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.     

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.     

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.     

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.     

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.     

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.     

CD3.TCR1, a human CD3 epitope expressed on viable gamma/delta lymphocytes exclusively.

T lymphocytes express either the alpha/beta or the gamma/delta receptor (TCR) in a mutually exclusive fashion. Both structures are associated on the cell membrane with the CD3 proteins which are thought to transduce signals resulting from antigen recognition. The CD3 complex is present in both alpha/beta and gamma/delta cells and includes at least five proteins (designated gamma, delta, epsilon, zeta and eta). We have developed here a novel mAb, anti-CD3.TCR1, which immunoprecipitates the CD3 molecules from both alpha/beta and gamma/delta cells lysates following solubilization with Triton X-100. While the SDS-PAGE migration profile of the material recognized by either anti-CD3.TCR1 or anti-OKT3 are superimposable in both cell types, this mAb recognizes viable untreated gamma/delta T lymphocytes exclusively. These findings further support the view that molecular interactions within the TCR/CD3 protein complex are distinct in the two T lymphocyte populations.     

Biochemical evidence for the presence of a single CD3delta and CD3gamma chain in the surface T cell receptor/CD3 complex

The T cell antigen receptor (TCR) consists of an alphabeta heterodimer and associated invariant CD3gamma, delta, epsilon, and zeta chains (TCR/CD3 complex). The general stoichiometry of the receptor complex, which is believed to be one molecule each of TCRalpha, TCRbeta, CD3gamma, and CD3delta and two molecules each of CD3epsilon and CD3zeta, is not clearly understood. Although it has been shown that there are two chains of CD3epsilon and CD3zeta, the stoichiometry of CD3gamma or CD3delta chains in the surface antigen receptor complex has not been determined. In the present study, transgenic mice expressing an altered form of mouse CD3delta and CD3gamma were employed to show that the surface TCR complexes contain one molecule each of CD3delta and CD3gamma. Thymocytes from wild type and CD3 chain transgenic mice on the appropriate knockout background were surface-biotinylated and immunoprecipitated using a specific antibody. The immunoprecipitates were resolved in two dimensions under nonreducing/reducing conditions to determine the stoichiometry of CD3delta and CD3gamma in the surface antigen receptor complex. Our data clearly show the presence of one molecule each of CD3delta and CD3gamma in the surface TCR/CD3 complex.     

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.     

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.     

Assembly, intracellular processing, and expression at the cell surface of the human alpha beta T cell receptor/CD3 complex. Function of the CD3-zeta chain

The TCR/CD3 complex is a multimeric protein complex composed of a minimum of seven transmembrane chains (TCR alpha beta-CD3 gamma delta epsilon zeta 2). Whereas earlier studies have demonstrated that both the TCR-alpha and -beta chains are required for the cell surface expression of the TCR/CD3 complex, the role of the CD3 chains for the TCR/CD3 expression have not been experimentally addressed in human T cells. In this study the function of the CD3-zeta chain for the assembly, intracellular processing, and expression of the TCR/CD3 complex in the human leukemic T cell line Jurkat was investigated. The results indicate that: 1) CD3-zeta is required for the cell surface expression of the TCR/CD3 complex; 2) the pentameric form (TCR alpha beta-CD3 gamma delta epsilon) of the TCR/CD3 complex and single TCR chains associated with CD3 (TCR alpha-CD3 gamma delta epsilon and TCR beta-CD3 gamma delta epsilon) are produced in the endoplasmic reticulum in the absence of CD3-zeta; 3) the CD3-zeta does not associate with TCR alpha-CD3 gamma delta epsilon or TCR beta-CD3 gamma delta epsilon complexes; 4) CD3-zeta associate with the pentameric form of the TCR/CD3 complex in the endoplasmic reticulum to form the heptameric complex (TCR alpha beta-CD3 gamma delta epsilon----TCR alpha beta-CD3 gamma delta epsilon 2); and 5) CD3-zeta is required for the export of the TCR/CD3 complex from the endoplasmic reticulum to the Golgi apparatus for subsequent processing.     

T cell development in mice lacking the CD3-zeta/eta gene.

The CD3-zeta and CD3-eta polypeptides are two of the components of the T cell antigen receptor (TCR) which contribute to its efficient cell surface expression and account for part of its transducing capability. CD3-zeta and CD3-eta result from the alternative splicing of a single gene designated CD3-zeta/eta. To evaluate the role of these subunits during T cell development, we have produced mice with a disrupted CD3-zeta/eta gene. The analysis of thymocyte populations from the CD3-zeta/eta-/- homozygous mutant mice revealed that they have a profound reduction in the surface levels of TCR complexes and that the products of the CD3-zeta/eta gene appear to be needed for the efficient generation and/or survival of CD4+CD8+ thymocytes. Despite the almost total absence of mature single positive thymocytes, the lymph nodes from zeta/eta-/- mice were found to contain unusual CD4+CD8- and CD4-CD8+ single positive cells which were CD3-. In contrast to the situation observed in the thymus, the thymus-independent gut intraepithelial lymphocytes present in zeta/eta-/- mice do express TCR complexes on their surface and these are associated with Fc epsilon RI gamma homodimers. These results establish an essential role for the CD3-zeta/eta gene products during intrathymic T cell differentiation and further emphasize the difference between conventional T cells and thymus-independent gut intraepithelial lymphocytes.     

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.     

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.     

The T cell receptor/CD3 complex is composed of at least two autonomous transduction modules.

Recent studies have demonstrated that the CD3-zeta subunit of the T cell antigen receptor (TCR) complex is involved in signal transduction. However, the function of the remaining invariant subunits, CD3-gamma, -delta, and epsilon, is still poorly understood. To examine their role in TCR function, we have constructed TCR/CD3 complexes devoid of functional zeta subunit and showed that they are still able to trigger the production of interleukin-2 in response to antigen or superantigen. These data, together with previous results, indicate that the TCR/CD3 complex is composed of at least two parallel transducing units, made of the gamma delta epsilon and zeta chains, respectively. Furthermore, the analysis of partially truncated zeta chains has led us to individualize a functional domain that may have constituted the building block of most of the transducing subunits associated with antigen receptors and some Fc receptors.     

The CD3gamma chain is essential for development of both the TCRalphabeta and TCRgammadelta lineages.

CD3gamma and CD3delta are the most closely related CD3 components, both of which participate in the TCRalphabeta-CD3 complex expressed on mature T cells. Interestingly, however, CD3delta does not appear to participate functionally in the pre-T-cell receptor (TCR) complex that is expressed on immature T cells: disruption of CD3delta gene expression has no effect on the developmental steps controlled by the pre-TCR. Here we report that in contrast with CD3delta, CD3gamma is an essential component of the pre-TCR. We generated mice selectively lacking expression of CD3gamma, in which expression of CD3delta, CD3epsilon, CD3zeta, pTalpha and TCRbeta remained undisturbed. Thus, all components for composing a pre-TCR are available, with the exception of CD3gamma. Nevertheless, T-cell development is severely inhibited in CD3gamma-deficient mice. The number of cells in the thymus is reduced to <1% of that in normal mice, and the large majority of thymocytes lack CD4 and CD8 and are arrested at the CD44-CD25+ double negative (DN) stage of development. Peripheral lymphoid organs are also practically devoid of T cells, with absolute numbers of peripheral T cells reduced to only 2-5% of those in normal mice. Both TCRalphabeta and TCRgammadelta lineages fail to develop effectively in CD3gamma-deficient mice, although absence of CD3gamma has no effect on gene rearrangements of the TCRbeta, delta and gamma loci. Furthermore, absence of CD3gamma results in a severe reduction in the level of TCR and CD3epsilon expression at the cell surface of thymocytes and peripheral T cells. The defect in the DN to double positive transition in mice lacking CD3gamma can be overcome by anti-CD3epsilon-mediated cross-linking. CD3gamma is thus essential for pre-TCR function.     

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.