Itit. J . Cancer: 17, 748-754 (1976)
SPECIFIC SENSITIZATION OF LYMPHOCYTES TO TUMOR ANTIGENS BY CO-CULTIVATION WITH PERITONEAL CELLS EXPOSED TO SUCH ANTIGENS lngegerd HELLSTROM and Karl Erik HELLSTROM Division of Immunology, Fred Hutchitison Cancer Research Center, I I24 Columbia Street, Seattle, Washington 98104; and Departments of Microbiology and Pathology, University of Washington, Seattle, Washington 9819.5. USA SUMMARY
Peritoneal cells from BALBIc mice were seeded into SO-mm plastic Petri dishes and exposed either to antigen extracts prepared from individual BALBIc sarcomas having unique tumor-specific antigens, to similar extractsprepared from 14- to 18-day-old mouse embryos, or to culture medium only. Lymphoid cells from BALBIc lymph nodes were then added to the peritioneal cells. Following 4-5 days of co-cultivation, the lymphoid cells were harvested and tested in a 30 h microcytotoxicity assay for reactivity against tumor cells carrying the respective sensitizing antigens as well as against control cells which consisted of either tumor cells lacking the sensitizing antigens or normal skin fibroblasts. By this approach sensitization to individually unique tumor antigens, as well as to antigens shared by mouse embryos and mouse sarcoma cells, was achieved.
Several recent reports indicate that lymphoid cells (LC) can be sensitized in vitro to tumor-specific antigens present on syngeneic neoplastic cells and become reactive to cells carrying the sensitizing antigens (Wagner and Rollinghoff, 1973 ; Warnatz and Scheiffarth, 1974; Kall and Hellstrom, 1975; Kall et a]., 1975; Plata et al., 1975; Martin-Chandon et al., 1975). The reported reactivity of the in vitro sensitized cells has varied from a n essentially tumorspecific one seen e.g. in the study of Kall and Hellstrom (1975) to a non-specific reactivity seen e.g. by Martin-Chandon et al. (1975). In most published experiments, the sensitizing material has been irradiated or mitomycin-C-treated cells derived from cultivated neoplasms. It would be advantageous, however, if antigen preparations, rather than whole cells, could also be used as immunogens. This would make it possible to sensitize against well-defined tumor antigens, to study how modifications of such antigens would influence sensitization, to work with antigens prepared directly from animal (or human) tumors, and to study neoplasms which do not easily adapt themselves to culture conditions. A procedure was recently developed by Treves et al. (1976), by which in vitro sensitization to tumor antigen preparations,
rather than to intact cells, indeed became possible. These workers exposed material containing tumor antigens to peritoneal cells (mostly macrophages) explanted in vitro; after incubation, LC were added to the antigen-exposed macrophages and co-cultured with these for 4-5 days. Under these conditions an effector cell population, which was cytocidal to target cells carrying the sensitizing antigens, was generated. Lymphocytes exposed to antigen alone or to antigen bound to fibroblasts did not become reactive. We present here attempts to confirm and extend some of the findings of Treves et al. (1976), using five BALB/c sarcomas of recent origin which have individually unique tumor-specific transplantation antigens. We have tried to establish the degree of specificity of any sensitization detected by carrying out most experiments in a " criss-cross " pattern against target cells from at least two different tumors. Tumor extracts, pre-tested for antigen activity by the leukocyte adherence inhibition (LAI) assay of Halliday et al. (1974), provided the sensitizing tumor antigens for our experiments. We also investigated whether LC exposed to peritoneal cells to which antigenic extracts from 14- to 18-day-old mouse embryos had been added could become reactive to putatively embryonic antigens shared by various mouse tumors (Baldwin, 1973; Hellstrom and Hellstrom, 1974). MATERIAL AND METHODS
Animals Brother-sister mated BALB/c mice, bred in our laboratory, were used. These mice permanently accept skin grafts within the strain. They were maintained on a standard pellet diet and water given ad libitum. Target cells for in vitro tests Five sarcomas, Nos. 1315, 1321, 1408, 1409 and 1410, were used. They had been induced by inoculating BALB/c mice intramuscularly with 0.1 mg methylcholanthrene dissolved in trioctanoin, and were kept by transplantation in BALB/c mice. Prior
Received: February 18, 1976.
LYMPHOCYTE SENSITIZATION TO TUMOR ANTIGENS
to the present experiments these tumors had been transferred three to six times in vivo. They were tested for tumor-specific transplantation antigenicity by immunization with living tumor, followed by its removal and subsequent challenge with a small tumorigenic dose of cells from either the immunizing tumor or from a separate one. Evidence was thereby obtained for individually unique transplantation antigens in these tumors; they were thus found to behave like other chemically induced sarcomas (Baldwin, 1973). Tumor suspensions prepared by trypsin izati on were cultivated in Waymouth's medium containing 20% fetal calf serum as previously described (Hellstrom et al., 1970). After two to five passages in vitro, the tumor cells were used as targets for microcytotoxicity assays. Fibroblasts used as control cells were established from explanted skin of newborn BALB/c mice; they were handled in the same way as the tumor cells. Peritioneal cell ( P C ) and lymphoid cell (LCJ donors
Peritoneal cells (PC) were obtained by rinsing the peritoneal cavity of approximately 2-month-old, previously untreated BALB/c mice with Waymouth's culture medium, containing 1 IU of heparin (Abbott Laboratories, Tukwila, Washington) per ml of medium. Approximately lo8cells were obtained from each mouse. These cells were used to establish the cell layer on which the LC could be sensitized (see below). Similarly obtained PC were also used for LA1 tests (see below). Mice which were to provide immune PC for LA1 tests were injected with approximately 1 x lo5 viable tumor cells. When the tumors reached about 8 mm in diameter, they were excised. One to 2 weeks later, immune PC were obtained as above. Lymphoid cells (LC) were obtained from lymph nodes of 2-month-old BALB/c mice and were brought into suspension by gently pressing the lymph nodes through a 60 mesh stainless steel screen. The cells were washed and suspended in Waymouth's medium. Preparation of antigen extracts
Antigen extracts were prepared according to a procedure described by Halliday et a / . (1974). Tumors (or 14- to 18-day-oldBALB/c embryos) were removed from mice, placed on ice and washed in cold phosphate buffered saline (PBS) to remove blood. Necrotic tissue (when present) was removed. The remaining tumor tissue was minced into fine pieces and pressed through a 60 mesh stainless steel screen. This tumor tissue was weighed, suspended in PBS (4 mI/g tumor tissue), and homogenized with a GKH electronic controlled laboratory mixer ( G . K. Heller Corp., Floral Park, New York, USA) on speed No. 2, and with a Thomas teflon pestle tissue homogenizer
749
(Arthur Thomas Co., Philadelphia, Pa., USA) size B304. The homogenized material was centrifuged for 30 min at 1,OOOxg at 4" C. The supernatant was harvested and centrifuged again for 30min at 20,OOOxg and 4" C with an RC-5 superspeed refrigerated centrifuge. It was then frozen overnight at -20" C. On the following day the material was thawed, centrifuged for 10 rnin at 1,000 x g , pipetted into tubes in 0.2 and 0.4 ml aliquots, and frozen at -70" C until used (the intermediary freezing at -20" C followed by centrifugation the following day was empirically found more often to give materia1 with specific reactivity in the LA1 tests). LAI tests of antigen preparations
A leukocyte adherence inhibition (LAO assay developed by Halliday et al. (1974), and modified by Holan et al. (1974), was used to pre-test the quality of the antigen preparations used for sensitization. Peritoneal cells (see above) from tumor-immune and untreated, control donors were suspended in cold Eagle's medium with heparin; in experiments with fetal antigens, PC from multiparous mice (pregnant at the time of the test) were studied as well. The PC were centrifuged at 200 x g for 10 rnin at 4" C , the supernatants discarded, and the pelleted cells transferred to 5-ml plastic tubes in approximately 3 ml of cold Eagle's medium, and centrifuged at 200 x g for 6 min. The supernatants were discarded. The washed PC were suspended in 1 ml of NH,Cl (0.15 M) at room temperature for 5 rnin in order to lyse red blood cells. The PC were washed twice in 4ml of Eagle's medium and adjusted to a concentration of 4 x 1O8 cells/ml. An antigen preparation (see above) was thawed, diluted (usually 15), in Eagle's medium, and dispensed in 0.15-ml aliquots to 10-ml glass tubes. To each tube 0.15 ml(6 x lo5cells) of the cell suspension (either control or immune) was added. The tubes were incubated in a horizontal position for 2 h at 37" C in air containing 5 % CO,. Triplicates were prepared for each determination. After incubation, the tubes were placed in a vertical position and their contents mixed gently with a pipet. A sample was withdrawn and the number of nonadherent cells counted in a hemocytometer. The percentage of non-adherent cells in the number of cells initially incubated was then calculated for both the control and the experimental groups. Percentage leukocyte adherence inhibiton (XLAI) was computed as the difference between the percentage nonadherent cells with immune as compared to control PC. A tumor extract was considered positive for tumor antigen when the number of non-adherent cells was significantly greater in the presence of the extract prepared from the specific tumor (i.e. the one to which the mice contributing immune cells were immunized) than in the presence of extract from a
750
HELLSTR6M AND HELLSTROM
different tumor. Fetal antigens were evaluated for their ability to give leukocyte adherence inhibition in conjunctim with peritoneal cells from multiparous BALB/c mice. Further information about our LA1 data will be published elsewhere (Hellstrom and Hellstrom, 1976). In vitro setisitization procedure Peritioneal cells from BALB/c mice were washed once in RPMI 1640 medium containing 10% heatinactivated fetal calf serum referred to below (as 10% RP 4 0 and the number of viable cells was determined. One million cells were suspended in 4 ml of 1076 RPMI, seeded into 50 mm Falcon plastic Petri dishes (Falcon Plastics, Oxnard, Calif., USA), and incubated for approximately 4 h at 37" C in air containing 5 0 4CO,. Each Petri dish was then rinsed with 2 ml of 37" C medium to remove any non-adherent cells. The cells remaining attached to the dish were mostly macrophages on morphological criteria, but no thorough studies as to their nature have yet been performed. To each dish 0.1 ml undiluted antigen extract (prepared and pretested as described above) and 2 ml lo:/, RPMI were added; control dishes received RPMI without added antigen. The dishes were incubated for 45min at 37°C with gentle rocking; the medium containing antigen was then removed, and a suspension of lo7LC in 4 ml of 10% RPMI was added to each dish. The cultures were incubated for 4 or 5 days at 37" C in a moist 5 % C 0 2in air atmosphere. During this time, they were fed daily with 0.5 ml of 10% RPMI. After 4-5 days' incubation, the medium containing the LC was removed from each dish. Each dish was rinsed with an additional 2 m l medium and the rinsing fluid pooled with the removed medium. The suspended LC were collected by centrifugation, washed once with 10% RPMI and resuspended in 6 ml 10% RPMI. The resuspended cells were then applied to a nylon wool column (Julius et a/., 1973) which was found to be useful for removing cells that would stick to the microtest plates in the microcytotoxicity assay. The column was prepared from 6 m l nylon wool which was pre-rinsed with 10% RPMI and placed in a 20ml disposable plastic syringe (18 mm diameter). The cells were allowed to flow quickly through the column (the passage took around 2 min). The column was then washed twice with 6-ml portions of 10% RPMI to remove loosely attached cells. The cells which had passed through the column were harvested by centrifugation, resuspended in 10% RPMI, and tested in microcytotoxicity assays. Between 8 and 10% of the cells originally seeded in the Petri dishes were recovered. The cells had a viability exceeding 90%. according to trypan blue exclusion tests.
Assays of lymphocyte-mediated cytotoxicity
Lymphoid cells sensitized on PC previously exposed to antigens were tested for their ability to destroy either target cells carrying the sensitizing antigens or control target cells lacking these antigens. A previously described microcytotoxicity test was used, in which target cells were plated in Falcon No. 3040 Microtest plates (Hellstrom and Hellstrom, 1971). One hundred target cells/well were plated. A pattern described elsewhere by Brown et al. (1976) for placing various LC suspensions into selected wells of the Microtest plate was used. This pattern had the advantage of minimizing the impact of the position of experimental and control group wells on the Microtest plates. After 12-18 h, when the target cells had become attached, effector cells were added to each well. The number of such cells per well varied from 588 to 9,400, representing ratios of approximately 6-100 LC/target-cell. These doses were chosen on the basis of pilot experiments, in which various doses from 150,000 LC/well were titrated at 1:2 dilutions. A dose of 9,400 LC/well was the highest in which there was still no detectable carry-over of cells attaching to the bottom of empty Microtest wells, when plated there as controls. A minimum of eight replicates per LC dose was used. The plates were incubated for another 30 h before staining. All plates were coded and counted without knowledge of the code. The percentage of target cells remaining attached after exposure to experimental group LC was calculated by comparison with groups receiving the same dose of control LC (sensitized on PC not exposed to antigen); a lower number of target cells remaining attached after exposure to experimental group LC than after exposure to control LC was considered to reflect a cytotoxic effect of the experimental group LC (Hellstrom and Hellstrom, 1971). The statistical significance of target cell destruction by experimental LC as compared to control LC was determined by Student's t-tests. KESULTS
Five experiments were performed in which LC were co-cultivated for 4-5 days with PC which had been pre-treated with tumor (or fetal) antigen extracts. The LC were then harvested and assayed with the microcytotoxicity test to determine whether they had become reactive against tumor cells of the lines from which the sensitizing antigens had been prepared, as well as against tumor cells from other lines (as controls). Most assays for tumor specific sensitization were carried out in a criss-cross pattern, with the same LC suspensions being tested on tumor cells carrying or lacking the sensitizing antigens. Since many tumors also contain cross-reacting,
75 1
LYMPHOCYTE SENSITIZATION TO TUMOR ANTIGENS
putatively fetal antigens (Baldwin, 1973), LC sensitized to extracts of embryos of 14-18 days' gestation were tested against cells from several tumors in vitro. In order to illustrate the lay-out used for this study, Table I presents (part of) one experiment in detail in which tumor-specific reactivity was demonstrated in a '' criss-cross " test with LC sensitized by PC exposed to either No. 1321 or 1409 tumor antigens. The Table also illustrates that sensitization was not totally specific, since some cross-reactivity was detected at the higher LC dose (9,400 LC/well); this cross-reactivity was not seen at the lower LC dose (4,700/well). Table I1 presents the five sensitization experiments carried out to date. It shows that the pattern of reactivity depicted in Table I holds true for the other experiments as well, i.e. that sensitization could be obtained with a relatively good (albeit not absolute) tumor specificity. In some experiments, generation of cytotoxic LC was also detected after the LC had been co-cultivated with PC pre-exposed to fetal antigens. The data presented in Table I1 are summarized in Table 111. It is apparent from the latter Table that statistically significant differences could be detected between groups in which LC were sensitized to the antigens of the target tumors as compared to antigens prepared from '' other " tumors. All but one of the antigen preparations used had been pretested with the LA1 assay and found to give leukocyte adherence inhibition in the presence of peritoneal cells from specifically immune tumors (Table In; however, there was n o clear correlation
between the degree of leukocyte adherence inhibition and the ability of an antigen preparation to sensitize. The LA1 data will be presented in detail elsewhere (Hellstrom and Hellstrom, 1976). DISCUSSION
The present findings confirm the report of Treves et a/. (1976) that lymphoid cells (LC) co-cultivated with peritoneal cells (PC) which have been preexposed to tumor antigens can become cytotoxic to neoplastic cells carrying the respective antigens. They extend the previous work by showing that the sensitization obtained is essentially tumor-specific, even if a certain cross-reactivity, particularly at higher LC: target-cell ratios, was seen in some tests. These results agree with in vivo data, according to which the tumors studied have individually unique tumor-specific transplantation antigens with little (if any) cross-reactivity (Baldwin, 1973 ; Hellstrom and Hellstrom, 1976). Our data also extend published work by indicating that sensitization to putatively embryonic antigens can be obtained in vitro. The latter data should, however, be viewed carefully until controls have been made in which LC are sensitized by co-ciiltivsitiorf"With PC to which antigenic extracts from various adult, normal tissues have been bound. The fact that we observed a relatively specific reactivity of the in vitro sensitized cells agrees with previous observations made by our group in experiments using mitomycin-C-treated sarcoma cells as sensitizers (Kall and Hellstrom, 1974; Kall et a]., 1975). It also agrees with reports by some other investigators (Warnatz and Scheiffarth, 1974;
TABLE I PRESENTATION OF (PART OF) ONE EXPERIMENT (No. 4 I N TABLE 11) SHOWING NUMBER OF REMAINING TARGET CELLSjWELL AT CONCLUSION OF MICROCYTOTOXICITY ASSAY WITH LYMPHOID CELLS (LC) SENSITIZED ON ANTIGEN-FED PERITONEAL CELLS I N VITRO 9,400 LC/well Target cells
Antigen to which LC sensitized
1321
None 1410 1321 1409
Fetal 1409
None 1410 1321 1409
Fetal ~~
.
Remaining target cellr/well. Mean fSE
4,700 LC/well
Percentage reduction
Remaining target cells/well. M e a n f s ~
Percentage reduction
39.3&2.5 38.613.1 20.3~ t 2 . 0 29.0&2.9 35.9h2.1
Control 1.8
36.353.5 35.9 1 2 . 0 26.3 & 1.9 34.1 *2.1 28.3 h2.3
Control 1.1 1
14.5k0.8 12.4k0.8 12.111 .o 7.4k1.2 11.6k 1.3
Control
20.8h1.5 22.0h 1 .o 24.451.7 15.451.9 20.1 k 2 . 0
Control
48.4*** 26.2** 8.7 14.5 16.6 49.0*** 20.0
28.4* 6.1 22.0
-5.8 -17.3 26.0* 3.4
_____
' Percentage reduction calculated from comparison with number of remaining target cells/well in the presence of LC co-cultivated with peritoneal cells not fed antigen. Statistical significance of difference between experimental and control groups calculated accordins to Student's t-test : * p 1 0 . 0 5 ; **p 10.02; *'*p
SPECIFIC SENSITIZATION OF LYMPHOCYTES TO TUMOR ANTIGENS BY CO-CULTIVATION WITH PERITONEAL CELLS EXPOSED TO SUCH ANTIGENS lngegerd HELLSTROM and Karl Erik HELLSTROM Division of Immunology, Fred Hutchitison Cancer Research Center, I I24 Columbia Street, Seattle, Washington 98104; and Departments of Microbiology and Pathology, University of Washington, Seattle, Washington 9819.5. USA SUMMARY
Peritoneal cells from BALBIc mice were seeded into SO-mm plastic Petri dishes and exposed either to antigen extracts prepared from individual BALBIc sarcomas having unique tumor-specific antigens, to similar extractsprepared from 14- to 18-day-old mouse embryos, or to culture medium only. Lymphoid cells from BALBIc lymph nodes were then added to the peritioneal cells. Following 4-5 days of co-cultivation, the lymphoid cells were harvested and tested in a 30 h microcytotoxicity assay for reactivity against tumor cells carrying the respective sensitizing antigens as well as against control cells which consisted of either tumor cells lacking the sensitizing antigens or normal skin fibroblasts. By this approach sensitization to individually unique tumor antigens, as well as to antigens shared by mouse embryos and mouse sarcoma cells, was achieved.
Several recent reports indicate that lymphoid cells (LC) can be sensitized in vitro to tumor-specific antigens present on syngeneic neoplastic cells and become reactive to cells carrying the sensitizing antigens (Wagner and Rollinghoff, 1973 ; Warnatz and Scheiffarth, 1974; Kall and Hellstrom, 1975; Kall et a]., 1975; Plata et al., 1975; Martin-Chandon et al., 1975). The reported reactivity of the in vitro sensitized cells has varied from a n essentially tumorspecific one seen e.g. in the study of Kall and Hellstrom (1975) to a non-specific reactivity seen e.g. by Martin-Chandon et al. (1975). In most published experiments, the sensitizing material has been irradiated or mitomycin-C-treated cells derived from cultivated neoplasms. It would be advantageous, however, if antigen preparations, rather than whole cells, could also be used as immunogens. This would make it possible to sensitize against well-defined tumor antigens, to study how modifications of such antigens would influence sensitization, to work with antigens prepared directly from animal (or human) tumors, and to study neoplasms which do not easily adapt themselves to culture conditions. A procedure was recently developed by Treves et al. (1976), by which in vitro sensitization to tumor antigen preparations,
rather than to intact cells, indeed became possible. These workers exposed material containing tumor antigens to peritoneal cells (mostly macrophages) explanted in vitro; after incubation, LC were added to the antigen-exposed macrophages and co-cultured with these for 4-5 days. Under these conditions an effector cell population, which was cytocidal to target cells carrying the sensitizing antigens, was generated. Lymphocytes exposed to antigen alone or to antigen bound to fibroblasts did not become reactive. We present here attempts to confirm and extend some of the findings of Treves et al. (1976), using five BALB/c sarcomas of recent origin which have individually unique tumor-specific transplantation antigens. We have tried to establish the degree of specificity of any sensitization detected by carrying out most experiments in a " criss-cross " pattern against target cells from at least two different tumors. Tumor extracts, pre-tested for antigen activity by the leukocyte adherence inhibition (LAI) assay of Halliday et al. (1974), provided the sensitizing tumor antigens for our experiments. We also investigated whether LC exposed to peritoneal cells to which antigenic extracts from 14- to 18-day-old mouse embryos had been added could become reactive to putatively embryonic antigens shared by various mouse tumors (Baldwin, 1973; Hellstrom and Hellstrom, 1974). MATERIAL AND METHODS
Animals Brother-sister mated BALB/c mice, bred in our laboratory, were used. These mice permanently accept skin grafts within the strain. They were maintained on a standard pellet diet and water given ad libitum. Target cells for in vitro tests Five sarcomas, Nos. 1315, 1321, 1408, 1409 and 1410, were used. They had been induced by inoculating BALB/c mice intramuscularly with 0.1 mg methylcholanthrene dissolved in trioctanoin, and were kept by transplantation in BALB/c mice. Prior
Received: February 18, 1976.
LYMPHOCYTE SENSITIZATION TO TUMOR ANTIGENS
to the present experiments these tumors had been transferred three to six times in vivo. They were tested for tumor-specific transplantation antigenicity by immunization with living tumor, followed by its removal and subsequent challenge with a small tumorigenic dose of cells from either the immunizing tumor or from a separate one. Evidence was thereby obtained for individually unique transplantation antigens in these tumors; they were thus found to behave like other chemically induced sarcomas (Baldwin, 1973). Tumor suspensions prepared by trypsin izati on were cultivated in Waymouth's medium containing 20% fetal calf serum as previously described (Hellstrom et al., 1970). After two to five passages in vitro, the tumor cells were used as targets for microcytotoxicity assays. Fibroblasts used as control cells were established from explanted skin of newborn BALB/c mice; they were handled in the same way as the tumor cells. Peritioneal cell ( P C ) and lymphoid cell (LCJ donors
Peritoneal cells (PC) were obtained by rinsing the peritoneal cavity of approximately 2-month-old, previously untreated BALB/c mice with Waymouth's culture medium, containing 1 IU of heparin (Abbott Laboratories, Tukwila, Washington) per ml of medium. Approximately lo8cells were obtained from each mouse. These cells were used to establish the cell layer on which the LC could be sensitized (see below). Similarly obtained PC were also used for LA1 tests (see below). Mice which were to provide immune PC for LA1 tests were injected with approximately 1 x lo5 viable tumor cells. When the tumors reached about 8 mm in diameter, they were excised. One to 2 weeks later, immune PC were obtained as above. Lymphoid cells (LC) were obtained from lymph nodes of 2-month-old BALB/c mice and were brought into suspension by gently pressing the lymph nodes through a 60 mesh stainless steel screen. The cells were washed and suspended in Waymouth's medium. Preparation of antigen extracts
Antigen extracts were prepared according to a procedure described by Halliday et a / . (1974). Tumors (or 14- to 18-day-oldBALB/c embryos) were removed from mice, placed on ice and washed in cold phosphate buffered saline (PBS) to remove blood. Necrotic tissue (when present) was removed. The remaining tumor tissue was minced into fine pieces and pressed through a 60 mesh stainless steel screen. This tumor tissue was weighed, suspended in PBS (4 mI/g tumor tissue), and homogenized with a GKH electronic controlled laboratory mixer ( G . K. Heller Corp., Floral Park, New York, USA) on speed No. 2, and with a Thomas teflon pestle tissue homogenizer
749
(Arthur Thomas Co., Philadelphia, Pa., USA) size B304. The homogenized material was centrifuged for 30 min at 1,OOOxg at 4" C. The supernatant was harvested and centrifuged again for 30min at 20,OOOxg and 4" C with an RC-5 superspeed refrigerated centrifuge. It was then frozen overnight at -20" C. On the following day the material was thawed, centrifuged for 10 rnin at 1,000 x g , pipetted into tubes in 0.2 and 0.4 ml aliquots, and frozen at -70" C until used (the intermediary freezing at -20" C followed by centrifugation the following day was empirically found more often to give materia1 with specific reactivity in the LA1 tests). LAI tests of antigen preparations
A leukocyte adherence inhibition (LAO assay developed by Halliday et al. (1974), and modified by Holan et al. (1974), was used to pre-test the quality of the antigen preparations used for sensitization. Peritoneal cells (see above) from tumor-immune and untreated, control donors were suspended in cold Eagle's medium with heparin; in experiments with fetal antigens, PC from multiparous mice (pregnant at the time of the test) were studied as well. The PC were centrifuged at 200 x g for 10 rnin at 4" C , the supernatants discarded, and the pelleted cells transferred to 5-ml plastic tubes in approximately 3 ml of cold Eagle's medium, and centrifuged at 200 x g for 6 min. The supernatants were discarded. The washed PC were suspended in 1 ml of NH,Cl (0.15 M) at room temperature for 5 rnin in order to lyse red blood cells. The PC were washed twice in 4ml of Eagle's medium and adjusted to a concentration of 4 x 1O8 cells/ml. An antigen preparation (see above) was thawed, diluted (usually 15), in Eagle's medium, and dispensed in 0.15-ml aliquots to 10-ml glass tubes. To each tube 0.15 ml(6 x lo5cells) of the cell suspension (either control or immune) was added. The tubes were incubated in a horizontal position for 2 h at 37" C in air containing 5 % CO,. Triplicates were prepared for each determination. After incubation, the tubes were placed in a vertical position and their contents mixed gently with a pipet. A sample was withdrawn and the number of nonadherent cells counted in a hemocytometer. The percentage of non-adherent cells in the number of cells initially incubated was then calculated for both the control and the experimental groups. Percentage leukocyte adherence inhibiton (XLAI) was computed as the difference between the percentage nonadherent cells with immune as compared to control PC. A tumor extract was considered positive for tumor antigen when the number of non-adherent cells was significantly greater in the presence of the extract prepared from the specific tumor (i.e. the one to which the mice contributing immune cells were immunized) than in the presence of extract from a
750
HELLSTR6M AND HELLSTROM
different tumor. Fetal antigens were evaluated for their ability to give leukocyte adherence inhibition in conjunctim with peritoneal cells from multiparous BALB/c mice. Further information about our LA1 data will be published elsewhere (Hellstrom and Hellstrom, 1976). In vitro setisitization procedure Peritioneal cells from BALB/c mice were washed once in RPMI 1640 medium containing 10% heatinactivated fetal calf serum referred to below (as 10% RP 4 0 and the number of viable cells was determined. One million cells were suspended in 4 ml of 1076 RPMI, seeded into 50 mm Falcon plastic Petri dishes (Falcon Plastics, Oxnard, Calif., USA), and incubated for approximately 4 h at 37" C in air containing 5 0 4CO,. Each Petri dish was then rinsed with 2 ml of 37" C medium to remove any non-adherent cells. The cells remaining attached to the dish were mostly macrophages on morphological criteria, but no thorough studies as to their nature have yet been performed. To each dish 0.1 ml undiluted antigen extract (prepared and pretested as described above) and 2 ml lo:/, RPMI were added; control dishes received RPMI without added antigen. The dishes were incubated for 45min at 37°C with gentle rocking; the medium containing antigen was then removed, and a suspension of lo7LC in 4 ml of 10% RPMI was added to each dish. The cultures were incubated for 4 or 5 days at 37" C in a moist 5 % C 0 2in air atmosphere. During this time, they were fed daily with 0.5 ml of 10% RPMI. After 4-5 days' incubation, the medium containing the LC was removed from each dish. Each dish was rinsed with an additional 2 m l medium and the rinsing fluid pooled with the removed medium. The suspended LC were collected by centrifugation, washed once with 10% RPMI and resuspended in 6 ml 10% RPMI. The resuspended cells were then applied to a nylon wool column (Julius et a/., 1973) which was found to be useful for removing cells that would stick to the microtest plates in the microcytotoxicity assay. The column was prepared from 6 m l nylon wool which was pre-rinsed with 10% RPMI and placed in a 20ml disposable plastic syringe (18 mm diameter). The cells were allowed to flow quickly through the column (the passage took around 2 min). The column was then washed twice with 6-ml portions of 10% RPMI to remove loosely attached cells. The cells which had passed through the column were harvested by centrifugation, resuspended in 10% RPMI, and tested in microcytotoxicity assays. Between 8 and 10% of the cells originally seeded in the Petri dishes were recovered. The cells had a viability exceeding 90%. according to trypan blue exclusion tests.
Assays of lymphocyte-mediated cytotoxicity
Lymphoid cells sensitized on PC previously exposed to antigens were tested for their ability to destroy either target cells carrying the sensitizing antigens or control target cells lacking these antigens. A previously described microcytotoxicity test was used, in which target cells were plated in Falcon No. 3040 Microtest plates (Hellstrom and Hellstrom, 1971). One hundred target cells/well were plated. A pattern described elsewhere by Brown et al. (1976) for placing various LC suspensions into selected wells of the Microtest plate was used. This pattern had the advantage of minimizing the impact of the position of experimental and control group wells on the Microtest plates. After 12-18 h, when the target cells had become attached, effector cells were added to each well. The number of such cells per well varied from 588 to 9,400, representing ratios of approximately 6-100 LC/target-cell. These doses were chosen on the basis of pilot experiments, in which various doses from 150,000 LC/well were titrated at 1:2 dilutions. A dose of 9,400 LC/well was the highest in which there was still no detectable carry-over of cells attaching to the bottom of empty Microtest wells, when plated there as controls. A minimum of eight replicates per LC dose was used. The plates were incubated for another 30 h before staining. All plates were coded and counted without knowledge of the code. The percentage of target cells remaining attached after exposure to experimental group LC was calculated by comparison with groups receiving the same dose of control LC (sensitized on PC not exposed to antigen); a lower number of target cells remaining attached after exposure to experimental group LC than after exposure to control LC was considered to reflect a cytotoxic effect of the experimental group LC (Hellstrom and Hellstrom, 1971). The statistical significance of target cell destruction by experimental LC as compared to control LC was determined by Student's t-tests. KESULTS
Five experiments were performed in which LC were co-cultivated for 4-5 days with PC which had been pre-treated with tumor (or fetal) antigen extracts. The LC were then harvested and assayed with the microcytotoxicity test to determine whether they had become reactive against tumor cells of the lines from which the sensitizing antigens had been prepared, as well as against tumor cells from other lines (as controls). Most assays for tumor specific sensitization were carried out in a criss-cross pattern, with the same LC suspensions being tested on tumor cells carrying or lacking the sensitizing antigens. Since many tumors also contain cross-reacting,
75 1
LYMPHOCYTE SENSITIZATION TO TUMOR ANTIGENS
putatively fetal antigens (Baldwin, 1973), LC sensitized to extracts of embryos of 14-18 days' gestation were tested against cells from several tumors in vitro. In order to illustrate the lay-out used for this study, Table I presents (part of) one experiment in detail in which tumor-specific reactivity was demonstrated in a '' criss-cross " test with LC sensitized by PC exposed to either No. 1321 or 1409 tumor antigens. The Table also illustrates that sensitization was not totally specific, since some cross-reactivity was detected at the higher LC dose (9,400 LC/well); this cross-reactivity was not seen at the lower LC dose (4,700/well). Table I1 presents the five sensitization experiments carried out to date. It shows that the pattern of reactivity depicted in Table I holds true for the other experiments as well, i.e. that sensitization could be obtained with a relatively good (albeit not absolute) tumor specificity. In some experiments, generation of cytotoxic LC was also detected after the LC had been co-cultivated with PC pre-exposed to fetal antigens. The data presented in Table I1 are summarized in Table 111. It is apparent from the latter Table that statistically significant differences could be detected between groups in which LC were sensitized to the antigens of the target tumors as compared to antigens prepared from '' other " tumors. All but one of the antigen preparations used had been pretested with the LA1 assay and found to give leukocyte adherence inhibition in the presence of peritoneal cells from specifically immune tumors (Table In; however, there was n o clear correlation
between the degree of leukocyte adherence inhibition and the ability of an antigen preparation to sensitize. The LA1 data will be presented in detail elsewhere (Hellstrom and Hellstrom, 1976). DISCUSSION
The present findings confirm the report of Treves et a/. (1976) that lymphoid cells (LC) co-cultivated with peritoneal cells (PC) which have been preexposed to tumor antigens can become cytotoxic to neoplastic cells carrying the respective antigens. They extend the previous work by showing that the sensitization obtained is essentially tumor-specific, even if a certain cross-reactivity, particularly at higher LC: target-cell ratios, was seen in some tests. These results agree with in vivo data, according to which the tumors studied have individually unique tumor-specific transplantation antigens with little (if any) cross-reactivity (Baldwin, 1973 ; Hellstrom and Hellstrom, 1976). Our data also extend published work by indicating that sensitization to putatively embryonic antigens can be obtained in vitro. The latter data should, however, be viewed carefully until controls have been made in which LC are sensitized by co-ciiltivsitiorf"With PC to which antigenic extracts from various adult, normal tissues have been bound. The fact that we observed a relatively specific reactivity of the in vitro sensitized cells agrees with previous observations made by our group in experiments using mitomycin-C-treated sarcoma cells as sensitizers (Kall and Hellstrom, 1974; Kall et a]., 1975). It also agrees with reports by some other investigators (Warnatz and Scheiffarth, 1974;
TABLE I PRESENTATION OF (PART OF) ONE EXPERIMENT (No. 4 I N TABLE 11) SHOWING NUMBER OF REMAINING TARGET CELLSjWELL AT CONCLUSION OF MICROCYTOTOXICITY ASSAY WITH LYMPHOID CELLS (LC) SENSITIZED ON ANTIGEN-FED PERITONEAL CELLS I N VITRO 9,400 LC/well Target cells
Antigen to which LC sensitized
1321
None 1410 1321 1409
Fetal 1409
None 1410 1321 1409
Fetal ~~
.
Remaining target cellr/well. Mean fSE
4,700 LC/well
Percentage reduction
Remaining target cells/well. M e a n f s ~
Percentage reduction
39.3&2.5 38.613.1 20.3~ t 2 . 0 29.0&2.9 35.9h2.1
Control 1.8
36.353.5 35.9 1 2 . 0 26.3 & 1.9 34.1 *2.1 28.3 h2.3
Control 1.1 1
14.5k0.8 12.4k0.8 12.111 .o 7.4k1.2 11.6k 1.3
Control
20.8h1.5 22.0h 1 .o 24.451.7 15.451.9 20.1 k 2 . 0
Control
48.4*** 26.2** 8.7 14.5 16.6 49.0*** 20.0
28.4* 6.1 22.0
-5.8 -17.3 26.0* 3.4
_____
' Percentage reduction calculated from comparison with number of remaining target cells/well in the presence of LC co-cultivated with peritoneal cells not fed antigen. Statistical significance of difference between experimental and control groups calculated accordins to Student's t-test : * p 1 0 . 0 5 ; **p 10.02; *'*p
