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.     

Biochemical characterization of the eta chain of the T-cell receptor. A unique subunit related to zeta

The T-cell antigen receptor is a multisubunit complex consisting of at least seven chains. Based upon structural and genetic considerations, we have divided these chains into three groups. The alpha and beta subunits (Ti) are the clonotypic chains responsible for antigen recognition. Three chains that are invariant among all T-cells define the CD3 complex. These include the CD3 gamma, delta, and epsilon chains. The zeta chain is a distinct component that, like the CD3 chains, is invariant among all T-cells. In the majority of receptors, zeta is found as a disulfide-linked homodimer. We have recently shown that approximately 10% of zeta is disulfide-linked to a chain which we have called eta. A preliminary model has been proposed, suggesting that there are two subclasses of receptors, depending upon the presence within the complex of either the zeta-zeta homodimer or the zeta-eta heterodimer. Evidence has been presented that these two subclasses may perform distinct signaling functions. In this paper the eta chain is characterized to determine whether it is structurally related to the zeta chain and, in particular, whether it might represent a post-translational modification of zeta. We can identify specific antigenic epitopes that are shared by both zeta and eta. However, not all antibodies raised against zeta can directly recognize eta. The apparent molecular mass of eta is 22 kDa, whereas zeta has a molecular mass of 16 kDa. We are unable to demonstrate any post-translational covalent modifications of eta to explain the difference in apparent molecular weight. These include phosphorylation, glycosylation, or sulfation. Amino acid incorporation studies demonstrate that the amino acid composition of eta is distinct from that of zeta. All of the eta in a T-cell is found in association with the rest of the components of the T-cell receptor. In addition, our anti-eta antibodies allow us to directly recognize human eta, which has an apparent molecular mass of approximately 23 kDa. Thus, eta and zeta appear to be related but distinct proteins, and we would propose that eta is the second member of the zeta group of components of the T-cell receptor.     

T cell receptor (TCR) beta chain homodimers on the surface of immature but not mature alpha, gamma, delta chain deficient T cell lines.

Transfected T cell receptor (TCR) beta chain genes are expressed as homodimers on the surface of immature (Sci/ET27F) but not on mature (58 alpha-beta-) T cell lines which lack TCR alpha, gamma and delta chains. The homodimer on Sci/ET27F cells is tightly bound to CD3 delta and CD3 epsilon while the association with CD3 gamma and CD3 zeta proteins is rather weak. Crosslinking of the TCR beta homodimers resulted in a strong and rapid calcium flux. In 58 alpha-beta- T cells the beta TCR chain could be easily visualized intracellularly but was not transported to the cell surface. The Scid cell lines considerably facilitate the molecular analysis of early differentiation events in the thymus which are likely to be regulated by the beta TCR homodimer.     

A fraction of CD3 epsilon subunits exists as disulfide-linked dimers in both human and murine T lymphocytes

In a T cell antigen receptor complex (TCR), the clonotypic disulfide-linked Ti heterodimer is noncovalently associated with the invariant CD3 polypeptides. The latter are composed of three monomeric subunits (gamma, delta, epsilon) and either a disulfide-linked homodimer (zeta zeta) or a disulfide-linked heterodimer (zeta eta). The exact stoichiometry of the Ti-CD3 subunits in a given complex is still largely unknown. Here, we report the presence of a CD3 epsilon dimer in a fraction of the TCR. When TCRs from both human and murine T lymphocytes were immunoprecipitated with monoclonal antibodies against either CD3 epsilon or Ti, a 40-kDa disulfide-linked dimer was coprecipitated with the other TCR subunits from digitonin lysates. Amino acid sequence analysis of peptides obtained by in situ CNBr cleavage of the 20-kDa product blotted to polyvinyl difluoride membranes from reducing/nonreducing two-dimensional gels identified human CD3 epsilon. Assuming this CD3 epsilon to derive from a homodimer, then either some TCRs contain more than one CD3 epsilon chain or several TCRs are covalently associated with one another via their CD3 epsilon subunits. Although it has been suggested that a putative TCR association with CD2 exists under similar conditions to those utilized to detect CD3 epsilon dimers, the CD2 molecule was not coimmunoprecipitated with the TCR by any of a series of anti-CD3 epsilon monoclonal antibodies. In conjunction with the fact that CD2 and the TCR do not colocalize during conjugate formation between T cells and antigen-presenting cells (Koyasu, S., Lawton, T., Novick, D., Recny, M. A., Siliciano, R. F., Wallner, B. P., and Reinherz, E. L. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2603-2607), we conclude that CD2 and the TCR are not physically associated on the T cell surface.     

T cell antigen receptor--structure, expression and function

T cell receptor complex is composed of at least 7 different polypeptides and is one of the most sophisticated receptor. There are two types of T cell receptor (TCR); alpha beta and gamma delta, both of which are composed of a heterodimer and associated with invariant CD3 complexes on the cell surface. T cells expressing alpha beta dimer recognize antigen-peptides in the context of self-MHC molecules, whereas the specificity and function of gamma delta T cells are largely unknown. Gene organization of alpha beta and gamma delta indicates the difference of mechanism to generate diversity. Whereas alpha and beta genes have a large number of V genes, those of gamma and delta genes are limited. However, especially for delta gene, the repertoire is largely produced by junctional diversity. There are increasing data showing new TCR heterodimers; such as beta delta heterodimer in human, beta homodimer in mouse and unknown new heterodimer in chicken, which are expressed on the cell surface in the association with CD3 complex. The characterization of these new receptor dimers and the function of cells expressing these receptors have to be determined. Among CD3 complex, zeta and eta chains are most important for signal transduction after antigen-recognition by TCR. eta gene is recently cloned and now found to be produced by an alternative splicing of a common gene with zeta chains gene. Tyrosine++ phosphorylation of zeta chain seems to be one of the earliest events of T cell activation. Since fyn, one of src oncogene family possessing tyrosine++ kinase function, is co-precipitated with TCR-CD3 complex, fyn seems to be involved in early phosphorylation for T cell activation. Positive and negative selection of thymocytes has been shown to occur via TCR using TCR-transgenic mice model. Molecular mechanism of the selection should be determined.     

Association of the human CD3-zeta chain with the alpha beta-T cell receptor/CD3 complex. Clues from a T cell variant with a mutated T cell receptor-alpha chain

The TCR for Ag, on the majority of human T cells, is a disulfide-linked heterodimer composed of TCR-alpha and -beta chains noncovalently associated with the monomorphic CD3 complex composed of the CD3-gamma, -delta, -epsilon, and -zeta chains. The interactions involved in the assembly of the various components of this multimeric protein complex are not fully understood. In this report, a variant of the human leukemic T cell line Jurkat that synthesized all of the known components of the TCR/CD3 complex but fails to express the TCR/CD3 complex at the cell surface is further characterized. This variant, J79, has a mutated TCR-alpha chain that does not affect the assembly of the pentameric form (TCR-alpha beta-CD3-gamma delta epsilon) of the TCR/CD3 complex but inhibits the assembly of the CD3-zeta homodimer with the rest of the complex (TCR-alpha beta-CD3-gamma delta epsilon----TCR-alpha beta-CD3-gamma delta epsilon zeta 2). Transfecting a wild-type TCR-alpha gene into J79 reconstituted expression of a complete functionally competent TCR/CD3 complex at the cell surface. The results indicate that the TCR-alpha chain plays a crucial role in the assembly of the CD3-zeta homodimer with the pentameric form of the TCR/CD3 complex.     

Pairwise, cooperative and inhibitory interactions describe the assembly and probable structure of the T-cell antigen receptor.

The T-cell antigen receptor (TCR) is a multi-subunit complex consisting of clonotypic heterodimers (TCR-alpha beta or TCR-gamma delta) that are non-covalently linked to at least four invariant chains (CD3-delta, -epsilon, -gamma; and zeta or eta). The ordered process of assembly and the final number of individual chains that comprise the TCR is unclear. In this study, we examined the molecular basis of subunit interactions and the component requirements leading to the formation of a complete TCR. Analysis of transient cotransfections in monkey kidney fibroblasts (COS cells) showed assembly between selective chain pairs. Multiple chain cotransfections demonstrated the formation of stable higher order partial complexes. Assembly of such subcomplexes was facilitated by cooperative interactions between clonotypic and invariant CD3 chains. When zeta was cotransfected with any TCR component, no pairwise interaction was detected. Only when there was coexpression of all of the other TCR chains (TCR-alpha, -beta, CD3- epsilon, -gamma, -delta) did zeta assemble with the TCR complex. Not all chain pairs formed stable heterodimers. For one such pair, lack of assembly is due to the inhibitory effects of negatively charged residues within their transmembrane domains. The combined effects of these interactions probably determine the assembly and the quaternary structure of the TCR complex.     

Differential regulation of T-cell receptor processing and surface expression affected by CD3 theta, an alternatively spliced product of the CD3 zeta/eta gene locus.

The T-cell receptor (TCR) is a multisubunit complex consisting of the clonotypic Ti alpha and beta (or Ti gamma and delta) subunits and the invariant CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, and CD3 eta subunits. Herein, we describe an additional product from the CD3 zeta/eta gene locus which we have termed CD3 theta. The cDNA derives from the first seven exons common to CD3 zeta and CD3 eta, 94 base pairs (bp) of the CD3 eta-specific exon 9 and an additional exon 10 encoding the carboxyl-terminal 15 amino acids and the 3'-untranslated region. The expression of CD3 theta is equivalent to that of CD3 eta in tissue distribution and level of expression as judged by RNase protection analysis. Despite the identity of the amino-terminal 121 amino acids of CD3 zeta, CD3 eta, and CD3 theta and an additional 31 amino acids shared between CD3 eta and CD3 theta, transfection of CD3 theta into the CD3 zeta- eta- T-cell hybridoma, MA5.8, failed to restore detectable surface TCR expression in contrast to transfection with CD3 zeta or CD3 eta. Analysis of the CD3 theta protein in transfectants indicated that CD3 theta is associated with the TCR intracellularly. However, unlike with CD3 zeta, Ti alpha-beta chains remain endoglycosidase H sensitive, suggesting a role for the unique COOH-terminal segment of CD3 theta in mediating TCR retention and/or degradation in a pre-Golgi compartment.     

Phosphorylation of multiple CD3 zeta tyrosine residues leads to formation of pp21 in vitro and in vivo. Structural changes upon T cell receptor stimulation.

T lymphocyte activation resulting from antigen recognition involves a protein tyrosine kinase pathway which triggers phosphorylation of several cellular substrates including the CD3 zeta subunit of the T cell receptor (TCR) to form pp21. The homologous TCR-associated protein, CD3 eta, is an alternatively spliced product of the same gene locus as CD3 zeta. CD3 eta lacks one of six cytoplasmic tyrosine residues (Tyr-132) found in CD3 zeta and is itself not phosphorylated. Site-directed mutagenesis in conjunction with in vitro and in vivo phosphorylation studies herein demonstrates that Tyr-132 is required for the formation of pp21. Moreover, the differential phosphorylation of CD3 zeta versus CD3 eta is not due to a selective association of the known TCR-associated protein tyrosine kinase, p59fyn; p59fyn but not p56lck or p62yes is associated with each of the three TCR isoforms containing CD3 zeta 2, or CD3 eta 2, or CD3 zeta-eta. This association occurs through components of the TCR complex distinct from CD3 zeta or CD3 eta. In addition, we show that pp21 formation is not only dependent on Tyr-132 but results from concomitant phosphorylation of other CD3 zeta residues including Tyr-121. Mutation of Tyr-90, -121, or -132 does not alter primary signal transduction as shown by the ability of individual CD3 zeta Tyr----Phe mutants to produce interleukin-2 upon TCR stimulation. Thus, the substantial structural changes in CD3 zeta upon TCR stimulation as reflected by alteration in its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis may affect subsequent events such as receptor desensitization, receptor movement, and/or protein associations.     

Role of CD3 gamma in T cell receptor assembly

The T cell receptor (TCR) consists of the Ti alpha beta heterodimer and the associated CD3 gamma delta epsilon and zeta 2 chains. The structural relationships between the subunits of the TCR complex are still not fully known. In this study we examined the role of the extracellular (EC), transmembrane (TM), and cytoplasmic (CY) domain of CD3 gamma in assembly and cell surface expression of the complete TCR in human T cells. A computer model indicated that the EC domain of CD3 gamma folds as an Ig domain. Based on this model and on alignment studies, two potential interaction sites were predicted in the EC domain of CD3 gamma. Site-directed mutagenesis demonstrated that these sites play a crucial role in TCR assembly probably by binding to CD3 epsilon. Mutagenesis of N-linked glycosylation sites showed that glycosylation of CD3 gamma is not required for TCR assembly and expression. In contrast, treatment of T cells with tunicamycin suggested that N-linked glycosylation of CD3 delta is required for TCR assembly. Site-directed mutagenesis of the acidic amino acid in the TM domain of CD3 gamma demonstrated that this residue is involved in TCR assembly probably by binding to Ti beta. Deletion of the entire CY domain of CD3 gamma did not prevent assembly and expression of the TCR. In conclusion, this study demonstrated that specific TCR interaction sites exist in both the EC and TM domain of CD3 gamma. Furthermore, the study indicated that, in contrast to CD3 gamma, glycosylation of CD3 delta is required for TCR assembly and expression.     

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 cytoplasmic domain of the T cell receptor zeta chain is sufficient to couple to receptor-associated signal transduction pathways.

The function of the T cell antigen receptor (TCR) invariant chains, CD3 gamma, delta, epsilon, and zeta, is poorly understood. Evidence suggests that CD3 couples receptor ligand binding to intracellular signaling events. To examine the role of the CD3 zeta chain in TCR-mediated signal transduction, a chimeric protein linking the extracellular and transmembrane domains of CD8 to the cytoplasmic domain of the zeta chain was constructed. The CD8/zeta chimera is expressed independently of the TCR and is capable of transducing signals that, by criteria of early and late activation, are indistinguishable from those generated by the intact TCR. These data indicate that CD8/zeta can activate the appropriate signal transduction pathways in the absence of CD3 gamma, delta, and epsilon, and suggest that the role of CD3 zeta is to couple the TCR to intracellular signal transduction mechanisms.     

A redundant role of the CD3 gamma-immunoreceptor tyrosine-based activation motif in mature T cell function

At least four different CD3 polypeptide chains are contained within the mature TCR complex, each encompassing one (CD3gamma, CD3delta, and CD3epsilon) or three (CD3zeta) immunoreceptor tyrosine-based activation motifs (ITAMs) within their cytoplasmic domains. Why so many ITAMs are required is unresolved: it has been speculated that the different ITAMs function in signal specification, but they may also serve in signal amplification. Because the CD3zeta chains do not contribute unique signaling functions to the TCR, and because the ITAMs of the CD3-gammadeltaepsilon module alone can endow the TCR with normal signaling capacity, it thus becomes important to examine how the CD3gamma-, delta-, and epsilon-ITAMs regulate TCR signaling. We here report on the role of the CD3gamma chain and the CD3gamma-ITAM in peripheral T cell activation and differentiation to effector function. All T cell responses were reduced or abrogated in T cells derived from CD3gamma null-mutant mice, probably because of decreased expression levels of the mature TCR complex lacking CD3gamma. Consistent with this explanation, T cell responses proceed undisturbed in the absence of a functional CD3gamma-ITAM. Loss of integrity of the CD3gamma-ITAM only slightly impaired the regulation of expression of activation markers, suggesting a quantitative contribution of the CD3gamma-ITAM in this process. Nevertheless, the induction of an in vivo T cell response in influenza A virus-infected CD3gamma-ITAM-deficient mice proceeds normally. Therefore, if ITAMs can function in signal specification, it is likely that either the CD3delta and/or the CD3epsilon chains endow the TCR with qualitatively unique signaling functions.     

Selective degradation of T cell antigen receptor chains retained in a pre-Golgi compartment

We have examined the fate of newly synthesized T cell antigen receptor (TCR) subunits in a T cell hybridoma deficient in expression of the clonotypic beta chain. Synthesis and assembly of the remaining chains proceed normally but surface expression of TCR chains is undetectable in these cells. A variety of biochemical and morphological techniques has been used to show that the TCR chains in these cells fail to be transported to any of the Golgi cisternae. Instead, they are retained in a pre-Golgi compartment which is either part of or closely related to the endoplasmic reticulum. The CD3-delta chain is degraded by a non- lysosomal process that is inhibited at temperatures at or below 27 degrees C. By contrast, the remaining chains (CD3-epsilon, CD3-gamma, and zeta) are very stable over 7 h. We propose possible mechanisms that may explain the differential fate of TCR chains retained in a pre-Golgi compartment.     

Human TCR that incorporate CD3zeta induce highly preferred pairing between TCRalpha and beta chains following gene transfer

TCR gene therapy is adversely affected by newly formed TCRalphabeta heterodimers comprising exogenous and endogenous TCR chains that dilute expression of transgenic TCRalphabeta dimers and are potentially self-reactive. We have addressed TCR mispairing by using a modified two-chain TCR that encompasses total human CD3zeta with specificities for three different Ags. Transfer of either TCRalpha:CD3zeta or beta:CD3zeta genes alone does not result in surface expression, whereas transfer of both modified TCR chains results in high surface expression, binding of peptide-MHC complexes and Ag-specific T cell functions. Genetic introduction of TCRalphabeta:zeta does not compromise surface expression and functions of an endogenous TCRalphabeta. Flow cytometry fluorescence resonance energy transfer and biochemical analyses demonstrate that TCRalphabeta:CD3zeta is the first strategy that results in highly preferred pairing between CD3zeta-modified TCRalpha and beta chains as well as absence of TCR mispairing between TCR:CD3zeta and nonmodified TCR chains. Intracellular assembly and surface expression of TCR:CD3zeta chains is independent of endogenous CD3gamma, delta, and epsilon. Taken together, our data support the use of TCRalphabeta:CD3zeta to prevent TCR mispairing, which may provide an adequate strategy to enhance efficacy and safety of TCR gene transfer.     

Developmental and functional impairment of T cells in mice lacking CD3 zeta chains.

CD3 zeta is a component of the T cell antigen receptor (TCR) complex and is important for signal transduction. We have established mice selectively lacking CD3 zeta but able to express CD3 eta, a polypeptide produced from the same locus through alternative splicing, using the method of gene targeting in embryonic stem cells. In homozygous mutant mice, the numbers of thymocytes and peripheral T cells were greatly reduced and the expression levels of TCR on these cells were 5-fold lower than those on wild-type cells. By contrast, TCR gamma delta+ intestinal intraepithelial lymphocytes were not obviously affected by the mutation. T cells from homozygous mutants exhibited an impaired proliferative response. These results imply that CD3 zeta has a critical role in the development and signal transduction of T cells in vivo.