Specific Immune Response Against Tumor-Associated of a Syngeneic Simian Virus 40-lnduced Sarcoma in

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1 Specific Immune Response Against Tumor-Associated of a Syngeneic Simian Virus 40-lnduced Sarcoma in Antigens Mice 1 Moshe Glaser 2, 3 ABSTRACT-Immune response against syngeneic simian virus 40 (SV40)-lnduced tumor-associated antigens In (BALB/c x C57BL/6)F, female mice was detected by In vivo tumor challenge and an In vivo tumor cell neutralization test (Wlnn) and by the In vitro S1Cr-release cytotoxicity assay. All mice Immunized with as low as 1X10' SV4G-transformed cells were protected against the growth of SV40-lnduced tumor, beginning at 3 days and up to 90 days after a single Immunization. In the tumor cell neutralization test, spleen cells (T-cells) from mice Immunized with 1X10' SV40- transformed cells prevented tumor growth at 10 days and up to 90 days after Immunization. The specificity of the Immune response was shown by the use of unrelated syngeneic tumor cells In crisscross experiments. In contrast to these In vivo assays, spleen cells from mice Immunized with a minimum of 20X10' SV40-transformed cells were cytotoxic In vitro against the relevant target cells In a 4-hour 51Cr-release cytotoxicity assay. Cytotoxic lymphocytes were detected 3 days after Immunization, reached peak activity at 8 days, and declined to low levels at 14 days after Immunization. At all points after Immunization, T -cells were needed for cytotoxic reactivity to be eliminated by specific antl-o serum and complement. The specificity of the cytotoxic reactivity was shown by the use of unrelated syngeneic tumor cells In crisscross experlments.-j Natl Cancer Inst 61: , Many tumors are now believed to possess specific antigens that are recognized in vivo by syngeneic hosts after appropriate immunization. Tests to measure in vitro reactivity to some of these antigens have included various types of cytotoxicity assays as well as assays of lymphocyte blastogenesis, macrophage migration inhibition, and delayed hypersensitivity (1). In many in vitro studies, some attempts have been made to correlate reactivity with tumor status (2); however, few in vitro studies have included careful examination of the kinetics of the immune response to tumors and the effector cells involved in relation to tumor growth and regression. The available data obtained by a particular assay are often conflicting, and only some studies show a relationship between reactivity and tumor growth (3, 4). This type of correlative study is essential for establishing the relevance of the results in a particular in vitro assay to in vivo resistance to tumor growth. For the past few years, extensive studies on immune response to SV 40-induced syngeneic transplantable mksa cells of BALB/c mice have been performed in our laboratory. This tumor has been shown to possess TAA (5-7). The TAA can be solubilized and partly purified, and immunization with these preparations as well as whole tumor cells can induce a specific transplantation rejection of mksa. Some in vitro assays (lymphocyte blastogenesis and macrophage migration inhibition) have been developed in our laboratory to measure the immune response in this system and to study the activity of solubilized and fractionated materials (8, 9). Until now development of an in vitro cell-mediated cytotoxicity assay in this system was difficult. In the present study, such an assay was developed and used to measure the immune response to SV40-induced TAA. The results obtained in this assay were compared to those obtained by in vivo direct challenge and tumor cell neutralization assays. The following points were considered: I) The SV 40- transformed cells of C57BL/6 mice (C57SV) are good targets in the in vitro 5lCr-release cytotoxicity assay, but these cells will not grow in vivo. 2) The SV40- transformed cells of BALB/c mice (mksa-tu5) grow and are good targets in vivo, but they are poor targets in vitro. 3) Cross-reactivity between C57SV and mksa Tu5 cells is strong. For these reasons, (BALB/c X C57BL/6) FI mice were used as recipients for comparative in vivo and in vitro experiments. MATERIALS AND METHODS Animals.-BALB/c, C57BL/6, and (BALB/c X C57BL/6)FI (CBFI) female mice, 2-3 months old, were obtained from the National Institutes of Health Breeding Colony. Tumors.-The mksa-tu5 tumor line was developed by transformation of the BALB/c kidney cell line with SV 40 (5). This line has been maintained in this laboratory for several years in tissue culture through more than 100 passages. An ascites form of mksa was developed by ip passage of lxi06 cells in BALB/c mice. The mksa (ASC) line grows as a solid tumor when injected sc and has a median tumor dose of 10 2 cells. Both the tissue culture and the ascites forms of the mksa tumor express the SV40 tumor (T) antigen and SV40 tumor-associated transplantation antigen, but are free of infectious virus (6). The C57SV line (SV40- transformed C57BL/6 mouse embryo fibroblasts) (10) was obtained from B. B. Knowles, Wistar Institute, Philadelphia, Pennsylvania. FBL-3, a Friend virus- ABBREVIATIONS USED: E:T=effector to target cell ratio; FBS=fetal bovine serum; SDS=sodium dodecyl sulfate; SV40=simian virus 40; TAA = tumor-associated antigen(s). 1 Received April ; accepted July Laboratory of Cell Biology. Immunology Program. Division of Cancer Biology and Diagnosis. National Cancer Institute. National Institutes of Health. Public Health Service. U.S. Department of Health. Education. and Welfare. Bethesda. Md I thank Dr. L. W. Law for his interest and support during the course of this work. C. Altman for technical help. and L. Brunson for helping with the preparation of this manuscript. VOL. 61. NO.5. NOVEMBER

2 1352 Glaser induced leukemia, and RBL-5, a Rauscher virus-induced leukemia, were maintained in C57BL/6 mice by weekly ip passage of lxi06 cells. The RBL-5 line was also maintained in tissue cultures in suspension. MSB is an adherent tissue culture line derived from a tumor induced by murine sarcoma virus in C57BL/6 mice. Immunizations.-Immunizations with C57SV and FBL-3 cells were performed by ip and sc routes, respectively, as described later. Preparation and purification of lymphoid cells. Spleens, lymph nodes, blood, thymuses, bone marrow, and peritoneal fluid were removed from the mice, and single-cell suspensions were prepared in RPMI medium supplemented with 100 Ilg streptomycin/ml, 100 U penicillin/ml, and 2 mm glutamine (all reagents from GIBCO, Grand Island, N.Y.). For removal of T cells, AKR anti-8 C3H antibody was obtained from H. Holden, National Cancer Institute, Bethesda, Maryland. The preparation of this serum and the treatment of lymphoid cells were performed according to the technique detailed in (11). This antiserum lysed more than 97% of C3H thymocytes and 35-40% of C3H spleen cells by try pan blue dye exclusion. The T-cell response of spleen cells to phytohemagglutinin was abolished after treatment, whereas the B-cell response to lipopolysaccharide was left intact. For removal of B-cells and macrophages, nylon wool columns were used according to a technique described by Julius et al. (12). About 30% of the starting spleen cell population was recovered in the nonadherent fraction (T-enriched); of these cells, 2-5% (as compared to 40-45% in the unfractionated population) were surface immunoglobulin-positive cells. Phagocytic cells 'were removed by the use of iron and magnet, as described in (4); this technique removed about 30% of the total cell population and eliminated most phagocytic cells, as assessed by late particle ingestion. Tumor cell neutralization assay (Winn).-Spleen cells were washed twice, mixed with freshly harvested mksa ascites cells, incubated for 30 minutes at 37 C, and injected sc in O.l-ml aliquots. Unless otherwise indicated, the E:T of spleen cells to tumor cells (lxi05) was 300: 1. This dose of tumor cells produces tumors in all mice given injections of tumor cells mixed with nonimmune spleen cells. Tumor growth was monitored until death of the animals. Spleen cells from 5-7 donors were used for each assay. Representative experiments are shown. Each experiment was repeated two or three times with similar results. Her-release assay. -The cytotoxicity test was performed in the wells of flat-bottom microtiter plates. Target cells were trypsinized, and 2XI04 cells and 5 IlCi 51Cr in 0.2 ml RPMI-1640 medium containing 5% FBS was added to each well. After 18 hours at 37 C in a 5% CO2 incubator, the target cells were washed and effector cells were added in 0.2 ml medium containing 10% FBS. After 4 hours' incubation at 37 C in a 5% C02 incubator, 0.1 ml supernatant was removed for counting. The target cells were lysed with 1 % SOS to measure the total releasable 51Cr in each well. Percent 51Cr specific release at an E:T of 200: 1 was calculated according to the formula: [(cpm 51Cr released from cells in the presence of immune cells-cpm 51Cr released from cells in the presence of normal cells)/cpm 51Cr released from cells in the presence of SOS]XIOO. The percent 51Cr release in the presence of nonimmune cells was always either very close or identical to the release in medium alone and ranged from 5 to 15% in the different target cells. Representative experiments are shown. Each experiment was repeated two or three times with similar results. Means and standard errors of replicate samples were compared with those of the controls and checked for significance by Student's t test. RESULTS Dose-Response Relationship In Immune Response to SV40-lnduced TAA CBF1 mice were untreated or immunized ip with different numbers of C57SV cells and then divided into 3 groups. The first group was challenged sc with lxi05 mksa cells 15 days later. The second group was killed 15 days after the immunization, and the spleen cells were mixed with lxi05 mksa cells in an E:T of 300: 1 and injected sc into normal CBF1 mice. All the mice were inspected until death from tumor growth (up to at least 3 mo). The third group was killed 8 days after the immunization, and the spleen cells were tested in a 4-hour 51Cr-release cytotoxicity assay against C57SV cells as targets. All mice immunized with as few as lxi05 C57SV cells were protected against mksa tumor growth in the direct challenge experiment (table 1). In the tumor cell neutralization assay, spleen cells from mice immunized with lxi06 C57SV cells prevented mksa tumor growth in all mice. In contrast to these in vivo assays, spleen cells from mice immunized with at least 20XlO6 C57SV cells were cytotoxic in vitro in the 51Cr-release assay. Little cytotoxic activity was observed after immunization with lower numbers of C57SV cells. On the basis of these results, in the following experiments all CBF1 mice were immunized ip with 20XlO6 C57SV cells. Kinetics of Immune Response to SV40--lnduced TAA CBF1 mice were immunized ip with 20XlO6 C57SV cells. At different times afterward, the mice were divided into 3 groups. The first group was challenged sc with lxlo5 mksa cells. The second group was killed, and the spleen cells were mixed with lxi05 mksa cells in 300: 1 E:T and injected sc into normal CBF1 mice. The spleen cells of the third group were tested in vitro in a 4-hour 51Cr-release assay against target C57SV cells. In the direct-challenge experiment, complete protection against mksa tumor growth was observed by 3 days after immunization, and the mice were still protected at 90 days (table 2). In the tumor cell neutralization assay, complete prevention of mksa tumor growth was first observed at 10 days after VOL. 61. NO.5. NOVEMBER 1978

3 No. of C57SV cells used for immunization None 60x x x x10 6 5x10 6 1x10 6 1x10 5 1x10' 1x10 3 Immune Response Against Syngeneic SV40-lnduced Tumor 1353 TABLE 1.-Dose-respcmse relationship in CBF 1 mice to SV40-induced TAA After challenge with 1 x 10 5 mksa cells 1 6/ Significantly different from control (P<0.05).. In Winn neutralization assay 5/8 Percent cytotoxicity (±SE) 12.5 (l.o) 39.5 (2.2) (2.0) (1.8)" 19.1 (1.4) 15.2 (1.4) 15.0 (1.2) 12.0 (1.0) 10.0 (0.7) 10.3 (0.9) Days after immunization o TABLE 2.-Kinetics of immune respcmse to SV40-induced TAA After challenge with mksa cells 1 1/10 " Significantly different from control (P<0.05). immunization, and activity remained up to 90 days. In the 5lCr-release assay, cytotoxic spleen cells were detected 3 days after immunization and reached a peak activity at 8 days, and then cytotoxicity declined to low levels by day 14. After a second injection of C57SV cells, even at 90 days after immunization, cytotoxic reactivity reappeared (not shown). Similar kinetics of cytotoxic activity was observed also with the use of effector cells from mesenteric lymph nodes, blood, and peritoneal exudate. Thymus and bone marrow had little cytotoxic reactivity (not shown). Specificity of Immune Response Against SV40-lnduced TAA CBFI mice were untreated or immunized ip with 20XlO6 C57SV cells or sc with 5XlO6 FBL-3 cells. Each group of mice was subdivided into 3 groups. The first one was challenged sc with IX105 mksa or RBL-5 cells 15 days after immunization. The second one was In Winn neutralization assay 2/8 Percent cytotoxicity (± SE ) 8.0 (0.9) 20.2 (2.0) (2.3) (3.6) (2.4) (1.5) 10.8 (1.3) 10.0 (1.2) 9.1 (0.9) 8.6 (0.9) killed 15 days after immunization, and the spleen cells were mixed with lxi05 mksa or RBL-5 cells and injected sc (E:T, 300: I) into normal CBF 1 mice. The spleen cells of the last group were tested in a 4-hour 5lCr-release assay against C57SV or MSB target cells 8 days after immunization with C57SV or 10 days after immunization with FBL-3 cells. All mice in the nonimmunized group developed mksa or RBL-5 tumors (table 3). Mice immunized with C57SV cells were protected against mksa but not against RBL-5 tumor growth. Mice immunized with FBL-3 cells were protected against the growth of RBL-5 but not against mksa. Similar results were obtained in the tumor cell neutralization assay. In the in vitro 5lCr-release assay, spleen cells from mice immunized with C57SV cells were cytotoxic against the relevant but not against the unrelated target cells. Spleen cells from mice immunized with FBL-3 cells were cytotoxic only against the corresponding target cells. The specificity of the cytotoxic reactivity was also maintained with mesenteric Cells used for immunization TABLE 3.-Specificity of immune response against SV40-induced TAA after challenge with: mksa cells RBL-5 cells None 9/10 8/10 C57SV 9/10 FBL-3 9/10 1/10 in Winn neutralization assay of: mksa cells RBL-5 cells 7/8 7/8 1/8 C57SV cells Percent cytotoxicity against: MSB cells VOL. 61, NO.5, NOVEMBER 1978

4 1354 Glaser TABLE 4.-Nature of cells needed for reactivity against SV40-induced TAA Treatment of immune spleen cells None Anti-8 serum plus complement Complement Nylon wool column Iron and magnet No. of mice dead/ total No. in Winn neutralization assay 1/8 7/8 1/8 Percent cytotoxicity lymph node cells, peritoneal exudate cells, and blood cells from C57SV-immune mice (19.2, 32.5, and 22.7% cytotoxicity, respectively; 1.8, 3.6, and 2.3% cytotoxicity against MSB cells). Nature of CeUs Needed for Reactivity Against SV40- Induced TAA CBFl mice were immunized ip with 20XI06 C57SV cells. At different times thereafter, their spleen cells were either left untreated, treated with specific anti-o serum and complement, treated with complement alone, passed over nylon wool columns, or treated with iron and magnet. After treatment, the various cell populations were used in vivo in a tumor cell neutralization assay with mksa cells, or they were used in vitro in the 4-hour 51Cr-release assay with C57SV target cells. Neither the mice given injections of untreated immune spleen cells and mksa (table 4) nor the mice given injections of immune spleen cells treated with complement alone, passed over nylon wool columns, or treated with iron and magnet developed tumors. However, all mice given injections of immune spleen cells treated with anti-o serum and complement developed tumors. In the 51Cr-release assay, cytotoxic activity against C57SV target cells was observed with. untreated immune spleen cells (table 4), spleen cells treated with complement alone, passed over nylon wool columns, or treated with iron and magnet. Cytotoxic activity was abrogated only after treatment of spleen cells with anti-o serum and complement. The results shown are with spleen cells tested for the tumor cell neutralization and the cytotoxicity assays at 15 and 8 days, respectively, after immunization. Similar results were obtained at all time points after immunization. DISCUSSION The purpose of the present study was to develop an in vitro cell-mediated cytotoxicity assay and to examine the relevance of the in vitro assay of cell-mediated immunity to in vivo resistance against SV 40-induced tumors in mice. It is difficult to evaluate this important issue directly, because in vivo systemic immunity is considerably more complex and subject to many more variables than is the individual in vitro assay. The focus of our experiments has been to determine the correlation between the ability of immune spleen cells to prevent tumor growth in normal mice in the tumor cell neutralization assay and the ability of the immune mice to reject the tumor in the direct-challenge experiments on the one hand, and the in vitro reactivity of the immune cells in the 51Crrelease assay on the other. The correlation was examined in regard to the antigenic specificity, the types of cells required, and the kinetics of the immune response. The transfer of cells from CBF1 mice immune to syngeneic SV40-transformed cells appeared to prevent specifically the growth of syngeneic SV 40-induced tumors in normal CBF1 mice. Similar results were obtained in the direct-challenge experiments. Challenge of mice immune to SV40-induced TAA with an unrelated tumor resulted in tumor growth. This observation of specific protection against the immunizing tumor agrees with our previous observation in an SV40 model but one in which a completely syngeneic system, i.e., mksa tumor in BALB/c mice, was used (13, 14). The specificity of these in vivo assays seemed to correlate with the results obtained in vitro. However, these results are not sufficient to determine the correlation between specificities detected in each assay system. Thus far, examination of the specificity of the 51Crrelease assay has been possible only by the use of the cytotoxicity inhibition assay (15, 16). More extensive specificity studies will be needed to determine the antigens involved in each reaction. The number of SV40-transformed cells needed to generate detectable immune response varied among the three assays. Tenfold more cells were needed for the tumor cell neutralization test to be positive than for the direct challenge, and 200-fold more cells were needed to detect cytotoxic lymphocytes in the 51Cr-release assay. The reasons for these differences are not clear, but obviously many more cells are needed to generate a detectable in vitro immune response. One should take into consideration the fact that both in vivo assays (as compared to the in vitro 51Cr-release assay) actually measure the secondary immune response (17, 18), which is more sensitive than the primary one. In other experiments (not shown), a strong secondary cytotoxic response after primary immunization with small numbers of C57SV cells was observed. The tumor cell neutralization assay and the 51Crrelease assay were found to be T-cell-dependent. B-cells or phagocytic cells did not appear to be required. However, it has not been firmly established that T-cells alone are responsible for the observed effects. This is particularly difficult to determine in the tumor cell neutralization assay, because the transferred cells might recruit recipient cells to become cytotoxic (13), and some of those active recipient cells might be B-cells (19) or macrophages (20). Furthermore, different subpopulations of T-cells might be involved in the different assays, or T-cells in different phases of differentiation might be responsible for the various effects. This last point may be directly relevant to differences In kinetics of the responses as discussed below. The analyses of the kinetics of the in vivo and in vitro assays appear to provide the best discrimination VOL. 61, NO.5, NOVEMBER 1978

5 Immune Response Against Syngeneic SV40-lnduced Tumor 1355 at pres em among the tests. Good correlation was found between the direct challenge and the tumor cell neutralization assay. Despite the fact that immunity in the direct challenge was noticed earlier than in the tumor cell neutralization assay, immunity in both assays was long-lasting. In comrast LO the in vivo assays, reactivity of the cylotoxic lymphocytes declined early aher immunization (14 days). These resuhs might be taken as an indication that the in vitro cylotoxicity assay is irrelevam to in vivo host resistance. However, a likely explanation for the discrepancy is that after immunization, i.e., more than 2 weeks later, large numbers of memory cells are presem in the spleen that, upon reexposure to tumor amigens, can rapidly become highly cyloloxic and thereby protective. In this system (not shown) and in other systems [see (21)], if immune spleen cells, long aher immunization with tumor amigens (when they are no longer direclly cytoloxic), were incubated for a few days with the relevam tumor cells, they developed high levels of cyloloxicity. In vivo challenge of the mice in the presem study (not shown) and in other studies (17, 18) long after immunization also led to the rapid appearance of cyloloxicity. Inasmuch as an imegral part of the tumor cell neutralization assay or the direct challenge is the challenge with tumor cells, a good opportunity exists for the lymphoid cells to be restimulated. REFERENCES (1) HERBERMAN RB: Cell-mediated immunity to tumor cells. Adv Cancer Res 19: (2) HELLSTR6M KE. HELLSTR6M I: Lymphocyte-mediated cytotoxicity and blocking serum activity to tumor antigens. Adv Immunol 18: (3) BANSAL SC. SJ6GREN HO: Counteraction of the blocking of cellmediated tumor immunity by inoculation of unblocking sera and splenectomy: Immunotherapeutic effects on primary polyoma tumor in rats. Int J Cancer 9: (4) GLASER M. LAVRIN DH. HERBERMAN RB: In vivo protection against syngeneic Gross virus-induced lymphoma in rats: Comparison with in vitro studies of cell-mediated immunity. J Immunol 16: (5) KIT S. KllRIMllRA T. DllBBS DR: Transplantable mouse tumor line induced by injection of SV-40 transformed mouse kidney cells. Int J Cancer 4: (6) DRAPKIN MS. ApPELLA E. LAw LW: Immunogenic properties of a soluble tumor-specific transplantation antigen induced by simian virus 40. J Natl Cancer Inst 52: (7) ApPELLA E. LAW LW. HENRIKSEN 0: Biological and biochemical properties of soluble tumor specific transplantation antigen (TSTA) of a simian virus 40 (SV-40)-induced neoplasm. Cancer Res 36: (8) DEAN JH. McCoy JL. LE'WIS D. et al: Studies of lymphocyte stimulation by intact tumor cells and solubilized tumor antigen. Int J Cancer 16: (9) McCoy JL. PADARATHSINGH M. DEAN JH. et al: Migration inhibition by an agarose microdroplet assay: Monitoring of tumor-associated antigens on a simian virus 40-induced sarcoma. J Immunol 119: (10) TRINCHIERI G. ADEN D. KNOWLES BB: Cell-mediated cytotoxicity to SV-40 specific tumour associated antigen. Nature 261: (11) HERBERMAN RB. NllNN ME. LAVRIN DH. et al: Effect of antibody to antigen on cell-mediated immunity induced in syngeneic mice by murine sarcoma virus. J Natl Cancer Inst 51: (12) JULIUS MH. SIMPSON H. HERZENBERG LA: A rapid method for the isolation of functional thymus-derived murine lymphocytes. Eur J Immunol 3: (13) HOWELL SB. DEAN JH. ESBER EC. et al: Cell interactions in adoptive immune rejection of a syngeneic tumor. Int J Cancer 14: (14) HOWELL SB. ESBER EC. LAW LW: Cellular immunity in mice with simian virus 40-induced mksa tumors: Comparison of three assays of tumor immunity. J Natl Cancer Inst 52: (15) ORTIZ DE LANDAWRI M. HERBERMAN RB: Specificity of cellular immune reactivity to virus-induced tumors. Nature [New Bioi] 238: (16) NllNN ME. DJHl JY. GLASER M. et al: Natural cytotoxic reactivity of rat lymphocytes against syngeneic Gross virus-induced lymphomas. J Natl Cancer Inst 56: (17) GLASER M. HERBERMAN RB: Secondary cell-mediated cytotoxic response to challenge of rats with syngeneic Gross virusinduced lymphoma. J Natl Cancer Inst 56: (18) HOLDEN HE. KIRCIINER H. HERBERMAN RB: Secondary cellmediated cytotoxic response to syngeneic mouse tumor challenge. J Immunol 115: (19) ORTIZ DE LANDAWRI M. KEIMR E. FAIlEY JL: Antibody-dependent cellular cytotoxicity to a syngeneic Gross virus-induced lymphoma. J Natl Cancer Inst 52: (20) EVANS R. ALEXANDER P: Rendering macrophages specifically cytotoxic by a factor released from immune lymphoid cells. Transplantation 12: (21) GLASER M. BONNARD GD. HERIIERMAN RB: In vitro generation. of secondary cell-mediated cytotoxic response against a syngeneic Gross virus-induced lymphoma in rats. J Immunol 116: VOL. 61. NO.5. NOVEMBER 1978