Human CD3-epsilon gene contains three miniexons and is transcribed from a non-TATA promoter.

The antigen receptor of the T lymphocyte consists of two variable T-cell receptor chains (either TCR-alpha, TCR-beta or TCR-gamma, TCR-delta) noncovalently linked to four different invariant membrane proteins (CD3-gamma, CD3-delta, CD3-epsilon, and the CD3-zeta homodimer). The CD3 genes are expressed early in thymocyte development, preceding the rearrangement and expression of the T-cell receptor genes. Here we report the isolation and structural analysis of the human CD3-epsilon gene. The gene consisted of nine exons. Three exons, encoding the junction of leader peptide and mature protein, were extremely small (21, 15, and 18 base pairs, respectively). The murine gene contained only two such miniexons, the sequences of which were not homologous to those of the three human miniexons. But from comparisons of intron sequences the regions surrounding the human miniexons III and IV appeared to be closely related to those surrounding the murine miniexons III and IV. The most-3' miniexon in the human gene (IVa) had no murine counterpart and appeared not to duplicate any of the other miniexons. Sequence analysis of CD3-epsilon cDNA clones isolated from four independent libraries gave no evidence for alternative use of these miniexons. Like CD3-delta, the CD3-epsilon gene was transcribed from a weak, nontissue-specific, TATA-less promoter. Pulsed-field electrophoresis showed that the human CD3-epsilon gene was separated from the CD3-gamma, CD3-delta gene pair by at least 30 kilobases, but by no more than 300 kilobases.     

Characterization and expression of the murine CD3-epsilon gene.

The receptor for antigen on the surface of T lymphocytes consists of a variable disulfide-bridged hetero-dimer (TCR-alpha/beta or -gamma/delta) associated with invariant CD3 proteins (CD3-gamma, -delta, -epsilon, and -zeta). The genes coding for the CD3 proteins are expressed in the earliest recognizable thymocytes, preceding the rearrangement and expression of the TCR genes. The isolation, characterization, and in vitro expression of the murine CD3-epsilon gene, as reported here, represent obligatory steps toward our understanding of the complex rules that govern T-cell-specific gene expression. The CD3-epsilon gene was transcribed from a non-TATA promoter and consisted of eight exons, two of which were unusually small (18 and 15 base pairs). The transmembrane exon was found to be homologous to the transmembrane exons of the CD3-gamma and CD3-delta genes. In transient-transfection experiments, a genomic fragment comprising 4 kilobases of upstream sequence and extending into the second exon sufficient to drive the expression of a reporter gene in murine T cells.     

Structure of the T-cell antigen receptor: evidence for two CD3 epsilon subunits in the T-cell receptor-CD3 complex.

The T-cell antigen receptor (TCR) consists of heterodimeric glycoproteins (TCR alpha beta or gamma delta) that demonstrate homology with immunoglobulins. Noncovalently associated with the alpha beta (or gamma delta) heterodimer are at least five nonvariant proteins (CD3-gamma, -delta, -epsilon, -zeta, and -eta), which together comprise the TCR-CD3 complex. The stoichiometry of the antigen receptor has been assumed to be either alpha beta gamma delta epsilon zeta zeta or alpha beta gamma delta epsilon zeta eta. In this paper we provide several lines of evidence that support the notion that the mature TCR-CD3 complex on the cell surface contains two CD3-epsilon polypeptide chains. Transfection of two murine T cell-T cell hybridomas with the human DNA encoding CD3-epsilon protein demonstrated that both murine and human CD3-epsilon chains were present within the same TCR-CD3 complex. Analysis of thymocytes isolated from transgenic mice that expressed high copy numbers of the human CD3-epsilon gene showed that the heterologous human CD3-epsilon subunits were coexpressed with murine CD3-epsilon in the same TCR-CD3 complex. Since CD3-epsilon was shown to form disulfide-linked homodimers both in human and murine T cells, the two CD3-epsilon subunits present in the TCR-CD3 complex were in direct contact with one another. The presence of two CD3-epsilon polypeptide chains in close proximity to one another in the TCR-CD3 complex may have important implications for its assembly and its signal transduction mechanisms.     

Instability of assembled T-cell receptor complex that is associated with rapid degradation of zeta chains in immature CD4+CD8+ thymocytes.

The intracellular fate of newly synthesized T-cell receptor (TCR) chains was compared in CD4+CD8+ (double positive; DP) thymocytes and in CD4+CD8- or CD4-CD8+ (single positive; SP) thymocytes. Purified DP and SP thymocytes from normal adult mice were analyzed by pulse-chase metabolic labeling and immunoprecipitation with specific anti-TCR antibodies. Biosynthesis of invariant chains (CD3 gamma, -delta, -epsilon, and zeta) was comparable between DP and SP thymocytes, whereas DP thymocytes synthesized TCR alpha and TCR beta chains at lower and higher levels than SP thymocytes, respectively. These newly synthesized TCR chains were degraded at different rates in SP thymocytes based on their sensitivities for degradation as previously reported: TCR alpha, TCR beta, CD3 gamma, and CD3 delta chains were rapidly degraded and CD3 epsilon and zeta chains were stable. Although the degradation rates of clonotypic and invariant CD3 chains were similar in DP and SP thymocytes, the zeta subunit was rapidly degraded in DP thymocytes (t1/2, approximately 1.5 hr). Degradation of zeta was inhibited by NH4Cl, implicating lysosomes as the site of degradation. Comparison of TCR subunit assembly in DP and SP thymocytes demonstrated that, despite the same relative rate of formation of TCR complexes in a pulse period (30 min), complete complexes were unstable and degraded during the subsequent 6 hr of chase in DP thymocytes. This contrasted with the stability and a progressive increase in the levels of completely assembled complexes in SP thymocytes. Thus, these results demonstrate that a unique posttranslational regulation operates in the formation of TCR complexes in DP thymocytes and that lack of stability of complete TCR complexes is a crucial mechanism that may account for the limited surface TCR expression on this thymocyte subset.     

Defective synthesis or association of T-cell receptor chains underlies loss of surface T-cell receptor-CD3 expression in enteropathy-associated T-cell lymphoma

Enteropathy-associated T-cell lymphoma, an often fatal complication of celiac disease, can result from expansion of aberrant intraepithelial lymphocytes in refractory celiac disease type II (RCD II). Aberrant intraepithelial lymphocytes and lymphoma cells are intracellularly CD3epsilon(+) but lack expression of the T-cell receptor (TCR)-CD3 complex on the cell surface. It is unknown what causes the loss of TCR-CD3 expression. We report the isolation of a cell line from an RCD II patient with the characteristic phenotype of enteropathy-associated T-cell lymphoma. We demonstrate that in this cell line the TCR-alpha and -beta chains as well as the CD3gamma, CD3delta, CD3epsilon, and zeta-chains are present intracellularly and that assembly of the CD3gammaepsilon, CD3deltaepsilon, and zetazeta-dimers is normal. However, dimerization of the TCR chains and proper assembly of the TCR-CD3 complex are defective. On introduction of exogenous TCR-beta chains, but not of TCR-alpha chains, assembly and functional cell surface expression of the TCR-CD3 complex were restored. Defective synthesis of both TCR chains was found to underlie loss of TCR expression in similar cell lines isolated from 2 additional patients. (Pre)malignant transformation in RCD II thus correlates with defective synthesis or defective association of the TCR chains, resulting in loss of surface TCR-CD3 expression.     

Crystal structure of a human CD3-epsilon/delta dimer in complex with a UCHT1 single-chain antibody fragment

The alpha/beta T cell receptor complex transmits signals from MHC/peptide antigens through a set of constitutively associated signaling molecules, including CD3-epsilon/gamma and CD3-epsilon/delta. We report the crystal structure at 1.9-A resolution of a complex between a human CD3-epsilon/delta ectodomain heterodimer and a single-chain fragment of the UCHT1 antibody. CD3-epsilon/delta and CD3-epsilon/gamma share a conserved interface between the Ig-fold ectodomains, with parallel packing of the two G strands. CD3-delta has a more electronegative surface and a more compact Ig fold than CD3-gamma; thus, the two CD3 heterodimers have distinctly different molecular surfaces. The UCHT1 antibody binds near an acidic region of CD3-epsilon opposite the dimer interface, occluding this region from direct interaction with the TCR. This immunodominant epitope may be a uniquely accessible surface in the TCR/CD3 complex, because there is overlap between the binding site of the UCHT1 and OKT3 antibodies. Determination of the CD3-epsilon/delta structure completes the set of TCR/CD3 globular ectodomains and contributes information about exposed CD3 surfaces.     

The T-cell receptor gamma chain-CD3 complex: implication in the cytotoxic activity of a CD3+ CD4- CD8- human natural killer clone

A subset of human T cells has recently been described. These cells express the CD3 complex but they do not carry the classical T-cell receptor (TCR)-alpha/-beta heterodimer on their surface (WT31- CD3+). Instead, they express a TCR-gamma chain associated with another type of polypeptide termed TCR-delta. We report here that a T-cell clone with natural killer (NK)-like activity, WM-14, had a disulfide bridged TCR-gamma homodimer associated with CD3 on its surface. The TCR-gamma chains of WM-14 cells were present in three different glycosylation forms of 43, 40, and 38 kDa, but they appeared to contain the same polypeptide backbone. Since cytotoxicity by WM-14 could be inhibited by anti-CD3 antibodies, we concluded that the TCR-gamma-CD3 complex was involved in the NK-like unrestricted killer activity. Although normal CD3-gamma, CD3-delta, and CD3-epsilon chains were present in this clone, the association with the TCR-gamma homodimer may be the cause of a complete processing of the N-linked oligosaccharides attached to the CD3-delta chain.     

The expression pattern of CD3 chain genes in fetal/maternal interface

In order to investigate the features of T-cell immune status in human placenta, the expression levels of CD3-gamma, -delta, -epsilon and -zeta chain genes in placenta were analyzed by real-time PCR. Umbilical cord blood obtained at delivery from the full-term healthy babies was used as a control. The beta2-microglobulin gene was employed as an endogenous reference, and the evaluations of mRNA expression level of each CD3 gene were used by the 2(-ΔC(t))×100% method. The expression level of CD3-gamma, -epsilon and -zeta genes (mean rank is 0.13, 0.34, and 0.49 respectively) from placenta were significantly lower than those from CB (P<0.0001). CD3-delta genes (mean rank is 5.71) expressed stronger from placenta than from CB (P = 0.0.895). Thus, the expression pattern of the four CD3 genes was presented as delta>zeta>epsilon> gamma from placenta and epsilon>gamma>zeta>delta from CB on the contrary. In conclusion, the present study characterized the expression pattern of CD3-gamma, -delta, -epsilon and -zeta chain genes from placenta, which contributes to further understanding of the features of T-cell immune status in placenta.     

Cytolytic and IFN-γ-producing activities of γδ T cells in the mouse intestinal epithelium are T cell receptor-β-chain dependent

We analyzed the cytolytic activity of intraepithelial T cells (IEL) isolated from the small intestines of 2- to 3-month-old mutant mice rendered deficient in different gene(s) in which the number of IEL expressing either T cell receptor (TCR)-alpha beta (alpha beta-IEL) or TCR-gamma delta (gamma delta-IEL) were absent or markedly diminished. When compared with wild-type littermates, cytolytic activity of gamma delta-IEL was sharply attenuated in TCR-beta mutant mice but remained unaltered in TCR-alpha mutant mice in which a minor population of dull TCR-beta+ (betadim)-IEL was also present. Cytolytic activity of gamma delta-IEL was maintained in mice doubly homozygous for beta2-microglobulin and transporter associated with antigen processing 1 gene mutations in which a conspicuous decrease was noted in absolute numbers of alpha beta-IEL. In contrast, both TCR-delta and IL-7 receptor-alpha gene mutations that lead to lack of gamma delta-IEL generation did not affect the development or cytolytic activity of the remaining alpha beta-IEL. The anti-CD3 and anti-TCR-gamma delta mAb-induced IFN-gamma production of gamma delta-IEL showed the same TCR-alpha and TCR-beta mutation-dependent variability. These results indicate that cytolytic and IFN-gamma-producing activities of gamma delta T cells in mouse intestinal epithelium are TCR-beta-chain-dependent.     

p59fyn-p125FAK cooperation in development of CD4+CD8+ thymocytes

p59fyn is an Src family nonreceptor tyrosine kinase that has been suggested to play an important role in T-cell development and function. p125FAK is a unique nonreceptor tyrosine kinase and has been known to respond to integrin-extracellular matrix interactions. To examine their roles in thymocytes, heterozygous fak mutation was introduced into homozygous Fyn deficiency. The double mutation, but neither Fyn deficiency nor FAK heterozygosity alone, displayed impaired development of CD4+CD8+ thymocytes with atrophy of the thymic cortex, suggesting a unique cooperation between p59fyn and p125FAK in CD4+CD8+ T-cell development.     

Development of CD4+ T cells expressing a nominally MHC class I-restricted T cell receptor by two different mechanisms

Differences in T cell receptor (TCR) signaling initiated by interactions among TCRs, coreceptors, and self-peptide-MHC complexes determine the outcome of CD4 versus CD8 lineage of T cell differentiation. The H-2Ld and Kbm3 alloreactive 2C TCR is positively selected by MHC class I Kb and a yet-to-be identified nonclassical class I molecule to differentiate into CD8+ T cells. Here we describe two mechanisms by which CD4+ 2C T cells can be generated in 2C TCR-transgenic mice. In the RAG-/- background, development of CD4+ 2C T cells requires the expression of both I-Ab and the TAP genes, indicating that both MHC class I and II molecules are required for positive selection of these T cells. Notably, only some of the 2C+ RAG-/- mice (approximately 30%) develop CD4+ 2C T cells, with frequencies in individual mice varying from 0.5% to as high as approximately 50%. In the RAG+ background, where endogenous TCRalpha genes are rearranged and expressed, CD4+ 2C T cells are generated because these cells express the 2C TCR as well as additional TCRs, consisting of the 2C TCRbeta and endogenous TCRalpha chains. Similarly, T cells expressing the OT-1 TCR, which is nominally MHC class I-restricted, can also develop into CD4+ T cells through the same two mechanisms. Thus, expression of two TCRs by a single thymocyte, TCR recognition of multiple MHC molecules, and heterogeneity of TCR, coreceptors, and peptide-MHC interactions in the thymus all contribute to the outcome of CD4 versus CD8 lineage development.     

Subcellular localization of T-cell receptor complexes containing tyrosine-phosphorylated zeta proteins in immature CD4+CD8+ thymocytes.

The T-cell antigen receptor (TCR) is a complex of at least six different proteins (alpha, beta, gamma, delta, epsilon, and zeta) that is assembled in the endoplasmic reticulum (ER) and transported to the cell surface. Unlike mature T cells, most immature CD4+CD8+ thymocytes retain within the ER and degrade greater than 90% of some of the TCR components they synthesize, resulting in low surface expression of TCR complexes. The few surface TCR complexes that most immature CD4+CD8+ thymocytes do express are only marginally capable of transducing signals mobilizing intracellular calcium. The inverse relationship with TCR expression and function suggested that phosphorylated zeta (P-zeta) molecules might function in CD4+CD8+ thymocytes either as an ER retention signal for newly synthesized TCR complexes or as a negative regulatory modification of TCR complexes present on the cell surface. The present study sought to evaluate these two possibilities by determining the subcellular location of TCR complexes containing P-zeta chains. We found that, unlike unmodified zeta chains, all P-zeta chains in CD4+CD8+ thymocytes existed in assembled TCR complexes and that all TCR complexes containing P-zeta molecules had undergone carbohydrate processing events indicative of transit through the Golgi apparatus. These results demonstrate that P-zeta chains are exclusively associated with mature TCR complexes, excluding the possibility that P-zeta serves as an ER retention signal in immature thymocytes. Although we could not directly determine the representation of P-zeta chains among surface TCR complexes, we found that 60-70% of surface TCR complexes on immature CD4+CD8+ thymocytes were associated with tyrosine-phosphorylated protein(s) and that this percentage was inversely correlated with their signaling competence. These results support the concept that tyrosine phosphorylation serves as a negative regulatory modification of certain TCR-associated proteins.     

The gene encoding the epsilon subunit of the T3/T-cell receptor complex maps to chromosome 11 in humans and to chromosome 9 in mice.

The T3 complex is composed of three polypeptide chains that are both structurally and functionally associated with the receptor for antigen on the surface of human T lymphocytes. In a series of experiments utilizing both somatic cell hybrids and chromosomal hybridization in situ, the genes encoding two members of the human T3 complex, T3-delta and T3-epsilon, were found to reside on the long arm of chromosome 11 in band q23. The murine T3-epsilon gene was localized to chromosome 9. The location of the T3-delta and T3-epsilon genes with respect to the Hu-ets-1 gene, which is also located in 11q23, is discussed. Recent assignments of several genes, preferentially expressed in human cells of hematopoietic and neuroectodermal origins, to band q23 of human chromosome 11 and the murine equivalents to murine chromosome 9 may define a conserved gene cluster important in cell proliferation and differentiation.     

Relationship between chromosomal breakpoint and molecular rearrangement of T-cell antigen receptors in adult T-cell leukaemia.

The relationship between chromosome breakpoints associated with T-cell antigen receptor (TCR) genes and TCR-alpha/beta/tau/delta rearrangements of peripheral leukaemic cells in 8 Japanese patients with the acute type of adult T-cell leukaemia (ATL) was examined. Break of the 14q11 region with the assigned locus of TCR-alpha/delta was revealed in 6 patients, interstitial deletion of the 7q32-36 region with assigned locus of TCR-beta in 1 patient, and break of the 7p15 region with assigned locus of TCR-tau in 2 patients. Molecular analysis revealed TCR-alpha rearrangement in 7 patients, TCR-beta rearrangement in all patients, and TCR-tau rearrangement in 5 patients. TCR-delta was deleted in all patients. These findings indicate a close relationship between 14q11 anomaly and TCR-alpha rearrangement, which may play an important role in the leukaemogenesis of ATL.