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Biomedical subjects

C E Rudd

Publications and source records attributed to C E Rudd.

85 records · Page 5Linked to original sources
PubMed1988

Role of the 2H4 molecule in the activation of suppressor inducer function.

The monoclonal antibody anti-2H4 recognizes a 220-kDa and 200-kDa glycoprotein and subdivides T4+ cells into distinct subpopulations: the T4+2H4+ inducer of suppression and the T4+2H4- inducer of help. The T4+2H4+ subset has been shown to play a crucial role in the activation of T8+ suppressor cells. In the present study, we attempted to determine whether the 2H4 molecule itself may be involved in initial triggering of suppressor inducer function. The results show that the addition of anti-2H4 antibody to a mixture of B and T cells resulted in a marked suppression of pokeweed mitogen-driven Ig synthesis. When anti-2H4 was added to B cell cultures containing T4+2H4+ or T4+2H4- cells but lacking T8 cells, no suppression was generated. In addition to the requirement for T8 cells, no suppression was generated if T4+2H4+ cells were absent. These results suggest that the anti-2H4 antibody contributes to the activation of the T4+2H4+ lymphocyte subset, which in turn induces T8 cells to suppress B cell Ig synthesis. Biochemical analysis of the T4+2H4+ subset of lymphocytes indicated that the in vitro addition of anti-2H4 antibody resulted in an increased expression of the 220-kDa and 200-kDa structure on T4 cells. We conclude that perturbation of the 2H4 molecule may potentiate the activation of the suppressor inducer subset and that the T200 molecule may be directly involved in suppressor inducer function.

Antibodies, Monoclonal↗
PubMed1988

The CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (pp58) from human T lymphocytes.

The CD4 (T4) antigen is a cell-surface glycoprotein that is expressed predominantly on the surface of helper T cells and has been implicated in the regulation of T-cell activation and in the associative recognition of class II antigens of the major histocompatibility complex. In addition, the CD4 antigen appears to serve as a receptor for the human immunodeficiency virus (HIV). An important question has been whether the CD4 receptor is linked to an intracellular mediator that could regulate the activation of the CD4+ subset. In this paper, we provide preliminary evidence that the CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (PTK) of 55-60 kDa, which is expressed specifically in T cells. The PTK is the human analogue of the murine pp56LSTRA (pp56lck) and has significant homology with c-src, c-yes, and other members of the src family. The identification of the PTK associated with CD4 receptor was made by use of an antiserum to a synthetic peptide that was deduced from the DNA sequence of PTK. Two-dimensional nonequilibrium pH gradient gel electrophoresis/NaDodSO4/PAGE revealed the kinase to focus as a heterogeneous collection of spots in the pH range of 4.0-5.0. Furthermore, in vitro phosphorylation revealed the phosphorylation of two additional polypeptides at 40 and 80 kDa, in addition to the autophosphorylation of the PTK at 55-60 kDa. The potential importance of the association between the CD4 receptor and the PTK of T cells is discussed in relation to T-cell activation and HIV infectivity.

Amino Acid Sequence↗
PubMed1987

The subdivision of the T4 (CD4) subset on the basis of the differential expression of L-C/T200 antigens.

The T4 (CD4) subset of T lymphocytes has been subdivided into two major subsets, a suppressor/inducer subset (T4+,2H4+) and a helper subset (T4+,2H4-) on the basis of the differential expression of the L-C/T200 (CD45) antigens. The 2H4 antigen itself comprises at least three distinct polypeptides at 125,200, and 220 X 10(3) Mr, of which the 200 and 220 X 10(3) Mr polypeptides constitute the highest Mr isoforms of a pool of five distinct L-C/T200 antigens. The T4+,2H4+ subset expresses at least four of these isoforms at 180, 190, 200, and 220 X 10(3) on the cell surface, while the T4+,2H4- subset expresses only the 180 and 190 X 10(3) Mr forms. Pulse-chase analysis and endoglycosidase treatment revealed that the 125 X 10(3) Mr chain of the 2H4 antigen is nonglycosylated, while the 200 and 220 X 10(3) polypeptides are structurally related and derived by N- and O-linked glycosylation from two nascent subunits at 150 and 160 X 10(3) Mr. The function of the T4+,2H4+ subset could be blocked only by an antibody reactive with the L-C/T200 isoforms enriched with O-linked oligosaccharides at 200 and 220 X 10(3) Mr.

Antibodies, Monoclonal↗
PubMed1987

Induction of suppression following autologous mixed lymphocyte reaction; role of a novel 2H4 antigen.

In this study, we have investigated the cellular and molecular basis for immunoregulatory function of T4+ cells after autologous mixed lymphocyte reaction (AMLR) activation. We demonstrated that the T4+ 2H4+ subset but not the T4+2H4- subset can proliferate maximally in response to autologous non-T cells. T4+ cells activated by AMLR exerted suppressor activity on pokeweed mitogen-driven IgG synthesis of autologous peripheral blood lymphocytes. The suppressor activity by AMLR-activated T4+ cells required the presence of fresh T8+ cells in the secondary culture, indicating that AMLR-activated T4+ cells functioned as a suppressor inducer rather than as a suppressor effector population. Following activation of T4+ cells in AMLR, it is the T4+2H4+ subset which induces suppression through the T8 population. Moreover, the treatment of AMLR-activated T4+2H4+ cells with anti-2H4 antibody, but not other antibodies, resulted in the abolishment of suppressor inducer function of such cells, suggesting that the 2H4 molecule itself may be involved in the suppressor inducer function. The 2H4 antigen on such cells was shown to be comprised of 220-kDa and 200-kDa glycoproteins. These results support the notion that the AMLR may play an important role in generating suppressor inducer signals and in down-regulating the immune response following self major histocompatibility complex recognition. More importantly, the present studies indicate that the 2H4 antigen on T4 cells serves not only as a phenotypic marker of suppressor inducer cells, but may have a functionally important role itself inducing suppression.

Antibodies, Monoclonal↗
PubMed1986

The role of the 2H4 molecule in the generation of suppressor function in Con A-activated T cells.

The molecular basis for the suppression generated in a concanavalin A (Con A)-activated T cell culture remains unknown. In this study, we have attempted to determine whether the 2H4 and 4B4 molecules on Con A-activated T cells play some role in the generation of suppression by such cells. We have shown that Con A-activated suppressor cells belong to the 2H4+ subset of T cells but not the 4B4+ (2H4-) subset. Con A-activated T cells exerted their optimal suppressor function on day 2 in culture, a time at which the expression of 2H4 on such cells was maximal and 4B4 was minimal. Furthermore, the stimulation of T cells with the higher concentration of Con A generated the stronger suppressor function. At the same time, both 2H4 expression and density were increased and 4B4 expression and density were decreased on such Con A-activated T cells. More importantly, the treatment of Con A-activated T cells with anti-2H4 antibody but not with anti-4B4, anti-TQ1, or anti-T4 antibodies can block the suppressor function of such cells. Taken together, the above results strongly suggest that the 2H4 molecule itself may be involved in the generation of suppressor function in Con A-activated T cells. The 2H4 antigen on such cells was shown to be comprised of 220,000 and 200,000 m.w. glycoproteins. Thus this study indicates that the 220,000 and 200,000 m.w. structure of the 2H4 molecule may itself play a crucial role in the generation of suppressor signals of Con A-activated cells.

Antibodies, Monoclonal↗
PubMed1986

Function of the LFA-1 and T4 molecules in the direct activation of resting human B lymphocytes by T lymphocytes.

Activated T lymphocytes can provide all of the signals necessary to induce the proliferation of resting B lymphocytes. The activation signal is presumably initiated through direct T-B lymphocyte contact. The role of the leukocyte function antigen-1 (LFA-1) and T4 molecules in the activation of purified, small B lymphocytes by mitomycin C-treated T lymphocytes was examined by using monoclonal antibodies that react with and inhibit the function of these molecules. Anti-LFA-1 antibody binding significantly inhibited T-B lymphocyte interactions that result in B lymphocyte proliferation. In contrast, the presence of anti-T4 antibodies at concentrations as high as 100 micrograms/ml did not inhibit this interaction. These results indicate that the B lymphocyte activation signal may not be mediated through the interaction of T4 molecules with major histocompatibility complex class II antigens of the B lymphocyte but is a cell-cell contact-dependent event that is facilitated by LFA-1 molecules.

Antibodies, Monoclonal↗
PubMed1985

HLA-D region antigen-associated invariant polypeptides as revealed by two-dimensional gel analysis. Glycosylation and structural inter-relationships.

Two-dimensional polyacrylamide gel analyses of immunoprecipitates of HLA-D region antigens prepared from [35S]methionine-labeled B lymphoblastoid cells revealed a number of invariant polypeptides (Ii and theta) that co-precipitate with the alpha and beta polypeptides of the class II (Ia) antigens. The invariant polypeptides comprised at least three Ii spots of Mr = 31,000 (Ii1-Ii3) and a series of six theta spots of Mr = 34,000 (theta 1-theta 6). The structural inter-relationships of these polypeptides have been investigated. Tryptic peptide fingerprints showed that Ii and theta have closely related amino acid sequences. In contrast, the fingerprints of the HLA-DR alpha and beta polypeptides clearly differed from those of theta and Ii as well as from each other. Analyses of immunoprecipitates prepared from cells cultured in the presence of tunicamycin revealed the presence of two N-linked oligosaccharides on each invariant polypeptide and suggested that the more acidic theta polypeptides (theta 1 and theta 2) differed from the other invariant polypeptides by the presence of sialic acid on one or both N-linked oligosaccharides. Removal of sialic acid by neuraminidase simplified the pattern of theta spots into three distinct Ii-related polypeptides. Endo-beta-N-acetylglycosaminidase H digestion indicated that the individual theta polypeptides represent stages in carbohydrate processing whereby Ii with two N-linked immature oligosaccharides are converted initially to theta 6-theta 3 with one immature and one complex, but nonsialylated, oligosaccharide and finally to theta 2-theta 1 with two complex oligosaccharides. Digestion of the theta polypeptides with N-acetylgalactosamine oligosaccharidase indicated that the theta spots are also derived by O-glycosylation from the Ii polypeptides. This assignment is supported by results obtained using monensin to block glycosylation within the Golgi. At least three spots persisted after complete removal of the N- and O-linked oligosaccharides, suggesting the presence of a family of invariant polypeptides differing in amino acid sequence.

Acetylglucosaminidase↗
PubMed1984

Isolation and characterization of a human T lymphocyte-associated glycoprotein (gp40).

The biosynthetic and structural characteristics of the human thymocyte/T cell antigen defined by the monoclonal antibody WT1 have been studied. WT1 identified a monomeric cell surface glycoprotein of Mr = 40,000 ( gp40 ). Cross-absorption experiments and two-dimensional gel analyses indicate that WT1 and another monoclonal antibody, 3A1, react with the same structure. This glycoprotein was asymmetrically inserted into the rough endoplasmic reticulum as a transmembrane structure. At this stage, the polypeptide chain possessed two N-linked, "high-mannose" type glycans; these were subsequently processed into endo-H-insensitive, complex oligosaccharides during intracellular transport to the cell surface. Inhibition of N-linked glycosylation with tunicamycin failed to block the processing of the nonglycosylated Mr = 29,000 polypeptide to a glycoprotein of Mr = 33,000. Cleavage of the mature Mr = 40,000 form with endo-F yielded a similar Mr = 33,000 product. The kinetics of synthesis of the Mr = 33,000 intermediate in conjunction with gal-NAc oligosaccharidase digestion indicated the presence of O-linked glycans in the mature cell surface WT1 antigen. The fully processed cell surface form of the polypeptide also contains covalently associated fatty acid, and was labeled by 32P phosphate, the predominantly labeled phosphoamino acid being phosphoserine. We also demonstrate biochemically that the reactivity of WT1 with cells from a few patients with acute myeloid leukemia reflects genuine expression of the gp40 structure on myeloid cells.

Adult↗
PubMed1981

Thymidine transport in human lymphocytes stimulated with concanavalin A: effect of colchicine.

Concanavalin A (Con A) was found to induce the appearance of time-dependent and saturable thymidine transport in human peripheral lymphocytes with a Km that ranged from 200 to 500 microM and a Vmax of between 5 to 8 pmols/cell/sec; transport of thymidine could not be demonstrated in resting cultures. Colchicine was found to cause the disappearance of microtubular networks in the majority of cells in the population, whereas lumicolchicine as expected had no significant effect on the presence of microtubules. Lumicolchicine caused 50% inhibition of thymidine transport and of DNA synthesis at a concentration of about 10(-4) M; the effect of this agent on the transport of thymidine could account for its ability to inhibit DNA synthesis. Colchicine also inhibited the transport of thymidine by 50% at about 10(-4) M. However, half-maximal DNA synthesis occurred at a colchicine concentration of 5.0 X 10(-8) M; thus, the effect of this drug on transport cannot account for its effect on DNA synthesis, since the latter process is some 3 orders of magnitude more sensitive than the former. The ability of colchicine to inhibit DNA synthesis in Con A-stimulated human lymphocytes would appear to be an indirect consequence of disassembly of microtubules.

Colchicine↗
PubMed1979

Microtubules, colchicine, and lymphocyte blastogenesis.

We have studied the time course of disassembly of microtubules of resting and stimulated mouse lymphocytes caused by the drug colchicine, as well as the effect of this compound on DNA and RNA synthesis of human and mouse lymphocytes. Fine-structure studies with the electron microscope showed a great increase in number of microtubules resulting from stimulation of mouse lymphocytes by the mitogenic lectin Con A. The presence of a network of microtubules was demonstrated in resting lymphocytes by use of the technique of immunofluorescence; this technique was not effective for the study of the microtubules of stimulated lymphocytes in the blast stage. The disappearance of microtubular networks in some cells (approximately 25%) was caused by the protocol of colchicine treatment used in many laboratories (30 min at 10(6) M); a 6- to 8-h treatment was required to cause all cells to lose their microtubules. It is indicated in these findings that there is need for extreme caution in implicating microtubule disruption as the cause of certain colchicine effects, such as that on the Con A-induced inhibition of receptor-ligand migration. The addition of colchicine to stimulated cells at varying times of culture caused marked inhibition of DNA synthesis provided that sufficient time (approximately 20 h for maximum inhibition) elapsed between addition of the drug to the stimulated culture and assay of DNA synthesis. Our data on the time course of inhibition of DNA synthesis by alpha-methyl mannoside (alpha MM) and by colchicine do not exclude the possibility that the latter compound may act partially by affecting the commitment of stimulated lymphocytes to DNA synthesis but they show that it can inhibit well after commitment is complete. The later the time of assay of thymidine incorporation, the more disparate were the curves relating the effects of alpha MM and colchicine to DNA synthesis of human cells. In the case of mouse splenic lymphocytes, there was no resemblance between the time course of the alpha MM and of the colchicine effects. Synthesis of RNA after 12 h of culture of stimulated human lymphocytes was also sensitive to colchicine.

Animals↗
PubMedDate not supplied

A novel epitope of the LFA-1 antigen which can distinguish killer effector and suppressor cells in human CD8 cells.

The CD4 subset of cells displays helper/inducer activity and recognizes class II antigens of the major histocompatibility complex (MHC), while the CD8 subset recognizes class I MHC antigens and exhibits cytotoxic or suppressor function. Considerable functional as well as corresponding phenotypic heterogeneity exists within the two major T cell subsets. Although the CD8+ population contains pre-cytotoxic, cytotoxic, pre-suppressor and suppressor effector T cells, these distinctions still rest largely on the use of functional assays. Attempts have been made to define the CD8+ precursor of the killer cell with new monoclonal antibodies. But more precise phenotypic distinctions between the functional subpopulations within CD8+ cells will be needed. We have now developed a monoclonal antibody, anti-S6F1 which can distinguish killer effector and suppressor effector cells in CD8 lymphocyte populations. The cell-surface structure defined by this antibody comprises two glycoproteins with relative molecular mass (Mr) 180K and 95K respectively. Also sequential immunoprecipitation studies and two dimensional gel electrophoresis indicate that anti-S6F1 recognizes a novel epitope on the LFA-1 antigen.

Animals↗