Znt. J. Cancer: 20, 381-387 (1977)
NATURAL CELL-MEDIATED CYTOTOXICITY IN MICE AGAINST NON-LYMPHOID TUMOR CELLS A N D SOME NORMAL CELLS M. E. NU", R. B. HERBERMAN, and H. T. HOLDEN Laboratory of Immunodiagnosis, National Cancer Institute, Bethesda, Md. 20014, USA.
Lymphocytes from normal mice were found t o have cell-mediated cytotoxicity, in a short-term 5'Cr release assay, against a variety of non-lymphoid tumor cells as well as against lymphomas. Some of the non-lymphoid tumors were as susceptible to natural cytotoxicity as the standardly used lymphoid lines. Some tissue culture cell lines and in vivo passaged tumor lines were susceptible to lysis, as were some primary virus-induced lymphomas. Tumors which arose in nude mice, which have high levels of natural cytotoxic activity, were all resistant to lysis. In addition t o the susceptibility of transformed cells t o natural cell-mediated cytotoxicity, some untranrformed cultured cells and cells from normal tissues were targets for this mechanism. Low levels of cytotoxicity were seen with normal thymus cells, bone-marrow cells, and short term cultures of macrophages, whereas normal spleen and lymph-node cells were completely resistant to lysis. These results indicate a broader spectrum for mouse natural cell-mediated cytotoxicity reactivity than has been previously recognized.
In the past few years, many investigators have observed natural cell-mediated cytotoxicity (designated NK for " natural killing ") against a variety of tumor cells. NK reactivity has been detected in mice (Herberman et a/., 1973, 1975; Kiessling et al., 1975), in rats (Holterman et a/., 1973; Nunn e f a/., 1976; Shellam and Hogg, 1977), and in humans (Takasugi, 1973; Oldham et al., 1973, 1975; Rosenberg et al., 1974). In studies on human NK, there have been a number of reports of reactivity in long-term cytotoxicity assays against tissue cultures of carcinomas, melanomas and non-lymphoid tumors (Takasugi et al., 1973; Oldham et a/., 1975; Pavie-Fisher et al., 1975) as well as reactivity in short-term assays against lymphoid and myeloid targets (Rosenberg e t a / . , 1974; West et al., 1977). In contmst, virtually all of the reported studies on NK reactivity in mice have been performed with lymphoid cell lines. The assumption has been made that only these lines were susceptible to lysis by the NK cells. Kiessling et a/. (1975) noted that NK reactivity could be seen against Moloney leukemia virus (MLV)induced lymphoma cells and they concluded that susceptibility to lysis was restricted to lymphomas induced by the MLV virus. Sendo and Aoki (1975) reported NK activity against a radiation-induced
leukemia in BALB/c mice and concluded.that NK activity was directed against X. 1' antigen associated with the expression of an endogenous type-C virus. Zarling et al. (1975) utilized a tumor induced by Gross leukemia virus and felt that NK reactivity was directed against Gross leukemia virus-associated antigens or embryonic antigens. Using these and other lymphoid cell lines as target cells and performing competitive inhibition assays, Herberman et al. (1975) concluded that NK reactivity was directed against several antigens associated with murine endogenous type-C viruses. To determine whether murine NK is analogous to human NK, we have examined a variety of' nonlymphoid cell lines for their susceptibility to NK. In our previous inhibition studies (Herberman et al., 1975), some non-lymphoid as well as lymphoid tumor cells gave positive results. This finding indicated that mouse NK might not be restricted to lymphoid target cells. Indeed, our results here indicate that many non-lymphoid tumor cells, harvested directly from in vivo tumors or from tissue culture cell lines, are susceptible to NK activity in a 4-h 5'Cr-release assay. In addition, we have found that some normal lymphoid and non-lymphoid cells are also susceptible to natural cytotoxicity.
MATERIAL AND METHODS
Mice C57BL/6N, BALB/c, CBA/N, NIH Swiss and NIH nude mice were obtained from the Rodent and Rabbit Production Section, Division of Research Services, National Institutes of Health (NIH), USA. BALB/c nude mice were obtained from the Mammalian Genetics Section, NCI, USA. AKR, NZB and CBA/J were obtained from Jackson Laboratories (Bar Harbor, Maine, USA).
Most of the mice studied were male, but sex did not appear to influence the reactivity of the NK cells. Unless otherwise stated, mice were tested for reactivity when 5-8 weeks of age. Mice more than 12 weeks old were injected with LCMV intra-
Received: June 20, 1977.
382
NUNN ET AL.
peritoneally and tested after 3 days (Herberman et al., 1977). Tumors Most of the tumor cells and the in vitro cultures derived from the tumors have been previously described (Herberman el ul., 1974). RLOl (Sendo and Aoki, 1975), a radiation-induced leukemia in BALB/c mice, was maintained as an ascites tumor by intraperitoneal passage. RBL-5 (McCoy et al., 1967), a Rauscher virus-induced leukemia in C57BL/6, was maintained as an ascites tumor by intraperitoneal passage. Sarcomas in BALB/c and nude mice (Outzen el a/., 1973, induced by methylcholanthrene (MCA), were kindly sent to us by Dr. Henry C. Outzen (Institute for Cancer Research, Fox Chase, Pa.). MCA tumors induced in CBA/H and CBA/H nude mice (Stutman, 1974) were kindly provided by Dr. Osias Stutman (Sloan-Kettering Institute for Cancer Research, New York, N.Y.). Mice bearing primary AKR spontaneous lymphomas were obtained from the Mammalian Genetics Section, NCI, USA. The B16 melanoma (Giovanella et a/., 1974) was obtained from Dr. Robert C. Ting and maintained as a solid tumor in C57BL/6 mice by subcutaneous injections. The 3LL Lewis lung carcinoma (Rotter and Trainin, 1975) was maintained in C57BL/6 mice by subcutaneous injections. The L5MF-22 lymphoma (Bonmassar et al., 1973), kindly provided by Dr. David Houchens, NCT, USA, were maintained in C57BL/6 and BlOA mice, respectively, by intraperitoneal passage. Tissue culture cells RLOl and RBL-5 were maintained in suspension cultures as previously described (Sendo and Aoki, 1975; Holden et al., 1977) and they are designated with the suffix TC. The other cell lines studied were maintained as monolayer cultures. All cell lines were grown in RPMI 1640 medium containing 20% fetal calf serum (Grand Island Biological Co., Grand Island, N.Y., USA). The embryo fibroblasts 3T12 and 3T3 type I CI 6, KA31, MSV-transformed non-producer line (Aoki et al., 1975; Ting and Herberman, 1971), were kindly provided by Drs. G. Todaro and C. C. Ting, NCI, USA. The E4, SV40-transformed 3T3 (Gillette and Fox, 1975), was provided by Dr. C. Boone, NCI, USA. The cell lines T-AI/N (fibroblasts, spontaneously transformed) and SV40-transformed SV-AL/N (Smith and Mora, 1972) were provided by Dr. Chungming Chang. The L8a (Parks and Scolnick, 1973) was derived from (C57BL/6 x A)F, mice and was provided by Dr. Wade Parks, NCI, USA. The TA3 (Sanford, 1967) was derived from an A strain mammary tumor and was obtained from Dr. C. C. Ting, NCI, USA.
Preparation of effector cell suspensions Effector cells from various lymphoid organs were prepared as previously described (Ortiz de Landazuri and Herberman, 1972). Preparation of target cells Target cells . obtained from monolayer tissue cultures were harvested with 0.25 % trypsin, washed with BSS (balanced salt solution) and prepared at the appropriate concentration (106/ml) in RPMI 1640 medium with 20% fetal calf serum for use in a 4-h Wr-release assay. In some experiments, after trypsinization of monolayer cells, they were maintained as single-cell suspensions in spinner cultures overnight before use as targets. Both techniques used gave similar results of NK reactivity and therefore we do not specify method of preparation in the " Results " section. Solid tumors of animals were placed in BSS and teased to yield suspensions of cells, which were washed with BSS. The peritoneal cavity of normal mice was washed with BSS and the cells collected, washed and put in Petri dishes in RPMI medium with 20% fetal calf serum for macrophage cultures. After overnight culture, the macrophages were scraped from the Petri dishes with a rubber policemen. Cells were washed and prepared for use as above. All target cells were labelled with "Cr as previously described (Nunn et al., 1976). Assay for cell-mediated cytotoxicity The assays were performed as previously described (Herberman et al., 1973, 1976~).Lymphoid cells, usually spleen cells, were incubated at a 200:l ratio with 51Cr-labelled target cells for 4 h at 37" C with rocking. The baseline 51Cr-release was determined with the use of unlabelled autologous target cells in place of effector cells. This neutral baseline was selected so that the same total number of cells would be present in each group and the activity of normal lymphoid cells could be detected (Nunn et al., 1976; Herberman et al., 19766). Most of the tests were performed with R L d l TC as a positive control for the other cell lines. To reduce variability in results among assays the RLOl TC were cryopreserved in multiple aliquots, according to procedures previously described (Holden et d., 1976). The baseline lysis of these cryopreserved target cells was 12-20% of the 61Cr incorporated into the cells. The baseline lysis of all other target cells was 5-15%. Levels of cytotoxicity 2 % or more above the baseline were invariably significantly different from the controls (p
NATURAL CELL-MEDIATED CYTOTOXICITY IN MICE AGAINST NON-LYMPHOID TUMOR CELLS A N D SOME NORMAL CELLS M. E. NU", R. B. HERBERMAN, and H. T. HOLDEN Laboratory of Immunodiagnosis, National Cancer Institute, Bethesda, Md. 20014, USA.
Lymphocytes from normal mice were found t o have cell-mediated cytotoxicity, in a short-term 5'Cr release assay, against a variety of non-lymphoid tumor cells as well as against lymphomas. Some of the non-lymphoid tumors were as susceptible to natural cytotoxicity as the standardly used lymphoid lines. Some tissue culture cell lines and in vivo passaged tumor lines were susceptible to lysis, as were some primary virus-induced lymphomas. Tumors which arose in nude mice, which have high levels of natural cytotoxic activity, were all resistant to lysis. In addition t o the susceptibility of transformed cells t o natural cell-mediated cytotoxicity, some untranrformed cultured cells and cells from normal tissues were targets for this mechanism. Low levels of cytotoxicity were seen with normal thymus cells, bone-marrow cells, and short term cultures of macrophages, whereas normal spleen and lymph-node cells were completely resistant to lysis. These results indicate a broader spectrum for mouse natural cell-mediated cytotoxicity reactivity than has been previously recognized.
In the past few years, many investigators have observed natural cell-mediated cytotoxicity (designated NK for " natural killing ") against a variety of tumor cells. NK reactivity has been detected in mice (Herberman et a/., 1973, 1975; Kiessling et al., 1975), in rats (Holterman et a/., 1973; Nunn e f a/., 1976; Shellam and Hogg, 1977), and in humans (Takasugi, 1973; Oldham et al., 1973, 1975; Rosenberg et al., 1974). In studies on human NK, there have been a number of reports of reactivity in long-term cytotoxicity assays against tissue cultures of carcinomas, melanomas and non-lymphoid tumors (Takasugi et al., 1973; Oldham et a/., 1975; Pavie-Fisher et al., 1975) as well as reactivity in short-term assays against lymphoid and myeloid targets (Rosenberg e t a / . , 1974; West et al., 1977). In contmst, virtually all of the reported studies on NK reactivity in mice have been performed with lymphoid cell lines. The assumption has been made that only these lines were susceptible to lysis by the NK cells. Kiessling et a/. (1975) noted that NK reactivity could be seen against Moloney leukemia virus (MLV)induced lymphoma cells and they concluded that susceptibility to lysis was restricted to lymphomas induced by the MLV virus. Sendo and Aoki (1975) reported NK activity against a radiation-induced
leukemia in BALB/c mice and concluded.that NK activity was directed against X. 1' antigen associated with the expression of an endogenous type-C virus. Zarling et al. (1975) utilized a tumor induced by Gross leukemia virus and felt that NK reactivity was directed against Gross leukemia virus-associated antigens or embryonic antigens. Using these and other lymphoid cell lines as target cells and performing competitive inhibition assays, Herberman et al. (1975) concluded that NK reactivity was directed against several antigens associated with murine endogenous type-C viruses. To determine whether murine NK is analogous to human NK, we have examined a variety of' nonlymphoid cell lines for their susceptibility to NK. In our previous inhibition studies (Herberman et al., 1975), some non-lymphoid as well as lymphoid tumor cells gave positive results. This finding indicated that mouse NK might not be restricted to lymphoid target cells. Indeed, our results here indicate that many non-lymphoid tumor cells, harvested directly from in vivo tumors or from tissue culture cell lines, are susceptible to NK activity in a 4-h 5'Cr-release assay. In addition, we have found that some normal lymphoid and non-lymphoid cells are also susceptible to natural cytotoxicity.
MATERIAL AND METHODS
Mice C57BL/6N, BALB/c, CBA/N, NIH Swiss and NIH nude mice were obtained from the Rodent and Rabbit Production Section, Division of Research Services, National Institutes of Health (NIH), USA. BALB/c nude mice were obtained from the Mammalian Genetics Section, NCI, USA. AKR, NZB and CBA/J were obtained from Jackson Laboratories (Bar Harbor, Maine, USA).
Most of the mice studied were male, but sex did not appear to influence the reactivity of the NK cells. Unless otherwise stated, mice were tested for reactivity when 5-8 weeks of age. Mice more than 12 weeks old were injected with LCMV intra-
Received: June 20, 1977.
382
NUNN ET AL.
peritoneally and tested after 3 days (Herberman et al., 1977). Tumors Most of the tumor cells and the in vitro cultures derived from the tumors have been previously described (Herberman el ul., 1974). RLOl (Sendo and Aoki, 1975), a radiation-induced leukemia in BALB/c mice, was maintained as an ascites tumor by intraperitoneal passage. RBL-5 (McCoy et al., 1967), a Rauscher virus-induced leukemia in C57BL/6, was maintained as an ascites tumor by intraperitoneal passage. Sarcomas in BALB/c and nude mice (Outzen el a/., 1973, induced by methylcholanthrene (MCA), were kindly sent to us by Dr. Henry C. Outzen (Institute for Cancer Research, Fox Chase, Pa.). MCA tumors induced in CBA/H and CBA/H nude mice (Stutman, 1974) were kindly provided by Dr. Osias Stutman (Sloan-Kettering Institute for Cancer Research, New York, N.Y.). Mice bearing primary AKR spontaneous lymphomas were obtained from the Mammalian Genetics Section, NCI, USA. The B16 melanoma (Giovanella et a/., 1974) was obtained from Dr. Robert C. Ting and maintained as a solid tumor in C57BL/6 mice by subcutaneous injections. The 3LL Lewis lung carcinoma (Rotter and Trainin, 1975) was maintained in C57BL/6 mice by subcutaneous injections. The L5MF-22 lymphoma (Bonmassar et al., 1973), kindly provided by Dr. David Houchens, NCT, USA, were maintained in C57BL/6 and BlOA mice, respectively, by intraperitoneal passage. Tissue culture cells RLOl and RBL-5 were maintained in suspension cultures as previously described (Sendo and Aoki, 1975; Holden et al., 1977) and they are designated with the suffix TC. The other cell lines studied were maintained as monolayer cultures. All cell lines were grown in RPMI 1640 medium containing 20% fetal calf serum (Grand Island Biological Co., Grand Island, N.Y., USA). The embryo fibroblasts 3T12 and 3T3 type I CI 6, KA31, MSV-transformed non-producer line (Aoki et al., 1975; Ting and Herberman, 1971), were kindly provided by Drs. G. Todaro and C. C. Ting, NCI, USA. The E4, SV40-transformed 3T3 (Gillette and Fox, 1975), was provided by Dr. C. Boone, NCI, USA. The cell lines T-AI/N (fibroblasts, spontaneously transformed) and SV40-transformed SV-AL/N (Smith and Mora, 1972) were provided by Dr. Chungming Chang. The L8a (Parks and Scolnick, 1973) was derived from (C57BL/6 x A)F, mice and was provided by Dr. Wade Parks, NCI, USA. The TA3 (Sanford, 1967) was derived from an A strain mammary tumor and was obtained from Dr. C. C. Ting, NCI, USA.
Preparation of effector cell suspensions Effector cells from various lymphoid organs were prepared as previously described (Ortiz de Landazuri and Herberman, 1972). Preparation of target cells Target cells . obtained from monolayer tissue cultures were harvested with 0.25 % trypsin, washed with BSS (balanced salt solution) and prepared at the appropriate concentration (106/ml) in RPMI 1640 medium with 20% fetal calf serum for use in a 4-h Wr-release assay. In some experiments, after trypsinization of monolayer cells, they were maintained as single-cell suspensions in spinner cultures overnight before use as targets. Both techniques used gave similar results of NK reactivity and therefore we do not specify method of preparation in the " Results " section. Solid tumors of animals were placed in BSS and teased to yield suspensions of cells, which were washed with BSS. The peritoneal cavity of normal mice was washed with BSS and the cells collected, washed and put in Petri dishes in RPMI medium with 20% fetal calf serum for macrophage cultures. After overnight culture, the macrophages were scraped from the Petri dishes with a rubber policemen. Cells were washed and prepared for use as above. All target cells were labelled with "Cr as previously described (Nunn et al., 1976). Assay for cell-mediated cytotoxicity The assays were performed as previously described (Herberman et al., 1973, 1976~).Lymphoid cells, usually spleen cells, were incubated at a 200:l ratio with 51Cr-labelled target cells for 4 h at 37" C with rocking. The baseline 51Cr-release was determined with the use of unlabelled autologous target cells in place of effector cells. This neutral baseline was selected so that the same total number of cells would be present in each group and the activity of normal lymphoid cells could be detected (Nunn et al., 1976; Herberman et al., 19766). Most of the tests were performed with R L d l TC as a positive control for the other cell lines. To reduce variability in results among assays the RLOl TC were cryopreserved in multiple aliquots, according to procedures previously described (Holden et d., 1976). The baseline lysis of these cryopreserved target cells was 12-20% of the 61Cr incorporated into the cells. The baseline lysis of all other target cells was 5-15%. Levels of cytotoxicity 2 % or more above the baseline were invariably significantly different from the controls (p
