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    Davignon, D., Martz, E., Reynolds, T., Kürzinger, K. & Springer, T.A. Lymphocyte function-associated antigen 1 (LFA-1): A surface antigen distinct from Lyt-2,3 that participates in T lymphocyte-mediated killing. Proc Natl Acad Sci USA 78, 7, 4535-4539 (1981).Abstract
    Davignon_1981_2360.pdf

    Monoclonal antibodies (MAb) have been used to probe the relationship of cytolytic T lymphocyte (CTL) surface molecules to CTL function. Rat MAb to mouse CTL were generated. Twelve MAb so obtained gave preferential binding to T cells as compared to B cells, and three of these recognized previously undescribed surface polypeptides. These Mab and more broadly reactive and previously obtained MAb were tested for their ability to block CTL-mediated killing in the absence of complement. To ensure that any observed blocking was due to binding of MAb to the effector cell rather than the target cell, a xenogeneic mouse CTL anti-rat BN lymphoma target cell system was utilized (MAb and target cells both of rat origin). Of 24 MAb tested here, 21 had little or no effect on CTL function, including those to H-2, Thy-1, Lyt-1, Ly 5, Ly 6, Lgp 100, and at least six other defined antigens. We confirmed inhibition of killing with two MAb to Lyt-2,3. Another MAb, M7/14, gave profound and consistent blockade of CTL function. It was confirmed that M7/14 MAb blocks killing by binding to the mouse CTL and does not bind to the rat lymphoma target cells used for the CTL assay. The findings suggest that the antigen defined by M7/14, termed a lymphocyte function-associated antigen, LFA-1, participates in or is closely associated with the mechanism of CTL-mediated killing. LFA-1 contains two polypeptide chains of 180,000 and 95,000 Mr and is distinct from other described lymphocyte glycoproteins. LFA-1 thus represents both a previously undescribed lymphocyte surface antigen and molecular site for blockade of CTL-mediated killing.

    Springer, T.A. Quantitation of light chain synthesis in myeloma x spleen cell hybrids and identification of myeloma chain loss variants using radioimmunoassay. J. Immunol. Methods 37, 2, 139-152 (1980).Abstract
    Springer_1980_814.pdf

    A radioimmunoassay specific for the MOPC 21 kappa (K) myeloma chain of NSI and X63 myeloma x spleen cell hybrids was used to study light chain secretion in myeloma-hybrid lines. The M1 series of rat spleen cell x NSI mouse myeloma hybrid lines was chosen to illustrate the application of the radioimmunoassay for K chain quantitation and identification of K chain loss variants. Most of these lines secrete H (specific heavy), L (specific light), and K (myeloma kappa) chains, i.e., are HLK lines. Assays specific for rat L chain and mouse K chain showed that the ratio of L/K chain secreted by 6 different hybrid HLK lines ranged from 1.1 to 12.4. Using the rapid radioimmunoassay screening procedure, HL clonal variants which had lost K chain secretion were isolated at a frequency of approximately 10(-2) and characterized. K chain loss was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of radiolabelled secreted products. Stability of one HL line and its HLK parent was examined during 9 months of growth in vitro. The HL line remained stable, while antibody secreted by the HLK line became inactive, apparently due to overgrowth by clonally dominant HK cells which no longer secreted specific L chains. The radioimmunoassay appears to detect MOPC 21 kappa chain variable region determinants. Therefore, although used here with rat-mouse hybrids, it should also be possible to use the assay to obtain mouse-mouse variant hybrid lines secreting antibody of improved homogeneity.

    Stern, P.L., et al. Monoclonal antibodies as probes for differentiation and tumor-associated antigens: A Forssman specificity on teratocarcinoma stem cells. Cell 14, 4, 775-783 (1978).Abstract
    Stern_1978_815.pdf

    A set of monoclonal antibodies derived by fusing P3-NS1/1-Ag4-1 myeloma cells with spleen cells from a rat immunized with mouse spleen were screened for activity against a tumor cell panel. One of these antibodies was found to react only with mouse embryonal carcinoma cells and no other tumor cell type tested, including differentiated derivatives of teratocarcinomas. In the adult mouse, this antigen is expressed by subpopulations of cells in the spleen, bone marrow, lymph node, brain, kidney and testes, although not in liver and thymus. This antigen has a species and tissue distribution consistent with that of Forssman antigen. The molecules which carry this specificity on the embryonal carcinoma cells appear to be glycolipids.

    Springer, T.A., Galfre, G., Secher, D.S. & Milstein, C. Monoclonal xenogeneic antibodies to murine cell surface antigens: identification of novel leukocyte differentiation antigens. Eur. J. Immunol. 8, 8, 539-551 (1978).Abstract
    Springer_1978_534.pdf

    Hybrid myeloma cell lines secreting monoclonal antibodies to mouse cell surface antigens have been prepared. Spleen cells from a DA rat immunized with B10 mouse spleen cells that had been enriched for T cells were fused to cells from a nonsecreting mouse myeloma line (NSI). The presence in the culture supernatants of antibodies binding to mouse spleen cells was tested by a binding assay with 125I-labeled anti-rat IgG. From a large number of positive cultures, ten independent hybrid clones were purified, each secreting a different antibody. Each antigenic target was analyzed by (a) gel electrophoresis of immunoprecipitated 125 I-labeled cell surface molecules, (b) heat stability, (c) strain and species distribution and (d) cross-inhibition of binding of different monoclonal antibodies. It was concluded that the ten monoclonal antibodies regognized four types of antigen. One was the heterophile, heat-stable, Forssman antigen. The second (mol.wt. 210 000) appears to be a major 125I-labeled lymphoid cell surface protein. The third, a minor component of spleen cells, was precipitated as two polypeptides of mol.wt. 190 000 and 105 000. Five IgG-secreting clones identify the fourth antigen, a heat-stable, possibly glycolipid component expressed on mouse red blood cells and also on thymocytes. Cross-inhibition studies suggest that these last monoclonal antibodies bind to overlapping, but not identical, determinants. The class and chain composition of the monoclonal antibodies were studied by gel electrophoresis, isoelectric focusing and ability to lyse red blood cells and thymocytes.

    Springer, T.A., Kaufman, J.F., Siddoway, L.A., Mann, D.L. & Strominger, J.L. Purification of HLA-linked B lymphocyte alloantigens in immunologically active form by preparative sodium dodecyl sulfate-gel electrophoresis and studies on their subunit association. J. Biol. Chem. 252, 6201-6207 (1977).Abstract
    Springer_1977_767.pdf

    The HLA-linked B cell alloantigen (p29,34) is composed of two subunits of 29,000 (p29) and 34,000 (p34) molecular weight. The partially purified HLA-linked B cell alloantigen was purified by a final step of preparative sodium dodecyl sulfate-gel electrophoresis. An antiserum was prepared against p29,34 which specifically lysed B lymphocytes. In sodium dodecyl sulfate at 21 degrees, p29 and p34 remained noncovalently associated and retained immunologic activity; subunit dissociation at higher temperatures correlated with loss of immunologic activity. Although the pI values of p29 and p34 are 6.1 and 5.2, respectively, the subunits co-electrofocus under nondenaturing conditions. In addition, cross-linking studies showed the B cell antigen has a (p29)1(p34)1 subunit structure.

    Strominger, J.L., et al. Structure of HLA-A and HLA-B antigens isolated from cultured human lymphocytes. Cold Spring Harbor Symp. Quant. Biol. 41, 323-329 (1977).Abstract
    Strominger_1977_2439.pdf

    From these data, a model was prepared which summarizes schematically our present knowledge of the structure and orientation of the HL-A antigenic molecule in the lymphocyte membrane (Fig. 3). It seems likely that the heavy chain spans the membrane, with the hydrophobic region inserted in the membrane and the hydrophilic C-terminus inside the cell. This C-terminal region bears one (possible two) SH residue which has the potential for forming interchain disulfides. Whether or not these are actually formed physiologically remains an interesting question. There is the attractive possibility that whatever the physiological functions of HL-A antigens are, structurally these molecules provide the potential for signaling from outside the cell to inside the cell because they span the membrane. It is even conceivable that this function might be expressed via the opening and closing of disulfide bridges.

    Springer, T.A., Mann, D.L., DeFranco, A.L. & Strominger, J.L. Detergent solubilization, purification, and separation of specificities of HLA antigens from a cultured human lymphoblastoid line, RPMI 4265. J. Biol. Chem. 252, 13, 4682-4693 (1977).Abstract
    Springer_1977_724.pdf

    HLA antigens have been purified to homogeneity after detergent solubilization from RPMI 4265, a human lymphoblastoid line. The inhibition of cytotoxicity assay for HLA antigen was modified, using preincubation with bovine serum albumin of antigen samples containing detergent to prevent lysis of target cells by detergent. Solubilization was tested with many types of detergents. A polyethyleneglycol oleyl ether nonionic detergent mixture, Brij 99:Brij 97 (2:1) was selected for solubilization, since it selectively solubilized HLA antigens, had a low absorbance at 280 nm and was uncharded. HLA antigens were then purified by Lens culinaris lectin affinity chromatography and Bio-Gel A-5m filtration. The antigen specifity HLA-A2 was separated from specificities HLA-B7,12 by isoelectric focusing. Purified HLA antigens contained a subunit of Mr=44,000 with NH2-terminal glycine, and a subunit of Mr=12,000, beta2-microglobulin, with NH2-terminal isoleucine.

    Springer, T.A., Robb, R.J., Terhorst, C. & Strominger, J.L. Subunit and disulfide structure of monomeric and dimeric forms of detergent-soluble HLA antigens. J. Biol. Chem. 252, 4694-4700 (1977).Abstract
    Springer_1977_1055.pdf

    The structure of monomeric and disulfide-bonded dimeric forms of HLA antigens has been studied. Detergent-soluble HLA antigen heavy chains contain one or two easily reduced sulfhydryl groups not found in papain-solubilized HLA antigens, as demonstrated by amino acid analysis (Springer, T. A., and Strominger, J.L. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2481-2485, and Terhorst, C., Parham, P., Mann, D.L., and Strominger, J.L. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 910-914) and by labeling with iodo[3H]acetate. Dimer formation occurred during purification, since it was prevented by pretreatment of membranes containing HLA antigen with iodoacetamide. Cross-linking studies showed that the non-disulfide-bonded form of HLA antigens contains one subunit each of the Mr = 44,000 heavy chain and the Mr = 12,000 light chain (beta2-microglobulin).

    Springer, T.A., Kaufman, J.F., Terhorst, C. & Strominger, J.L. Purification and structural characterisation of human HLA-linked B-cell antigens. Nature 268, 5617, 213-218 (1977).Abstract
    Springer_1977_1054.pdf

    The human B cell-specific alloantigen which is closely linked genetically to HLA contains two non-covalently associated, sialogycoprotein subunits of molecular weight (MW) 29,000 (p29), and 34,000 (p34). Although p29 and p34 have different amino-terminal sequences, their tyrosine peptide maps indicate considerable similarity in other portions of their polypeptide chains. Thus the genes for their proteins may have evolved by duplication of a common ancestral gene. Another lymphocyte cell surface protein of MW 16,000 (p16) has also been characterised. Both p16 and p44 (the heavy chain of HLA-A,B antigens) have been compared with p29 and p34.

    Springer, T.A. & Strominger, J.L. Detergent-soluble HLA antigens contain a hydrophilic region at the COOH-terminus and a penultimate hydrophobic region. Proc. Natl. Acad. Sci. U. S. A. 73, 2481-2485 (1976).Abstract
    Springer_1976_357.pdf

    Purified, detergent-soluble HLA antigens (p44,12) are composed of a glycoprotein of molecular weight 44,000 (p44) and a peptide of molecular weight 12,000 (p12), beta2-microglobulin. Upon digestion with papain, p44,12 is converted to p39,12, then to p34,12, which retains antigenic activity. The NH2-terminal amino acid sequences of p34 and p44 are identical. p44, p39, and p34 were purified, and comparison of their amino acid compositions showed that the COOH-terminal peptide removed by the first papain cleavage is hydrophilic and contains cysteine that can be alkylated after mild reduction. The penultimate COOH-terminal peptide removed by the second papain cleavage is hydrophobic, and presumably anchors HLA antigens to the membrane. This correlates with the observation that p44,12 and p39,12 bind detergent, while p34,12 does not. The orientation and integration of HLA antigensin the lymphocyte membrane were thus defined, and the structure suggests that HLA antigens span the plasma membrane.

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