CD3 Gamma Delta Epsilon Research Articles

Masking of the CD3 gamma di-leucine-based motif by zeta is required for efficient T-cell receptor expression.

The T-cell receptor (TCR) is a multimeric receptor composed of the Ti alpha beta heterodimer and the noncovalently associated CD3 gamma delta epsilon and zeta(2) chains. All of the TCR chains are required for efficient cell surface expression of the TCR. Previous studies on chimeric molecules containing the di-leucine-based endocytosis motif of the TCR subunit CD3 gamma have indicated that the zeta chain can mask this motif. In this study, we show that successive truncations of the cytoplasmic tail of zeta led to reduced surface expression levels of completely assembled TCR complexes. The reduced TCR expression levels were caused by an increase in the TCR endocytic rate constant in combination with an unaffected exocytic rate constant. Furthermore, the TCR degradation rate constant was increased in cells with truncated zeta. Introduction of a CD3 gamma chain with a disrupted di-leucine-based endocytosis motif partially restored TCR expression in cells with truncated zeta chains, indicating that the zeta chain masks the endocytosis motif in CD3 gamma and thereby stabilizes TCR cell surface expression.

The CD3-γδε Transducing Module Mediates CD38-induced Protein-tyrosine Kinase and Mitogen-activated Protein Kinase Activation in Jurkat T Cells

We have examined the ability of the CD3-gamma delta epsilon and CD3-zeta signaling modules of the T cell receptor (TCR) to couple CD38 to intracellular signaling pathways. The results demonstrated that in TCR+ T cells that express the whole set of CD3 subunits CD38 ligation led to complete tyrosine phosphorylation of both CD3-zeta and CD3-epsilon polypeptide chains. In contrast, in TCR+ cells with a defective CD3-zeta association CD38 engagement caused tyrosine phosphorylation of CD3-epsilon but not of CD3-zeta. Despite these differences, in both cell types CD38 ligation resulted in protein-tyrosine kinase and mitogen-activated protein kinase activation. However, in cells expressing chimerical CD25-zeta or CD25-epsilon receptors or in a TCR-beta- Jurkat T cell line, CD38 ligation did not result in tyrosine phosphorylation of the chimeric receptors, or CD3 subunits, or protein-tyrosine kinase or mitogen-activated protein kinase activation. In summary, these results support a model in which CD38 transduces activating signals inside the cell by means of CD3-epsilon and CD3-zeta tyrosine phosphorylation. Moreover, these data identify the CD3-gamma delta epsilon signaling module as a necessary and sufficient component of the TCR/CD3 complex involved in T cell activation through CD38.

Engagement of T cell receptor triggers its recruitment to low-density detergent-insoluble membrane domains.

T-cell receptors (TCRs) upon binding to peptide-MHC ligands transduce signals in T lymphocytes. Tyrosine phosphorylations in the cytoplasmic domains of the CD3 (gammadeltaepsilon) and zeta subunits of the TCR complex by Src family kinases initiate the signaling cascades via docking and activation of ZAP-70 kinase and other signaling components. We examined the role of the low-density detergent-insoluble membranes (DIMs) in TCR signaling. Using mouse thymocytes as a model, we characterized the structural organization of DIMs in detail. We then demonstrated that TCR engagement triggered an immediate increase in the amount of TCR/CD3 present in DIMs, which directly involves the engaged receptor complexes. TCR/CD3 recruitment is accompanied by the accumulation of a series of prominent tyrosine-phosphorylated substrates and by an increase of the Lck activity in DIMs. Upon TCR stimulation, the DIM-associated receptor complexes are highly enriched in the hyperphosphorylated p23 zeta chains, contain most of the TCR/CD3-associated, phosphorylation-activated ZAP-70 kinases and seem to integrate into higher order, multiple tyrosine-phosphorylated substrate-containing protein complexes. The TCR/CD3 recruitment was found to depend on the activity of Src family kinases. We thus provide the first demonstration of recuitment of TCR/CD3 to DIMs upon receptor stimulation and propose it as a mechanism whereby TCR engagement is coupled to downstream signaling cascades.

ＮＫ細胞の増殖性疾患

Natural killer (NK) cells are a distinct non-T, non-B lineage of lymphocytes that mediate major histocompatibility complex-unrestricted cytotoxicity. Morphologically they are large granular lymphocytes, and phenotypically they commonly express CD16 and CD56 antigens, without expressing cell surface CD3. Although the developmental pathway of NK cells is not fully understood, they arise from CD34+ hematopoietic stem cells and, at least in part, differentiate in the bone marrow. They gain byctoplasmic CD3 gamma delta epsilon zeta antigens during maturation, and lose cytoplasmic CD3 gamma delta epsilon thereafter until the terminal maturation. Lymphoproliferative disorders of NK cells include NK cell-lineage granular lymphocyte-proliferative disorders (NK-GLPD), NK-cell lymphoma, and acute leukemia of NK-cell lineage. NK-GLPD are relatively rare. Most patients exhibit a chronic indolent clinical course, and do not require specific treatment. However, some patients exhibit an aggressive clinical course, and die of the disease despite extensive chemotherapy. This aggressive type NK-GLPD is caused by Epstein-Barr virus (EBV). Patients with NK-cell lymphoma are rare, and often exhibit necrotic lesion and angiocentric morphology. This tumor is mainly found in the nasal tract, but the true incidence of NK-cell lymphoma in nasal lymphomas is not known. Probably many lymphomas arising from the nasal cavity, but not from paranasal sinuses, are of NK-cell lineage. NK-cell lymphoma is also caused by EBV, and is resistant to combination chemotherapy. Acute leukemia of NK-cell lineage is very rare. Several cases of acute lymphoblastic leukemia and a single case of blast crisis of chronic myelogenous leukemia have been documented to have leukemic blasts characteristic of NK cells. However, the precise lineage and differentiation stage of the leukemic blasts have not been delineated.

The alpha beta T cell receptor clustering upon superantigen/MHC recognition.

Antigen recognition by T cells is the key event for the antigen specific immune responses to be triggered. This recognition is initiated by the binding of the T cell receptor (TCR) to antigen peptide/major histocompatibility complex (MHC) on the surface of the antigen presenting cells. TCR on most of the T cells is a heterodimer composed of alpha and beta chains which are associated with CD3 gamma delta epsilon as well as zeta chains, the signal transmission molecules. The dynamics of this TCR complex upon antigen/MHC recognition, however, has not been well understood. In this paper the authors analyse the configuration of TCR complex on T cells from a TCR beta chain gene transgenic mouse (TGM) strain. Unlike many other TGM strains reported, a considerable proportion of T cells from this TGM expresses both transgene-encoded (V beta 3) and endogenous TCR beta chains on their surface. By immunoprecipitation and immunoblotting analysis of T cells stimulated with a superantigen, staphylococcal enterotoxin B (SEB), the authors found that V beta 3 was coprecipitated with V beta 8, demonstrating the clustering of TCR alpha beta upon superantigen/MHC recognition.

T cell receptor alpha-chain tail is required for protein kinase C-mediated down-regulation, but not for signaling.

Antigen stimulation through the T cell receptor (TCR) induces phosphorylation of the associated CD3 gamma delta epsilon- and zeta-chain cytoplasmic tails. These events lead to the induction of the intracellular signaling pathways with concomitant receptor down-regulation. The TCR is down-regulated from the cell surface by the activation of protein kinase, C (PKC) and subsequent serine phosphorylation of the CD3 gamma-chain. We report here that the TCR alpha-chain cytoplasmic tail is also necessary for PKC-mediated internalization of the TCR complex. The requirement for the TCR alpha-chain cytoplasmic tail is specific for internalization of the TCR complex, since down-regulation of CD4 is still intact in hybridoma cells expressing a tailless TCR alpha-chain. The absence of TCR internalization directly correlates with defective PKC-mediated phosphorylation of the CD3 gamma-chain. Despite deficient PKC-mediated TCR down-regulation, the tailless alpha beta TCR still transduces antigenic signals resulting in the production of interleukin-2. Although the TCR tails are not obviously required for signal transduction, the TCR alpha-tail may serve as a targeting domain for PKC-mediated down-regulation of the TCR complex.

Calnexin Associates Exclusively with Individual CD3δ and T Cell Antigen Receptor (TCR) α Proteins Containing Incompletely Trimmed Glycans That Are Not Assembled into Multisubunit TCR Complexes

Most T lymphocytes express on their surfaces an oligomeric protein complex consisting of clonotypic alpha beta polypeptides associated with invariant CD3-gamma delta epsilon and zeta chains, designated the T cell antigen receptor (TCR) complex. Assembly and intracellular transport of nascent TCR proteins is believed to be assisted by their interaction with the molecular chaperone calnexin, which for certain molecules functions as a lectin for monoglucosylated glycans. However, as most of our knowledge about calnexin-TCR protein associations has been obtained under conditions of limited TCR assembly, the role of calnexin in the formation of nascent TCR complexes is unclear. Here, we studied the role of glucose (Glc) trimming and calnexin association in the oligomerization of TCR alpha and CD3 delta glycoproteins in murine splenic T lymphocytes, a model cell type for efficient assembly of complete TCR complexes. We show that removal of Glc residues from both CD3 delta proteins and TCR alpha proteins occurred prior to their association with any other TCR components and that calnexin specifically interacted with unassembled TCR alpha and CD3 delta proteins containing incompletely trimmed oligosaccharides. Interestingly, we found that removal of Glc residues from glycan chains was necessary for efficient association of calnexin with TCR alpha glycoproteins but not with CD3 delta glycoproteins. These studies define Glc trimming and calnexin association as initial molecular events in the translation of CD3 delta and TCR alpha proteins occurring coincident with or immediately after their translocation into the endoplasmic reticulum and preceding the ordered pairing of TCR chains. In addition, these data document that calnexin assembly with CD3 delta and TCR alpha glycoproteins involves both glycan-dependent and glycan-independent mechanisms.

Lymphoproliferative disorders of natural killer cells.

Natural killer (NK) cells are a distinct non-T, non-B lineage of lymphocytes that mediate major histocompatibility complex-unrestricted cytotoxicity. Morphologically they are large granular lymphocytes, and phenotypically they commonly express CD16 and CD56 antigens, without expressing cell surface CD3. Although the developmental pathway of NK cells is not fully understood, they arise from CD34+ hematopoietic stem cells and, at least in part, differentiate in the bone marrow. They gain byctoplasmic CD3 gamma delta epsilon zeta antigens during maturation, and lose cytoplasmic CD3 gamma delta epsilon thereafter until the terminal maturation. Lymphoproliferative disorders of NK cells include NK cell-lineage granular lymphocyte-proliferative disorders (NK-GLPD), NK-cell lymphoma, and acute leukemia of NK-cell lineage. NK-GLPD are relatively rare. Most patients exhibit a chronic indolent clinical course, and do not require specific treatment. However, some patients exhibit an aggressive clinical course, and die of the disease despite extensive chemotherapy. This aggressive type NK-GLPD is caused by Epstein-Barr virus (EBV). Patients with NK-cell lymphoma are rare, and often exhibit necrotic lesion and angiocentric morphology. This tumor is mainly found in the nasal tract, but the true incidence of NK-cell lymphoma in nasal lymphomas is not known. Probably many lymphomas arising from the nasal cavity, but not from paranasal sinuses, are of NK-cell lineage. NK-cell lymphoma is also caused by EBV, and is resistant to combination chemotherapy. Acute leukemia of NK-cell lineage is very rare. Several cases of acute lymphoblastic leukemia and a single case of blast crisis of chronic myelogenous leukemia have been documented to have leukemic blasts characteristic of NK cells. However, the precise lineage and differentiation stage of the leukemic blasts have not been delineated.

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.

Structure of the T cell receptor in a Ti alpha V beta 2, alpha V beta 8-positive T cell line.

The T cell receptor (TcR) is composed of at least six different polypeptide chains consisting of the clonotypic Ti heterodimer (Ti alpha beta or Ti gamma delta) and the noncovalently associated CD3 chains (CD3 gamma delta epsilon zeta). The exact number of subunits constituting the TcR is still not known; however, it has been suggested that each TcR contains two Ti dimers. To gain insight into the structure of the TcR we constructed a Ti alpha V beta 2, alpha V beta 8-positive T cell line which expressed the endogenous human TiV beta 8 and the transfected mouse TiV beta 2 both in association with the endogenous Ti alpha and CD3 chains at the cell surface. Preclearing experiments with radioiodinated cell lysate prepared with digitonin lysis buffer demonstrated that depleting the lysate of Ti alpha V beta 8 by immunoprecipitation with anti V beta 8 monoclonal antibody (mAb) did not reduce the amount of Ti alpha V beta 2 in the lysate, and likewise, depleting the lysate of Ti alpha V beta with anti-V beta 2 mAb did not reduce the amount of Ti alpha V beta 8. Comodulation experiments showed that V beta 8 and V beta 2 did not comodulate with each other. Furthermore, functional tests demonstrated that TcR containing V beta 8 and TcR containing V beta 2 mediated transmembrane activation signals independently of each other. These data demonstrate that mouse V beta 2 and human V beta 8 were not expressed in the same TcR in agreement with a TcR model where each TcR contains only one Ti dimer.

Assembly of the T-cell antigen receptor. Participation of the CD3 omega chain.

The human TCR is composed of the Ti alpha beta heterodimer in association with the CD3 chains CD3 gamma delta epsilon zeta 2. Another chain, referred to as CD3 omega, has recently been described in T cells. CD3 omega is an intracellular protein transiently associated with the CD3 complex during the assembly of the TCR in the endoplasmic reticulum (ER) and it is not expressed on the cell surface. The function of CD3 omega is unknown but it has been suggested that it plays an important role in the assembly of the TCR. We have studied the possible function of CD3 omega in the human leukemic T-cell line Jurkat and different variants of this cell line. Cells were metabolically labeled, subjected to lysis, immunoprecipitated, and analyzed by SDS-PAGE. The results indicate that: 1) CD3 omega associates primarily with the CD3 delta epsilon complex; 2) CD3 omega is not associated with single Ti alpha or Ti beta chains, but is present in complexes composed of both the CD3 and the Ti chains; 3) CD3 omega is part of the complete, intracellular receptor complex Ti alpha beta/CD3 gamma epsilon delta omega zeta 2; and 4) CD3 omega dissociates from the Ti/CD3 complex in the ER before maturation of the Ti alpha beta heterodimer. On the basis of these results, we propose a model for the assembly and subunit stoichiometry of the TCR complex which includes the participation of the CD3 omega chain.

Restoration of T cell development in RAG-2-deficient mice by functional TCR transgenes.

Introduction of TCR alpha transgene, TCR beta transgene, or both into RAG-2-/-mice differentially rescues T cell development. RAG-2-/- mice have small numbers of TCR-CD4-CD8-(double negative, DN) thymocytes that express CD3 gamma delta epsilon and zeta proteins intracellularly. Introduction of a TCR beta transgene, but not a TCR alpha transgene, into the RAG-2-/- background restored normal numbers of thymocytes. These cells were CD4+CD8+ (double positive, DP) and expressed small amounts of surface TCR beta chain dimers in association with CD3 gamma delta epsilon but not zeta. RAG-2-/- mice that expressed alpha and beta TCR transgenes developed both DP and single positive thymocytes. Thus, the TCR beta subunit, possibly in association with a novel CD3 complex, participates in the DN to the DP transition.

Characterization of T cell receptor assembly and expression in a Ti γδ‐positive cell line

T cell antigen receptor (TcR) heterodimers of both the Ti-alpha beta and Ti-gamma delta types are expressed at the surface of T cells noncovalently associated with the CD3 complex composed of the monomorphic chains gamma, delta, epsilon and zeta. The structural relationship and assembly of the various components of this multimeric protein complex is still not fully understood. In this report, the human leukemic T cell line Lyon which expresses a Ti-gamma delta/CD3 complex, was characterized and compared to another human leukemic T cell line Jurkat (Ti-alpha beta/CD3). Membrane TCR-/CD3- variants of the T cell Lyon were induced and found to produce all of the Ti/CD3 components, with the exception of Ti-delta. Biochemical analysis indicated that: (1) Ti-gamma/CD3 gamma, delta, epsilon complexes were formed in the endoplasmic reticulum in the absence of Ti-delta; (2) the CD3-zeta chain did not associate with the Ti-gamma/CD3 gamma delta epsilon complex and (3) the Ti-delta chain was required for cell surface expression of the Ti-gamma delta/CD3 complex. Introduction of Jurkat wild-type Ti-alpha cDNA into Lyon T cells resulted in Ti-alpha beta/CD3 expression and abrogated Ti-gamma delta/CD3 expression. In contrast, the expression of the Ti-gamma delta/CD3 complex was not affected by transfection of a mutated Ti-alpha cDNA into Lyon cells. The mutated Ti-alpha chain formed complexes with Ti-beta and CD3 gamma delta epsilon, but the CD3-zeta chain did not associate with these complexes. Taken together analysis of Lyon cells transfected with either wild-type or mutated Ti-alpha suggested that the CD3-zeta chain may have higher affinity for Ti-alpha beta/CD3 complexes than for Ti-gamma delta/CD3 complexes.

Structure of the T‐Cell Receptor in a Tiαβ, Tiγδ Double Positive T‐Cell Line

The multichain T-cell receptor is composed of at least six different polypeptide chains. The clonotypic Ti heterodimer (Ti alpha beta or Ti gamma delta) is non-covalently associated with the CD3 chains (CD3 gamma delta epsilon zeta). The exact number of subunits constituting the T-cell receptor is still not known. It has been suggested that each T-cell receptor contains two Ti dimers. To gain insight into the structure of the T-cell receptor we constructed a Ti alpha beta, Ti gamma delta double positive T-cell line which contained four functional Ti chains (Ti alpha, beta, gamma, and delta). The data demonstrated an absence of Ti dimers containing mixtures of chains other than the typical Ti alpha beta and Ti gamma delta combinations. Furthermore, by co-modulation experiments we demonstrated that the Ti alpha beta and the Ti gamma delta dimers were not expressed in the same T-cell receptor. Our data indicate that the T-cell receptor does not contain two Ti dimers.

TCR isoform containing the Fc receptor gamma chain exhibits structural and functional differences from isoform containing CD3 zeta.

The structure and function of the TCR-CD3 complex containing a homodimer of the gamma chain of the high affinity receptor for IgE (FcR gamma) (FcR gamma+ TCR) was investigated by transfecting the FcR gamma gene into a CD3 zeta-, CD3 eta-, FcR gamma- T cell line. Introduction of FcR gamma, as well as CD3 zeta, induced a high expression of the TCR-CD3 complex on the cell surface. Transfected FcR gamma formed a homodimer and associated firmly with the TCR alpha beta dimer but only weakly with the CD3 gamma delta epsilon. Stimulation of both FcR gamma and CD3 zeta transfectants by antibodies against TCR or CD3 induced accumulation of inositol phosphates, the Ca2+ response, IL-2 production, and growth inhibition. On the other hand, antigen stimulation of transfectants expressing FcR gamma as well as CD3 zeta induced IL-2 production, but only the latter exhibited the antigen-induced growth inhibition. In vitro kinase assay suggested that the CD3 zeta dimer but not the FcR gamma dimer associates with the Fyn kinase. These results indicate that the FcR gamma homodimer is able to form a functional TCR complex but that the mode of assembly and the signaling function of FcR gamma+ TCR, including its association with tyrosine kinase(s), may differ from the TCR-CD3 complex containing CD3 zeta homodimers (zeta+ TCR). This provides an example which illustrates that different TCR isoforms mediate distinct signals and functions.

Expression of cytoplasmic CD3 epsilon proteins in activated human adult natural killer (NK) cells and CD3 gamma, delta, epsilon complexes in fetal NK cells. Implications for the relationship of NK and T lymphocytes.

NK cells have been defined as CD3-, CD16+, and/or CD56+ lymphocytes that mediate MHC-unrestricted cytotoxicity against certain tumors and virus-infected cells. Although CD3 epsilon transcripts have been detected in some NK clones, it has generally been thought that NK cells do not express CD3 proteins other than zeta which is associated with CD16 (Fc gamma RIII). We demonstrate that adult peripheral blood NK cell lines and clones express cytoplasmic CD3 epsilon proteins, but not CD3 delta or gamma. CD3 epsilon proteins were detected by immunoprecipitation, Western blot analysis, and immunofluorescence using antiserum directed against the cytoplasmic domain of CD3 epsilon. Although resting, adult peripheral blood NK cells have essentially undetectable levels of CD3 epsilon protein, expression was increased substantially after activation. In contrast to adult NK cells, NK cell clones established from human fetal liver express CD3 gamma, delta, and epsilon protein subunits that associate and form CD3 epsilon, gamma and CD3 epsilon, delta complexes in the cytoplasm, but are apparently unable to be transported to the cell surface. These results indicate that expression of CD3 gamma delta epsilon subunits is not restricted to T lymphocytes and supports the possibility that NK and T cells may be derived from common origins.

Structural mutations of C-domains in members of the Ig superfamily. Consequences for the interactions between the T cell antigen receptor and the zeta 2 homodimer.

Several molecules belonging to the Ig superfamily are expressed together with noncovalently associated subunits. This applies for membrane-bound IgM and IgD, some of the FcR, and the Ti dimers of the TCR. The interactions between members of the Ig superfamily and their associated subunits are still not fully understood. We locate critical amino acid residues for TCR assembly in the Ti-alpha and -beta extracellular C-domains. A point mutation (phenylalanine195----valine) in a highly conserved residue in the Ti-alpha chain of the Jurkat variant J79 was identified by DNA sequencing. This mutation did not prevent cytoplasmic association of Ti alpha beta and CD3 gamma delta epsilon, but abolished binding of the zeta 2 homodimer to the rest of the TCR. The consequences of this mutation for TCR assembly were confirmed by transfection of a site-directed mutagenized Ti-alpha chain into a Ti-alpha-deficient Jurkat variant. Computer model analysis showed that the Ti-alpha phenylalanine195 directly contributed to the beta-sheet facing away from the Ti-beta chain, indicating that it could be directly involved in the interactions between one or more of the CD3 chains or the zeta 2 dimer. Site-directed mutagenesis of the corresponding residue in the Ti-beta chain demonstrated that a phenylalanine216----valine substitution had similar effects on TCR assembly as the Ti-alpha mutation, whereas a phenylalanine216----histidine substitution allowed TCR assembly and expression. Whether the consequences for TCR assembly of the Ti-alpha and -beta mutations were due to any direct effects on the interaction between zeta and the Ti alpha beta dimer or to indirect effects are discussed.

Failure to synthesize the CD3-gamma chain. Consequences for T cell antigen receptor assembly, processing, and expression.

The TCR consists of the Ti alpha beta heterodimer and the associated CD3 chains, CD3 gamma delta epsilon zeta 2 or zeta eta. The structural relationships between the subunits of the Ti/CD3 complex are still not fully understood. To explore the roles of the individual CD3 chains for the assembly, intracellular processing, and expression of the TCR, mutants of the T cell line Jurkat were isolated. One variant, JGN, was found to produce all the Ti/CD3 components with the exception of CD3-gamma. The results indicate that: 1) the tetrameric form (Ti alpha beta-CD3 delta epsilon) of the Ti/CD3 complex is produced in the endoplasmic reticulum in the absence of CD3-gamma; 2) CD3-zeta does not associate with the Ti alpha beta-CD3 delta epsilon complex; 3) the Ti alpha beta-CD3 delta epsilon complex is not exported from the endoplasmic reticulum to the Golgi apparatus; and 4) CD3-gamma is required for cell surface expression of the Ti/CD3 complex. Transfection of the wild-type CD3-gamma gene into JGN reconstituted expression of functional Ti/CD3 complexes, and analysis of T cell lines producing different amounts of CD3-gamma indicated that CD3-gamma and CD3-delta competed for the binding to CD3-epsilon.

The high affinity Fc epsilon receptor gamma subunit (Fc epsilon RI gamma) facilitates T cell receptor expression and antigen/major histocompatibility complex-driven signaling in the absence of CD3 zeta and CD3 eta

The T cell receptor (TCR) is a molecular complex formed by at least seven transmembrane proteins: the antigen/major histocompatibility complex recognition unit (Ti alpha-beta heterodimer) and the invariant CD3 chains (gamma, delta, epsilon, zeta, and eta). In addition to targeting partially assembled Ti alpha-beta CD3 gamma delta epsilon TCR complexes to the cell surface, CD3 zeta appears to be essential for interleukin-2 production after TCR stimulation with antigen/major histocompatibility complex. The gamma chain of the high affinity Fc receptor for IgE (Fc epsilon RI gamma) has significant structural homology to CD3 zeta and the related CD3 eta subunit. To identify the functional significance of sequence homologies between CD3 zeta and Fc epsilon RI gamma in T cells, we have transfected a Fc epsilon RI gamma cDNA into a T cell hybridoma lacking CD3 zeta and CD3 eta proteins. Herein we show that a Fc epsilon RI gamma-gamma homodimer associates with TCR components to up-regulate TCR surface expression. A TCR composed of Ti alpha-beta CD3 gamma delta epsilon Fc epsilon RI gamma-gamma is sufficient to restore the coupling of TCR antigen recognition to the interleukin-2 induction pathway, demonstrating the functional significance of structural homology between the above receptor subunits. These results, in conjunction with the recent finding that CD3 zeta, CD3 eta, and Fc epsilon RI gamma are coexpressed in certain T cells as subunits of an unusual TCR isoform, suggest that Fc epsilon RI gamma is likely to play a role in T cell lineage function.

Characterization of thymus-derived lymphocytes expressing Ti alpha-beta CD3 gamma delta epsilon zeta-zeta, Ti alpha-beta CD3 gamma delta epsilon eta-eta or Ti alpha-beta CD3 gamma delta epsilon zeta-zeta/zeta-eta antigen receptor isoforms: analysis by gene transfection.

To characterize the function of the CD3 eta subunit of the T cell receptor (TCR), we have used cDNAs encoding CD3 zeta, CD3 eta, or both to reconstitute a variant of a cytochrome c-specific, I-Ek-restricted murine T cell hybridoma, termed MA5.8, which lacks CD3 zeta and CD3 eta proteins. We provide direct evidence that assembly and surface expression of TCRs can be mediated by either of these subunits separately or together. However, the level of TCR expression on zeta transfectants is up to one order of magnitude greater than that on eta transfectants, implying that CD3 eta is weakly associated with the pentameric Ti alpha-beta CD3 gamma delta epsilon complex and/or inefficient at salvaging the incomplete TCR from lysosomal degradation. As a component of the TCR, the CD3 eta subunit preferentially forms a heterodimer with CD3 zeta, but is also able to form a CD3 eta-eta homodimer. Crosslinking of Ti alpha-beta CD3 gamma delta epsilon zeta-zeta, Ti alpha-beta CD3 gamma delta epsilon eta-eta, or Ti alpha-beta CD3 gamma delta epsilon zeta-zeta/zeta-eta TCR isotypes with anti-CD3 epsilon monoclonal antibody or a cytochrome c peptide epitope on I-Ek antigen-presenting cells mediates signal transduction resulting in reversible cell-cycle arrest of transfected clones. Given the potential for diversity of signals generated by these functional TCR isotypes and the expression of the CD3 eta gene product in the thymus, CD3 eta is likely to play a role in selection and/or activation of thymocytes during development.