Immunology 1979 38 757
Induction of cell-mediated cytotoxicity by lipoprotein containing histocompatibility antigens
G. DENNERT Department ofCancer Biology, The Salk Institutefor Biological Studies, San Diego, Ca, U.S.A.
Acceptedfor publication 12 July 1979
surface (Lesley, Hyman & Dennert, 1974). Direct biochemical evidence, however, demonstrating that purified histocompatibility antigens bind to T killer cells is still lacking. While T-cell killing can be specifically inhibited by competing cold target cells carrying the relevant H-2 antigens (Ortiz de Landazuri & Herberman, 1972), specific inhibition by subcellular fractions has been a controversial issue. Thus while in some laboratories no inhibition of cell-mediated cytotoxicity by subcellular fractions of target cells was observed (Brunner, Mauel, Cerottini & Chapuis, 1968; Brunner, Nordin & Cerottini, 1971), others reported specific inhibition by such preparations (Bonavida, 1974; Wagner & Boyle, 1972; Wekerle & Feldman, 1974). In a recent communication Linna, Engers, Cerottini & Brunner (1978) reported that subcellular fractions may inhibit cell mediated cytotoxicity non-specifically or specifically, depending on the preparation. Others, however, could only observe non-specific inhibition by preparations containing serologically detectable H-2 antigen (Plata & Levy, 1974; Sendo, Aoki & Buafo, 1974; Todd, Stulting & Amos, 1975). In another approach to this problem it was recently reported (Nabholz et al., 1974) that H-2K and I region gene products may absorb to lymphocytes activated in a mixed lymphocyte reaction (Elliott, Nagy, Nabholz & Pernis, 1977; Nagy, Elliott & Nabholz, 1976a; Nagy, Elliott, Nabholz, Krammer & Pernis, 1976b). Yet while the binding of such solubilized antigenic material appeared to be specific (Elliott et al., 1977; Nagy et al., 1976a, b) and also capable of
Summary. Lipoprotein was isolated from tumour cells by sonication and ultracentrifugal flotation on KBr gradients. It contained H-2 antigen detectable by antibody binding and induced a primary or secondary cell-mediated cytotoxic response in vitro which was H-2 specific. In a syngeneic model only a secondary cell-mediated response was stimulated and no competitive inhibition of the effector step of cellmediated lysis could be demonstrated. The implications of these findings are discussed.
INTRODUCTION
Cytotoxic thymus derived lymphocytes (T-killer cells) interact with the major histocompatibility antigens on the target cell during the lytic reaction. This has been shown by genetic experiments making use of H-2 recombinant mouse strains (Alter, Schendel, Bach, Bach, Klein & Stimpfling, 1973; Nabholz, Vives, Young, Meo, Miggiano, Rijnbeek & Shreffler, 1974) and by employing targets either lacking H-2 histocompatibility antigens (Dennert & Hyman, 1977) or displaying various amounts of these antigens on their cell Abbreviations: BSS, Hanks's balanced salt solution; a/t, attacker to target cell ratio; FCS, foetal calf serum. Correspondence: Dr G. Dennert, Department of Cancer Biology, The Salk Institute for Biological Studies, Post Office Box 1809, San Diego, Ca 92112, U.S.A. 0019-2805/79/1200-0757 S02.00
© 1979 Blackwell Scientific Publications 757
758
G. Dennert
inhibiting killer cell-target cell binding specifically (Gilmer, McDevitt & McConnell, 1978; Whisnant, Singer & Amos, 1978), no inhibition of cytotoxicity could be demonstrated. Similarly unsatisfying are experiments in which it was tried to induce cellmediated cytotoxicity with subcellular preparations of target cells in vitro: while a secondary cell-mediated response can be readily stimulated by such preparations (Wagner & Boyle, 1972; Engers, Thomas, Cerottini & Brunner, 1975; Corley, Dawson & Amos, 1975; Manson & Palmer, 1975; Lemonnier, Mescher, Sherman & Burakoff, 1978; Mescher, Sherman, Lemonnier & Burakoff, 1978; Fast & Fan, 1978; Engelhard, Strominger, Mescher & Burakoff, 1978; Finberg, Mescher & Burakoff, 1978; Alaba & Law, 1978), they fail to induce a primary response for unknown reasons. Because of the importance of demonstrating specific binding of histocompatibility antigens to T cells, we studied a recently developed procedure making use of ultracentrifugal flotation of lipoproteins containing histocompatibility antigens (Allison, Pellegrino, Ferrone, Callahan & Reisfeld, 1977) to see if it was possible to enrich cell surface antigens able to interact with the receptors of T killer cells. Experiments reported in this communication show that lipoproteins which contain H-2 antigen are able to stimulate allogeneic killer cells in a primary response and syngeneic killer cells in a secondary response. MATERIALS AND METHODS Animals C57B1/6 (H-2b) and BALB/c (H-2d) mice were obtained from Frederick Cancer Research Center (Md). Cell lines The following cell lines were grown in Dulbecco's modified Eagle's minimal essential medium containing 10% heat-inactivated horse serum. The BALB/c myeloma S194 (H-2d), the BALB/c thymic lymphoma S49 (H-2d), the C58 thymic lymphoma RI(TL+) (H-2k), the C57BI/6 thymic lymphoma E14 (H-2b), and the DBA/2 mastocytoma P815 (H-2d). P815 was also grown in DBA/2 mice as ascites.
Induction and assay of cytotoxic T cells For the induction of cell-mediated cytotoxicity, spleen cells (107/ml) were cultured with either irradiated spleen (1000 rads), tumour cells (10,000 rads) or lipo-
somes in 3 cm tissue culture dishes in RPMI 1640 medium containing 5% foetal calf serum (FCS) and 5 x 10-5 M mercaptoethanol (Dennert & Hyman, 1977). After five days the cytotoxic activity was assayed on 5'Cr-labelled target cells in RPMI 1640 medium containing 10% FCS as described previously (Dennert & Hyman, 1977). The following equation was used to express cytotoxicity: Experimental 51Cr release spontaneous 5'Cr release cytotoxicity = 100 Maximal 5'Cr releasespontaneous 5'Cr release Maximal release was determined by freezing and thawing the target cells three times. The mean values of duplicate samples, as well as the standard errors of the means (SEM), were calculated.
0%
Serological assays To determine the antibody binding activity of the lipoprotein fractions, 5'Cr-labelled target cells, diluted anti-target cell antibody, rabbit complement and serial dilutions of lipoprotein were mixed and incubated for 45 min at 370. After centrifugation radioactivity in the supernatant was determined. H-2d and H-2b specific antisera were kindly provided by Dr R. Hyman (Salk Institute). Rabbit complement was from a rabbit selected for low toxicity to mouse cells and was used at a final dilution of 1: 20. Protein determinations were done using the Lowry method. Preparation of H-2 containing lipoprotein fractions from tumour cells S 194 (H-2d) or P815 (H-2d) cells were suspended in five volumes of Hanks's balanced salt solution (BSS) and sonicated in ice for 10 min. To the sonicate KBr was added to a density of 1-2 g/ml, followed by centrifugation for 48 h at 150,000 g (Allison et al., 1977). Lipoprotein on top of the gradient was removed and suspended in BSS by sonication. Lipoprotein preparations were assayed for their protein content and antibody binding activity. Usually a five- to ten-fold enrichment of H-2 antigens in the lipoprotein fraction on top of the KBr gradient was obtained. Antibody binding assays revealed that presumably due to the intrinsic stickiness of such preparations to cell surfaces (Berke, 1977) there was only a relatively narrow range in which specific inhibition of antibody-dependent complement lysis of the specific target was observed (Table 1).
759
Induction of cell-mediated cytotoxicity Table 1. Specific inhibition of antibodydependent complement lysis of S194 targets by a lipoprotein preparation from P815 Live cells (%)t
Lipoprotein*
(Pg/ml) 16 32 64 160 320 800 1600
S194 (H-2d) E14 (H-2b)
Induction of cell-mediated cytotoxicity by lipoprotein containing histocompatibility antigens
G. DENNERT Department ofCancer Biology, The Salk Institutefor Biological Studies, San Diego, Ca, U.S.A.
Acceptedfor publication 12 July 1979
surface (Lesley, Hyman & Dennert, 1974). Direct biochemical evidence, however, demonstrating that purified histocompatibility antigens bind to T killer cells is still lacking. While T-cell killing can be specifically inhibited by competing cold target cells carrying the relevant H-2 antigens (Ortiz de Landazuri & Herberman, 1972), specific inhibition by subcellular fractions has been a controversial issue. Thus while in some laboratories no inhibition of cell-mediated cytotoxicity by subcellular fractions of target cells was observed (Brunner, Mauel, Cerottini & Chapuis, 1968; Brunner, Nordin & Cerottini, 1971), others reported specific inhibition by such preparations (Bonavida, 1974; Wagner & Boyle, 1972; Wekerle & Feldman, 1974). In a recent communication Linna, Engers, Cerottini & Brunner (1978) reported that subcellular fractions may inhibit cell mediated cytotoxicity non-specifically or specifically, depending on the preparation. Others, however, could only observe non-specific inhibition by preparations containing serologically detectable H-2 antigen (Plata & Levy, 1974; Sendo, Aoki & Buafo, 1974; Todd, Stulting & Amos, 1975). In another approach to this problem it was recently reported (Nabholz et al., 1974) that H-2K and I region gene products may absorb to lymphocytes activated in a mixed lymphocyte reaction (Elliott, Nagy, Nabholz & Pernis, 1977; Nagy, Elliott & Nabholz, 1976a; Nagy, Elliott, Nabholz, Krammer & Pernis, 1976b). Yet while the binding of such solubilized antigenic material appeared to be specific (Elliott et al., 1977; Nagy et al., 1976a, b) and also capable of
Summary. Lipoprotein was isolated from tumour cells by sonication and ultracentrifugal flotation on KBr gradients. It contained H-2 antigen detectable by antibody binding and induced a primary or secondary cell-mediated cytotoxic response in vitro which was H-2 specific. In a syngeneic model only a secondary cell-mediated response was stimulated and no competitive inhibition of the effector step of cellmediated lysis could be demonstrated. The implications of these findings are discussed.
INTRODUCTION
Cytotoxic thymus derived lymphocytes (T-killer cells) interact with the major histocompatibility antigens on the target cell during the lytic reaction. This has been shown by genetic experiments making use of H-2 recombinant mouse strains (Alter, Schendel, Bach, Bach, Klein & Stimpfling, 1973; Nabholz, Vives, Young, Meo, Miggiano, Rijnbeek & Shreffler, 1974) and by employing targets either lacking H-2 histocompatibility antigens (Dennert & Hyman, 1977) or displaying various amounts of these antigens on their cell Abbreviations: BSS, Hanks's balanced salt solution; a/t, attacker to target cell ratio; FCS, foetal calf serum. Correspondence: Dr G. Dennert, Department of Cancer Biology, The Salk Institute for Biological Studies, Post Office Box 1809, San Diego, Ca 92112, U.S.A. 0019-2805/79/1200-0757 S02.00
© 1979 Blackwell Scientific Publications 757
758
G. Dennert
inhibiting killer cell-target cell binding specifically (Gilmer, McDevitt & McConnell, 1978; Whisnant, Singer & Amos, 1978), no inhibition of cytotoxicity could be demonstrated. Similarly unsatisfying are experiments in which it was tried to induce cellmediated cytotoxicity with subcellular preparations of target cells in vitro: while a secondary cell-mediated response can be readily stimulated by such preparations (Wagner & Boyle, 1972; Engers, Thomas, Cerottini & Brunner, 1975; Corley, Dawson & Amos, 1975; Manson & Palmer, 1975; Lemonnier, Mescher, Sherman & Burakoff, 1978; Mescher, Sherman, Lemonnier & Burakoff, 1978; Fast & Fan, 1978; Engelhard, Strominger, Mescher & Burakoff, 1978; Finberg, Mescher & Burakoff, 1978; Alaba & Law, 1978), they fail to induce a primary response for unknown reasons. Because of the importance of demonstrating specific binding of histocompatibility antigens to T cells, we studied a recently developed procedure making use of ultracentrifugal flotation of lipoproteins containing histocompatibility antigens (Allison, Pellegrino, Ferrone, Callahan & Reisfeld, 1977) to see if it was possible to enrich cell surface antigens able to interact with the receptors of T killer cells. Experiments reported in this communication show that lipoproteins which contain H-2 antigen are able to stimulate allogeneic killer cells in a primary response and syngeneic killer cells in a secondary response. MATERIALS AND METHODS Animals C57B1/6 (H-2b) and BALB/c (H-2d) mice were obtained from Frederick Cancer Research Center (Md). Cell lines The following cell lines were grown in Dulbecco's modified Eagle's minimal essential medium containing 10% heat-inactivated horse serum. The BALB/c myeloma S194 (H-2d), the BALB/c thymic lymphoma S49 (H-2d), the C58 thymic lymphoma RI(TL+) (H-2k), the C57BI/6 thymic lymphoma E14 (H-2b), and the DBA/2 mastocytoma P815 (H-2d). P815 was also grown in DBA/2 mice as ascites.
Induction and assay of cytotoxic T cells For the induction of cell-mediated cytotoxicity, spleen cells (107/ml) were cultured with either irradiated spleen (1000 rads), tumour cells (10,000 rads) or lipo-
somes in 3 cm tissue culture dishes in RPMI 1640 medium containing 5% foetal calf serum (FCS) and 5 x 10-5 M mercaptoethanol (Dennert & Hyman, 1977). After five days the cytotoxic activity was assayed on 5'Cr-labelled target cells in RPMI 1640 medium containing 10% FCS as described previously (Dennert & Hyman, 1977). The following equation was used to express cytotoxicity: Experimental 51Cr release spontaneous 5'Cr release cytotoxicity = 100 Maximal 5'Cr releasespontaneous 5'Cr release Maximal release was determined by freezing and thawing the target cells three times. The mean values of duplicate samples, as well as the standard errors of the means (SEM), were calculated.
0%
Serological assays To determine the antibody binding activity of the lipoprotein fractions, 5'Cr-labelled target cells, diluted anti-target cell antibody, rabbit complement and serial dilutions of lipoprotein were mixed and incubated for 45 min at 370. After centrifugation radioactivity in the supernatant was determined. H-2d and H-2b specific antisera were kindly provided by Dr R. Hyman (Salk Institute). Rabbit complement was from a rabbit selected for low toxicity to mouse cells and was used at a final dilution of 1: 20. Protein determinations were done using the Lowry method. Preparation of H-2 containing lipoprotein fractions from tumour cells S 194 (H-2d) or P815 (H-2d) cells were suspended in five volumes of Hanks's balanced salt solution (BSS) and sonicated in ice for 10 min. To the sonicate KBr was added to a density of 1-2 g/ml, followed by centrifugation for 48 h at 150,000 g (Allison et al., 1977). Lipoprotein on top of the gradient was removed and suspended in BSS by sonication. Lipoprotein preparations were assayed for their protein content and antibody binding activity. Usually a five- to ten-fold enrichment of H-2 antigens in the lipoprotein fraction on top of the KBr gradient was obtained. Antibody binding assays revealed that presumably due to the intrinsic stickiness of such preparations to cell surfaces (Berke, 1977) there was only a relatively narrow range in which specific inhibition of antibody-dependent complement lysis of the specific target was observed (Table 1).
759
Induction of cell-mediated cytotoxicity Table 1. Specific inhibition of antibodydependent complement lysis of S194 targets by a lipoprotein preparation from P815 Live cells (%)t
Lipoprotein*
(Pg/ml) 16 32 64 160 320 800 1600
S194 (H-2d) E14 (H-2b)
