DISCUSSION PAPER: I N VZTRO AUGMENTATION OF TUMOR IMMUNITY IN A MURINE METHYLCHOLANTHRENE SARCOMA SYSTEM * Melvin A. Yarlott, Jr. and Charles F. McKhann Departments of Surgery and Microbiology University of Minnesota Minneapolis, Minnesota 55455

INTRODUCTION Adoptive immunotherapy is accomplished by transfusion of lymphocytes sensitized to tumor-associated antigens to increase the specific immune reaction against a malignancy. The effectiveness of adoptive immunotherapy depends on the ability of the infused lymphocytes to survive in the recipient’s circulation and to implement a tumor immune response. The former consideration discourages use of allogeneic HLA-incompatible lymphocytes, whose effectiveness is limited by the recipient’s transplantation reaction. Ideally, the patient’s own lymphocytes, treated in vitro to augment their tumoricidal effect, are the best source of “educated” lymphocytes. In numerous experimental tumor models, normal spleen cells have been sensitized to tumor antigens completely in vitro.’?4 , 6 , l o , In the clinical situation, however, the patient’s lymphocytes always have had previous exposure to tumor antigens in vivo. The investigations of Rollinghoff,12 Plata et al.,IrJand Cerottini et aL3 show that the production of cytotoxic lymphocytes in vitro is accelerated if the lymphocytes have had a prior exposure to tumor antigens in vivo. The secondary immune response appears to involve the conversion of “educated” noncytotoxic lymphocytes (“memory cells”) into potent tumoricidal cells. Furthermore, in vitro sensitization of lymphocytes may be more efficient than in vivo sensitization.7, * , l o With a murine chemically induced tumor system, we have attempted to measure cell-mediated tumor immunity after tumor resection and to enhance the cytotoxicity of spleen cells recovered from these mice by in vitro incubation with the same tumor.

MATERIALS AND METHODS Fibrosarcomas were induced in 8-1 2-week-old C3H/ Hej mice (Jackson Laboratories) by intramuscular injection of 0.05 mg of 3-methylcholanthrene in crotin oil. The tumors were passaged in vivo by serial injection of normal mice subcutaneously with 1 0 viable tumor cells prepared by treating tumor fragments with 0.25 % trypsin at room temperature with continuous stirring for 30-min intervals. The cells were collected from the supernatant by centrifugation (1000 rpm for 10 min) and were washed three times in media.

* Supported by American Cancer Society Grant IM-25G and Contract NOl-CP92061 from the National Cancer Institute.

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Continuous culture lines of two fibrosarcomas, MC43 and MC93, were established and carried in minimal essential medium (MEM) plus 10% fetal calf serum (FCS) (International Scientific Ind). and used as target cells in the microcytotoxicity assay. Mice were “immunized” by injecting loG tumor cells into the right hind leg, followed by leg amputation 9 days later. Single-cell suspensions of lymphocytes were obtained by teasing apart sterilely resected spleens with forceps and lysis of erythrocytes with Tris-buffered ammonium chloride (0.75 % ) . Lymphocytes were cultured in Hanks’ balanced salt solution with 0.5% isologous mouse serum, MEM essential amino acids (SOX), MEM nonessential amino acids ( l o o x ) , nucleic acid precursors (25x1,MEM vitamins (IOOX), sodium pyruvate (100 mM), L-glutamine (200 mM), penicillin-streptomycin (200 mM) , 2-mercaptoethanol (4 >( lo-$ M), sodium bicarbonate (7.5%) and Hepes buffer (20 mM) at a concentration of 5 X los cells/ml in 60-mm culture dishes kept at 37” C in 5% CO,. Lymphocytes were cultured in media alone or in the presence of “inactivated” live whole tumor cells at a 1ymphocyte:tumor cell ratio of 2OO:l-1O:l. Tumor cells were “inactivated” by mitomycin C treatment (30 pg/cm3 for 1 hr) or irradiation (5000 R ) . Selective concentration of specifically immune lymphocytes was accomplished by incubating 10* spleen cells, obtained from mice 3 weeks post-tumor resection, over a confluent monolayer of tumor cells isologous to the in vivo sensitizing tumor. The cells were cocultivated in MEM plus 10% FCS in 100-mm culture dishes at 37”C in 5% CO, for 5 hr in a stationary position. Nonadherent lymphocytes were aspirated, and the adherent layer of cells were washed three times with warm media. The tumor cells and remaining adherent lymphocytes were recovered by treatment with 5 mM ethylenediaminetetraacetate (EDTA) for 1 min and resuspended in MEM plus 10% FCS with vigorous pipetting. They were then carefully layered over a continuous Ficoll gradient constructed according to the method of Pretlow.Il Briefly, a cushion of 45% Ficoll was placed in a sterile 100-ml pdycarbonate centrifuge tube. With a Gilson pump, continuously diluted 5% Ficoll was placed over the cushion to a final volume of 79 ml. The Ficoll concentration at the cushiongradient interface was 5 % , and that at the sample-gradient interface was 2.7%. Cells (5.0X 10‘) of the tumor cell-adherent spleen cell mixture were placed on the gradient and centrifuged at 800 rpm for 13 min. The first 15 rnl of the gradient were discarded; the next 30 ml were collected, centrifuged at 1500 rpm for 15 min, washed, and resuspended in fresh media. Twenty percent of the lymphocytes placed on the gradient were recovered and were 299% pure lymphocytes. The microcytotoxicity assay used in this study was modified from that described by Shiku et d . 1 3 A monolayer of cultured tumor cells was prelabeled by adding [SHIleucine (50 pCi/ml) to the culture media (MEM 10% FCS) for 5 hr. The labeled tumor cells were harvested by treatment with trypsin-Versend (0.02%),washed three times, and plated (1000 tumor cells/ well) in Falcon 2-3040 microtest plates (Falcon Plastics) with a Hamilton syringe. Lymphocytes (105) to be tested for cytotoxicity were immediately added to the microtest plate, so that the total volume in each well was 0.2 ml. The plates were incubated in a stationary position for 24 hr at 37” C in 5 % CO,. ‘This assay was terminated by inversion and thorough washing of the plate with warm phosphate-buffered saline plus 5% FCS. The plates were air dried, sprayed with fluro-Glide@ (Chemoplast Inc.), and the well bottoms

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punched out into polyethylene vials. TS- 1 solubilizer (Research Products International) and liquid scintillation fluid [toluene with 2,5-diphenyloxazole and p-bis(2,5-phenyloxazolyl)benzene] were added to the vials. The plastic disk and residual adherent tumor cells dissolved, assuring complete recovery of the isotope. The vials were kept at 4”C overnight, and the amount of isotope, which was proportional to the number of residual tumor cells adherent to the plastic disk, was determined by liquid scintillation counting. The data in this report are expressed in counts per minute in the log scale to the base 10 to facilitate statistical analysis. Differences in mean log counts per minute between normal controls and sensitized lymphocytes were significant when the percentage reduction from normal controls in the sensitized lymphocyte samples was equal to or greater than 8-lo%, depending on the mean variance of each assay. Mean values for each population were derived from 16 individual samples.

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FIGURE1. Cytotoxicity of spleen cells from C3H/Hej mice at various times I ter resection of a p;ogressively growing MCA sarcoma. Shaded bars indicate a significant difference at the 95% confidence level in mean counts per minute of 16 samples of spleen cells from mice post-tumor resection (“immune”) compared to normal spleen cells. Numbers above bars indicate values derived from samples assayed at the same time. RESULTS Cytotoxicity of Spleen Cells f r o m Mice After Tumor Resection

C3H/Hej mice were injected with lo5 fibrosarcoma cells. Nine days later, the progressively growing tumors were amputated. One to 5 weeks after tumor resection, the animals were sacrificed, the spleens were resected, and the cytotoxicity of the spleen cells was measured in an in vitro microcytotoxicity assay. For 2-3 weeks after tumor resection, significant cytotoxicity was seldom measured. Spleen cells were most consistently cytotoxic 3 weeks after tumor resection, although significant cytotoxicity was also present at 4 and 5 weeks. In three of six assays, after peak cytotoxicity was measured, there was a progressive diminution of lymphocytotoxicity with time. The results are summa1. Overall, significant cytotoxicity at any time was measured rized in FIGURE

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in only 40% of the assays. This led to the investigation of various methods that would increase the cytotoxicity of spleen cells in mice after tumor resection. Selective Concentration of Cytotonic Spleen Cells by Imriiunoadherence

Stulting and Berke," who used the murine EL4 lymphoid leukemia system, reported that a subpopulation of immune spleen cells adherent to target tumor cells was more cytotoxic than were nonadherent lymphocytes or the whole spleen cell preparation. We, therefore, attempted to increase the reactivity of spleen cell preparations in the microcytotoxicity assay by concentrating adherent lymphocytes. Three weeks after tumor resection, spleen cells were incubated over a monolayer of tumor cells isologous to the in vivo sensitizing tumor for 5 hr. Nonadherent cells were aspirated, and adherent lymphocytes and tumor cells were removed with 5 mM EDTA. Subsequently, the adherent lymphocytes were separated from the tumor cells on a continuous Ficoll gradient. FIGURE2 summarizes our results in three assays. In one of three assays, significant cytotoxicity was observed in the whole population, In none of the assays was the nonadherent population cytotoxic. In contrast, the adherent lymphocytes were consistently cytotoxic. However, selective concentration of adherent cells did not yield a cytotoxic population in all assays where the numbers of immune lymphocytes were too few to be effectively concentrated.

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FIoune 2. Cytotoxicity of spleen cells after selective concentration by immunoadherence. Shaded bars indicate significant differences at the 95% confidence level. Spleen cells were recovered from mice 3 weeks after tumor resection.

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Days of Preincubation FIGURE 3. Cytotoxicity of spleen cells secondarily exposed to tumor cells in vitro 8 weeks after tumor resection. Open bars indicate normal minus “immune” spleen cells; both were incubated in media alone for 72 hr prior to use in the assay. Shaded bars indicate normal minus “immune” spleen cells; both were incubated in the presence of mitomycin C-treated (30 pg/cma for 1 hr) tumor cells isologous to the in vivo tumor for 7 2 hr at a spleen cel1:tumor cell ratio of 25: I prior to use of spleen cells in the assay. Asterisks denote significant differences at the 95% confidence level.

Secondary Expossure of lrnrnuite Spleen Cells io lsologous Tumor Cells in Vitro

In a lymphocyte population in which few cells are immediately reactive toward the target tumor cells, there may be a subpopulation of cells that can respond to a secondary exposure to tumor antigens. To test this hypothesis, spleen cells recovered from mice 8 weeks after tumor resection were incubated in vitro in media alone or in the presence of mitomycin C-blocked isologous tumor cells. The lymphocytes were incubated for 1-4 days prior to use in the microcytotoxicity assay. FIGURE3 reveals that, when compared to normal spleen cells treated in an identical manner, only “immune” spleen cells incubated in the presence of tumor cells for 3 days or longer became significantly cytotoxic. In a dose-response study of mitomycin C-treated tumor cells, “immune” spleen cells 8 weeks after tumor resection, were preincubated with tumor cells at 1ymphocyte:tumor cell ratios of 200: 1-1 0: 1. Peak cytotoxicity occurred at a ratio of 25 :I (FIGURE 4 ) . Thus, to consistently generate a secondary immune response in vitro in our tumor system, lymphocytes and tumor cells had to be preincubated for 72 hr or longer at a 1ymphocyte:tumor cell ratio of 25: 1. Specificity of Secondary Cell-Mediated Immune Response

In the process of restoring the cytotoxicity of a spleen cell population, lymphocytes previously exposed to a tumor antigen in vivo must have the ability to recognize the same specific tumor antigen in vitro and generate an

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Preineubation Lymphocyte /Tumor Ratio

FIGURE4. Cytotoxicity of spleen cells stimulated by tumor cells in vitro 8 weeks after tumor resection; effect of difFerent spleen:tumor cell ratios during preincubation. Double dagger denotes normal minus “immune” spleen cells; both were incubated in media alone or in the presence of mitomycin C-treated tumor cells that were isologous to the in vivo tumor at spleen cel1:tumor cell ratios of 2OO:l-1O:l for 72 hr prior to use of spleen cells in the assay. Shaded bars indicate significant differences at the 95% confidence level. accelerated immune response. Spleen cells were recovered from mice who had carried a MC93 fibrosarcoma 5 weeks earlier. The spleen cells were incubated with both mitomycin C-treated and irradiated MC93 and MC43 tumor cells for 3 days at a 1ymphocyte:tumor cell ratio of 25: 1. As shown in FIGURE 5, spleen cells “immune” to MC93 responded to “inactivated” MC93 tumor cells by generating a cytotoxic population but were not affected by in vitro exposure to a different syngeneic fibrosarcoma, MC43. Thus, it appears that the recognition of tumor antigen and subsequent generation of an effector cell population reflect the individual specificity of tumor antigens commonly associated with chemically induced tumors. DISCUSSION The purpose of the present study was twofold. First, we wanted to measure, with an in vitro microcytotoxicity assay, the tumor immune response after resection of a progressively growing murine sarcoma. Second, we wanted to develop in vitro techniques that would enhance immunity after tumor removal. Our data showed that significant cytotoxicity was present 3-5 weeks after tumor removal. Before or after this period of time, significant cytotoxicity was seldom observed. Le Francois et d . , 5 Whitney et ai.,lG and Barski and Younl all reported on the effect of tumor resection on the development of cytotoxicity in murine syngeneic tumor systems by use of the colony growth inhibition test. These investigators reported the presence of significant cytotoxicity 23, 5 , and 12 days, respectively, after tumor removal. Lymphocytes remained cytotoxic for 50, 26, and 56 days, respectively. In the present study, similar to those reported above, an “eclipse“ period was seen after tumor resection, during which time the lymphocytes remained refractory prior to the development of peak cytotoxicity. In contrast, however, we found that the magnitude of the

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cellular immune response diminished rapidly 2 1-35 days after tumor resection, whereas Le Francois et U I . ~and Barski and Youn observed a strong immune response 50 and 56 days, respectively, after tumor removal. This apparent conflict in data is probably due to the use of different in vitro cellular cytotoxicity assays. The microcytotoxicity assay used in our study was incubated for 24 hr and measured the effect of lymphocytes immediately reactive toward the target tumor cells. Le Francois et uL5 and Barski and Youn both used the colony growth inhibition (CGI) test, where lymphocytes and target tumor cells are incubated together for 7-15 days, so that the CGI test probably measures not only immediate reactivity but also the ability of a population of lymphocytes to generate cells with lytic properties during prolonged exposure in vitro to target tumor cells. While we found that spleen cells 5 weeks o r longer after tumor removal were not cytotoxic in our assay, the same spleen cell preparations could generate cytotoxic lymphocytes if cocultivated with “inactivated” tumor cells for 72 hr or more at a preincubation 1ymphocyte:tumor cell ratio of 25: 1. Rollinghoff investigated the in vitro secondary antitumor response with the “Cr release assay in the syngeneic mouse plasma cell tumor system. At a 1ymphocyte:tumor cell ratio of 60:l during preincubation, peak cytotoxicity occurred at 4-5 days during the secondary response, whereas peak cytotoxicity did not occur until 7 days during the primary response. Plata et al.1° compared the response of normal C57BW6 spleen cells to that of C57BL/6 spleen cells from mice that had rejected a murine sarcoma virus (MSV) -induced sarcoma 15-30 days earlier when both were incubated in vitro with irradiated MSV sarcoma cells

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Preincubot ion FIGURE5 . Specificity of cytotoxicity of spleen cells stimulated by tumor cells in vitro S weeks after resection of an MC93 sarcoma. Bars indicate the difference in counts per minute of normal minus “immune” spleen cells; both were incubated in media alone or in the presence of “inactivated” MC93 or MC43 tumor cells at a spleen cel1:tumor cell ratio of 25:l prior to use of spleen cells in the assay. Tumor cells were “inactivated” by mitomycin C treatment (30 pg/cma for 1 hr) or by irradiation (SO00 R ) . Shaded bars indicate significant differences at the 95% confidence

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at a 1ymphocyte:tumor cell ratio of 25:l. With the W r release assay, lytic activity was present by Day 5 in the primary culture and by Day 3 in the secondary culture. Lytic activity on Day 7 was 23-fold higher in the secondary culture. Both Rollinghoff l 2 and Plata et aI.1O found that the secondary in vitro reaction was specific, and both postulated that a population of antigen-reactive cells, referred to as “memory cells,” were involved in the generation of cytotoxic lymphocytes. Our data support this hypothesis, because spleen cells resected 5-8 weeks post-tumor removal were seldom cytotoxic, but the same cells could generate an effector cell population when incubated in vitro with tumor cells isologous to the in vivo sensitizing tumor at an accelerated rate, compared to a population of normal spleen cells treated in an identical manner. Furthermore, we also found this secondary reaction to be specific. If the secondary reaction involves an “antigen-specific memory cell,” the magnitude of the response may depend on the availability of this cell type in the spleen cell population. We know that cytotoxic lymphocytes are present in the spleen 2-5 weeks after tumor resection. Noncytotoxic lymphocytes responsive to secondary tumor antigen exposure have been observed at 5 and 8 weeks after tumor resection. Experiments are presently underway to measure the magnitude of the secondary reaction from 1 to 12 weeks post-tumor resection in an effort to define the ideal time to induce a secondary reaction in this murine tumor system. It is of interest that Plata et a1.lo observed that the secondary response of spleen cells collected 5 days after MSV inoculation was already five times greater than that of normal spleen cells. Furthermore, Le Francois et aL5 reported that the “eclipse” period prior to the development of cytotoxic lymphocytes after tumor resection could be significantly shortened if the mice were inoculated 4 days after surgery with their own irradiated tumor cells. We hope that these studies will help resolve the perplexing clinical questions of when and in what manner it may be beneficial to intervene immunotherapeutically after resection of a progressively growing tumor. The feasibility of using in vitro sensitized autologous lymphocytes for adoptive immunotherapy is worthy of additional study. REFERENCES 1. BARSKI, G. & J. K. YOUN.1969. J. Nat. Cancer Inst. 43: 111. 2. BURTON, R.,J. THOMPSON & N. L. WARNER. 1975. J. Immunol. Methods 8: 133. J., H. D. ENGERS, H. R. MACDONALD & K. T. BRUNNER.1974. J. 3. CEROTTINI, Exp. Med. 140: 703. 4. KALL,M.A. & I. HELLSTROM. 1975. J. Immunol. 114: 1083. D., J. K. YOUN,J. BELEHRADEK, JR. & G . BARSKI. 1971. J. Nat. 5. LE FRANCOIS, Cancer Inst. 46: 981. 6. LUNDAK, R. L. & D. J. RAIDT. 1973. Cell. Immunol. 9 60. I. W., E. ESBER& J. R. WUNDERLICH. 1973. Fed. Proc. 32: 173. 7. MARTIN, J. W.,J. R. WUNDERLICH & J. MACDONALD. 1973. Israel J. Med. Sci. 8. MARTIN, 9: 324. A. B. & F. H. BACH. 1973. J. Immunol. Methods 3: 147. 9. PECK, 10. PLATA, F., J. C. CEROTTINI 8c K. T. BRUNNER. 1975. Unpublished manuscript. 11. PRETLOW, T. G. 1971. Ann. Biochem. 41: 248. 12. ROLLINGHOFF, M. 1974. I. Immunol. 112: 1718. 1975. J. Nat. Cancer 13. SHIKU,H., M. A. BEAN,L. J. OLD& H. F. OETTGBN. Inst. 54: 415. 14. S T U L T ~R. G D. , &G.BERKE. 1973. J. Exp. Med. 137: 932. R. B., J. G. LEVY & A. G. SMITH. 1974. J. Nat. Cancer Inst. 53: 111. 15. WHITNEY,

Discussion paper: in vitro augmentation of tumor immunity in a murine methylcholanthrene sarcoma system.

DISCUSSION PAPER: I N VZTRO AUGMENTATION OF TUMOR IMMUNITY IN A MURINE METHYLCHOLANTHRENE SARCOMA SYSTEM * Melvin A. Yarlott, Jr. and Charles F. McKha...
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