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Results for “CD3 Complex”

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PubMed1996

Redirecting the complete T cell receptor/CD3 signaling machinery towards native antigen via modified T cell receptor.

We show that a chimeric T cell receptor (TCR) beta chain consisting of a single-chain Fv portion derived from a monoclonal antibody and the full TCR beta chain is able to assemble functionally with endogenous TCR/CD3 components and transfer the antibody specificity as well as the TCR specificity into TCR beta- as well as into TCR beta+ T cells. This allows the incorporation new non-major histocompatibility complex-restricted ligand specificities into the intact TCR/CD3 complex which can exploit the full range of biological activities of the endogenous TCR signaling machinery. This approach can provide wider opportunities to redirect T cells to virus or tumor antigen-bearing cells.

Amino Acid Sequence↗

PubMed1990

BMA031, a monoclonal antibody suited to identify the T-cell receptor alpha beta/CD3 complex on viable human T lymphocytes in normal and disease states.

Two types of T lymphocytes can be discriminated on the basis of expression of either the classical T-cell receptor (TCR) alpha beta or the more recently identified TCR gamma delta. Whereas TCR alpha beta + lymphocytes are known to respond to recognition of antigen in the context of major histocompatibility complex molecules by proliferation, lymphokine secretion, and/or cytotoxicity, the potential ligand specificities and functions of TCR gamma delta + cells have not been completely unraveled. Antibodies specific for either receptor type are important tools to elucidate the role TCR gamma delta + cells play in the immune system. They can be used to quantify TCR gamma delta + cells and TCR alpha beta + cells in normal and disease states, to isolate both T-cell subsets, and to perform in vitro functional assays. Only few antibodies reactive with common determinants on either TCR alpha beta or TCR gamma delta are available. Generally, the monoclonal antibody (mAb) WT31 is used for definition of viable human TCR alpha beta + cells. However, WT31 has recently been shown to cross-react with TCR gamma delta. We describe an mAb, BMA031, that combines the unique features of reactivity with intact viable cells and true specificity for a common determinant on the TCR alpha beta/CD3 complex. Its performance in immunofluorescence staining and immunochemistry has been compared with that of WT31 and anti-TCR gamma delta mAbs, using TCR alpha beta and TCR gamma delta expressing cells isolated from blood and bone marrow of healthy individuals and immunodeficient patients.

Animals↗

PubMed1998

T cell receptor zeta allows stable expression of receptors containing the CD3gamma leucine-based receptor-sorting motif.

The leucine-based motif in the T cell receptor (TCR) subunit CD3gamma constitutes a strong internalization signal. In fully assembled TCR this motif is inactive unless phosphorylated. In contrast, the motif is constitutively active in CD4/CD3gamma and Tac/CD3gamma chimeras independently of phosphorylation and leads to rapid internalization and sorting of these chimeras to lysosomal degradation. Because the TCRzeta chain rescues incomplete TCR complexes from lysosomal degradation and allows stable surface expression of fully assembled TCR, we addressed the question whether TCRzeta has the potential to mask the CD3gamma leucine-based motif. By studying CD4/CD3gamma and CD16/CD3gamma chimeras, we found that CD16/CD3gamma chimeras associated with TCRzeta. The CD16/CD3gamma-TCRzeta complexes were stably expressed at the cell surface and had a low spontaneous internalization rate, indicating that the leucine-based motif in these complexes was inactive. In contrast, the CD4/CD3gamma chimeras did not associate with TCRzeta, and the leucine-based motif in these chimeras was constitutively active resulting in a high spontaneous internalization rate and low expression of the chimeras at the cell surface. Thus, our data demonstrate that TCRzeta allows stable cell surface expression of receptors containing CD3gamma leucine-based motifs by its potential to mask such motifs.

Amino Acid Sequence↗

PubMed1990

Ia-independent binding of peptide antigen to the T cell receptor.

We have investigated the potential for direct interaction between a peptide Ag, human fibrinopeptide B (hFPB), and the TCR on an hFPB-specific murine T hybridoma. Fluoresceinated hFPB binds specifically to hFPB-responsive T cells, but not to unrelated T hybrids. Among variant subclones of the original hybridoma, ability to bind hFPB correlates with hFPB-specific response and expression of the CD3/TCR complex, indicating that hFPB is binding to the TCR. This TCR-hFPB interaction has an affinity of approximately 6.6 microM, reflecting slow association and rapid dissociation of the Ag from its receptor. These findings confirm the potential for direct Ag-TCR interaction and indicate an Ag recognition mechanism that is not initiated by Ag-MHC interaction.

Animals↗

PubMedDate not supplied

A T3-like protein complex associated with the antigen receptor on murine T cells.

Antigen recognition by human T lymphocytes and initiation of T-cell activation are mediated by a group of integral membrane proteins, the T-cell antigen receptor (TCR) and the T3 complex. The polypeptides which comprise T3 (a gamma-chain of relative molecular mass (Mr) 25,000 (25K), and delta and epsilon chains of 20K each) are physically associated with the TCR chains. Surface expression of the complex requires the presence of all the component T3 and TCR proteins. In contrast to the human system, murine T3 has not been identified using antibodies. Here we describe a murine T3-like protein complex. It appears to be more complicated than human T3, containing three monomeric glycoproteins (21-28K), two of which have N-linked carbohydrate side chains and a novel family of TCR-associated homo- and heterodimers. The 28K protein is identified as the murine T3 delta-chain. The 21K protein is phosphorylated on cell activation with concanavalin A (Con A).

Animals↗

PubMed1999

Extracellular signal-regulated kinase (ERK) activation by the pre-T cell receptor in developing thymocytes in vivo.

The first checkpoint in T cell development occurs between the CD4(-)CD8(-) and CD4(+)CD8(+) stages and is associated with formation of the pre-T cell receptor (TCR). The signaling mechanisms that drive this progression remain largely unknown. Here, we show that extracellular signal-regulated kinases (ERKs)-1/2 are activated upon engagement of the pre-TCR. Using a novel experimental system, we demonstrate that expression of the pre-TCR by developing thymocytes induces ERK-1/2 activation within the thymus. In addition, the activation of this pre-TCR signaling cascade is mediated through Lck. These findings directly link pre-TCR complex formation with specific downstream signaling components in vivo.

Animals↗

PubMed2004

Inhibition of T cell receptor signal transduction by tyrosine kinase-interacting protein of Herpesvirus saimiri.

T cells play a central role in orchestrating immunity against pathogens, particularly viruses. Thus, impairing T cell activation is an important strategy employed by viruses to escape host immune control. The tyrosine kinase-interacting protein (Tip) of the T lymphotropic Herpesvirus saimiri (HVS) is constitutively present in lipid rafts and interacts with cellular Lck tyrosine kinase and p80 endosomal protein. Here we demonstrate that, due to the sequestration of Lck by HVS Tip, T cell receptor (TCR) stimulation fails to activate ZAP70 tyrosine kinase and to initiate downstream signaling events. TCR zeta chains in Tip-expressing T cells were initially phosphorylated to recruit ZAP70 molecule upon TCR stimulation, but the recruited ZAP70 kinase was not subsequently phosphorylated, resulting in TCR complexes that were stably associated with inactive ZAP70 kinase. Consequently, Tip expression not only markedly inhibited TCR-mediated intracellular signal transduction but also blocked TCR engagement with major histocompatibility complexes on the antigen-presenting cells and immunological synapse formation. These results demonstrate that a lymphotropic herpesvirus has evolved a novel mechanism to deregulate T cell activation to disarm host immune surveillance. This process contributes to the establishment and maintenance of viral latency.

Antigen-Presenting Cells↗

PubMed1987

An in vivo functional immune system lacking polyclonal T-cell surface expression of the CD3/Ti(WT31) complex.

A polyclonal T-cell receptor complex (TCR) expression defect (as detected with monoclonal antibody WT31) has been found in two children belonging to an otherwise healthy Spanish family. One of the sibs (V, who had been vaccinated with attenuated poliomyelitis virus) showed clinical signs of immunodeficiency with an autoimmune syndrome, but the other (older) sib (D, vaccinated with attenuated rubella, measles, mumps, and poliomyelitis viruses) has been symptomless throughout life. In contrast to both sibs' normal expression of other peripheral leucocyte markers, as measured by flow cytometry (including CD1, CD2, CD4, CD8, and CD16), only about 6% of CD2+ polyclonal T cells expressed surface antigen-specific T-cell receptor (Ti/WT31), and only about 23% weakly expressed surface CD3 determinants. On the remaining CD2+ T cells in each sib the expression of Ti and CD3 was undetectable; the defect in CD3 expression is very likely secondary to the defect in Ti expression. Natural killer (NK) activity was not increased in any of the sibs, ruling out a high content of NK cells among their CD2+ lymphocytes. Functional data indicate that CD3-mediated T-cell activation with anti-CD3 monoclonals and Ti-mediated responses to allogeneic and tetanus toxoid antigens were severely depressed, whereas activation via CD2 was normal in the T lymphocytes of both sibs. Genes encoding for Ti alpha, beta, and gamma chains did not show major alterations by southern blot analysis, and polyclonal beta chain genes rearrangements were detected in both children's T-cell blasts. Family clustering suggests a genetic pathogenesis, but linkage to HLA or other blood group markers has not been found. Sib V had a concomitant autoimmune disease and died after a severe autoimmune haemolytic anaemia, indicating a relationship between the TCR and generation of autoimmune clones. However, the resistance of both individuals to infection and to vaccination with attenuated viruses, and the fact that sib D has been symptomless to date questions the relative importance of the TCR in the immune response against infection, and suggests that alternative T-cell activation pathways and non-specific defence mechanisms (external surfaces--bound and/or cellular) may suffice under certain circumstances.

Adult↗

PubMed1998

Ca2+ signaling in T-lymphocytes.

Ca2+ signaling in response to antigenic stimulation is essential for proliferation of T cells and therefore is one of the important early events in T-lymphocyte signal transduction. Several aspects of T cell receptor/CD3 complex stimulated Ca2+ signaling are reviewed: generation, metabolism, function, and intracellular targets of the Ca(2+)-mobilizing second messengers inositol 1,4,5-trisphosphate and cyclic ADP-ribose, the mechanism of Ca2+ entry, and the generation of Ca2+ oscillations on the single cell level. In addition, Ca2+ signaling induced by further stimuli is discussed, including other T-lymphocyte surface receptors (e.g., CD4 or beta 1-integrins, lipids, and physical stimuli).

Animals↗

PubMed1991

Trispecific F(ab')3 derivatives that use cooperative signaling via the TCR/CD3 complex and CD2 to activate and redirect resting cytotoxic T cells.

To investigate whether the retargeting of resting CTL can benefit from cooperative signaling between the TCR/CD3 complex and various accessory molecules, such as CD2, CD4, CD5, and CD8, we have constructed a series of trispecific F(ab')3 derivatives. Each derivative was designed to engage effector T lymphocytes with two Fab' arms, and tumor cells with a single Fab' arm. They were constructed by selective coupling of three mAb Fab' fragments, primarily via their hinge-region sulfhydryl groups, using the cross-linker o-phenylenedimaleimide. En route to the production of trispecific F(ab')3 antibodies a range of bispecific F(ab')2 derivatives was first prepared which could bind simultaneously to two different receptor molecules on T cells. Bispecific derivatives containing specificities for (CD2 (T11(1)) x CD3), (CD3 x CD4), (CD3 x CD8) or two epitopes on CD2, ((T11(1) x (T11(3)), all yielded two to three times the uptake of [3H]thymidine with fresh PBMC to that seen with intact IgG from anti-CD3 (OKT3). The exception to these findings was a bispecific F(ab')2 derivative with specificities for (CD3 x CD5) which caused slightly less proliferation than the control reagent, OKT3 IgG. When these bispecific antibodies were converted into trispecific antibodies (TsAb) by the addition of a Fab' from anti-CD37 they were then all able to retarget resting, unprimed, T cells from fresh PBMC for lysis of CD37+ tumor cells. However, the cytotoxic activity of these reagents fell into two distinct groups: group one, containing (anti-CD3 x anti-CD4 x anti-CD37), (anti-CD3 x anti-CD5 x anti-CD37), and (anti-CD3 x anti-CD8 x anti-CD37), gave minimal lysis and behaved in a similar way to the BsAb, (anti-CD3 x anti-CD37), i.e., no evidence of cooperative signaling for lysis; and group two, containing (anti-T11(1) x anti-CD3 x anti-CD37) and (anti-T11(1) x anti-T11(3) x anti-CD37), which were highly cytotoxic and gave up to 80% specific 51Cr-release. The failure of group one TsAb, in particular (anti-CD3 x anti-CD8 x anti-CD37) which should recruit CD8+ CTL, to give efficient lysis despite having anti-T cell arms that were mitogenic as a bispecific antibody, indicates that the cooperative signaling for proliferation is probably distinct from the signal(s) provided by group two TsAb that activate for both proliferation and lysis.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibody Specificity↗

PubMed1994

Developmental control of T-cell receptor internalization.

Since TCR/CD3 modulation may be involved in induction of T cell tolerance to self antigens, we compared ligand-induced TCR/CD3 internalization by a CTL clone and by immature thymocytes and mature T cells from mice bearing the same TCR alpha beta as transgene. The ligand used is a monoclonal antibody (mAb) specific for the receptor expressed by the clone and transgenic mice (anti-Ti mAb). CD8+ splenocytes triggered by anti-Ti mAb internalize the ligand-TCR/CD3 complex at a low rate, through a mechanism inhibited by the protein tyrosine kinase (PTK) inhibitor genistein and by staurosporine, a potent but non selective protein kinase C (PKC) inhibitor. This pattern of inhibition was similar to that observed in the CTL clone. Anti-Ti mAb induced TCR/CD3 internalization in CD4+CD8+ thymocytes at a high rate, through a mechanism which was insensitive to either genistein or staurosporine. In the CTL clone, genistein was shown to inhibit TCR/CD3 surface redistribution preceeding internalization. To characterize the PTK possibly involved in this step, we analyzed TCR/CD3 associated kinases in mature T splenocytes and thymocytes. Kinase activities present in anti-Ti mAb immunoprecipitates phosphorylated the CD3 components gamma, delta, epsilon, and zeta in both cell types although the intensity was stronger in splenic than in thymocyte extracts, whereas the phosphorylation of 70, 14 and 12kD substrates was more pronounced in thymocytes than in splenocytes. Comparable amounts of CD3 components were coprecipitated with and phosphorylated by p56lck and p59fyn respectively, in both cell types.

Alkaloids↗

PubMed1993

Biosynthesis of Tcr-alpha, beta and Tcr-gamma, delta/CD3 complexes.

Jurkat J76 clone, LYON L12.37 clone and L12.37 cells transfected with J76-alpha cDNA or J76 Tcr-alpha mutated cDNA (J79) were analysed for membrane expression of Tcr/CD3 complex using WT31 mAb (Tcr-alpha, beta) or Tcr-delta 1 mAb (Tcr-gamma, delta): LYON cells express V beta 9 bearing Tcr-beta chains. J76 Tcr-alpha cDNA transfected LYON cells have intracellular Tcr-gamma, delta chains and J79 Tcr-alpha cDNA transfected LYON cells have intracellular Tcr-alpha (M), beta chains.

Amino Acid Sequence↗

PubMed1989

Recognition of cluster of differentiation 1 antigens by human CD4-CD8-cytolytic T lymphocytes.

Human cluster-of-differentiation 1 (CD1) is a family of cell surface glycoproteins of unknown function expressed on immature thymocytes, epidermal Langerhans cells and a subset of B lymphocytes. Three homologous proteins, CD1a, b and c, have been defined serologically, and the CD1 gene locus on human chromosome 1 contains five potential CD1 genes. Analysis of the predicted amino-acid sequences of CD1 molecules reveals a low but significant level of homology to major histocompatibility complex (MHC) class I and class II molecules, and, like MHC class I molecules, CD1 molecules are associated non-covalently with beta 2-microglobulin. These structural similarities to known antigen-presenting molecules, together with the expression of CD1 on cells capable of antigen presentation, suggest a role for CD1 molecules in antigen recognition by T cells. Here we demonstrate the specific recognition of CD1a by a CD4-CD8- alpha beta T-cell receptor (TCR) expressing cytolytic T lymphocyte (CTL) line and the specific recognition of CD1c by a CD4-CD8- gamma delta TCR CTL line. The interaction of CD1-specific CTLs with CD1+ target cells appeared to involve the CD3-TCR complex, and did not show evidence of MHC restriction. These results suggest that for a subset of T cells, CD1 molecules serve a function analogous to that of MHC class I and II molecules.

Antibodies, Monoclonal↗

PubMed1999

Platelet-activating factor plays a role in the mechanism of major histocompatibility complex in T lymphocytes.

In recent studies, using a swine model of single lung transplantation, we demonstrated that IRI alone increased MHC II expression in the host's peripheral T lymphocytes. The inhibition of increased MHC II expression with TCV-309, a specific platelet-activating factor (PAF) antagonist suggested that PAF might play a role in the mechanism of increased MHC II expression. The purpose of the current study was two fold: 1) to investigate the mechanism of PAF-induced increased expression of MHC II in T lymphocytes, 2) to determine whether a specific PAF-antagonist, TCV-309, is capable of inhibiting the increased expression in an in vitro system. This study was subdivided, using four in vitro conditions: 1) purified resting T cells, 2) purified proliferating T cells, 3) PBL treated with PAF, and 4) PBL preincubated with TCV-309 and treated with PAF. The level of MHC II on T cells were measured by two color flow cytometry analysis (swine anti-CD3, MHC II-DR-(beta)antibodies). Both MHC II intensity and the number of CD3+MHC+ T cells did not change in resting purified T cells once treated with PAF, Furthermore, MHC II intensity did not change in purified proliferating T cells treated with PAF. The number of CD3+MHC+ T cells, however, increased significantly (p<0.05) from day 1 to day 4 as compared with pre-treatment value (day 0) for purified proliferating T cells. Treatment of PBL with PAF (10(-7)M) resulted in a significant (p<0.05) increase in MHC II expression from day 2 to day 4 post-treatment. The number of CD3+MHC+ T cells in PBL, however, did not change significantly upon treatment with PAF. The results of this study indicated that PAF did not have a direct effect on increased MHC II expression in resting or proliferating purified T lymphocytes. However, the mechanism of PAF-induced increased expression of MHC II in T cells may be via an indirect pathway involving accessory cells. TCV-309, a specific PAF receptor antagonist, is capable of inhibiting this PAF-induced increased expression of MHC II in T cells.

Animals↗

PubMed1998

Endogenous inflammatory response to dermal wound healing in the fetal and adult mouse.

The recruitment of inflammatory cells to a wound may play an important role in the resulting cellular processes and ultimately the quality of the healing response in the fetus (scar-free healing) or the adult (scar-forming healing). Using a range of antibodies to monocytes and macrophages and also to different activation markers of activated macrophages, we have compared the inflammatory profile of scar-free healing E16 mouse fetal wounds to those of scarring adult wounds. In the fetal wound, small numbers of monocyte derived cells (MOMA-2 and F4/80 positive) are recruited to the wound by 3 hr post-wounding. No Mac-1 positive cells indicative of activated macrophages were observed until 18 hr post-wounding. Eventually all types of macrophages studied were recruited to both adult and fetal wound sites but the numbers and persistence of these cells are lower in the fetus than in the adult. B cells were detected in healing adult wounds but not in the fetal wounds. This absence of H-21-A positive (B) cells in murine fetal wounds could be associated with the low levels of activated Mac-1 positive macrophages at the murine fetal wound site. Activated macrophages in addition to releasing growth factors may also release signals to recruit B cells. Thus, the E16 mouse fetus can mount an inflammatory response to wounding. This response differs from that of the adult in the numbers of inflammatory cells recruited to the wound and the subpopulations of activated cells found at the wound site. This study indicates that there are complex differences between the inflammatory responses elicited in adult and fetal murine dermal wounds. These differences may determine the profile of growth factors and cytokines released at fetal and adult wound sites. Manipulation of either the numbers or the activation states of inflammatory cells at the adult wound site may be an approach to the control of scarring during adult wound healing.

Animals↗

PubMed1993

Ligation of B7 with CD28/CTLA-4 on T cells results in CD40 ligand expression, interleukin-4 secretion and efficient help for antibody production by B cells.

It has been extensively shown that when T cells are co-stimulated with B7-CD28 interaction, a strong proliferative as well as cytolytic T cell response can be induced. In contrast, there exists only indirect evidence that the B7-CD28 interaction is of importance for the induction of T cell helper functions in B cell responses. Here we have used mouse fibroblasts transfected with the human Fc gamma receptor type II and human B7 to address this issue. We found that T cells, when activated through the T cell receptor (TcR)/CD3 complex with monoclonal antibodies and co-stimulated by B7-CD28 interaction, can provide efficient help for the induction of both IgM and IgG production by resting B cells. This helper activity is, at least in part, mediated by the interaction between the CD40 ligand on the T cells and CD40 on the B cells. We also demonstrate that more than one signal to the T cell is required for the induction of the CD40 ligand, one being delivered through the TcR/CD3 complex and the second by ligation of CD28 with the B7 molecule. In addition to the induction of cognate T helper function, we provide evidence that co-stimulation of T cells with B7-CD28 interaction can result in the secretion of both Th1- and Th2-type lymphokines.

Adult↗

PubMed2003

The flotillins are integral membrane proteins in lipid rafts that contain TCR-associated signaling components: implications for T-cell activation.

Lipid rafts play an important role in signal integration and cellular activation by the T-cell antigen receptor (TCR). We demonstrate that flotillin-1 and flotillin-2 are important structural raft components, which redistribute to the site of TCR engagement. An antibody to flotillin-1 was able to immobilize other TCR-associated raft components. Although rafts purified from unstimulated cells demonstrated abundant Lck but inabundant LAT, rafts from stimulated cells include an abundance of both components. This suggests dynamic changes in lipid raft composition during CD3/CD28 costimulation. Stimulation of primary human CD4(+) T cells leads to increased GM1 and flotillin-1 expression in the surface membrane, where these components colocalize. This may reconstitute new signaling complexes required for T-cell activation. Altered lipid raft composition and function may play a role in the decline of antigen responsiveness in senescent T cells. In this regard, we observed an increase in the raft-associated gangliolipid, GM1, in resting human CD4(+) and CD8(+) lymphocytes with aging.

Adaptor Proteins, Signal Transducing↗

PubMed2000

All infiltrating T-lymphocytes in Hodgkin's disease express immunohistochemically detectable T-cell receptor zeta-chains in situ.

AIM: We studied the expression of TCR zeta-chain on tumour-infiltrating lymphocytes in EBV-positive and EBV-negative cases of Hodgkin's disease (HD), to assess whether downregulation of TCR zeta-chain on tumour-infiltrating lymphocytes might be a mechanism for immune escape of the neoplastic cells. METHODS AND RESULTS: By immunohistochemistry we investigated tissue of 27 cases of primary HD, both paraffin embedded and frozen, for the presence of T-cell receptor complex zeta-chain and other T-cell markers on the reactive cells. Strong membranous staining of TCR zeta-chain was present in all cases in frozen tissue. In contrast, in paraffin-embedded material substantial loss of TCR zeta-chain was detected in old (> 6 years) tissues. However, no differences in either the number of positive cells or their staining intensity were observed in EBV-positive and negative cases of HD as detected in frozen tissue. Storage of paraffin-embedded tissue leads to a rapid and substantial loss of TCR zeta-chain reactivity compared to frozen material of the same HD cases. Staining reactivity of other T-cell markers (CD3, CD4 and CD8) on paraffin-embedded material remained unaffected. Immunofluorescent double-staining confirmed colocalization and coexpression of TCR zeta-chain and CD3. CONCLUSIONS: In frozen biopsies of primary HD TCR zeta-chain was expressed on all reactive CD3-positive cells, both in EBV-positive and EBV-negative cases. This suggests that zeta-chain downregulation is not a likely mechanism whereby neoplastic cells of HD can escape immune surveillance.

Adolescent↗