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Specific MIF release by rat lymphocytes following incubation with syngeneic anti-tumor “Immune” RNA
CELLULAR
IMMUNOLOGY
Specific
18, 24&250 (1975)
MIF Release by Rat Lymphocytes Following Incubation with Syngeneic Anti-Tumor “Immune” RNA ‘9 ’ STEPHEN
R.
WALDMAN
s AND
YOSEF
H.
PILCH
of Oncology, Departnaent of Surgery, University of California, Los Angeles, California 90024, and Tumor Immunology Laboratory, Veteralzs Administration Hospital, Sepulveda, California 91343
Division
Received January 6,1975
Other investigators have previously shown that normal nonimmune lymphoid cells, after incubation with “Immune” RNA, will release MIF when these cells are incubated with the specific antigen used to immunize the RNA donor. This conversion can be detected with the macrophage migration inhibition assay. These observations have been confirmed in a system involvin, r the transfer of immune response to tumor associated antigens with syngeneic “Immune” RNA. Syngeneic “Immune” RNA was extracted from the spleens of Fischer 344/N rats bearing growing transplants of one or another of two syngeneic chemically induced sarcomas. Normal, nonimmune Fischer 344/N spleen cells were incubated with these RNA preparations. When these
RNA-incubated spleen cells were exposed to solubilized antigens from that particular tumor used to immunize the RNA donor, MIF was released.RNAse treatment of the “Immune” RNA abrogated the response,while DNAse or pronase treatment did not. RNA extracted from lymphoid organs of immune donors as well as from macrophages exposed to antigens, in vitro, has been demonstrated to convert normal nonimmune lymphocytes to immunoreactive status. These “Immune” RNA preparations have been shown to mediate cellular immune responses as well as antibody synthesis (l-8). Within the last few years, several groups of investigators have demonstrated the release of macrophage inhibitory factor (MIF) by human (5) and animal (6-8) lymphocytes following incubation with “Immune” RNA extracted from lymphoid cells of immune donors. Thus, an in vitro assay which correlates with delayed cutaneous hypersensitivity responses in viva was successfully employed to quantitate conversion of lymphoid cells to immunoreactive status following incubation with “Immune” RNA. Also, release of leukocyte inhibitory factor (LIF) (9) and MIF (10) by lymphocytes obtained from human cancer patients and obtained from animals following rejection of tumors (11) has been demonstrated in the presence of tumor antigen preparations. 1 Supported in part by Grant No. CA-14846 from the National Institutes of Health, Grant No. ET-28 from the American Cancer Society and by the Veterans Administration. 2 Reprint requests should be sent to Dr. Y. H. Pilch, Harbor General Hospital, 1000 W. Carson Street, Torrance, California 90509. s Research Scientist, City of Hope National Medical Center, Duarte, California 91010. 246 Copyright Q 1975 by Academic Press, Inc. All lights of reproduction in any form reserved.
SHORT
COhfMUNICATlONS
247
Recently, a quantitative microcytotoxicity assay for lymphocyte mediated cytotoxicity was employed to demonstrate that normal, nonimmune Fischer F344/N spleen cells, preincubated with RNA extracted from spleens of syngeneic rats bearing growing tumor isografts, were cytotoxic to tumor cells, in vitro (12). This was the first report of the transfer of immunity to tumor associated antigens with “Immune” RNA extracted from lymphoid tissues of tumor-bearing animals rather than hyperimmunized animals. In this report, the same model system was studied by demonstrating the release of MIF, in order to confirm that RNA extracted from tumor-bearing donors converts normal lymphoid cells to specific immunoreactive status. MATERIALS
AND METHODS
Two fibrosarcomas, one of which was induced in female Fischer 344/N rats with methylcholanthrene and the other induced by benzapyrene, were serially transplanted in syngeneic hosts. Tumor isografts of from 1 to 2 cm were removed from tumor bearing rats which had been injected with tumor cells taken from the 10th to the 20th passage. The tumor tissue was minced in RPM1 1640 and membrane antigens were prepared by extraction in hypertonic potassium chloride. The soluble antigen preparation was partially purified by repeated centrifugation and dialysis steps as previously described (13). Antigen preparations were sterilized by membrane filtration and protein concentration was measured by the method of Lowry et al. (14). “Immune” RNA was prepared from spleens of Fischer rats bearing lto 2-cm tumors by the hot phenol method previously described (15). RNA, DNA, and protein concentrations were measured and RNA preparations characterized by ultracentrifugation in continuous 5-20s sucrose density gradients as described previously (15). I n some experiments, “Immune” RNA, at a concentration of 1 mg/ml, was treated with 50 pg/ml RNAse-A for 30 min at 37”C, or with RNAsefree DNAse at an enzyme to substrate ratio of 1 to 20 for 30 min at 37”C, or with pronase at an enzyme to substrate ratio of 1 to 10 for 30 min at 37°C (15). Lymphocytes were prepared from spleens of normal Fischer rats. Spleens were removed aseptically, minced, and gently pressed through 40 mesh and 120 mesh stainless steel sieves. Erythrocytes were lysed by a 5-min exposure to 0.84% NHICl, followed by two washings with RPM1 1640 Medium. Viability was always greater than 95% as judged by trypan blue exclusion. Cell concentration was adjusted to 1 x lo* cells/ml in RPMI. One-half milliliter of cells and 500 pg RNA were adjusted to a l-ml vol with RPM1 and incubated together for 20 min at 37°C. Control lymphocyte preparations were incubated without RNA, but otherwise were treated identically to lymphocytes incubated with RNA. Cells were washed twice with RPM1 and resuspended to 1 X lo7 cells/ml in RPM1 1640 medium supplemented with 50 units/ml penicillin, 50 pg/ml streptomycin, 2 m&1 glutamine, 0.05% amphatericin B, and 15% heat inactivated, filtered guinea pig serum. Guinea pig serum was obtained by cardiac puncture of anesthetized English Smooth Hair guinea pigs (Hilltop Laboratories, Philadelphia, Pennsylvania). Aliquots containing 2 ml of each cell suspension were added to sterile 5-ml plastic culture tubes (Falcon 3033). Each cell suspension derived from an RNA incubation was aliquoted equally into two tubes. Tumor antigen preparation at a final concentration of 50 pg/ml was added to one tube. No antigen was added to the other tube until after the incubation period. In some experiments there were
248
SHORT
COMMUNICATIONS
two experimental tubes each containing a different tumor antigen preparation. Tubes were incubated stationary for 48 hr in 5% COa atmosphere at 37°C. Culture tubes were centrifuged, an equal concentration of tumor antigen was added to the culture supernatants of control tubes, as mentioned previously, and all culture supernatants were stored at -70°C. The assay was performed as previously described by David et al. (16) with the following modifications. Peritoneal exudate cells were obtained from English Smooth Hair guinea pigs 3 days following the intraperitoneal injection of 30 ml mineral oil. After washing cells twice in RPMI, presealed and prescored glass melting point tubes were filled with the cell suspension using a Zl-gauge spinal needle. The cells were sedimented to the sealed ends of the tubes by centrifugation and the tubes divided at the score mark. Two tubes were added to each chamber of an eight chamber slide (Lab Tek Chamber Slides, Lab Tek Division, Miles Laboratories, Naperville, Illinois) and 0.25 ml of a test culture supernate was added to each of four chambers and 0.25 ml of a control supernate was added to each of the other four chambers. The chambers were incubated overnight in a 5% COz atmosphere at 37°C the culture fluid decanted and the slides dipped in distilled water in order to remove media prior to drying. Areas of migration were traced from a projected image and measured with a planimeter. Mean areas of migration were calculated and standard errors were usually within 10% of the mean. Groups were compared for significance using Student’s t test for unpaired data. Migration index (MI) is expressed as follows : MI =
(area of migration with antigen) (area of migration no antigen)
x 100.
RESULTS In order to standardize the assay and the quality of tumor antigen preparations, a group of rats were immunized to tumors by cautery of tumor bearing limb and challenged two times at weekly intervals with tumor isografts (17). Spleen cells prepared from immunized rats were incubated with or without antigen preparations and release of MIF was demonstrated with a MI of 0.50 for MC3-R system and MI of 0.61 for BP-R system. The release of MIF by cells that have been incubated with RNA from rats bearing MCS-R tumor and the inhibitory effect of RNAse treatment on “Immune” RNA activity is demonstrated in Table 1. The range of migration indices for lymphocytes incubated with RNA extracted from spleens of f344 tumor bearing rats was from 60.2 to 72.5. As demonstrated by many other investigators the activity of “Immune” RNA preparations is not decreased by prior treatment of RNA with DNAse.or pronase. However, RNAse treatment of “Immune” RNA abrogates the response. In Table 1, Expts 4 and 5, this pattern of sensitivity to RNAse but not DNAse and pronase is verified. It will be noted that standard errors are usually within 10% of the mean of the replicates. The release of MIF by lymphocytes incubated with RNA extracted from spleens of syngeneic Fischer rats bearing l-2 cm diam BP-R tumors is indicated in Table 2 ; 50 pg/ml BP-R antigen and 50 pg/ml MC3-R antigen were added separately to two of three tubes. Following the incubation period, BP-R antigen was added to culture fluid of the third tube from each incubation. There was no difference in
SHOKT
TABLE RELEASE
OF
MIF
1
2
3
4
5
1
BY SYNGENEIC LYMPHOID CELLS INCUBATED WITH F344 RATS SPLEENS OF MC3-R TUMOR-BEARING
Culture
Expt
249
COMMUNICATIONS
No antigen
Control MC3-R RNA
51.0 f 48.6 f
Control MC3-R RNA MC3-R RNA (RNAse) MC3-R RNA (DNAse)
MC3-R antigen
3.6 4.8
74.1 f 1.3 97.0 f 8.6 91.5 f 10.3 100.2 f 2.2
RNA
EXTRACTED
Migration Index
41.0 f 4.2 36.0 f. 1.5
80.5 70
71.1 f 58.3 f 82.0 f 81.0 f
4.3 3.0 9.9 2.8
96.0 60.2 90.0 79.0
Control MC3-R RNA MCS-R RNA (RNAse)
19.3 f 30.1 f 26.0 f
1.1 2.0 3.0
23.4 f 2.6 21.8 f 2.7 28.0 f 3.1
122.0 72.5 107.0
Control MC3-R RNA MC3-R RNA (DNAse)
92.4 f 77.1 f 72.4 f
6.3 3.4 3.2
89.2 f 7.7 48.0 f 3.3 33.0 f 6.0
96.0 62.0 45.0
Control MC3-R RNA MC3-R RNA (RNAse) MC3-R RNA (Pronase)
40.0 f 58.6 f 36.3 f 63.7 f
3.2 6.4 4.5 2.9
39.0 * 46.0 f 46.6 f 42.6 f
4.7 2.4 3.9 7.5
99.0 78.0 125.0 67.0
FKOM
P
<0.025
migration of macrophages exposed to culture fluids of normal lymphocytes incubated with either BP-R or MC3-R antigen preparations. In Expt 6 and in Expt 7 there was substantial inhibition of migration by culture fluid of lymphocytes preincubated with “Immune” RNA in the presence of BP-R antigen. No release of MIF by cells incubated with MC3-R antigens was noted nor did inhibition occur in cultures incubated with “Immune” RNA previously treated with RNAse, TABLE
2
RELEASE OF MIF BY SYNGENEIC LYMPHOID CELLS INCUBATED WITH RNA EXRTACTED FROM SPLEENS OF BP-R TUMOR-BEARING F344 RATS
Expt
Culture
6
Control BP-R RNA BP-R RNA (RNAse)
57.3 f 3.9 67.3 f 7.5 73.3 f 5.4
7
Control BP-R RNA BP-R RNA (RNAse)
100.8 f 9.7 78.2 f 4.0 52.3 f 3.0
a P < 0.001. b Not done.
No antigen
MC3-R antigen 79.5 xt 2.8 59.1 f 7.1 61.5 f 3.6
N.D.6
Migration Index
BP-R antigen +
Migration Index
134 87.8 84.2
62.8 f. 3.8 24.2 f 3.6 79.0 f 6.7
110 35.90 107.8
N.D.
90.6 i 7.9 53.5 f 3.9 70.1 f 8.9
90 68.4a 134
250
SHORT
COMMUNICATIONS
DISCUSSION Prior investigations have adequately demonstrated that the macrophage migration inhibition assay detects conversion of normal lymphoid cells to release MIF following incubation with “Immune” RNA only when those cells are incubated with the specific antigen used to immunize RNA donors (5-S). In the studies reported here, we have confirmed these observations in a system involving immune responses to tumor associated antigens. We have also confirmed, utilizing yet another in vitro assay of cellular immunity, the transfer of immune responses to tumor associated antigens with “Immune” RNA. Our studies serve to confirm and expand upon the recent work of Paque et al. (8). The MIF assay employed in our study was a two-step procedure involving a 4%hr incubation of splenic lymphoid cells in the presence of antigen and the subsequent addition of culture supernatants to macrophage-filled capillaries. We were able to use small quantities of antigen in this assay. The efficacy of “Immune” RNA extracted from spleens of tumor bearing rats is consistent with observations of cell mediated immunity directed to tumor associated antigens in tumor bearing hosts as demonstrated by many investigators studying concomitant immunity (18). It appears clear, from these studies, as well as from demonstration of the observed immune cytolysis of tumor cells by lymphocytes preincubated with “Immune” RNA extracted from lymphoid organs of syngeneic RNA can specifically augment cell tumor-bearing rats (1.2)) that “Immune” mediated immune responses to tumor associated antigens. Experiments are now in progress to determine whether or not lymphoid cells preincubated with “Immune” RNA from tumor-bearing rats will synthesize anti-tumor antibodies in culture in addition to mediating cellular anti-tumor immune responses. ACKNOWLEDGMENTS We.wish to thank Sheldon Silver and Terry Matsumoto for expert technical assistance.
REFERENCES Mannick, J. A., and Egdahl, R. H., Ann. Surg. 156, 356, 1962. Wilson, 0. B., and Wecker, E. E., J. Immunol. 97, 512, 1966. Bondevick, H., and Mannick, J. A., Proc. Sot. Exp. Biol. Med. 129, 264, 1968. Ramming, K. P., and Pilch, Y. H., J. Nat. Cancer Inst. 46, 735, 1971. Thor, D. E., and Dray, S., J. Zmmunol. 101, 51, 1968. Paque, R. E., and Dray, S., J. Immunol. 105, 134, 1970. Likhite, V., Sabbadini, E., and Sehon, A., Proc. N. Y. Acad. Sci. 207, 389, 1973. Paque, R. E., Meltzer, M. S., Zbar, B., Rapp, H. J., and Dray, S., Cancer Res. 33, 3165, 1973. 9. Halliday, W. J., Maluish, A., and Isbister, W. H., Brit. J. Cancer 29, 31, 1974. 10. Hilberg, R. W., Balarzak, S. P., and LoBuglio, A. F., Cell. Immunol. 7, 1.52,1973. 11. Halliday, W. J., Maluish, A., and Miller, S., Cell. Immunol. 10, 467, 1974. 12. Kern, D. H., Drogemuller, C. R., and Pilch, Y. H., J. Nat. Cancer Inst. 52, 299, 1974. 13. Reisfeld, R. A., Pellegrino, M. A., and Kahan, B. D., Sc’ience 172, 1134, 1971. 14. Lowry, 0. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem. 193, 265, 1951. 7, 296, 1969. 15. Ramming, K. P., and Pilch, Y. H., Transplantation 16. David, J. R., Al-Askari, D., Lawrence, H. S., and Thomas, L., J. Immunol. 93, 264, 1964. 17. Deckers, P. J., and Pilch, Y. H., Cancer Res. 32, 839, 1972. 18. Deckers, P. J., Edgerton, B. W., Thomas, B. S., and Pilch, Y. H., Cancer Res. 31, 734, 1971. 1. 2. 3. 4. 5. 6. 7. 8.
IMMUNOLOGY
Specific
18, 24&250 (1975)
MIF Release by Rat Lymphocytes Following Incubation with Syngeneic Anti-Tumor “Immune” RNA ‘9 ’ STEPHEN
R.
WALDMAN
s AND
YOSEF
H.
PILCH
of Oncology, Departnaent of Surgery, University of California, Los Angeles, California 90024, and Tumor Immunology Laboratory, Veteralzs Administration Hospital, Sepulveda, California 91343
Division
Received January 6,1975
Other investigators have previously shown that normal nonimmune lymphoid cells, after incubation with “Immune” RNA, will release MIF when these cells are incubated with the specific antigen used to immunize the RNA donor. This conversion can be detected with the macrophage migration inhibition assay. These observations have been confirmed in a system involvin, r the transfer of immune response to tumor associated antigens with syngeneic “Immune” RNA. Syngeneic “Immune” RNA was extracted from the spleens of Fischer 344/N rats bearing growing transplants of one or another of two syngeneic chemically induced sarcomas. Normal, nonimmune Fischer 344/N spleen cells were incubated with these RNA preparations. When these
RNA-incubated spleen cells were exposed to solubilized antigens from that particular tumor used to immunize the RNA donor, MIF was released.RNAse treatment of the “Immune” RNA abrogated the response,while DNAse or pronase treatment did not. RNA extracted from lymphoid organs of immune donors as well as from macrophages exposed to antigens, in vitro, has been demonstrated to convert normal nonimmune lymphocytes to immunoreactive status. These “Immune” RNA preparations have been shown to mediate cellular immune responses as well as antibody synthesis (l-8). Within the last few years, several groups of investigators have demonstrated the release of macrophage inhibitory factor (MIF) by human (5) and animal (6-8) lymphocytes following incubation with “Immune” RNA extracted from lymphoid cells of immune donors. Thus, an in vitro assay which correlates with delayed cutaneous hypersensitivity responses in viva was successfully employed to quantitate conversion of lymphoid cells to immunoreactive status following incubation with “Immune” RNA. Also, release of leukocyte inhibitory factor (LIF) (9) and MIF (10) by lymphocytes obtained from human cancer patients and obtained from animals following rejection of tumors (11) has been demonstrated in the presence of tumor antigen preparations. 1 Supported in part by Grant No. CA-14846 from the National Institutes of Health, Grant No. ET-28 from the American Cancer Society and by the Veterans Administration. 2 Reprint requests should be sent to Dr. Y. H. Pilch, Harbor General Hospital, 1000 W. Carson Street, Torrance, California 90509. s Research Scientist, City of Hope National Medical Center, Duarte, California 91010. 246 Copyright Q 1975 by Academic Press, Inc. All lights of reproduction in any form reserved.
SHORT
COhfMUNICATlONS
247
Recently, a quantitative microcytotoxicity assay for lymphocyte mediated cytotoxicity was employed to demonstrate that normal, nonimmune Fischer F344/N spleen cells, preincubated with RNA extracted from spleens of syngeneic rats bearing growing tumor isografts, were cytotoxic to tumor cells, in vitro (12). This was the first report of the transfer of immunity to tumor associated antigens with “Immune” RNA extracted from lymphoid tissues of tumor-bearing animals rather than hyperimmunized animals. In this report, the same model system was studied by demonstrating the release of MIF, in order to confirm that RNA extracted from tumor-bearing donors converts normal lymphoid cells to specific immunoreactive status. MATERIALS
AND METHODS
Two fibrosarcomas, one of which was induced in female Fischer 344/N rats with methylcholanthrene and the other induced by benzapyrene, were serially transplanted in syngeneic hosts. Tumor isografts of from 1 to 2 cm were removed from tumor bearing rats which had been injected with tumor cells taken from the 10th to the 20th passage. The tumor tissue was minced in RPM1 1640 and membrane antigens were prepared by extraction in hypertonic potassium chloride. The soluble antigen preparation was partially purified by repeated centrifugation and dialysis steps as previously described (13). Antigen preparations were sterilized by membrane filtration and protein concentration was measured by the method of Lowry et al. (14). “Immune” RNA was prepared from spleens of Fischer rats bearing lto 2-cm tumors by the hot phenol method previously described (15). RNA, DNA, and protein concentrations were measured and RNA preparations characterized by ultracentrifugation in continuous 5-20s sucrose density gradients as described previously (15). I n some experiments, “Immune” RNA, at a concentration of 1 mg/ml, was treated with 50 pg/ml RNAse-A for 30 min at 37”C, or with RNAsefree DNAse at an enzyme to substrate ratio of 1 to 20 for 30 min at 37”C, or with pronase at an enzyme to substrate ratio of 1 to 10 for 30 min at 37°C (15). Lymphocytes were prepared from spleens of normal Fischer rats. Spleens were removed aseptically, minced, and gently pressed through 40 mesh and 120 mesh stainless steel sieves. Erythrocytes were lysed by a 5-min exposure to 0.84% NHICl, followed by two washings with RPM1 1640 Medium. Viability was always greater than 95% as judged by trypan blue exclusion. Cell concentration was adjusted to 1 x lo* cells/ml in RPMI. One-half milliliter of cells and 500 pg RNA were adjusted to a l-ml vol with RPM1 and incubated together for 20 min at 37°C. Control lymphocyte preparations were incubated without RNA, but otherwise were treated identically to lymphocytes incubated with RNA. Cells were washed twice with RPM1 and resuspended to 1 X lo7 cells/ml in RPM1 1640 medium supplemented with 50 units/ml penicillin, 50 pg/ml streptomycin, 2 m&1 glutamine, 0.05% amphatericin B, and 15% heat inactivated, filtered guinea pig serum. Guinea pig serum was obtained by cardiac puncture of anesthetized English Smooth Hair guinea pigs (Hilltop Laboratories, Philadelphia, Pennsylvania). Aliquots containing 2 ml of each cell suspension were added to sterile 5-ml plastic culture tubes (Falcon 3033). Each cell suspension derived from an RNA incubation was aliquoted equally into two tubes. Tumor antigen preparation at a final concentration of 50 pg/ml was added to one tube. No antigen was added to the other tube until after the incubation period. In some experiments there were
248
SHORT
COMMUNICATIONS
two experimental tubes each containing a different tumor antigen preparation. Tubes were incubated stationary for 48 hr in 5% COa atmosphere at 37°C. Culture tubes were centrifuged, an equal concentration of tumor antigen was added to the culture supernatants of control tubes, as mentioned previously, and all culture supernatants were stored at -70°C. The assay was performed as previously described by David et al. (16) with the following modifications. Peritoneal exudate cells were obtained from English Smooth Hair guinea pigs 3 days following the intraperitoneal injection of 30 ml mineral oil. After washing cells twice in RPMI, presealed and prescored glass melting point tubes were filled with the cell suspension using a Zl-gauge spinal needle. The cells were sedimented to the sealed ends of the tubes by centrifugation and the tubes divided at the score mark. Two tubes were added to each chamber of an eight chamber slide (Lab Tek Chamber Slides, Lab Tek Division, Miles Laboratories, Naperville, Illinois) and 0.25 ml of a test culture supernate was added to each of four chambers and 0.25 ml of a control supernate was added to each of the other four chambers. The chambers were incubated overnight in a 5% COz atmosphere at 37°C the culture fluid decanted and the slides dipped in distilled water in order to remove media prior to drying. Areas of migration were traced from a projected image and measured with a planimeter. Mean areas of migration were calculated and standard errors were usually within 10% of the mean. Groups were compared for significance using Student’s t test for unpaired data. Migration index (MI) is expressed as follows : MI =
(area of migration with antigen) (area of migration no antigen)
x 100.
RESULTS In order to standardize the assay and the quality of tumor antigen preparations, a group of rats were immunized to tumors by cautery of tumor bearing limb and challenged two times at weekly intervals with tumor isografts (17). Spleen cells prepared from immunized rats were incubated with or without antigen preparations and release of MIF was demonstrated with a MI of 0.50 for MC3-R system and MI of 0.61 for BP-R system. The release of MIF by cells that have been incubated with RNA from rats bearing MCS-R tumor and the inhibitory effect of RNAse treatment on “Immune” RNA activity is demonstrated in Table 1. The range of migration indices for lymphocytes incubated with RNA extracted from spleens of f344 tumor bearing rats was from 60.2 to 72.5. As demonstrated by many other investigators the activity of “Immune” RNA preparations is not decreased by prior treatment of RNA with DNAse.or pronase. However, RNAse treatment of “Immune” RNA abrogates the response. In Table 1, Expts 4 and 5, this pattern of sensitivity to RNAse but not DNAse and pronase is verified. It will be noted that standard errors are usually within 10% of the mean of the replicates. The release of MIF by lymphocytes incubated with RNA extracted from spleens of syngeneic Fischer rats bearing l-2 cm diam BP-R tumors is indicated in Table 2 ; 50 pg/ml BP-R antigen and 50 pg/ml MC3-R antigen were added separately to two of three tubes. Following the incubation period, BP-R antigen was added to culture fluid of the third tube from each incubation. There was no difference in
SHOKT
TABLE RELEASE
OF
MIF
1
2
3
4
5
1
BY SYNGENEIC LYMPHOID CELLS INCUBATED WITH F344 RATS SPLEENS OF MC3-R TUMOR-BEARING
Culture
Expt
249
COMMUNICATIONS
No antigen
Control MC3-R RNA
51.0 f 48.6 f
Control MC3-R RNA MC3-R RNA (RNAse) MC3-R RNA (DNAse)
MC3-R antigen
3.6 4.8
74.1 f 1.3 97.0 f 8.6 91.5 f 10.3 100.2 f 2.2
RNA
EXTRACTED
Migration Index
41.0 f 4.2 36.0 f. 1.5
80.5 70
71.1 f 58.3 f 82.0 f 81.0 f
4.3 3.0 9.9 2.8
96.0 60.2 90.0 79.0
Control MC3-R RNA MCS-R RNA (RNAse)
19.3 f 30.1 f 26.0 f
1.1 2.0 3.0
23.4 f 2.6 21.8 f 2.7 28.0 f 3.1
122.0 72.5 107.0
Control MC3-R RNA MC3-R RNA (DNAse)
92.4 f 77.1 f 72.4 f
6.3 3.4 3.2
89.2 f 7.7 48.0 f 3.3 33.0 f 6.0
96.0 62.0 45.0
Control MC3-R RNA MC3-R RNA (RNAse) MC3-R RNA (Pronase)
40.0 f 58.6 f 36.3 f 63.7 f
3.2 6.4 4.5 2.9
39.0 * 46.0 f 46.6 f 42.6 f
4.7 2.4 3.9 7.5
99.0 78.0 125.0 67.0
FKOM
P
<0.025
migration of macrophages exposed to culture fluids of normal lymphocytes incubated with either BP-R or MC3-R antigen preparations. In Expt 6 and in Expt 7 there was substantial inhibition of migration by culture fluid of lymphocytes preincubated with “Immune” RNA in the presence of BP-R antigen. No release of MIF by cells incubated with MC3-R antigens was noted nor did inhibition occur in cultures incubated with “Immune” RNA previously treated with RNAse, TABLE
2
RELEASE OF MIF BY SYNGENEIC LYMPHOID CELLS INCUBATED WITH RNA EXRTACTED FROM SPLEENS OF BP-R TUMOR-BEARING F344 RATS
Expt
Culture
6
Control BP-R RNA BP-R RNA (RNAse)
57.3 f 3.9 67.3 f 7.5 73.3 f 5.4
7
Control BP-R RNA BP-R RNA (RNAse)
100.8 f 9.7 78.2 f 4.0 52.3 f 3.0
a P < 0.001. b Not done.
No antigen
MC3-R antigen 79.5 xt 2.8 59.1 f 7.1 61.5 f 3.6
N.D.6
Migration Index
BP-R antigen +
Migration Index
134 87.8 84.2
62.8 f. 3.8 24.2 f 3.6 79.0 f 6.7
110 35.90 107.8
N.D.
90.6 i 7.9 53.5 f 3.9 70.1 f 8.9
90 68.4a 134
250
SHORT
COMMUNICATIONS
DISCUSSION Prior investigations have adequately demonstrated that the macrophage migration inhibition assay detects conversion of normal lymphoid cells to release MIF following incubation with “Immune” RNA only when those cells are incubated with the specific antigen used to immunize RNA donors (5-S). In the studies reported here, we have confirmed these observations in a system involving immune responses to tumor associated antigens. We have also confirmed, utilizing yet another in vitro assay of cellular immunity, the transfer of immune responses to tumor associated antigens with “Immune” RNA. Our studies serve to confirm and expand upon the recent work of Paque et al. (8). The MIF assay employed in our study was a two-step procedure involving a 4%hr incubation of splenic lymphoid cells in the presence of antigen and the subsequent addition of culture supernatants to macrophage-filled capillaries. We were able to use small quantities of antigen in this assay. The efficacy of “Immune” RNA extracted from spleens of tumor bearing rats is consistent with observations of cell mediated immunity directed to tumor associated antigens in tumor bearing hosts as demonstrated by many investigators studying concomitant immunity (18). It appears clear, from these studies, as well as from demonstration of the observed immune cytolysis of tumor cells by lymphocytes preincubated with “Immune” RNA extracted from lymphoid organs of syngeneic RNA can specifically augment cell tumor-bearing rats (1.2)) that “Immune” mediated immune responses to tumor associated antigens. Experiments are now in progress to determine whether or not lymphoid cells preincubated with “Immune” RNA from tumor-bearing rats will synthesize anti-tumor antibodies in culture in addition to mediating cellular anti-tumor immune responses. ACKNOWLEDGMENTS We.wish to thank Sheldon Silver and Terry Matsumoto for expert technical assistance.
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