Robert E. Cone

Isolation of membrane associated immunoglobulins from T lymphocytes by non-ionic detergents

Abstract Lactoperoxidase-catalyzed radioiodination was employed to incorporate 125 I into membrane proteins of living thymocytes or splenic lymphocytes. Radiolabelled cells were lysed in Triton X-100 and Triton X-100 soluble membrane proteins were mixed with rabbit antiserum to murine Ig light chains and sheep anti-rabbit Ig serum to precipitate radiolabelled membrane Ig. T cell membrane Ig was detected when 1 × 10 7 cells were lysed with 1 ml of 0.01–0.1% Triton X-100, 0·05–0.1% being optimal. T cell Ig was not detected when thymocytes were lysed with detergent concentrations exceeding 0.1%. The loss in detectability of T cell Ig at high detergent concentrations was reversible upon dilution of the detergent and correlated with the critical micelle concentration (CMC) of the detergent. Detection of B cell membrane Ig was not significantly affected by detergent concentrations above the CMC. PAGE analysis of reduced and alkylated membrane Ig of spleen cells revealed light chains and heavy chains with the mobility of Ig μ chains and heavy chains with an apparent mol. wt of 60–65,000 daltons. T cell Ig isolated by detergent alone possessed light chains and heavy chains with an apparent mol. wt of 45,000 daltons. The heavy and light chains were not linked by interchain disulfide bonds. The results suggest that membrane immunoglobulins of T and B lymphocytes are structurally distinct and that T cell Ig may be more hydrophobic than its B cell counterpart.

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.

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.

Analysis of detergent-extracted membrane immunoglobulins of T and B lymphocytes by ultracentrifugation and column chromatography.

Abstract Lactoperoxidase-catalyzed radioiodination was used to incorporate 125I or 131I into membrane proteins of murine thymocytes, spleen cells or spleen cells from mice depleted of T cells by adult thymectomy, X-irradiation and reconstitution with T cell-depleted bone-marrow cells (ATXBM mice). Radiolabelled cells were lysed with 0.05% Triton X-100 and the lysates were fractionated by ultracentrifugation through sucrose gradients and/or chromatography in Sephadex or Sepharose gels. Membrane immunoglobulins in the fractionated lysate were detected with rabbit antiserum to murine Ig L-chains. Membrane Ig associated with thymocytes sedimented more slowly than serum IgG in sucrose gradients but had a molecular size of 180,000 or 360,000d based on column chromatography. Eighty % of M-Ig obtained from B cells of ATXBM mice sedimented in the 8–10s region of sucrose gradients and the sedimentation rate of normal spleen cell M-Ig was split into two populations sedimenting slower or faster than IgG. The results provide further support to the proposal that T cell M-Ig and B cell M-Ig are structurally distinct.

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.

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.

Analysis of normal and neoplastic lymphocyte surface-labeled proteins by two-dimensional polyacrylamide gel electrophoresis☆

Normal and neoplastic murine and human lymphocytes were surface-labeled by lactoperoxidase-catalyzed radioiodination, and the cell lysates were subjected to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analyses, combining isoelectric focusing in the first dimension and sodium dodecyl sulfate-PAGE (SDS-PAGE) in the second dimension. 2D-PAGE autoradiogram patterns were reproducible and reflected differences in cell types. A string of spots with a Mr of 100K was tentatively identified as a new T-cell marker (Tp100) which was present in all murine and human T cells examined including human T lymphomas. Murine and human B cells displayed markers characteristic to B cells of each species with some similarities between them. Human lymphomas and murine cell lines showed markers which were absent or only weakly visible in normal cells. Thus, 2D-PAGE analysis of lymphocyte surface proteins proved to be a method useful for searching for various markers.

Isolation of t cell membrane proteins (IgT) with antisera to non-isotypic determinants of immunoglobulins: Evidence thaT IgT ‘Light’ chains are not identical to B cell kappa chains☆

Abstract Radiolabelled cell membrane proteins obtained by detergent lysis of murine T cells labelled by lactoperoxidase-catalysed radioiodination were specifically immunoprecipitated with an antiserum to immunoglobulin Fab determinants. Absorption of the antiserum by myeloma proteins or normal immunoglobulins coupled to Sepharose and the use of antibody proteins eluted from these absorbents indicated that the antibody activity in the serum responsible for binding detergent-soluble T cell membrane proteins was directed towards variable region and not to the constant region determinants of kappa light chains. Immunoglobulin light chain sized polypeptides isolated from T cells with this antiserum were found to possess serological characteristics which differed from serum or B cell membrane immunoglobulin kappa chains. Evidence is presented which suggests that a portion of the light chain sized polypeptides isolated from T cells may be proteolytic fragments of a larger polypeptide chain. The results suggest that immunoglobulin-like detergent-soluble T cell membrane proteins isolated with antisera to immunoglobulins (IgT) possess neither immunoglobulin heavy nor light chain constant regions determinants but do carry determinants associated with immunoglobulin variable regions.