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Rat lymphocytes stimulated with insoluble concanavalin-A release mediators which render mouse macrophages nonspecifically tumoricidal
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phokines. (3) The kinetics of PA production reaches a maximum after 48-72 hr. The results show that PA production by macrophages can be used as a parameter of immune activation. PA production by activated macrophages also opens hitherto unknolvn pathways from lymphokines to other humoral effector systems, such as the complement system. Rat Lymphocytes Stinmlatcd zwith Insoluble Concamwa/irz-A Rclcase Xediators zwhich Rerkdcr illolrse dfacroplragrs Il’o~zspecijically Tzc+noricidal. ISAIAH J. FIDLER ATD JOHN H. DARNELL, Basic Research Program, NC1 Frederick Cancer Research Center, Frederick, Maryland. Lymphocytes from normal Fischer rats were stimulated ilk vitro with insoluble concanavalin A (Con A). Lymphocyte culture supernatants were assayed for macrophage-activating factors (MAF) by incubation with normal C57BL/6 mouse macrophages. Macrophages were then Optimal tested for their cytotoxicity in oitro to mouse target cells prelabeled with ‘YUDR. MAF levels were achieved when lymphocytes were incubated for 48 hr with Sepharose-bound Con A. A brief (2 hr) exposure to MAF was sufficient to initiate activation, but cytotoxicity was not fully expressed unless tumor cells were added 46 hr later. A distinctive property of the MAF activity described here was its resistance to temperatures reaching 100°C. Control experiments established that a true lymphocyte product (lymphokine), not residual, unbound Con A, was responsible for the induction of macrophage cytotoxicity. The spectrum of cytotoxicity exhibited by macrophages activated with MAF from Con A-stimulated normal rat lymphocyte was similar to that observed with specific antigen-induced M.4F from presensitized rat lymphocytes. Rat Con A X4F rendered normal mouse macrophages cpto:oxic to several syngeneic and allogeneic tumor cell lines ; normal syngeneic or allogeneic cells xvere not harmed even in the presence of susceptible tumor cells. Apparently, this discrimination is independent of tumor or transplantation antigens. Mzkma+t Lefrkocyte Migratiokk Inhibition Factor (LIF) Activity ix Culture Supmkatakkts from Mixed Lymphocyte Tkruzor Cell Extract Interactions (MLTI). J. H. DEAN, A. KIBKITE, J. L. MCCOY, G. B. CANNON, and R. B. HERBERMAN, Department of Immunology, Litton Bionetics, Inc., Kensington, Maryland and Laboratory of Immunodiagnosis, National Cancer Institute, Bethesda, Maryland. Lymphocytes from lung and breast carcinoma patients were incubated with autologous tumor cell extracts, recall antigen, and mixed leukocyte cultures (MLC), and lymphocyte proliferation (LP) and LIF production in culture fluids (CF) were measured. Studies indicated optimal conditions for LIF generation were 2 X 10’ Ficoll-Hypaque mononuclear cells per culture, overnight triggerin g with antigen followed by a 3-day incubation without antigen, and assay of Cl? on purified polymorphonuclear indicator cells in an indirect agarose microdroplet assay after overnight incubation. Approximately 52% of lung carcinoma and 41% of breast carcinoma patients gave positive LP responses in MLTI to autologous tumor extracts, but not to normal tissue extracts. LIF activity was detected in CF at dilutions of up to 1: 10 of most patients demonstrating positive t3H]TdR responses in autologous MLTI, to recall antigen and in MLC, but some discordance between LIF generation and positive [“H]TdR responses was observed. Experiments are in progress to characterize the lymphocyte population (s) responding in MLTI with LP and I-IF production. 11%Vitro
and k Viva Ezalkkation of Macrophage Fzljlction in Man. E. M. HERSH, B. PAPERC. GSCHWIND, A. RIOS, G. M. MAVLIGIT, AND J. U. GUTTERXAN, M. 1). Anderson Hospital, Houston, Texas. MASTER,
Normal human macrophage (M) function was evaluated ilz vitro by measurement of [“Clglucosamine and [3H]leucine incorporation before and after addition of control or concanavalin A induced M activating factor (MAF) and by inhibition of [‘Hlleucineor [“Hlthymidinelabeled target cell lines (HeLa, lymphoblastoid, lymphoma, and melanoma). Iti z&o, M function was evaluated by the induration induced by intradermal injection of a dose range of lymphokine extracted from the supernatant of the 1788 lymphoblastoid cell line. Twenty-two . . . normal subjects were studled tfk vztro and 20 cancer patlcnts were studled irk vizto. Protein synthesis increased l.lO- to S-fold with both MAF preparations. Both increased glucosamine
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phokines. (3) The kinetics of PA production reaches a maximum after 48-72 hr. The results show that PA production by macrophages can be used as a parameter of immune activation. PA production by activated macrophages also opens hitherto unknolvn pathways from lymphokines to other humoral effector systems, such as the complement system. Rat Lymphocytes Stinmlatcd zwith Insoluble Concamwa/irz-A Rclcase Xediators zwhich Rerkdcr illolrse dfacroplragrs Il’o~zspecijically Tzc+noricidal. ISAIAH J. FIDLER ATD JOHN H. DARNELL, Basic Research Program, NC1 Frederick Cancer Research Center, Frederick, Maryland. Lymphocytes from normal Fischer rats were stimulated ilk vitro with insoluble concanavalin A (Con A). Lymphocyte culture supernatants were assayed for macrophage-activating factors (MAF) by incubation with normal C57BL/6 mouse macrophages. Macrophages were then Optimal tested for their cytotoxicity in oitro to mouse target cells prelabeled with ‘YUDR. MAF levels were achieved when lymphocytes were incubated for 48 hr with Sepharose-bound Con A. A brief (2 hr) exposure to MAF was sufficient to initiate activation, but cytotoxicity was not fully expressed unless tumor cells were added 46 hr later. A distinctive property of the MAF activity described here was its resistance to temperatures reaching 100°C. Control experiments established that a true lymphocyte product (lymphokine), not residual, unbound Con A, was responsible for the induction of macrophage cytotoxicity. The spectrum of cytotoxicity exhibited by macrophages activated with MAF from Con A-stimulated normal rat lymphocyte was similar to that observed with specific antigen-induced M.4F from presensitized rat lymphocytes. Rat Con A X4F rendered normal mouse macrophages cpto:oxic to several syngeneic and allogeneic tumor cell lines ; normal syngeneic or allogeneic cells xvere not harmed even in the presence of susceptible tumor cells. Apparently, this discrimination is independent of tumor or transplantation antigens. Mzkma+t Lefrkocyte Migratiokk Inhibition Factor (LIF) Activity ix Culture Supmkatakkts from Mixed Lymphocyte Tkruzor Cell Extract Interactions (MLTI). J. H. DEAN, A. KIBKITE, J. L. MCCOY, G. B. CANNON, and R. B. HERBERMAN, Department of Immunology, Litton Bionetics, Inc., Kensington, Maryland and Laboratory of Immunodiagnosis, National Cancer Institute, Bethesda, Maryland. Lymphocytes from lung and breast carcinoma patients were incubated with autologous tumor cell extracts, recall antigen, and mixed leukocyte cultures (MLC), and lymphocyte proliferation (LP) and LIF production in culture fluids (CF) were measured. Studies indicated optimal conditions for LIF generation were 2 X 10’ Ficoll-Hypaque mononuclear cells per culture, overnight triggerin g with antigen followed by a 3-day incubation without antigen, and assay of Cl? on purified polymorphonuclear indicator cells in an indirect agarose microdroplet assay after overnight incubation. Approximately 52% of lung carcinoma and 41% of breast carcinoma patients gave positive LP responses in MLTI to autologous tumor extracts, but not to normal tissue extracts. LIF activity was detected in CF at dilutions of up to 1: 10 of most patients demonstrating positive t3H]TdR responses in autologous MLTI, to recall antigen and in MLC, but some discordance between LIF generation and positive [“H]TdR responses was observed. Experiments are in progress to characterize the lymphocyte population (s) responding in MLTI with LP and I-IF production. 11%Vitro
and k Viva Ezalkkation of Macrophage Fzljlction in Man. E. M. HERSH, B. PAPERC. GSCHWIND, A. RIOS, G. M. MAVLIGIT, AND J. U. GUTTERXAN, M. 1). Anderson Hospital, Houston, Texas. MASTER,
Normal human macrophage (M) function was evaluated ilz vitro by measurement of [“Clglucosamine and [3H]leucine incorporation before and after addition of control or concanavalin A induced M activating factor (MAF) and by inhibition of [‘Hlleucineor [“Hlthymidinelabeled target cell lines (HeLa, lymphoblastoid, lymphoma, and melanoma). Iti z&o, M function was evaluated by the induration induced by intradermal injection of a dose range of lymphokine extracted from the supernatant of the 1788 lymphoblastoid cell line. Twenty-two . . . normal subjects were studled tfk vztro and 20 cancer patlcnts were studled irk vizto. Protein synthesis increased l.lO- to S-fold with both MAF preparations. Both increased glucosamine