Biomedical subjects

C Terhorst

Publications and source records attributed to C Terhorst.

At least 163 records · Page 9Linked to original sources

PubMed1985

The T3/T cell receptor complex: antigenic distinction between the two 20-kd T3 (T3-delta and T3-epsilon) subunits.

Clonally distributed (clonotypic) antigen receptors on human T lymphocytes (alpha and beta chains) are associated with three invariable T3 polypeptide chains (T3 gamma, delta and epsilon), together forming the T3/T cell receptor complex. Monoclonal antibodies prepared against the two 20-kd T3 polypeptide chains demonstrated that T3-delta and T3-epsilon are distinct polypeptide chains. Only one monoclonal antibody (anti-T3-delta chain) reacted with the T cell surface as judged by indirect immunofluorescence, and by its mitogenicity for quiescent peripheral blood lymphocytes. Immunohistological staining and immunoprecipitation experiments showed that both the T3-delta and T3-epsilon chains are T cell-specific. As seen with the anti-alpha/beta chain reagent WT-31, anti-T3-delta chain monoclonal antibodies stained medullary thymocytes more intensely than cortical thymocytes, whereas the difference between the staining of cortical and medullary thymocytes was generally not apparent with anti-T3-epsilon chain antibodies. Because of this specificity and their ability to react with both the denatured and the native forms of each polypeptide chain, these new monoclonal reagents will be useful tools in studies of the biosynthesis and cell surface expression of the T3/T cell receptor complex during normal and malignant thymic differentiation.

Antibodies, Monoclonal↗

PubMed1985

Stimulation of the T3-T cell receptor complex induces a membrane-potential-sensitive calcium influx.

Three monoclonal antibodies selected for their recognition of parts of the T3-T cell receptor complex on human T lymphocytes were found to induce an increase in cytoplasmic free Ca2+ (Ca2+i) in the T cell leukemia line HPB-ALL as measured by Quin2 fluorescence. These reagents are directed against T3 (OKT3), a nonvariable T3-associated structure (WT-31) and the variable region of the T3-associated antigen receptor (T40/25) of this cell line. The rise in Ca2+i was dependent on the presence of extracellular Ca2+, occurred within 30 sec of stimulation, and was sustained for at least 10 min. Fab fragments of OKT3 also caused a rapid increase in Ca2+i, indicating that cross-linking is not necessary to induce a Ca2+ response. Alterations in plasma membrane potential and La3+ blocked the Ca2+ influx induced by OKT3 and T40/25. These data suggest that the T3-T cell receptor complex of human T lymphocytes may be an antigen-regulated Ca2+ channel.

Aminoquinolines↗

PubMed1985

beta 2-Microglobulin from serum associates with several class I antigens expressed on the surface of mouse L-cells.

Bovine beta 2-microglobulin (beta 2-m) present in fetal calf serum (FCS) is able to replace endogenous beta 2-m associated with several class I antigens from human and mouse cells maintained in culture [Bernabeu et al. (1984) Nature, Lond. 308, 642-645]. Here we show that human HLA-A2 and HLA-B7, as well as mouse H-2Ld and H-2Dd heavy chains expressed after gene transfer in mouse L-cells, associate to a large extent with bovine beta 2-m. We also demonstrate that bovine beta 2-m associated with the endogenous H-2Kk/Dk heavy chains generates an antibody response when L-cells are injected into syngeneic C3H mice.

Animals↗

PubMed1985

Assignment of the gene coding for the T3-delta subunit of the T3-T-cell receptor complex to the long arm of human chromosome 11 and to mouse chromosome 9.

The gene encoding the 20-kDa glycoprotein of the T3-T-cell receptor complex (T3-delta chain) has been mapped to human chromosome 11 by hybridization of a T3-delta cDNA clone (pPGBC#9) to DNA from a panel of human-rodent somatic cell hybrids. In Southern blotting experiments with DNAs of somatic cell hybrids that contained segments of chromosome 11, we were able to assign the T3-delta gene to the distal portion of the long arm of human chromosome 11 (11q23-11qter). By use of a newly developed cDNA clone (pPEM-T3 delta) that codes for the murine T3-delta chain, the mouse T3-delta gene was mapped on chromosome 9. The importance of the T3-delta map position and its relationship to the other genes on the long arm of human chromosome 11 and to those on mouse chromosome 9 is discussed.

Animals↗

PubMed1985

Characteristics of a monoclonal antibody (WT-31) that recognizes a common epitope on the human T cell receptor for antigen.

We describe a monoclonal antibody, WT-31, that reacted with all human T lymphocytes. Electrophoretic analysis of the material reacting with WT-31 revealed that it precipitated predominantly an 80-kD disulfide-linked heterodimer from the cell surface-labeled T leukemic cell line HPB-ALL. This heterodimer was identical to the one precipitated with a recently described monoclonal reagent, T40/25, which recognizes a clonotypic structure on HPB-ALL. The target antigen of WT-31 comodulated with T3 after incubation of T cells with excess anti-T3 antibody, indicating that the WT-31 target antigen is associated with T3. We also found that anti-T3 reagents, but not the clonotypic reagent T40/25, blocked binding of FITC-labeled WT-31 to HPB-ALL cells. This indicates that the T cell receptor epitope recognized by WT-31 is located close to the epitopes recognized by the anti-T3 reagents anti-Leu-4 and SPV-T3b but distal from the clonotypic T40/25 epitope. Functional studies showed that WT-31 reacts similar to anti-T3 antibodies. It is mitogenic for resting T cells, blocks cytolysis mediated by alloantigen-specific CTL clones, and induces antigen-nonspecific cytolysis by CTL clones against Daudi target cells. WT-31 did not inhibit the formation of conjugates, but it blocked cytolysis just before or during the Ca2++-dependent programming for lysis. We conclude that WT-31 is an antibody that recognizes a common determinant on the T cell receptor for antigen. The present results support the notion that the two chains of the T cell receptor (alpha and beta) form a functional protein ensemble with the three invariable T3 polypeptide chains (T3-gamma-, delta-, epsilon).

Antibodies, Monoclonal↗

PubMed1985

Structure and function of the T3/T cell receptor complex.

The T3/T cell receptor complex is a protein ensemble inserted in the plasma membrane of human thymus derived lymphocytes. Whereas the alpha and beta chains are variable elements (i.e. between different T cell clones, alpha and beta chains are distinct), the gamma, delta and epsilon chains are invariant between human T cell clones. Recently, genes coding for the alpha, beta and delta chains have been isolated. Functional studies in several laboratories, including our own, suggest a "division of labor" between the alpha/beta chain heterodimer (T cell receptor) and the three other polypeptide chains (T3 gamma, delta, epsilon): The T cell receptor alpha and beta chains together recognize antigen and MHC product on the antigen presenting or target cell and an activating signal is transduced to the cytoplasmatic side of the plasma membrane via the T3 complex. Thus, T cell responses are initiated immediately upon binding of ligand. In our model, Ca+2 influx plays a pivotal role in the signal transducing event. In fact, we postulate that the T3-epsilon chain itself might be a calcium gate.

Animals↗

PubMed1985

Structure and function of the T cell receptor/T3 complex.

The T cell receptor/T3 complex is a protein ensemble inserted in the plasma membrane of human thymus derived lymphocytes. Whereas the alpha and beta chains are variable elements (i.e. between different T cell clones alpha and beta chains are related but distinct), the gamma, delta and epsilon chains are invariant between human T cell clones. Recently, genes coding for the alpha, beta and delta chains have been isolated. Functional studies in several laboratories, including our own, suggest a "division of labor" between the alpha/beta chain heterodimer (T cell receptor) and the three other polypeptide chains (T3 gamma, delta, epsilon): The cell receptor alpha and beta chains together recognize antigen and MHC product on the antigen presenting target cell and an activating signal is transduced to the cytoplasmatic side of the plasma membrane via the T3 complex. Thus, T cell responses are initiated immediately upon binding of ligand. In our model, Ca2+ influx plays a pivotal role in the signal transducing event. In fact, we postulate that the T3-epsilon chain itself might be a calcium gate.

Animals↗

PubMed1985

The T-cell receptor/T3 complex.

The T-cell receptor/T3 complex is a protein ensemble inserted in the plasma membrane of human thymus-derived lymphocytes. Whereas the alpha and beta chains are variable elements (i.e. between different T-cell clones, the alpha and beta chains are variable elements (i.e. between different T-cell clones, the alpha and beta chains are related but distinct), the gamma, delta and epsilon chains are invariant between T-cell clones. Recently, genes coding for the alpha, beta and delta chains have been isolated. Functional studies in several laboratories, including our own, suggest a 'division of labor' between the alpha/beta-chain heterodimer (T-cell receptor) and the 3 other polypeptide chains (T3-gamma, -delta, -epsilon): the T-cell receptor alpha and beta chains together recognize antigen and MHC product on the antigen-presenting target cell and an activation signal is transduced to the cytoplasmic side of the plasma membrane via the T3 complex. Thus T-cell responses are initiated immediately upon binding of ligand. In our model, Ca2+ influx plays a pivotal role in the signal-transducing event. In fact, we postulate that the T3-epsilon chain itself might be a calcium gate.

Animals↗

PubMed1984

Recognition of HLA-A2 by cytotoxic T lymphocytes after DNA transfer into human and murine cells.

A gene coding for the major histocompatibility antigen HLA-A2 was transferred into human HLA-A2 negative M1 cells and murine L cells. Following transfection, these cells expressed molecules at the cell surface that are biochemically indistinguishable from HLA-A2 antigens on the human cell line JY from which the HLA-A2 gene was isolated. The M1A2 cells were recognized and lysed by a cytolytic T-cell clone specific for HLA-A2. The transfected L cells which express HLA-A2 in association with human beta 2-microglobulin were not lysed by this T-cell clone. The specific cytolysis of M1A2 cells could be inhibited by monoclonal antibodies to HLA-A2, and monoclonal antibodies to T3, T8, and LFA-1 on cytotoxic T lymphocytes. These results suggest that killing by allospecific T cells requires HLA-A2 antigens as well as other species-specific structures on the target cell surface.

Animals↗

PubMed1984

Characterization of the two heavy chains of the T3 complex on the surface of human T lymphocytes.

The T3 complex has been defined by a group of monoclonal antibodies which react with all human peripheral blood T lymphocytes and a subpopulation of thymocytes. This membrane structure includes glycoproteins of 44 (alpha), 37 (beta), 25 (gamma), and 20 kDa (delta) as well as a nonglycosylated polypeptide of 20 kDa (epsilon). The characterization of the alpha and beta chains has been of particular interest because they may constitute the T cell receptor for antigen. Here we show that the T3 complex prepared by immunoprecipitation from T lymphocytes of a leukemic patient (Sezary syndrome) displays an unusually strong association of the alpha and beta chains with the 20/25-kDa T3 proteins. The alpha and beta chains (48 and 44 kDa) were co-precipitated by anti-20-kDa T3 monoclonal antibodies as a disulfide-linked 90-kDa heterodimer. A minor 220-kDa multimer composed of proteins similar to the alpha and beta chains was also present in these immunoprecipitates. This multimer could be independently precipitated with a new monoclonal antibody WT-31, which detects the larger polypeptide chains of the T3 complex on all human T lymphocytes. After removal of N-linked oligosaccharides, both the alpha and beta chain were found to have 33-kDa peptide backbones with distinct isoelectric points. Using a monoclonal reagent T40/25, a 90-kDa heterodimer, consisting of 40- and 49-kDa chains with peptide backbones of 34 kDa was found to be T3-associated on the T leukemic cell line HPB-ALL. When the alpha and beta chains from the Sezary patient were compared with the corresponding chains from HPB-ALL by peptide mapping, large differences were observed. Taken together, the data presented here provide strong evidence that the T cell receptor for antigen is part of the T3 complex on the surface of human T lymphocytes.

Acetylglucosaminidase↗

PubMed1984

HLA-DC antigens can serve as recognition elements for human cytotoxic T lymphocytes.

The specificity of four cytotoxic T lymphocyte (CTL) clones which recognize class II major histocompatibility complex (MHC) antigens was analyzed. All clones recognized antigens associated with the serologically defined HLA-DRw6 specificity. The activity of two of these clones, JR-2-2 and JR-2-10, could be inhibited by a monoclonal antibody Q 5/13 specific for a monomorphic determinant present on HLA-DR. In contrast, the activity of the two other CTL clones, JR-2-19 and JR-2-26, was not blocked by Q 5/13, but by a new monoclonal reagent, SPV-L3. This latter monoclonal antibody precipitated a two-chain structure of 28 kDa and 33 kDa and reacts with a monomorphic determinant. The molecular weight of the polypeptides precipitated with SPV-L3 was slightly less than those precipitated with a HLA-DR-specific monoclonal reagent. In addition two-dimensional gel electrophoresis showed that the antigen precipitated by SPV-L3 differed in charge from those precipitated with the anti-HLA-DR antibody. These results indicate that SPV-L3 recognizes a class II MHC product different from HLA-DR. This observation was confirmed by partial amino acid sequence analysis of the two chains which revealed that the molecule precipitated by SPV-L3 is homologous to HLA-DC/DS molecules. Therefore this report provides the first evidence that human cytotoxic T cells can recognize HLA-DC/DS antigens.

Amino Acid Sequence↗

PubMed1984

Human cutaneous dendritic cells express two glycoproteins T6 and M241 which are biochemically identical to those found on cortical thymocytes.

Monoclonal antibodies anti-T6 and anti-M241 define unique cell populations within different lineages: cortical thymocytes and dendritic cells in the skin. T6 positive cutaneous dendritic cells are located predominantly in the epidermis and belong to the Langerhans/indeterminate lineage, whereas, most of the M241 positive cells are located in the perivascular regions of the dermis. Biochemical analysis of thymocytes and cutaneous dendritic cells was performed in order to determine whether the reactivity of these antibodies with these cell types is due to sharing of antigenic determinants by two unrelated proteins, or whether similar proteins are present on cells of different lineages. Our results indicate that T6 antigens are borne by the same glycoprotein (49K) on cortical thymocytes and Langerhans/indeterminate cells. Similarly, M241 antigens isolated from thymocytes and cutaneous dendritic cells are found on the same glycoprotein (43K).

Antibodies, Monoclonal↗

PubMed1984

Deficiency of a leukocyte surface glycoprotein (LFA-1) in two patients with Mo1 deficiency. Effects of cell activation on Mo1/LFA-1 surface expression in normal and deficient leukocytes.

Mo1, a phagocyte surface glycoprotein heterodimer, is involved in a number of phagocyte adhesion functions such as binding and ingestion of serum-opsonized particles, zymosan-induced degranulation, and superoxide generation. Deficiency of this antigen in humans has been associated with increased susceptibility to recurrent bacterial infections. The beta subunit of Mo1 is shared by another surface glycoprotein named LFA-1, which is involved in lymphocyte proliferation, cytolytic T cell, and natural killing activities. Two unrelated patients with Mo1 deficiency were found to be deficient in LFA-1 as well as in the common beta subunit. Investigation of lymphocyte functions in these two patients revealed normal mixed leukocyte culture-generated cytolytic T cell and natural killing activities and significantly reduced proliferative response to phytohemagglutinin. LFA-1-deficient cells also proliferated in response to soluble antigen and different alloantigens. These responses were partially blocked by anti-LFA-1 antibody. Whereas LFA-1 was undetectable by immunofluorescence and immunoprecipitation on the patients' resting T cells, significantly reduced (approximately 5% of normal) but detectable amounts of the heterodimeric LFA-1 antigen were found on mitogen and alloantigen-activated T cells. On granulocytes, Mo1 surface expression was also dependent on the state of cellular activation. The amount of surface Mo1 present on resting normal granulocytes increased by 3-10-fold following exposure to stimuli that induced degranulation, suggesting the presence of a major intracellular pool for this antigen. Analysis of subcellular fractions from granulocytes showed that intracellular Mo1 is located primarily in the specific granule fraction. Activated granulocytes had little or no increase in their surface expression of LFA-1 antigen. Deficient granulocytes had significantly increased numbers of Mo1 antigen expressed on the surface following stimulation with calcium ionophore (1 microM). However, the amount expressed continued to be significantly reduced compared with normal cells. Quantitation of surface Mo1 on granulocytes exposed to calcium ionophore (1 microM) showed that both parents in one family but only the mother in the other family had significantly reduced levels of Mo1, suggesting heterogeneity in the inheritance of this disorder. Whereas LFA-1 deficiency on lymphocytes was associated with normal alloantigen-induced cytolytic T cell and natural killing activities in these two patients, functions which were in part dependent on small amounts of detectable LFA-1 antigen, the Mo1 deficiency state led to significant defects in phagocyte adhesion functions. Hence, the clinical symptoms associated with this combined deficiency state reflect a more profound phagocyte than lymphocyte disorder.

Animals↗

PubMed1984

Purification and N-terminal amino acid sequence of the human T cell surface antigen T8.

Monoclonal antibodies allow for the detection of structures on the cell surface of human cytotoxic T lymphocytes (CTL) that are involved in their effector function. Among these cell surface components, T8 is of particular interest because it is required during the recognition of target cells by a subset of CTL. An understanding of its role during CTL:target adhesion requires detailed biochemical structural analysis of the T8 molecule. This has been hindered by the small amounts of protein currently available. Here we describe the development of a purification scheme that will permit the accumulation of larger quantities of T8. We studied the binding of T8 to several lectins and determined that one of these, wheat germ agglutinin, bound T8 quantitatively. Experiments designed to test the properties of T8 in a phase separation system with the use of Triton X-114 were performed. These indicated that T8 partitions into the aqueous phase rather than the detergent phase during this procedure. With this in mind, we developed a protocol that resulted in a significant purification of T8 after affinity chromatography. Upon preparative SDS-PAGE followed by electroelution, the T8 antigen was purified to homogeneity and used for N-terminal acid acid sequencing. This analysis yielded the amino terminal 22 amino acids of T8. On purification, it was observed that the protein existed as two bands of Mr 33 and 34 kilodaltons after SDS-PAGE analysis. The relationship between these chains was investigated by limited radio-sequencing and tryptic peptide map analysis; our results indicated that the two polypeptides were identical. The two chains were treated with trifluoromethane sulfonic acid to determine whether carbohydrates accounted for the difference in m.w. This reagent, which cleaves both N-linked and O-linked sugars, cleaved approximately 2000 daltons of oligosaccharides from the T8 molecule. These oligosaccharides are most likely of the O-linked rather than the N-linked variety.

Amino Acid Sequence↗

PubMed1984

Coexpression of the human HLA-A2 or HLA-B7 heavy chain gene and human beta 2-microglobulin gene in L cells.

L cells expressing human HLA-A2 or HLA-B7 class I antigen heavy chains are not recognized by human cytotoxic T lymphocytes directed at HLA-A2 or HLA-B7 antigens. To test whether the absence of human beta 2-m was the cause of the lack of recognition by the human cytotoxic T lymphocytes, coexpression of the human beta 2-m gene and the HLA-A2 or HLA-B7 heavy chain in L cells ("double transfectants") was obtained. In addition, L cells expressing HLA-A2 or HLA-B7 antigens in association with human beta 2-m were obtained by an exchange reaction, in which human beta 2-m from serum replaced the endogenous murine beta 2-m. Both types of transfectant cells were used in 51Cr-release assays and cold target inhibition assays for human cytotoxic T cell clones which were directed at HLA-A2 or HLA-B7. Neither human CTL clones nor a mixture of CTL specific for HLA-A2 and HLA-B7 were able to recognize these cells. Several alternative explanations for these observations are discussed.

Animals↗

PubMed1983

The T8 antigen is a multimeric complex of two distinct subunits on human thymocytes but consists of homomultimeric forms on peripheral blood T lymphocytes.

Monoclonal antibodies allow for the detection of structures on the cell surface of human cytotoxic thymus-derived lymphocytes, which are involved in their function. Among these cell surface components, T8 is of particular interest, since it is required during the recognition of target cells by a subset of human cytotoxic T lymphocytes. The only other cell types on which T8 has been detected are functionally inert thymocytes. Here we demonstrate that T8 isolated from peripheral blood T lymphocytes is different from thymocyte T8. On peripheral blood T lymphocytes T8 was found in multimeric forms of a 34-kDa glycoprotein. These forms were also found on thymocytes, but in addition multimeric complexes of the 34-kDa T8 with a 46-kDa protein were detected. Preliminary chemical analyses showed that the 34-kDa and the 46-kDa forms differ in both their protein and carbohydrate structures. The 46-kDa structure was found to be distinct from the major thymocyte antigen T10. The interchain disulfide bridges between the 34-kDa T8 polypeptide are located in the 24-kDa CNBr fragment, which contains a hydrophobic region. Two (14 kDa and 20 kDa) of the three CNBr fragments of the 46-kDa T8 subunit were found to be involved in interchain disulfide bridging with the 34-kDa T8 species. We suggest that the switch from heteromultimers to homomultimers may occur concomitantly with the last step in T lymphocyte maturation.

Antigens, Surface↗

PubMed1983

The T3 complex on human T lymphocytes involves four structurally distinct glycoproteins.

Monoclonal antibodies allow for the detection of antigens which are specific for human thymus-derived lymphocytes. Among these antigens, the T3 complex is of particular interest since it is involved in several T cell functions. The main target antigen of the anti-T3 reagents is borne by a 20-kDa glycoprotein. In addition, glycoproteins of 25-28, 37, and 44 kDa are found in anti-T3 immunoprecipitates derived from surface-labeled cells. The four antigens appeared to be strongly associated with each other in detergent-containing solutions. Comparative studies of the four proteins, facilitated by the use of endo-beta-N-acetylglycosaminidase F, revealed that their polypeptide backbones have different molecular weights and pI values. Moreover, peptide maps of the 20-kDa T3 and the 25-28-kDa T3 were quite different. Metabolic labeling experiments suggested that the 25-28-kDa protein might become associated with the 20-kDa T3 antigen during biosynthesis. The 37-kDa and 44-kDa proteins could not, however, be detected and, therefore, might become associated with the 20-kDa T3 on the cell surface. Evidence has been found for the existence of a fifth member of the T3 complex, namely an unglycosylated 20-kDa T3 species.

Antibodies, Monoclonal↗

PubMed1983

The T3 complex on human thymus-derived lymphocytes contains two different subunits of 20 kDa.

The human cell surface antigen T3 is involved in several T lymphocyte specific functions, as determined by the effect of monoclonal antibodies (OKT3, anti-Leu-4, UCHT1) directed at this molecular structure. The main target antigen of these monoclonal antibodies is a glycoprotein of 20 kDa. It is associated with four, less predominant, structurally distinct glycoproteins of 25-28 kDa, 37 kDa and 44 kDa. Of these molecules only the 20-kDa T3 antigen could be labeled with the hydrophobic reagent 5-iodonaphthyl-1-azide (INA). Here we present evidence that the main 20-kDa T3 antigen is comprised of, in fact, two structurally different molecules. One of these is a glycoprotein with a protein backbone of 14 kDa, the other is an unglycosylated protein of 20 kDa. This unglycosylated protein is labeled specifically with INA. Additional evidence for the existence of two different 20-kDa T3 antigens is provided by studies using the enzymes endo-beta-N-acetylglucosaminidase H and endo-beta-N-acetylglucosaminidase F and the drug tunicamycin. We hypothesize that the specific susceptibility to labeling with INA of the unglycosylated 20-kDa T3 form reflects a positioning in the lipid bilayer different from that of the glycosylated 20-kDa T3 form.

Antibodies, Monoclonal↗