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R E Cone

Publications and source records attributed to R E Cone.

At least 55 records · Page 3Linked to original sources
PubMed1989

An approach to the unification of suppressor T cell circuits: a simplified assay for the induction of suppression by T cell-derived, antigen-binding molecules (T-ABM).

A system is presented in which the in vitro response to sheep red blood cells (SRBC) can be regulated using antigenic determinants coupled to SRBC and T cell-derived antigen-binding molecules (T-ABM) directed against the coupled determinants. T suppressor-inducer factors (TsiF's) are composed of two molecules, one of which is a T-ABM and one which bears I-J determinants (I-J+ molecule). Using two purified T-ABM which have not previously been shown to have in vitro activity, we produced antigen-specific TsiF's which were capable of inducing the suppression of the anti-SRBC response. Suppression was found to require both the T-ABM and the I-J+ molecule, SRBC conjugated with the antigen for which the T-ABM was specific, and a population of Ly-2+ T cells in the culture. Two monoclonal TsiF (or TsF1) were demonstrated to induce suppression of the anti-SRBC response in this system, provided the relevant antigen was coupled to the SRBC in culture. The results are discussed in terms of the general functions of T-ABM in the immune system. This model will be useful in direct, experimental comparisons of the function of T-ABM and suppressor T cell factors under study in different systems and laboratories.

Animals↗
PubMed1989

A monoclonal antigen-binding T cell immunoprotein: antigenic relatedness to T cell receptor beta chain FR1 V and J peptide segments: physicochemical distinctiveness from classical immunoglobulins and T cell receptor heterodimers.

The monoclonal murine T cell hybridoma, 51H7D, was previously shown to bind the arsazobenzene hapten and to produce a soluble antigen-binding molecule. In this paper we characterize this antigen-binding immunoprotein for its relationship to known T cell receptors serologically, using antibodies specific for variable region framework, or joining region peptides predicted from gene sequence and by biochemical means. The 51H7D cell expresses a protein with subunit size of approximately 31,000, that reacts antigenically with affinity-purified antibodies directed against synthetic first framework and joining segment peptides, corresponding to the gene sequence of the T cell receptor beta chain, YT35. This molecule does not react with affinity-purified antibodies directed against murine immunoglobulin, framework 1 sequences of alpha and gamma T cell receptors, or with antibodies against synthetic heavy chain joining segments. The subunit of mol. wt. 31,000 can form higher aggregates, notably in the mol. wt range of 60,000-70,000, depending upon extraction conditions. The soluble form of the antigen-binding molecule bears the J beta cross-reactive determinant and occurs predominantly as a charge restricted molecular species of approximate mol. wt 60,000-70,000. The purified molecule has a blocked N-terminus, but quantitative statistical analysis of its amino acid composition indicates a closer relatedness to T cell receptor beta chains and other antigen-binding T cell products, than it has to alpha, gamma or delta TCR chains. No evidence for more than one type of polypeptide chain was found and the polymerization is not dependent upon the formation of disulfide bonds. These studies raise the possibility that antigen-binding soluble T cell molecules might belong to a new family of immunoproteins, that is related to, but distinct from, classical immunoglobulins and alpha beta or gamma delta heterodimers.

Amino Acids↗
PubMed1988

Characterization of the molecules involved in thymocyte thymic epithelial cell adhesion.

Cell to cell adhesion is important for mechanisms of cellular recognition, growth, and differentiation. The identification of molecules involved in these interactions is necessary in order to understand the molecular basis of these processes. We have previously described the development of two different thymic epithelial cell lines (TECS and TECL). Using an assay with radiolabeled thymocytes we found that thymocytes can adhere specifically to these thymic epithelial cells. This adhesion is trypsin sensitive, suggesting that involvement of specific cell surface proteins. In the present study we further characterize and begin identification of the molecules involved in this interaction. We found that thymocyte binding to thymic epithelial cells requires the presence of Ca2+ and Mg2+ and is mediated by a molecule greater than 10,000 MW. Also, we identified several antibodies which inhibit the adhesion of thymocytes to TECS. The membrane nature of the molecule mediating this interaction was confirmed by the ability of thymocyte membranes to block the inhibitory antibodies.

Animals↗
PubMed1987

Quantitation of T cell antigen-binding molecules (TABM) in the sera of nonimmunized, immunized, and desensitized mice.

Heterologous antibodies to non-MHC-restricted T cell antigen-binding molecules (TABM) were used in an ELISA to detect and quantitate TABM levels in the sera of nonimmune mice and in mice immunized and/or desensitized to methylated bovine serum albumin (MBSA). TABM were detected at low levels in sera from nonimmune and immune mice. Amounts of TABM rose 300-fold in MBSA-immune mice within 12 hr of a desensitizing injection of MBSA, and rapidly returned to pre-desensitization levels. Serum TABM were purified by precipitation with 50% (NH4)2SO4 and chromatography in carboxymethylcellulose. The purified TABM were Mr 30,000 to 37,000 reduced, 72,000 nonreduced, and had a pI range of 5.5 to 7.1. A portion of the elevated TABM in desensitized mice bound MBSA specifically, whereas TABM from the sera of nonimmune/nondesensitized mice did not bind MBSA. The timing of the appearance of TABM early in desensitization suggests the possibility that they may play a role in the subsequent appearance of suppressor cells in later stages of this phenomenon. In addition, the results presented here raise the possibility that fluctuations in TABM levels might prove to be a sensitive indicator of immunoregulatory status in general.

Animals↗
PubMed1986

Isolation of antigen-binding membrane molecules from an antigen-specific T-cell hybrid.

Cell surface-radioiodinated proteins of a murine T-cell hybrid specific for and able to bind azobenzenearsonate were isolated by adsorption to Sepharose beads conjugated with a rabbit antiserum to murine T-cell antigen-binding molecules. These isolated proteins, Mr 72,000, were found to bind specifically azobenzenearsonate while proteins isolated in this manner from the tumor parent BW5147 did not bind azobenzenearsonate. Similar cell surface proteins were isolated by affinity for antigen and immunoprecipitated with an antiserum to T-cell antigen-binding molecules. The results suggest that antigen-binding T cells express T-cell antigen-binding molecules as membrane receptors for antigen.

Animals↗
PubMed1986

Phenotypic similarity between T-cell antigen binding molecules.

T-cell antigen binding molecules (TABM) specific for trinitrophenol (TNP), oxazalone, azobenzenearsonate or sheep erythrocytes were purified by affinity to antigen, adsorption to monoclonal antibodies to antigen binding molecules or were synthesized by translation of immunopurified mRNA for TABM in vitro. These molecules and a T-cell line, BW5147, membrane protein bound by rabbit antibodies to TABM were radiolabeled by 125I, digested with Staphylococcus V8 protease, and peptides of the proteolytic digest were resolved by 2D-gel peptide mapping. Comparison of the peptide maps of these proteins and amino acid analysis of T-cell antigen binding molecules specific for TNP or sheep erythrocytes indicate similarities and distinctions suggesting variable and constant domains in these molecules.

Amino Acids↗
PubMed1985

Antigen-binding molecules of T-cells charge heterogeneity and structural lability.

T-cell products released by immune cells during culture and which bind specifically the nominal antigens, trinitrophenol (TNP) or oxazalone, were isolated from culture media by hapten-affinity chromatography and compared by isoelectric focusing and 2D-gel analysis. These proteins and an azobenzenearsonate-specific T-cell product synthesized in vitro by translation of mRNA from an azobenzenearsonate-specific T-cell hybrid were also compared for structural lability of the polypeptides. Polyclonal T-cell antigen-binding molecules (TABM) specific for TNP or oxazalone showed marked charge heterogeneity and distinctions in isoelectric focusing in an acidic pH gradient, while the azobenzenearsonate-specific, clonal T-cell product displayed restricted focusing. All TABM studied showed dissociation of Mr 70,000 polypeptides to Mr 45,000 and 25,000 polypeptides after treatment with guanidine. The results provide further evidence for distinctions and similarities between TABM.

Animals↗
PubMed1985

Comparison of altered expression of histocompatibility antigens with altered immune function in murine spleen cells treated with ultraviolet radiation and/or TPA.

Previous studies in our laboratory demonstrated that several treatments that inhibited the ability of cells to stimulate the mixed lymphocyte reaction (MLR) also blocked the shedding of histocompatibility antigens and Ia antigens from murine spleen cells. In the present studies, one of these treatments, ultraviolet radiation (UV), was shown to cause an initial loss in the density of H-2K, IA, and IE antigens prior to the block in shedding observed after culture of these cells. Further analysis revealed that the UV-induced loss of antigens could be prevented by the presence of colchicine during irradiation. Biosynthetic analyses revealed the IA antigen synthesis was also inhibited in the UV-irradiated cells. Examination of the effects of a second agent, 12-0-tetradecanoylphorbol-13-acetate (TPA) on the turnover of histocompatibility antigens revealed that the biosynthesis and shedding of these antigens were accelerated by this agent. However, addition of TPA to UV-irradiated cells did not result in a reversal of the UV-induced block in biosynthesis of IA antigens. Results of immune function assays correlated with the biochemical studies: UV-irradiation inhibited the generation of the MLR, but TPA enhanced this reaction, and addition of TPA to mixed lymphocyte cultures with UV-irradiated stimulators did not reverse the UV-induced inhibition. These results suggest that, although the turnover of histocompatibility antigens may be affected by TPA and UV in an antagonistic fashion, additional factors other than the expression of histocompatibility antigens are operating in the inhibition of stimulation of an MLR by UV radiation or its enhancement by TPA.

Animals↗
PubMed1985

Cell sorting of lymphocyte subpopulations by biotinylated antibody and avidin-Sepharose beads.

A method has been developed to divide lymphocyte populations into subsets based on the affinity of avidin for biotinylated antibodies. Murine splenic lymphocytes were mixed with biotin-conjugated anti-immunoglobulin, Thy-1 or anti-Lyt antibodies. Cells bound by these reagents were adsorbed to avidin-sepharose beads and by this negative selection procedure effluent cell populations enriched for T cells, B cells or Lyt subsets were prepared reproducibly and rapidly.

Animals↗
PubMed1984

Production and purification of monoclonal T lymphocyte antigen binding molecules (TABM).

T lymphocyte antigen binding molecules are distinct from B cell immunoglobulins. These proteins were produced by an antigen-specific T cell hybrid as monoclonal products and these cells were successfully grown as an ascites. The T cell antigen binding proteins in ascites were harvested and purified in milligram amounts. The procedure we describe should provide a valuable source of large quantities of pure T lymphocyte antigen binding molecules. This method will allow for studies into their mechanisms of function in regulation of the immune response, biochemistry and molecular biology as B cell myelomas and monoclones provided for the study of immunoglobulins.

Animals↗
PubMed1984

Two-dimensional peptide mapping by polyacrylamide-gel electrophoresis with limited proteolysis in SDS.

The peptide mapping method described by Cleveland, et al. (1) was improved to a two-dimensional analysis applicable to minute amounts (less than 0.5 microgram) of proteins. Radioiodinated proteins for analysis were purified by electrophoretic elution of the proteins from polyacrylamide gels into buffer containing 0.1% sodium dodecyl sulfate. The proteins were digested enzymatically in the presence of 0.1% sodium dodecyl sulfate and an excess of nonlabeled bovine serum albumin (0.2 mg/ml) relative to labeled substrate in order to attain reproducibility by maintaining a consistent substrate concentration among different samples. The peptides of these limited proteolytic products were resolved by two-dimensional polyacrylamide gel electrophoresis (isoelectric focusing followed by SDS-gels). The resulting 2D-peptide maps of murine and bovine albumin and a murine lymphocyte membrane protein, Tp100, showed excellent resolution and reproducibility.

Animals↗
PubMed1984

Messenger RNA for an antigen-specific binding molecule from an antigen-specific T-cell hybrid.

The T-cell hybridoma 51H7D specifically binds the hapten azobenzenearsonate (ABA). An antiserum (anti-PCIF) specific for antigenic determinants shared by antigen-binding molecules of T cells (TABM) was used to precipitate polyribosomes containing mRNA for TABM from this T-cell hybridoma. The resultant mRNA was translated in vitro. The translated product (TrP51H7D) bound specifically ABA and was bound by both anti-PCIF and anti-T-cell suppressor factor. The parent lymphoma BW5147 yielded a similar translated product with the same antiserum used to isolate specific mRNA containing polysomes. This product (TrPBW) was bound by the antiserum used but did not bind ABA. The specific translated protein from both cells had a pI of approximately equal to 5.0, and apparent molecular weight of 71,000 (reduced) or 145,000 (nonreduced). Both protein products, when treated with guanidine to break down all noncovalent bonds, revealed an elemental peptide of Mr 23,500. The cDNA made from the isolated mRNA had 600-900 bases. mRNA of this size is expected for a protein of Mr 25,000. Our data indicate that a TABM specific for ABA is composed of peptides of Mr 23,500.

Animals↗
PubMed1984

Physical and pharmacologic inhibition of the shedding of I-A antigens.

Lactoperoxidase-catalyzed cell surface radioiodination was employed to radiolabel murine splenic lymphocyte membrane Ia antigens (I-Ak, I-Ek) and IgM. The catabolism of labeled membrane proteins was monitored by in vitro culture of the labeled cells, detergent lysis at intervals during culture, and immunoprecipitation of labeled antigens. Treatment of cells with UV irradiation, heat treatment, glutaraldehyde fixation, and colchicine reduces the shedding and/or expression of cell surface antigens and in particular those of the I-A subregion. Considered in physiological terms, we suggest that in addition to the amount of I-A antigen present on antigen-presenting cells, turnover and shedding of I-A antigens may be important for the stimulation of an immune response by allogeneic cells and in allowing a cell with receptors for foreign antigen (x) as well as for self I-A to find antigen x on I-A antigen-bearing cells.

Animals↗
PubMed1983

Properties of reticulum cell sarcomas in SJL/J mice. VIII. Prominent role of RCS cell I-A antigens in the stimulation of syngeneic T cells.

While T cells from SJL and from F1 hybrids of SJL that do not express I-E antigens give strong proliferative responses to RCS, T cells from F1 hybrids expressing surface I-E do not. The nature of the stimulating antigen on the RCS cell surface was examined using monoclonal antibodies. Complete inhibition of the T-cell proliferative response was obtained with antibodies to I-A antigens, whereas antibodies to I-E antigens did not inhibit at all. This inhibition was mediated via an effect of the antibodies on the stimulating cells. Biochemical characterization of immunoprecipitated 125I-and 35S-labeled RCS antigens was performed using two-dimensional gel electrophoresis. Using this technique, I-A antigens were readily detected. However, neither Ia.7-specific antibodies nor antibodies specific for E alpha: E beta complexes precipitated any E alpha or E beta chains. Comparison of I-A antigens from RCS and normal SJL spleen cells revealed minor mobility differences in the gels, possibly due to differences in glycosylation, the significance of which needs to be further evaluated. Examination of RNA extracted from RCS, using E alpha and A alpha cDNA probes showed that RCS cells do not transcribe the E alpha gene as has been shown previously for normal H-2s cells. Furthermore, DNA from RCS cells showed a defect in the E alpha gene similar to that known to exist in normal H-2s cells. Our findings exclude the presence of E alpha on RCS cells and suggest a major role for I-A, either alone or in conjunction with another as yet unidentified cell surface antigen, in the stimulation of T cells.

Animals↗
PubMed1983

Affinity-purified antigen-specific products produced by T cells share epitopes recognized by heterologous antisera raised against several different antigen-specific products from T cells.

Heterologous antisera to murine or rat T-cell antigen-binding molecules (T-ABM) were raised in rabbits or sheep. The T-ABM used for immunization were purified by affinity for antigen and did not bear known immunoglobulin isotypes. T-ABM and anti-T-ABM were raised in three separate laboratories. Antisera to T-ABM were exchanged and tested for binding to T-ABM in three separate laboratories. Thus antisera to at least three distinct T-ABM were tested directly for binding to T-ABM or by adsorption of biological activity. Rabbit antisera to murine trinitrophenol (TNP)-specific T-ABM or rat AgB-specific T-ABM bound both murine or rat T-ABM, indicating evolutionary conservation of T-ABM. Similar results were found with sheep antisera to murine T-ABM. In addition, all heterologous anti-T-ABM antisera used bound murine T-ABM specific for TNP, 4-hydroxy-3-nitrophenyl acetate (NP), SRBC, or T-cell membrane proteins with similar structure. Thus, there is a commonality of antigenic determinants between various T-ABM and T-cell membrane homologues which may be T-cell surface receptors for foreign antigen.

Animals↗