Brief Communication: Inhibition of Cell-Mediated Cytotoxicity Against Tumor-Associated Antigens by Suppressor Cells From Tumor-Bearing Mice 1 Moshe Glaser/· 3 Holger Kirchner,2 Howard T. Holden,2 and Ronald B. Herberman 2 SUMMARY-Spleen cells from mice bearing primary tumors induced by Moloney strain of murine sarcoma virus (M-MuSY) strongly inhibited the in vitro generation of specific secondary cell-mediated cytotoxic response of spleen cells from M-MuSY regressor mice. These suppressor cells were resistant to treatment with anti-theta serum and complement or to X-irradiation. It appeared that suppressor cells may have had a role in limiting the host's immune response against tumor growth.J Nat! Cancer Inst 56: 86~7, 1976.
Suppressor cells in spleens of tumor-bearing mice have inhibited the in vitro lymphoproliferative responses to mitogens, alloantigens, and tumor-associated antigens (1-4). However, in the murine sarcoma virus (Moloney) (M-MuSV) system that we have been studying, the peak suppressor cell activity, 14 days after virus inoculation, was coincident with the peak primary, specific, cellmediated cytotoxicity (2). In vitro removal of the suppressor cells of lymphoproliferation did not enhance cytotoxicity of splenic T lymphocytes, and addition of suppressor cells did not inhibit cytotoxicity (3). Thus the suppressor cells are obviously without effect on the effector phase of specific cytotoxicity. However, we have presented evidence suggesting that the suppressor cell activity may be restricted to inhibition of proliferationdependent lymphocyte functions. Therefore, the suppressor cells may have the potential of interfering with the generation of cytotoxic effector cells, but in the M-MuSV system, cytotoxic T cells may be generated before a significant number of suppressor cells has developed. A technique of generating an in vitro secondary cytotoxic response in the M·MuSV system has been recently described (5). This assay seems an appropriate way to determine whether the described suppressor cells can inhibit the generation of specifically cytotoxic effector cells. We found that spleen cells from M-MuSV tumor-bearing mice suppress the in vitro secondary cytotoxic response of spleen cells from M-MuSV regressor mice. MATERIALS AND METHODS
Tumor system.-The M-MuSV tumor system as used in this study was described in detail previously (6). Male C57BL/6N mice 8-12 weeks old (Division of Research Services, National Institutes of Health) were inoculated im in the right hind leg with 0.05 ml M-MuSV. Tumors were first detected at about 7 days, reached peak size at 12-16 days, and routinely regressed after 21 days. As a target for cytotoxicity tests RBL-5 ascites lymphoma cells were used. RBL-5 is a Rauscher strain of murine leukemia virus-induced tumor line of C57BL/6 mice and carries the relevant antigens of the M-MuSV tumor system (6). Primary cytotoxici~y again.st RBL-5 cells could be detected 7-21 days after moculatIOn of M-MuSV; thereafter it declined to very low levels (5). We could detect a marked in vitro secondary cytotoxic response against RBL-5 cells for at least 180 days by culturing
spleen cells from regressors with RBL5 for 5 days. In this series of experiments, spleen cells were tested for their in vitro secondary response, 30 days after virus inoculation (M-MuSV-30 spleen cells). In vitro secondary cytotoxic response.-Spleen cell suspensions were cultivated in medium RPMI-1640 containing 5 X 1()-5 M 2-mercaptoethanol (Schwarz/Mann, Orangeburg, N.Y.) and supplemented with 5% fetal bovine serum, 2 mM glutamine, 100 U penicillin/ml, and 100 p.g streptomycin/ml (all tissue culture reagents from Grand Island Biological Co., Grand Island, N.Y.) for 5 days at a total cell number of 20 X I ()6/5 ml in 16-ounce tissue culture flasks (Falcon Plastics, Cockeysville, Md.), which were kept upright in an atmosphere of 5% CO2 and 95% air. To elicit secondary cytotoxic response, we incubated spleen cells from regressor mice with 0.5x 1()6 RBL-5 cells inactivated by treatment with 100 p.g mitomycin C/ml (Nutritional Biochemical Corp., Cleveland, Ohio) for 30 minutes; this completely inhibited their proliferation. Control cultures were .incubated without added RBL-5 cells. After 5 days, cells were collected by centrifugation and used as effector cells in the 4-hour 51Cr-release assay (CRA). For demonstration of suppressor cell activity, various doses of spleen cells from tumor-bearing mice were added to a fixed number of spleen cells from regressor mice. To eliminate cytotoxic effector cells from suppressor cell populations, we treated them by anti-theta serum plus complement or by 2,500 rads of X-irradiation. These techniques previously described (2, J). Normal spleen cells, treated identically, served as controls. After 5 days, the number of viable cells in cultures not containing mixtures of cells was about 20% (by trypan blue exclusion) of the original number of cells, before culture. The percentages of viable cells in cultures containing M-MuSV-30 spleen cells plus the cells to be tested for suppressive activity were 30, 40, and 60, corresponding to the increase in the cells added to the fixed number of M-MuSV-30-infected cells. Since these latter cultures originally contained the same number of M-MuSV-30 spleen cells as the controls, and since the added cell populations had no activity by themselves (not shown), dilutions were made in the "mixed" cultures, identical to those made in the M-MuSV-30 cultures, for testing in the CRA. The procedure of the CRA was detailed in (6). RBI:-5 cells were used as targets, and effector/target cell ratIos of 50: 1, 25: 1, and 12.5: 1 were routinely tested. Results were expressed as percent specific '51Cr-release by subttaction of the background release (6). The means of quadruplicate determinations are given. Received September 22, 1975; accepted November 18, 1975. Laboratory of IDlIDunodiagnosis. National Cancer Institute (NCI). National Institutes of Health. Public Health Service. U.S. Department of Health. Education. and Welfare. Bethesda. Md. 20014. 3 Address reprint requests to present address: Laboratory of Cell Biology, NCI. 1
2
JOURNAL OF THE NATIONAL CANCER INSTITUTE. VOL. 56, NO.4. APRIL 1976
865
866
GLASER, KIRCHNER, HOLDEN, AND HERBERMAN
RESULTS
The spleen cells from M-MuSV-infected regressor mice 30 days after virus inoculation and about 10 days after tumor regression (designated M-MuSV-30 spleen cells) had low or insignificant levels of primary cytotoxic reactivity against RBL-5 cells in the CRA (6). However, when they were incubated for 5 days in the presence of mitomycin C-treated RBL-5 cells, a high secondary cytotoxic response was detected (text-fig. 1; table I). The cells to be tested for suppressive activity were spleen cells from M-MuSV-infected tumor-bearing mice 14 days after virus inoculation (designated M-MuSV-14 spleen cells), when peak. suppressor cell activity had been demonstrated (2). However, in M-MuSV-14-infected spleens, primary cytotoxic effector cells were also present (6). To eliminate these (7) and thereby obtain an enriched population of suppressor cells (2), we treated M-MuSV-14 spleen cells with anti-theta serum plus complement. Alternatively, they were treated with 2,500 rads, since suppressor cells are resistant to this level of X-irradiation, whereas most cytotoxic activity was removed by 75
60
45
30
15
12.5:1 50:1 25:1 EFFECTOR CELUTARGET CEll RATIO TEXT'FIGUJU; I.-Effect of X-irradiated (2,500 rads) M-MuSV-14 spleen cells on the in vitro secondary cytotoxic response of M·MuSV-30 spleen cells to RBL-5 lymphoma cells. ~=M-MuSV-30 spleen cells alone; O=M·MuSV·30 spleen cells plus an equal number of irradiated normal spleen cells; 0= M-MuSV-30 spleen cells plus an equal number of irradiated M·MuSV·14 spleen cells_ AU con· trol cultures without added mitomycin C-treated RBL-5 cells had less than 10% cytotoxicity. TABLE I.-Effect of M-MuSV-14 spleen cells treated by anti-theta serum plus complement on the in vitro secondary cytotoxic response of M-MuSV-30 spleen cells to RBL-5 lymphoma cells Stimulating cells Number and source of cells .. None _______________________________ _ 40 X 10' normal spleen cells ____________ _ 40XI01 M-MuSV-14 spleen cells _______ _ 20X 10' normal spleen cells ____________ _ 20XI0' M-MuSV-14 spleen cells _______ _ 10 XI0' normal spleen cells ____________ _ 10XI0' M-MuSV-14 spleen cells _______ _
None
RBL-5
8.2 7.8 7.3 8.7 9.1 8.0 8.4
71.2 64.1 17.3
66.7
29_8 70.2 59.6
• Treated with anti-theta serum and complement and added to 20 X1()f M-MuSV30 apleen cells_ M-MuSV-3O cells alone or with varying numbers of anti-thetatreated M-MuSV-14 or normal spleen cells were cultivated with or without 0.5 X 101 mitomycin C-treated RBL-5 cells and aubsequently tested for their ability to lyse RBL-5 oella in a 4-br eRA in an effector/target ratio of so: 1. Results are expreoeed as mean specific cytotoxicity of quadruplicate samples (81C
Suppressor cells in spleens of tumor-bearing mice have inhibited the in vitro lymphoproliferative responses to mitogens, alloantigens, and tumor-associated antigens (1-4). However, in the murine sarcoma virus (Moloney) (M-MuSV) system that we have been studying, the peak suppressor cell activity, 14 days after virus inoculation, was coincident with the peak primary, specific, cellmediated cytotoxicity (2). In vitro removal of the suppressor cells of lymphoproliferation did not enhance cytotoxicity of splenic T lymphocytes, and addition of suppressor cells did not inhibit cytotoxicity (3). Thus the suppressor cells are obviously without effect on the effector phase of specific cytotoxicity. However, we have presented evidence suggesting that the suppressor cell activity may be restricted to inhibition of proliferationdependent lymphocyte functions. Therefore, the suppressor cells may have the potential of interfering with the generation of cytotoxic effector cells, but in the M-MuSV system, cytotoxic T cells may be generated before a significant number of suppressor cells has developed. A technique of generating an in vitro secondary cytotoxic response in the M·MuSV system has been recently described (5). This assay seems an appropriate way to determine whether the described suppressor cells can inhibit the generation of specifically cytotoxic effector cells. We found that spleen cells from M-MuSV tumor-bearing mice suppress the in vitro secondary cytotoxic response of spleen cells from M-MuSV regressor mice. MATERIALS AND METHODS
Tumor system.-The M-MuSV tumor system as used in this study was described in detail previously (6). Male C57BL/6N mice 8-12 weeks old (Division of Research Services, National Institutes of Health) were inoculated im in the right hind leg with 0.05 ml M-MuSV. Tumors were first detected at about 7 days, reached peak size at 12-16 days, and routinely regressed after 21 days. As a target for cytotoxicity tests RBL-5 ascites lymphoma cells were used. RBL-5 is a Rauscher strain of murine leukemia virus-induced tumor line of C57BL/6 mice and carries the relevant antigens of the M-MuSV tumor system (6). Primary cytotoxici~y again.st RBL-5 cells could be detected 7-21 days after moculatIOn of M-MuSV; thereafter it declined to very low levels (5). We could detect a marked in vitro secondary cytotoxic response against RBL-5 cells for at least 180 days by culturing
spleen cells from regressors with RBL5 for 5 days. In this series of experiments, spleen cells were tested for their in vitro secondary response, 30 days after virus inoculation (M-MuSV-30 spleen cells). In vitro secondary cytotoxic response.-Spleen cell suspensions were cultivated in medium RPMI-1640 containing 5 X 1()-5 M 2-mercaptoethanol (Schwarz/Mann, Orangeburg, N.Y.) and supplemented with 5% fetal bovine serum, 2 mM glutamine, 100 U penicillin/ml, and 100 p.g streptomycin/ml (all tissue culture reagents from Grand Island Biological Co., Grand Island, N.Y.) for 5 days at a total cell number of 20 X I ()6/5 ml in 16-ounce tissue culture flasks (Falcon Plastics, Cockeysville, Md.), which were kept upright in an atmosphere of 5% CO2 and 95% air. To elicit secondary cytotoxic response, we incubated spleen cells from regressor mice with 0.5x 1()6 RBL-5 cells inactivated by treatment with 100 p.g mitomycin C/ml (Nutritional Biochemical Corp., Cleveland, Ohio) for 30 minutes; this completely inhibited their proliferation. Control cultures were .incubated without added RBL-5 cells. After 5 days, cells were collected by centrifugation and used as effector cells in the 4-hour 51Cr-release assay (CRA). For demonstration of suppressor cell activity, various doses of spleen cells from tumor-bearing mice were added to a fixed number of spleen cells from regressor mice. To eliminate cytotoxic effector cells from suppressor cell populations, we treated them by anti-theta serum plus complement or by 2,500 rads of X-irradiation. These techniques previously described (2, J). Normal spleen cells, treated identically, served as controls. After 5 days, the number of viable cells in cultures not containing mixtures of cells was about 20% (by trypan blue exclusion) of the original number of cells, before culture. The percentages of viable cells in cultures containing M-MuSV-30 spleen cells plus the cells to be tested for suppressive activity were 30, 40, and 60, corresponding to the increase in the cells added to the fixed number of M-MuSV-30-infected cells. Since these latter cultures originally contained the same number of M-MuSV-30 spleen cells as the controls, and since the added cell populations had no activity by themselves (not shown), dilutions were made in the "mixed" cultures, identical to those made in the M-MuSV-30 cultures, for testing in the CRA. The procedure of the CRA was detailed in (6). RBI:-5 cells were used as targets, and effector/target cell ratIos of 50: 1, 25: 1, and 12.5: 1 were routinely tested. Results were expressed as percent specific '51Cr-release by subttaction of the background release (6). The means of quadruplicate determinations are given. Received September 22, 1975; accepted November 18, 1975. Laboratory of IDlIDunodiagnosis. National Cancer Institute (NCI). National Institutes of Health. Public Health Service. U.S. Department of Health. Education. and Welfare. Bethesda. Md. 20014. 3 Address reprint requests to present address: Laboratory of Cell Biology, NCI. 1
2
JOURNAL OF THE NATIONAL CANCER INSTITUTE. VOL. 56, NO.4. APRIL 1976
865
866
GLASER, KIRCHNER, HOLDEN, AND HERBERMAN
RESULTS
The spleen cells from M-MuSV-infected regressor mice 30 days after virus inoculation and about 10 days after tumor regression (designated M-MuSV-30 spleen cells) had low or insignificant levels of primary cytotoxic reactivity against RBL-5 cells in the CRA (6). However, when they were incubated for 5 days in the presence of mitomycin C-treated RBL-5 cells, a high secondary cytotoxic response was detected (text-fig. 1; table I). The cells to be tested for suppressive activity were spleen cells from M-MuSV-infected tumor-bearing mice 14 days after virus inoculation (designated M-MuSV-14 spleen cells), when peak. suppressor cell activity had been demonstrated (2). However, in M-MuSV-14-infected spleens, primary cytotoxic effector cells were also present (6). To eliminate these (7) and thereby obtain an enriched population of suppressor cells (2), we treated M-MuSV-14 spleen cells with anti-theta serum plus complement. Alternatively, they were treated with 2,500 rads, since suppressor cells are resistant to this level of X-irradiation, whereas most cytotoxic activity was removed by 75
60
45
30
15
12.5:1 50:1 25:1 EFFECTOR CELUTARGET CEll RATIO TEXT'FIGUJU; I.-Effect of X-irradiated (2,500 rads) M-MuSV-14 spleen cells on the in vitro secondary cytotoxic response of M·MuSV-30 spleen cells to RBL-5 lymphoma cells. ~=M-MuSV-30 spleen cells alone; O=M·MuSV·30 spleen cells plus an equal number of irradiated normal spleen cells; 0= M-MuSV-30 spleen cells plus an equal number of irradiated M·MuSV·14 spleen cells_ AU con· trol cultures without added mitomycin C-treated RBL-5 cells had less than 10% cytotoxicity. TABLE I.-Effect of M-MuSV-14 spleen cells treated by anti-theta serum plus complement on the in vitro secondary cytotoxic response of M-MuSV-30 spleen cells to RBL-5 lymphoma cells Stimulating cells Number and source of cells .. None _______________________________ _ 40 X 10' normal spleen cells ____________ _ 40XI01 M-MuSV-14 spleen cells _______ _ 20X 10' normal spleen cells ____________ _ 20XI0' M-MuSV-14 spleen cells _______ _ 10 XI0' normal spleen cells ____________ _ 10XI0' M-MuSV-14 spleen cells _______ _
None
RBL-5
8.2 7.8 7.3 8.7 9.1 8.0 8.4
71.2 64.1 17.3
66.7
29_8 70.2 59.6
• Treated with anti-theta serum and complement and added to 20 X1()f M-MuSV30 apleen cells_ M-MuSV-3O cells alone or with varying numbers of anti-thetatreated M-MuSV-14 or normal spleen cells were cultivated with or without 0.5 X 101 mitomycin C-treated RBL-5 cells and aubsequently tested for their ability to lyse RBL-5 oella in a 4-br eRA in an effector/target ratio of so: 1. Results are expreoeed as mean specific cytotoxicity of quadruplicate samples (81C
