be induced by strong but not weak agonists. Here, we propose a thermodynamic model whereby the specificity of the TCR-CD3-pMHC interaction is explained by its multivalent nature. We also propose that the free energy barriers involved in the change in conformation of the receptor impose a response threshold and determine the kinetic properties of recognition. Finally, we suggest that multivalent TCR-CD3s can amplify signals by spreading them from pMHC-engaged TCR-CD3s to unengaged complexes as a consequence of the cooperativity in the system.

Humans↗

PubMed1990

Phosphorylation of the ETS-2 protein: regulation by the T-cell antigen receptor-CD3 complex.

Phosphorylation of the human ets-2 protein in response to mitogenic signals to T lymphocytes was investigated in Jurkat cells. Activation of the cells by antibodies against the T-cell antigen receptor-CD3 complex or by concanavalin A was followed within 5 min by increased phosphorylation of the protein, as shown by a mobility shift of the protein from 54 to 56 kilodaltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and increased incorporation of 32P. The Ca2+ ionophores A23187 and ionomycin were able to mimic this effect, suggesting that this phosphorylation is mediated by Ca2+.

Antigens, Differentiation, T-Lymphocyte↗

PubMedDate not supplied

Altered signaling triggered by ligation of the TCR/CD3 receptor in T lymphocytes from patients with systemic lupus erythematosus: the road from anergy to autoimmunity.

Some of the signaling phenotypic abnormalities observed in peripheral blood T cells from patients with systemic lupus erythematosus resemble those seen in cells anergized by stimulation through the TCR/CD3 complex, in the absence of costimulatory signals, or by stimulation with altered ligands. The potential association of this "anergic" phenotype and the triggering of autoimmune responses in human lupus is discussed.

Autoimmunity↗

PubMed1990

Regulation of expression of interleukin 2 receptors upon triggering of the TCR-CD3 complex on human T lymphocytes.

Monoclonal antibodies reactive with CD3 molecular complex can induce antigen-associated early biochemical changes in purified, monocyte-depleted resting T cell populations and synergize with interleukin 2 (IL2) in the induction of T-cell proliferation. Interleukin 2 mediates its effects via two receptor molecules of apparent 70-75 kD (p70/p75) and 50-55 kD (p50/55) molecular weights respectively. Using radioactive IL2 and bi-functional cross-linking chemistry, we are able to determine that incubation of purified, monocyte-depleted, resting T cells with anti-CD3 (OKT3) antibody induces a significant and selective increase in the expression of p70/75 IL2 receptors from their low constitutively expressed levels. This event occurs in the complete absence of cellular proliferation. Although IL2 also causes the upregulation of p70/75 molecules, it is the synergistic action of both antibody and lymphokine which is needed for the induction of significant amounts of the p50/55 IL2 receptors and the concomitant cellular proliferation. The effect of anti-CD3 on p70/75 receptor expression is specific, as determined by the inability of a non-related (anti-CD2) monoclonal antibody of the same subclass (IgG2a) to induce a similar effect. The Ca++ ionophore ionomycin, under conditions that cause significant intracellular Ca++ influx cannot by itself mediate upregulation of IL2 receptor expression in T cells. Since anti-CD3 itself can induce intracellular Ca++ increase in purified T cells, the finding with the ionophore suggests that the intracellular Ca++ accumulation alone cannot account for the IL2 receptor molecular events described here. Addition of PMA induces both p70/75 and p50/55 IL2 receptor upregulation, as well as IL2-dependent proliferation. Although resting T cells constitutively express p70/75 receptors, under our experimental conditions and with the concentration of IL2 used, these molecules cannot transduce the lymphokine signal efficiently. Thus, in a physiologic context, a simple interpretation of our data could be that upon interaction of the TCR/CD3 with antigen a selective upregulation of p70/75 IL2 receptors renders them competent of not only binding the lymphokine, but also transducing its signal. The latter event leads to the expression of p50/55 receptors and subsequent proliferation. Whether an increase in the numbers of these receptors is all that is needed or additional events are necessary merits further investigation.

Antibodies, Monoclonal↗

PubMed1990

Activation of human tumor-infiltrating lymphocytes by monoclonal antibodies directed to the CD3 complex.

We have used high concentrations of recombinant-methionyl human interleukin 2 (rIL-2) for the initial growth and expansion of human tumor-infiltrating lymphocytes (TIL). Early in the life of the TIL bulk culture, cytotoxicity was non-major histocompatibility complex restricted. Under these culture conditions antitumor cytotoxicity was observed to decline with increasing age of the bulk culture. In addition, TIL became refractory to rIL-2-induced expansion. We have used solid-phase anti-CD3 antibodies for TIL activation followed by culture in reduced concentrations of rIL-2 to reactivate TIL previously grown in high concentrations of rIL-2. TIL refractory to rIL-2 in terms of growth and antitumor cytotoxicity proved sensitive to anti-CD3 activation. The use of solid-phase anti-CD3 was also more effective than high concentrations of rIL-2 in the expansion of TIL when used at the start of culture. Finally, TIL could be induced to secrete IL-2 following solid-phase activation with anti-CD3. These data suggest that human TIL are susceptible to activation by signals directed at the CD3 complex of the TIL cell surface.

Antibodies, Monoclonal↗

PubMed1996

Stimulation of the T-cell antigen receptor-CD3 complex signaling pathway by the tyrosine phosphatase inhibitor pervanadate is mediated by inhibition of CD45: evidence for two interconnected Lck/Fyn- or zap-70-dependent signaling pathways.

The tyrosine phosphatase specific inhibitor pervanadate is a potent activator of T lymphocytes through induction of tyrosine phosphorylation and downstream events of the activation cascade. Using CD45- or CD3-negative variants of the Jurkat leukemic T-cell line we show that the different biochemical events induced by pervanadate appeared to be dependent on the presence at the cell surface of either CD45 or CD3. CD45-dependent events such as tyrosine phosphorylation of Shc, activation of nuclear factor-kappa B (NF-kappa B), activator protein-1 (AP-1), transcription factors, and stimulation of interleukin-2 (IL-2) promoter and of CD69 and CD25 surface expression paralleled activation of the tyrosine kinases lck and fyn. By contrast, stimulation of calcium influx, a CD3-dependent event, paralleled zap-70 activation. The data demonstrate that the T-cell antigen receptor-CD3 (TcR-CD3) complex is functionally linked to two different protein tyrosine kinase (PTK) modules with separate specific functions and that CD45 may be an important regulator of this coupling.

Base Sequence↗