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.     

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 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.     

The CD3-gamma and CD3-delta subunits of the T cell antigen receptor can be expressed within distinct functional TCR/CD3 complexes.

The T cell receptor for antigen (TCR) consists of two glycoproteins containing variable regions (TCR-alpha/beta or TCR-gamma/delta) which are expressed on the cell surface in association with at least four invariant proteins (CD3-gamma, -delta, -epsilon and -zeta). CD3-gamma and CD3-delta chains are highly homologous, especially in the cytoplasmic domain. The similarity observed in their genomic organization and their proximity in the chromosome indicate that both genes arose from duplication of a single gene. Here, we provide several lines of evidence which indicate that in human and murine T cells which expressed both the CD3-gamma and CD3-delta chains on their surface, the TCR/CD3 complex consisted of a mixture of alpha beta gamma epsilon zeta and alpha beta delta epsilon zeta complexes rather than a single alpha beta gamma delta epsilon zeta complex. First, a CD3-gamma specific antibody failed to co-immunoprecipitate CD3-delta and conversely, several CD3-delta specific antibodies did not coprecipitate CD3-gamma. Secondly, analysis of a panel of human and murine T cell lines demonstrated that CD3-gamma and CD3-delta were expressed at highly variable ratios on their surface. This suggested that these chains were not expressed as a single complex. Thirdly, CD3-gamma and CD3-delta competed for binding to CD3-epsilon in transfected COS cells, suggesting that CD3-gamma and CD3-delta formed mutually exclusive complexes. The existence of these two forms of TCR/CD3 complexes could have important implications in the understanding of T cell receptor function and its role in T cell development.     

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.     

Differential biological role of CD3 chains revealed by human immunodeficiencies

The biological role in vivo of the homologous CD3gamma and delta invariant chains within the human TCR/CD3 complex is a matter of debate, as murine models do not recapitulate human immunodeficiencies. We have characterized, in a Turkish family, two new patients with complete CD3gamma deficiency and SCID symptoms and compared them with three CD3gamma-deficient individuals belonging to two families from Turkey and Spain. All tested patients shared similar immunological features such as a partial TCR/CD3 expression defect, mild alphabeta and gammadelta T lymphocytopenia, poor in vitro proliferative responses to Ags and mitogens at diagnosis, and very low TCR rearrangement excision circles and CD45RA(+) alphabeta T cells. However, intrafamilial and interfamilial clinical variability was observed in patients carrying the same CD3G mutations. Two reached the second or third decade in healthy conditions, whereas the other three showed lethal SCID features with enteropathy early in life. In contrast, all reported human complete CD3delta (or CD3epsilon) deficiencies are in infants with life-threatening SCID and very severe alphabeta and gammadelta T lymphocytopenia. Thus, the peripheral T lymphocyte pool was comparatively well preserved in human CD3gamma deficiencies despite poor thymus output or clinical outcome. We propose a CD3delta > CD3gamma hierarchy for the relative impact of their absence on the signaling for T cell production in humans.     

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.     

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.     

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.     

Physical linkage of three CD3 genes on human chromosome 11.

T-cell antigen receptors are associated on T cell surfaces with a complex of proteins called CD3 (formerly T3). Human CD3 consists of at least four proteins, gamma, delta, epsilon and zeta, and all but the latter have been cloned as cDNA. Using standard cloning techniques, together with field inversion gel electrophoresis, we have demonstrated the physical linkage of three CD3 genes. The genes for CD3 gamma and CD3 delta are situated close together, about 1.6 kb apart, organized in a head-to-head orientation. The gene encoding CD3 gamma has been sequenced, and is split into seven exons spread over 9 kb of DNA. Like CD3 delta, CD3 gamma gene has an unusual promoter which lacks a TATA-box and potential Sp1 binding sites. The CD3 gamma-CD3 delta gene pair is within 300 kb of the CD3 epsilon gene, and therefore these genes form a tightly linked cluster in chromosome 11 band q23. The clustering of the CD3 genes may be significant in terms of their simultaneous activation during T-cell development.     

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.     

Human T cell leukemia virus type I prevents cell surface expression of the T cell receptor through down-regulation of the CD3-gamma, -delta, -epsilon, and -zeta genes

Infection and transformation by human T cell leukemia virus type I (HTLV-I) up-regulates expression of several inducible genes including those coding for cytokines involved in the proliferation of normal and leukemic T cells. We demonstrate that HTLV-I can also shut off expression of the CD3-gamma, delta, epsilon, and zeta genes that code for the constant elements of the TCR for Ag. In addition, the T cell-specific CD3-epsilon enhancer was found to be inactive in a HTLV-I-infected T cell clone. This HTLV-I-infected T cell clone (827-p19-II) that could be cultured in the absence of IL-2 lacked the CD3 proteins but did express the TCR-alpha and -beta proteins intracellularly. In the absence of the CD3-gamma, delta, epsilon, and zeta polypeptide chains the disulfide bridged TCR-alpha/beta heterodimer was not formed and the Ag receptor did not appear at the cell surface. These results allowed two major conclusions: first, HTLV-I infection has an effect on the T cell specific regulatory elements that coordinately regulate CD3-gamma, delta, epsilon, and zeta expression and second, the CD3-gamma, delta, epsilon, and zeta proteins are necessary for formation and routing the variable TCR-alpha/beta (or -gamma/delta) heterodimer to the human 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.     

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.     

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.     

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.