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.     

Characterization of immature thymocyte lines derived from T-cell receptor or recombination activating gene 1 and p53 double mutant mice.

The T-cell receptor (TCR) beta chain is instrumental in the progression of thymocyte differentiation from the CD4-CD8- to the CD4+CD8+ stage. This differentiation step may involve cell surface expression of novel CD3-TCR complexes. To facilitate biochemical characterization of these complexes, we established cell lines from thymic lymphomas originating from mice carrying a mutation in the p53 gene on the one hand and a mutation in TCR-alpha, TCR-beta, or the recombination activating gene 1 (RAG-1) on the other hand. The cell lines were CD4+CD8+ and appeared to be monoclonal. A cell line derived from a RAG-1 x p53 double mutant thymic lymphoma expressed low levels of CD3-epsilon, -gamma, and -delta on the surface. TCR-alpha x p53 double mutant cell lines were found to express complexes consisting of TCR-beta chains associated with CD3-epsilon, -gamma, and -delta chains and CD3-zeta zeta dimers. These lines will be useful tools to study the molecular structure and signal transducing properties of partial CD3-TCR complexes expressed on the surface of immature thymocytes.     

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.     

Deficiency of CD3gamma, delta, epsilon, and zeta expression in T cells from AML patients

In order to elucidate the feature of T-cell receptor (TCR) signal transduction in T-cells from acute myeloid leukemia (AML), the expression levels of CD3gamma, delta, epsilon and zeta chain genes in CD3+ T cells were analyzed using real-time PCR. CD3+ T cells sorted from peripheral blood of 10 AML patients and 10 healthy donors were used in the study. Significantly lower expression levels of all four CD3gamma, delta, epsilon, and zeta chain genes were found in the AML samples. The expression pattern of the four CD3 chains was epsilon>gamma>delta>zeta in CD3+ T cells from AML samples, which was different from the healthy control group. In conclusion, the results provide a global gene expression profile of CD3gamma, delta, epsilon, and zeta chains in AML patients. Deficiency of all four CD3 gene expression levels might represent the feature related to T-cell immunodeficiency.     

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.     

Impaired expression of the CD3-zeta chain in peripheral blood T cells of patients with chronic myeloid leukaemia results in an increased susceptibility to apoptosis

In patients with myeloid malignancies, cell-mediated immunity is often suppressed, being most profound in those with advanced disease. Such immune dysfunction, as demonstrated in many patients with chronic lymphocytic leukaemia (CLL) and myelodysplastic syndrome (MDS), may, at least in part, be due to altered expression of the CD3-zeta chain, which is an important component of the T-cell receptor (TCR). We speculated that impaired expression of the TCR-zeta chain would be evident in peripheral blood T cells of patients with chronic myeloid leukaemia (CML) and that such an abnormality would result in an increased ex vivo susceptibility to apoptosis. In this study, we demonstrated that, compared with normal controls, zeta chain expression was significantly downregulated in all of the T-cell subsets (P < 0.009) in more than 90% of CML patients. In addition, there was a significantly lower expression of the CD3-epsilon chain (P < 0.001) in patients than in controls. In those patients with abnormal zeta chain expression, the proportion of lymphocytes with spontaneous DNA fragmentation, as determined by terminal deoxynucleotide transferase-mediated dUTP-biotin nick-end labelling (TUNEL) assays, was also significantly higher (P < 0.002) than controls. From all of the patients tested, it was possible to upregulate partially zeta chain expression and hence to reduce the susceptibility to apoptosis by cross-linking the T cells with interleukin (IL)-2, interferon (IFN)-alpha or immobilized CD3. In addition, such cross-linked T cells showed a significantly higher capacity to proliferate than the native CML T cells.     

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.     

Tumor-associated macrophages and CD3-ζ expression of tumor-infiltrating lymphocytes in human esophageal squamous-cell carcinoma

The clinical significance of tumor-associated macrophages (TAMs) and CD3-zeta chain expression of tumor-infiltrating lymphocytes (TILs), and their correlation in human esophageal squamous cell carcinoma (SCC) are not very clear. Serial histological slides from 137 esophageal SCC patients who had undergone tumor resection were immunohistochemically studied with anti-CD68, anti-CD3-zeta and anti-CD3-epsilon antibodies. TAMs infiltration (expressed as macrophage index, M(phi)I) and CD3-zeta expression (judged by Z/E = CD3-zeta+ cells/CD3-epsilon+ cells ratio) in different tissue compartments were observed. We found that the total tumor tissue region had significantly higher macrophage density and lower CD3-zeta expression (mean +/- SD: M(phi)I(normal): 225.3 +/- 85.9; Z/E(total): 0.52 +/- 0.25; n = 137) relative to adjacent histologically normal esophageal squamous epithelium (M(phi)I(normal): 60.5 +/- 31.7, P < 0.001; Z/E(normal): 0.79 +/- 0.35, P = 0.001; n = 70). Significantly higher M(phi)I(stroma) (P = 0.006) and lower Z/E(total) (P = 0.016) were detected in patients with lymph node metastasis than in those without. Patients with high M(phi)I(total) and M(phi)I(cancer) but low Z/E(total) had poorer surgical outcomes. Univariate analysis of M(phi)I(total) and multivariate analysis of M(phi)I(total) with specific clinico-pathological parameters demonstrated M(phi)I(total) to be an independent prognostic factor for survival in esophageal SCC patients (Cox proportional hazard model, P = 0.029 and P = 0.031, respectively).     

Expression feature of CD3, FcεRIγ, and Zap-70 in patients with chronic lymphocytic leukemia

In leukemia patients, T-cell function has been suppressed with the disease progress. Patients with chronic lymphocytic leukemia (CLL) are all to a degree immunodeficient. In order to elucidate the feature of T-cell receptor signal transduction in CLL, the expression levels of CD3γ, δ, ε, and ζ chain, FcεRIγ, and Zap-70 genes in peripheral blood mononuclear cells (PBMCs) were analyzed. Real-time polymerase chain reaction with SYBR Green technique was used for detecting the gene expression level in PBMCs from 13 patients with CLL, 13 healthy individuals, and 10 B-cell acute lymphocytic leukemia (B-ALL) served as control. The β2-microglobulin gene was used as an endogenous reference. Relative mRNA expression level of genes was analyzed by using the 2(-ΔCt) × 100% method. Significant lower expression levels of CD3γ, ε, and ζ chain genes, as well as FcεRIγ gene were found in CLL samples. Moreover, there was lost the negative correlation of the expression levels between CD3ζ and FcεRIγ genes. The expression level of Zap-70 in CLL was lower than those from healthy controls, while higher than those from B-ALL group. There was no significant correlation between the expression levels of CD3ζ and Zap-70 genes neither in the healthy group nor in the CLL group. In conclusion, the results provide a global gene expression profile of CD3γ, δ, ε, and ζ chains, and the CD3ζ-related genes FcεRIγ and Zap-70 in CLL. Deficiency of these gene expression levels might represent the feature related to T-cell immunodeficiency. The study might contribute to better understand the cellular immune features in CLL patients.     

Identification of a murine CD28 dileucine motif that suppresses single-chain chimeric T-cell receptor expression and function

Recent preclinical and clinical trials have demonstrated the therapeutic potential of T lymphocytes redirected with genetically engineered T-cell receptor (TCR) surrogates against infected, cancerous, or autoreactive cells. These surrogate TCRs link a ligand-recognition domain to signaling regions from the TCR. We previously compared the function of surrogate TCRs that include TCR or TCR and CD28 signaling regions. We found that primary murine T cells modified to specifically target Kb-restricted CD8+ T cells using either Kb-zeta or Kb-CD28-zeta receptors had similar functional activities, although the CD28-zeta receptor showed a 2-fold to 4-fold decreased expression. We have now identified a previously unrecognized dileucine motif in the murine CD28 signaling domain that accounts for this reduced expression. Inactivation of this motif increased chimeric receptor surface expression 2- to 5-fold. T cells expressing the dileucine-mutated CD28-zeta chimeric receptor demonstrated enhanced proliferation, cytokine production, and cytolytic activities. Further, cells expressing this dileucine-mutated receptor were highly effective in eliminating antigen-specific CD8+ T lymphocytes in vivo. These results therefore identify a critical motif limiting the function of receptor-modified T lymphocytes, demonstrate that inactivation of this motif enhances chimeric receptor function, and illustrate a potential novel application of receptor-modified T lymphocytes in the induction of immune tolerance.     

Stages of T-cell receptor protein expression in T-cell acute lymphoblastic leukemia.

In this study five monoclonal antibodies (MoAbs) to T-cell receptor (TCR) proteins (WT31, alpha F1, beta F1, TCR delta-1 and delta TCS-1) were used to identify discrete maturative stages in 40 cases of T-cell acute lymphoblastic leukemia (T-ALL). These MoAbs reacted exclusively with CD3+ T cells and did not label B-lineage and myeloid cells. In 17 of the 40 T-ALL cases studied the leukemic blasts lacked membrane and cytoplasmic TCR chains (group I). In 12 cases cells did not have membrane CD3/TCR but expressed cytoplasmic TCR proteins heterogenously: nine cases had cytoplasmic TCR beta chains (beta F1+, alpha F1-; group II), one case had cytoplasmic TCR alpha chains (alpha F1+, beta F1-; group III), and two cases were labeled by both alpha F1 and beta F1 MoAbs (group IV). The remaining 11 cases were mCD3+: nine were TCR alpha beta+ (group Va) and two exhibited TCR gamma delta (TCR delta-1+, delta TCS-1+; group Vb). The analysis of the TCR beta, -gamma, and -delta gene configurations in 23 of the 40 T-ALLs showed that: (1) the lack of TCR protein expression was due to the lack of TCR gene rearrangements only in one of nine cases; (2) five of five TCR beta+, TCR alpha- cases studied had germline TCR alpha genes (ie, no detectable TCR delta gene deletions); (3) seven of eight cases with TCR delta gene deletions expressed TCR alpha proteins, whereas in 12 of 20 of the T-ALLs with TCR beta gene rearrangements the synthesis of the corresponding protein occurred; only 2 of 16 cases with rearranged TCR delta genes expressed TCR delta chains. The T-ALL categories identified with anti-TCR MoAbs did not have additional characteristic phenotypic patterns and may correspond to the normal stages of T-cell development more precisely than those defined by other differentiation antigens.     

Replicating Ad-recombinants encoding non-myristoylated rather than wild-type HIV Nef elicit enhanced cellular immunity

OBJECTIVE: To determine if immunization with non-myristoylated nef would elicit enhanced cellular immune responses resulting from improved presentation of Nef peptides by MHC-I on the cell surface, and enhanced T-cell help. DESIGN: The myristoylation site of HIV and SIV Nef is required for several Nef functions that modulate the immune response in an infected host, including downregulation of MHC-I, MHC-II, and CD4, and increased expression of the invariant chain on the cell surface. We constructed replication-competent Ad5- and Ad7-HIV recombinants encoding wild-type nef (nefWT) or a nef mutant (nefNM) lacking 19 amino-terminal amino acids, including the myristoylation site, and sequentially immunized chimpanzees mucosally, first with Ad5-HIVnef recombinants and subsequently with Ad7-HIVnef recombinants. METHODS: Peripheral blood lymphocytes were evaluated over the immunization course for Nef-specific cellular immune responses by interferon (IFN)-gamma ELISPOT and T-cell proliferation assays. Nef-specific CD4 and CD8 memory T cells that produced intracellular IFN-gamma, interleukin-2, and tumor necrosis factor (TNF)-alpha were assessed by flow cytometry. RESULTS: In comparison to immunization with Ad-HIVnefWT, Ad-HIVnefNM elicited statistically significant increases in numbers of IFN-gamma-secreting cells after the Ad7-HIVnefNM immunization and increased T-cell proliferative responses following both Ad5- and Ad7-HIVnefNM immunizations. Nef-specific CD4 and CD8 memory T-cell populations secreting TNF-alpha were also significantly increased in the Ad-HIVnefNM immunization group. CONCLUSIONS: The results support the hypothesis that immunization with Ad-recombinants encoding HIVnefNM rather than HIVnefWT elicits enhanced cellular immunity resulting from improved antigen presentation and greater T-cell help.     

Reconstitution of the T-cell repertoire following treatment with alemtuzumab (anti-CD52 monoclonal antibody) in patients with B-cell chronic lymphocytic leukaemia

In this pilot study, T-cell receptor B-variable (TCR-BV) gene usage in CD4 and CD8 T cells was assessed, by real-time polymerase chain reaction, as well as complementarity-determining region 3 (CDR3)-length polymorphism, before and after therapy in five patients with B-cell chronic lymphocytic leukaemia who received alemtuzumab (anti-CD52 monoclonal antibody) as first-line therapy. A decline in expression of most BV family genes in both CD4 and CD8 T cells was observed after alemtuzumab treatment, which was followed by a gradual increase in most BV families during long-term follow-up. After treatment, CDR3-length polymorphism showed an even more restricted pattern in CD4 T cells compared with pretreatment, with a shift towards a monoclonal/oligoclonal pattern. The clonally restricted pattern was significantly reduced in CD4 (P < 0.01) but not in CD8 T cells. This was followed by a gradual increase in the number of peaks within the CDR3 region of the different TCR-BV families, i.e. a polyclonal repertoire, during long-term follow-up. A restricted CDR3 pattern became even more restricted after treatment, but normalised during unmaintained follow-up. These results indicate that perturbations in the T-cell alterations following alemtuzumab are complex and include not only changes in CD4/CD8 T-cell numbers but also a highly restricted T-cell repertoire especially in CD4 T cells.     

Amyocarditic coxsackievirus B3 causes myocarditis in immunocompromised mice

In the present study, we investigated the role of the immune status of the host in the pathogenesis and development of coxsackievirus B3 myocarditis. We compared the disease expression in myocarditic coxsackievirus B3 (CB3M)-infected BALB/c wild-type mice and severe combined immunodeficient (SCID) mice and in amyocarditic coxsackievirus B3 (CB3O)-infected BALB/c wild-type mice untreated or treated with immunosuppressive agents and SCID mice. There were no differences in viral growth in vitro between CB3M and CB3O. Severe myocarditis developed in CB3M-infected wild-type and SCID mice, CB3O-infected SCID mice and CB3O-infected wild-type mice with total immunosuppression. However, myocarditis was not induced in CB3O-infected wild-type mice untreated and treated with partial immunosuppression. There were no changes in myocardial virus titres among these groups of mice. In addition, myocarditis was induced in CB3O-infected wild-type mice treated with Thy 1.2 (pan T) or Lyt 2 (CD8) antibody but not in those mice treated with L3T4 (CD4) antibody. Thus, the CB3O variant did not induce myocarditis in wild-type mice associated with the induction of the CD8⁺ lymphocyte subset but was shown to have the genetic capability to induce myocarditis if the host was in an almost total immunosuppressive or CD8-depleted state. The results suggest that induction of myocarditis by the amyocarditic strain of coxsackievirus B3 may occur and partially depends on the immune status of the host, and that myocarditis is due in part to an immunopathogenic mechanism.