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C Terhorst

Publications and source records attributed to C Terhorst.

At least 127 records · Page 7Linked to original sources
PubMed1989

Surface expression of CD3 in the absence of T cell receptor (TcR): evidence for sorting of partial TcR/CD3 complexes in a post-endoplasmic reticulum compartment.

The T cell receptor (TcR) for antigen, on the majority of T cells, is a disulfide-linked heterodimer composed of the alpha and beta chains, noncovalently associated with the CD3 complex of polypeptides (gamma, delta, epsilon and zeta). In this report, two murine thymoma cell lines are described which synthesized incomplete TcR/CD3 complexes and expressed low levels of CD3 on their surface in the absence of the TcR chains. The partial TcR/CD3 complexes were composed primarily of the inherently metabolically stable CD3 gamma and epsilon subunits. These results were in contrast to previous studies, which suggested that synthesis of all of the component chains of the TcR/CD3 complex is required for the successful transport of any of the chains to the cell surface. The efficiency of transport of the partial TcR/CD3 complexes from the endoplasmic reticulum (ER) to medial Golgi in the two thymomas was similar to complete complexes. However, the transport of the incomplete receptors was impaired at some point between the medial Golgi and the plasma membrane. Taken together with previous studies, these results suggested that T cells have mechanisms to retain partial TcR/CD3 complexes intracellularly both in the ER and in an undefined post-ER compartment. However, the transport of low levels of partial TcR/CD3 complexes to the cell surface in some T cell lines implied that the retention mechanisms may not always be completely efficient. Cross-linking of the surface, partial TcR/CD3 complexes with anti-CD3 epsilon antibodies did not stimulate interleukin 2 (IL 2) production. It is possible, however, that the partial TcR/CD3 complexes have some function which is unrelated to the stimulation of IL 2 production.

Animals↗
PubMed1989

The CD3 delta gene encodes multiple transcripts regulated by transcriptional and post-transcriptional mechanisms.

CD3 is a multi-subunit complex of proteins noncovalently associated with the T cell receptor (TcR) for antigen. Considerable evidence indicates a role for CD3 molecules in the transduction of activation signals in T cells. The murine CD3 delta gene encodes a 0.7-kb transcript present in mature T cells. Here we report the characterization of several additional CD3 delta transcripts; two nuclear transcripts, 4-4.5 kb in size, and two predominamtly cytoplasmic transcripts of 1.5 kb and 2.5 kb. Both T lymphoma cell lines and normal thymocytes express the 1.5-kb and 2.5-kb CD3 delta transcripts. These cytoplasmic transcripts have long 3'-untranslated sequences which extend beyond the polyadenylation site of the predominant 0.7-kb transcript. The protein synthesis inhibitor cycloheximide (CHX) increases the expression of all three cytoplasmic CD3 delta transcripts, indicating that their level of expression may be regulated by a labile inhibitor protein(s). The CHX elicited increase in CD3 delta mRNA appears to result from post-transcriptional events since the rate of CD3 delta gene transcription remains constant. In contrast to CHX, the calcium ionophore A23187 increases the rate of CD3 delta gene transcription and, like CHX, also increases the level of cellular CD3 delta mRNA. The immunosuppressive agent cyclosporin A inhibits A23187-mediated stimulation of transcription, but has no effect on the CHX-mediated induction of CD3 delta mRNA. We conclude that both transcriptional and post-transcriptional mechanisms can regulate the amount of all three cytoplasmic CD3 delta transcripts.

Animals↗
PubMed1989

An enhancer located in a CpG-island 3' to the TCR/CD3-epsilon gene confers T lymphocyte-specificity to its promoter.

The gene encoding the CD3-epsilon chain of the T cell receptor (TCR/CD3) complex is uniquely transcribed in all T lymphocyte lineage cells. The human CD3-epsilon gene, when introduced into the mouse germ line, was expressed in correct tissue-specific fashion. The gene was then screened for T lymphocyte-specific cis-acting elements in transient chloramphenicol transferase assays. The promoter (-228 to +100) functioned irrespective of cell type. A 1225 bp enhancer with strict T cell-specificity was found in a DNase I hypersensitive site downstream of the last exon, 12 kb from the promoter. This site was present in T cells only. The CD3-epsilon enhancer did not display sequence similarity with the T cell-specific enhancer of CD3-delta, a related gene co-regulated with CD3-epsilon during intrathymic differentiation. The CD3-epsilon enhancer was unusual in that it constituted a CpG island, and was hypomethylated independent of tissue type. Two HTLV I-transformed T cell lines were identified in which the CD3-epsilon gene was not expressed, and in which the enhancer was inactive.

Animals↗
PubMed1989

Isolation and characterization of a cDNA and gene coding for a fourth CD1 molecule.

The CD1 locus encodes a family of major histocompatibility complex (MHC) antigen-like glycoproteins which associate with beta 2-microglobulin and are expressed on immature thymocytes and Langerhans cells. Three CD1 molecules have been identified by monoclonal antibodies and molecular cloning: CD1a, -b, and -c. We have isolated a cDNA coding for a fourth CD1 molecule from a human thymocyte library and termed this molecule CD1d. Reported here are the complete nucleotide sequence and genomic organization of CD1d. They predict that this molecule is related to the previously identified CD1a, -b, and -c molecules and to MHC class I molecules, with three external domains, a transmembrane domain, and a short cytoplasmic tail. The sequence of CD1d is the most divergent among the CD1 molecules in the membrane-distal alpha 1 and alpha 2 domains and in the 5' untranslated region. In contrast, all four CD1 molecules are highly homologous in the membrane-proximal alpha 3 domain, which is likely involved in beta 2-microglobulin binding. A comparison of CD1 and MHC class I sequences suggests that these molecules each evolved to interact with a distinct set of cell surface proteins.

Amino Acid Sequence↗
PubMed1988

Transfection of genes encoding the T cell receptor-associated CD3 complex into COS cells results in assembly of the macromolecular structure.

The T cell antigen receptor (TCR) consists of a disulfide-linked TCR-alpha/beta heterodimer that is both structurally and functionally associated with a set of four non-covalently linked membrane proteins termed CD3-gamma, -delta, -epsilon, and -zeta. An additional protein described recently, CD3-omega, has been suggested to play a role in assembly of the CD3 complex on the basis of its transient association with the CD3 proteins early during biosynthesis. Association of all the proteins seems to be a prerequisite for intracellular transport, since mutants lacking either the TCR-alpha or -beta protein do not express the CD3 complex on the cell surface. CD3-cDNAs were transfected into COS cells in order to study the protein-protein interactions ruling the assembly of the CD3 macromolecular structure. CD3-delta-epsilon, CD3-gamma-epsilon, and CD3-gamma-delta-epsilon intermediates could be detected. These data indicated that a CD3 core structure could be formed in the absence of the other members of the complex (CD3-zeta, -omega, TCR-alpha, and -beta). Both the individual CD3 chains and the assembled CD3.gamma.delta.epsilon complexes could not be detected on the cellular surface but in an intracellular compartment, probably the endoplasmic reticulum or the cis Golgi. The transfection experiments allowed us to identify the 25-kDa member of the murine CD3 complex as CD3-epsilon m. Furthermore, a 23-kDa glycoprotein seen upon metabolic labeling of human T cells was shown to be an immature form of the CD3-gamma h protein.

Animals↗
PubMed1988

Abnormal T cells from MRL/Mp-lpr/lpr mice do not respond to anti-T-cell-receptor antibody or tumor promoter and calcium ionophore A23187 despite the presence of the T-cell-receptor complex and protein kinase c.

Abnormal T cells from autoimmune MRL/Mp-lpr/lpr mice express T-cell-receptor complexes on their surfaces. These receptors are nonfunctional, since monoclonal anti-T-cell-receptor antibody-conjugated beads, which normally activate receptor-bearing T cells, were unable to activate these abnormal T cells. In addition, these abnormal T cells are unresponsive to the synergistic effect of phorbolmyristate acetate (PMA) and calcium ionophore A23187, as quantitated by proliferation, interleukin-2(IL-2) production, and the expression of IL-2 receptors. The failure of abnormal T cells to respond to PMA is not due to the absence of PMA receptors since Scatchard plot analysis reveals that there are 1-1.5 X 10(5) PMA-binding sites/cell with a Kd of 6-10 nM on these abnormal T cells. Similar to normal T lymphocytes, protein kinase c activity can be readily detected in the cytosolic fraction of these abnormal T cells. More importantly, in vitro culture of the abnormal T cells with PMA results in translocation of protein kinase c activity from the cytosolic fraction to the membrane fraction. From these experiments we concluded that the defect in the signal-transducing machinery in MRL/Mp-lpr/lpr T cells is not due to the lack of PMA receptors or the absence of protein kinase c activity, but may result from events which occur after the activation and translocation of protein kinase c. However, whether defects in response to a physiological stimulus (i.e., anti-receptor antibody) could occur in a step prior to protein kinase c activation remains to be determined.

Animals↗
PubMed1988

Assembly of the human T cell receptor-CD3 complex takes place in the endoplasmic reticulum and involves intermediary complexes between the CD3-gamma.delta.epsilon core and single T cell receptor alpha or beta chains.

Functionally mature human T lymphocytes express a cell-surface receptor for antigen (T cell receptor (TCR)-CD3) composed of at least six polypeptides (TCR-alpha and -beta; T3-gamma, -delta, -epsilon, and -zeta). Immature thymocytes and variants of T cell lines lacking one of the TCR.CD3 polypeptide chains fail to express surface receptor and accumulate the other chains intracellularly. Here we show that the assembly of the TCR.CD3 complex within the endoplasmic reticulum (ER) began with a core of CD3-gamma, -delta, and -epsilon to which TCR-alpha and -beta bound. A recently described intracellular protein, CD3-omega, participated in the assembly since it was found to be associated with the free TCR-alpha or -beta chains or with the CD3 chains. CD3-omega dissociated as TCR.CD3 complexes were formed in the ER. Association of non-disulfide-linked TCR-alpha and -beta chains with CD3 was detected before that of disulfide-bridged TCR-alpha/beta heterodimers. These data suggest that during assembly, the association of TCR-alpha and -beta chains with the CD3 complex precedes the formation of a TCR-alpha/beta dimer. The existence of intermediates consisting of CD3-gamma, -delta, and -epsilon chains and a single TCR-alpha or -beta chain was also confirmed by using a series of variant T cell lines lacking the TCR-beta or -alpha chain, respectively. Once the single TCR-alpha and -beta chains were associated with CD3, disulfide linkages were formed, and a 70-kDa form of the TCR was detected within the ER. This intracellular precursor of the TCR.CD3 complex was subsequently processed into the mature 90-kDa TCR as the TCR.CD3 complex passed through the Golgi apparatus. Assembly of the TCR.CD3 complex is a rather rapid process, whereas export from the ER occurs at a slow rate. After 1 h, 75% of the receptor complex remained within the ER.

Cell Line↗
PubMed1988

The transmembrane orientation of the epsilon chain of the TcR/CD3 complex.

The antigen receptor of the T lymphocytes is one of the most complex eukaryotic membrane structures studied to date. The T cell receptor (TcR) consists of two disulfide-linked glycoprotein chains (alpha/beta or gamma/delta) and is noncovalently associated with a group of small and invariable CD 3 proteins. Four CD 3 chains have been recognized: two highly homologous glycoproteins CD 3 gamma and delta, the more distantly related nonglycosylated CD 3 epsilon chain, and the nonglycosylated CD 3 zeta, the latter being present as a homodimer. The unraveling of the architecture of the TcR/CD 3 complex is crucial to our understanding of the processes underlying its assembly, recognition and transmembrane signaling. The transmembrane orientation of the TcR chains and of CD 3 gamma and CD 3 delta can be directly inferred from their primary structure, based on the presence of concensus N-linked glycosylation sites N-terminal of their transmembrane domains. This prediction can not be made, however, for nonglycosylated molecules like the CD 3 epsilon chain. In order to determine the transmembrane orientation of CD 3 epsilon, anti-peptide antisera directed against the N-termini of the human and murine CD 3 epsilon chains were generated in rabbits. Both antisera stained intact T cells, demonstrating that the N-terminus of the CD 3 epsilon chain was located at the outer surface of the plasma membrane. The anti-human CD 3 epsilon peptide antiserum was found to be mitogenic for peripheral blood T cells, a finding previously reported only for monoclonal anti-TcR/CD 3 reagents. Using a novel transient expression system in murine T lymphocytes, the human CD 3 epsilon chain could be expressed on the surface of CD 3+, but not CD 3- murine T cells, as indicated by fluorescence staining with the anti-peptide antiserum. This experiment confirmed the specificity of the anti-peptide antiserum and, perhaps more importantly, indicated that the human CD 3 epsilon chain was correctly assembled in the murine CD 3 complex. Moreover, the anti-human CD 3 monoclonal antibody UCHT1 was found to stain T cells expressing the human CD 3 epsilon chain.

Amino Acid Sequence↗
PubMed1988

A T cell-specific enhancer is located in a DNase I-hypersensitive area at the 3' end of the CD3-delta gene.

During intrathymic differentiation, the genes coding for the T cell receptor/CD3 (TCR/CD3) complex are expressed in stages resulting in surface expression of competent receptors on the most mature T cells. The CD3-delta, gamma and epsilon proteins, which are expressed intracellularly in the earliest detectable cells of T lineage, form the core of the TCR/CD3 complex. In the present investigation aimed at understanding the tissue specific expression of the murine CD3-delta gene, a T cell specific DNase I hypersensitive site was found 0.6 kb downstream of the polyadenylation site of the gene. A 0.4 kb genomic fragment encompassing this hypersensitive site exhibited properties of a strictly T cell-specific transcriptional enhancer which acts in a position- and orientation-independent manner. However, the activity of the promoter region was not found to be T cell restricted. Within this novel T cell-specific enhancer region, sequence motifs were found which are shared with the promoters of both the mouse and human CD3-delta genes; in this region we also found sequence homologies to the enhancer core element of the thymotropic viruses SL3 and RadLV. These findings suggest that regulatory elements 3' of the CD3-delta gene are at least in part responsible for its exclusive expression in thymus-derived lymphocytes.

Animals↗
PubMed1988

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.

Amino Acid Sequence↗
PubMed1988

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.

Amino Acid Sequence↗
PubMed1988

Cytoplasmic expression of the CD3 antigen as a diagnostic marker for immature T-cell malignancies.

The expression of cytoplasmic CD3 (CyCD3) was analyzed in 45 leukemias, five thymus cell samples, five peripheral blood (PB) samples, and ten cell lines. All T cell acute lymphoblastic leukemias (T-ALL) that did not express surface membrane CD3 (SmCD3) appeared to express CyCD3. Furthermore, the majority of SmCD3+ T-ALL also expressed CyCD3. Analogous results were obtained with thymus cell samples in that about 95% of the thymocytes expressed CyCD3 whereas 60% to 75% of the thymocytes also expressed SmCD3. In normal peripheral blood only prominent SmCD3 expression was found. These data indicate that immature T cells express CyCD3 only, that the combined expression of CyCD3 and SmCD3 is characteristic for intermediate differentiation stages, and that mature T cells express prominent SmCD3. All (precursor) B cell leukemias, acute myeloid leukemias, and non-T cell lines tested did not express CyCD3. On the basis of these data, we conclude that CyCD3 expression is restricted to the T cell lineage and can be used as a diagnostic marker for immature SmCD3- T cell malignancies. Therefore, we evaluated which fixative is optimal for CyCD3 staining, and we determined by immunofluorescence staining and Western blotting which anti-CD3 monoclonal antibody (MoAb) can be used for the detection of CyCD3. In our opinion, acid ethanol was the best fixative for the cytocentrifuge preparations. Furthermore, we demonstrated that CyCD3 can be easily detected by use of MoAbs raised against denaturated CD3 chains such as those of the SP series (SP-6, SP-10, SP-64, and SP-78). In addition we tested 22 anti-CD3 MoAbs of the Oxford CD3 panel that were raised against native SmCD3, and it appeared that only four (UCHT1, VIT-3b, G19-41 and SK7/Leu-4) of them were able to detect CyCD3. In Western blot analysis all four MoAbs recognized the CD3-epsilon chain only.

Acute Disease↗
PubMed1987

Murine natural killer cells stimulated in vivo do not express the T cell receptor alpha, beta, gamma, T3 delta, or T3 epsilon genes.

Murine blast natural killer (NK) cells responding to in vivo stimulation were isolated and characterized as to their expression of the T cell receptor (TCR) variable genes alpha, beta, and gamma and the TCR constant genes T3 delta and T3 epsilon. In vivo stimulated blast T cells were isolated for comparison. RNA extracted from highly purified blast cell populations elicited in vivo was probed for TCR transcripts by Northern blot analysis. In contrast to the blast T cells which expressed high levels of the alpha, beta, delta, and epsilon genes, blast NK cells expressed very low to not detectable levels of these genes. Blast NK cells isolated from euthymic mice were comparable to those isolated from athymic mice and both populations had profoundly reduced levels of transcripts for TCR genes. The expression of gamma-chain genes was extremely low in the in vivo elicited blast T cells as well as the blast NK cells. Highly purified NK cells isolated from unmanipulated mice and propagated in culture with recombinant interleukin 2 for short periods of time also had extremely low levels of delta and epsilon, whereas T cells expanded in culture had high level expression of these genes. As delta and epsilon appear to be required for expression of a functional recognition structure on the T cell surface and are expressed early in T cell ontogeny, these results indicate that the functional NK cells responding to proliferation signals do not form a conventional T cell recognition structure. Furthermore, the results support a separation of the T cell and NK cell lineages early in ontogeny.

Animals↗
PubMed1987

T3-p28 is a protein associated with the delta and epsilon chains of the T cell receptor-T3 antigen complex during biosynthesis.

The human T cell receptor-T3 antigen complex is composed of at least five polypeptide chains. In addition to the 45-kDa/50-kDa heterodimer (alpha and beta chains) of the T cell receptor, the complex includes 25-kDa (T3-gamma) and 20-kDa (T3-delta) glycoproteins and a nonglycosylated 20-kDa (T3-epsilon) protein. Here we report that in pulse-chase biosynthetic labeling experiments we detect a new polypeptide chain (T3-p28) which is associated with the T3-delta and T3-epsilon chains during biosynthesis but not on the cell surface. T3-p28, which is not recognized by anti-T3 antibodies, can be chemically distinguished from the previously described T3-gamma chain. The carboxylic ionophore monensin blocks the apparent dissociation of T3-p28 from the T3-delta and T3-epsilon chains. Peripheral blood lymphocytes as well as all T cell leukemic lines tested contain T3-p28, except one HPB-ALL subline. Since the T3-p28 protein is only observed early in biosynthesis of T3-delta and T3-epsilon, it may function in intracellular transport or assembly of the T cell receptor-T3 complex.

Antigens, Surface↗