Transfer of Tumor-Specific Immunity With RNA: Demonstration by Immune Cytolysis of Tumor Cells In Vitro 1

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1 Transfer of Tumor-Specific Immunity With RNA: Demonstration by Immune Cytolysis of Tumor Cells In Vitro 1 KENNETH P. RAMMING and YOSEF H. PILCH, Surgery Branch, National Cancer Institute,2 Bethesda, Maryland SUMMARY-Extracts rich in RNA were prepared From the spleens of inbred strain-2 guinea pigs that had been immunized with a 3-methylcholanthrene-induced, strain-2 liposarcoma (MCA-A). Normal, non immune, strain-2 lymphoid cells incubated with these RNA preparations caused specific immune cytolysis of MCA-A cells in vitro. Treatment of the active RNA preparations with ribonuclease resulted in their complete inactivation. RNA from the spleens of guinea pigs not exposed to the tumor was inerfective. Incubation of lymphoid cells with extracts of MCA-A rich in solubilized tumor-specific antigens did not reproduce the immune cytolysis caused by the active RNA preparations. Spleen cells incubated in RNA extracted from strain-2 guinea pigs immunized with MCA-A did not cause significant immune cytolysis when applied to monolayers of MCA-25, a strain-2 sarcoma whose tumor-specific transplantation antigens do not cross-react with MCA-A.-J Nat Cancer Inst 45: ,1970. NORMAL, NONIMMUNE lymphoid cells can be converted to specific immunologic competency by incubation in vitro with RNA-rich preparations extracted from lymphoid tissues or macrophages previously exposed to specific antigens in vitro or in vivo (1-4). Lymphoid cells treated with RNA extracts prepared from lymphoid tissues of appropriately immunized animals can produce antibody to the specific antigen used to immunize the RNA donor. This response has been determined by the serologic titration of specific antibody (5-8). That previously nonimmune lymphoid cells are transformed into specific antibody-forming cells after incubation with the RNA extracts has also been confirmed by the detection of zones of specific hemolysis in gels (9-11). In addition, such RNA-incubated lymphoid cells inhibit macrophage 1l r migration in capillary tubes when exposed to specific antigen (12-15) and mediate allograft immunity (16, 17). Recent reports from our laboratory described the inhibited growth of isografts of a chemically induced murine tumor in inbred mice after intraperitoneal injections of syngeneic spleen cells; these spleen cells had been incubated in vitro with RNA-rich preparations extracted from the lymph nodes and spleens of guinea pigs previously immunized with the same mouse tumor (18, 19). The response was, at least partially, tumor specific, in that RNA from guinea pigs immunized with 1 Received March 20, 1970; accepted May 20, National Institutes of Health, Public Health Service, U.S. Department of Health, Education, and Welfare; 543

2 544 RAMMING AND PILCH normal mouse tissues caused no statistically significant inhibition of the growth of tumor isografts. Treatment of these RNA extracts in vitro with ribonuclease before incubation with spleen cells inactivated these preparations. To study further the mediation of tumor-specific transplantation immunity by such RNA-rich extracts, a completely syngeneic system was needed in which the RNA donor, the spleen cell donor, and the tumor-host system were of the same inbred species. Experiments performed within such a system form the subject of this report. The specific immune cytolysis in vitro of tumor cells grown as monolayers in tissue culture by RNA-incubated, syngeneic lymphoid cells was used to detect immune responses to tumor-specific antigens. MATERIALS AND METHODS Tumor.-A liposarcoma (MCA-A) was induced by 3-methylcholanthrene and carried in inbred, adult, female Sewall-Wright strain-2 guinea pigs obtained from the Animal Production Unit of the National Institutes of Health. MCA-A contains tumor-specific transplantation antigens.and is strongly immunogenic in syngeneic animals, as reported by Morton et al. (20) and Holmes et al. (21). Trypsinized cells from the seventh transplant generation of MCA-A were grown in 75-cm 2 plastic tissue-culture flasks (Falcon Plastics, Los Angeles, Calif.) and serially subcultured. Medium was RPMI 1640 with 20% fetal calf serum, 25 p.g/ml amphotericin B, 50 p.g/ml chlortetracycline, and 50 p.g/ml kanamycin. The monolayers were obtained from the fourth to thirteenth subcultures. In tissue culture, MCA-A has maintained all the growth and morphologic characteristics of the original tumor, wit~out fibroblastic degeneration, and washed cells from the,thirteenth passage promptly grew into palpable tumors when injected into normal, female strain-2 guinea pigs. Immunization.-Adult, female strain-2 guinea pigs were given a single intradermal injection in the flank of 10 7 viable MCA-A cells suspended in medium 199. When.2-3 cm in diameter, tumors were excised. Mter days, 5 X 10 6 viable tumor cells were injected intradermally. This inoculum, which grows to palpable and eventually lethal tumors in over 90% of unimmunized strain-2 guinea pigs, never resulted in tumor growth in the guinea pigs immunized by the excision of growing tumors as described above. Subsequent intradermal injections of 5 X 10 6 MCA-A cells were given at 2-week intervals for a minimum of 3 months. At least 2 intraperitoneal injections of 5 X 106 tumor cells were given at biweekly intervals at the end of this period. RNA extraction.-spleens of immunized strain-2 guinea pigs (or, for controls, unimmunized strain-2 guinea pigs) were excised and immediately frozen in dry ice. RNA was extracted by a hot phenol method as described in (22). Mter precipitation overnight in ethanol at -20 C, the RNA was washed with 3: I ethcj.nol-water solution, and the precip~tate dissolved in Earle's balanced salt solution (BSS) to an approximate concentration of 500 p.g/ml. Some RNA solutions were treated,with ribonuclease (Bovine Pancreatic Ribonuclease, Worthington Biochemical Corp., Freehold, N.].) at 20 p.g/ml for 30 minutes at 37 C. All RNA solutions were made 0.35 M with respect to sucrose before use. Preparation oj spleen cell suspensions and incubation with RNA.-Spleens were excised, minced with with scissors in cold BSS, and passed through #40 mesh stainless-steel screens. The suspension was then fil tered through #80 mesh stainless-steel screens and centrifuged 'at 200 X g for 10 minutes. The cells were washed once with cold BSS. For incubation with RNA, the cell button was resuspended in the RNA solution at a concentration of cells/ml. The mixtures of RNA and spleen cells were then incubated at 37 C for 20 minutes in a gyratory shaking water bath, washed once with cold BSS, counted, and suspended in RPM I [640 with 20% fetal calf serum at a concentration of 4 X 10 7 viable lymphoid cells/ml. Viability was determined by trypan blue exclusion. Immune cytolysis of plaque-forming assay.-under a tissue culture hood, 0.25 ml portions of the washed RNA-incubated spleen cells containing 10 7 cells were dropped onto confluent monolayers of MCA- A cells through a 3%-inch #15 needle. The cells dropped through the media and diu used into circular areas 1 or 2 cm in diameter on the tumor cell monolayers. This' procedure is illustrated in figure 1. In some experiments, phytohemagglutinin (Phytohemagglutinin-P, Difco Labora- JOURNAL OF THE NATIONAL CANCER INSrrnJTE

3 TRANSFER OF TUMOR-SPECIFIC IMMUNITY WITH RNA 545 tories, Detroit, Mich.), 1 :50, was added to the tissue culture medium before application of the droplets of RNA-incubated spleen cells. Great care was taken not to disturb or jar the tissue culture flasks for several hours. The flasks were then carefully placed into an incubator at 37 C. After 48 hours, they were shaken, and the spleen cell droplets dislodged. The monolayers were washed once with fresh tissue culture medium and inspected. Clear, acellular, macroscopic zones of lysis, or plaques, within the tumor cell monolayer and corresponding to the areas covered by the spleen cells, were considered evidence of immune cytolysis (fig. 2). Without immune cytolysis, the tumor cell monolayer remained intact after elution of the droplets (fig. 3). Soluble tumor antigen preparation.-soluble antigens were extracted from MCA-A by the method of Holmes et al. (21). The tumor was excised, weighed, and minced with scissors. The rest of the procedure was carried out at 4 C. In sucrose-tris-hcl buffer, ph 7.47, the tumor was passed through a #20 and then a #40 stainless-steel screen. This mixture was centrifuged at 10,000 X g for 10 minutes. The pellet was washed and resuspended in buffer to a concentration of 3-6 X 107 cells/mi. Fifty ml portions of this suspension were then placed in a Raytheon Membrane Sonicator (Raytheon Manufacturing Co., Waltham, Mass.). Ultrasonic vibration was carried out until 95% of the cells w~re eliminated. The suspension was then centrifuged at 100,000 X g for I hour. The supernatant was passed successively through and 0.20-~ membrane filters (Nalge Co., Rochester, N.Y.), concentrated tenfold by pressure dialysis against phosphatebuffered saline, ph 7.4, and stored in liquid nitrogen. This preparation had protein concentrations of 3.5 mg/ml, as determined by the method of Lowry et ai. (23). Soluble MCA-A antigens also were extracted, by the method ofoettgen et ai. (24). A 20% solution of minced tumor in phosphate-buffered saline, ph 7.4, was homogenized for 6 minutes in a Sorvall Omnimixer (Ivan Sorvall, Inc., Norwalk, Conn.). The suspension was centrifuged at 105,000 X g for 15 minutes, and the supernatant containing visible debris was aspirated and discarded. The remaining suspension was passed through a 0.45-~ filter and subjected to pressure dialysis to concentrate it to one-fourth the initial volume. Protein concentration of the extracts was 45.3 mg/ml. RESULTS Initial experiments were designed 1) to demonstrate the immune status of the immunized pigs whose spleens were to be used for RNA extraction, 2) to determine the immunoreactivity of cells from these spleens in terms of their ability to mediate immune cytolysis of MCA-A cells in vitro, and 3) to establish and standardize the immune cytolysis or plaque-forming assay. Cell suspensions were prepared from the spleens of guinea pigs that had been immunized with MCA-A as described above. The last dose of MCA-A was given 5-7 days before the spleen cells were obtained. The cells were washed with BSS and suspended in tissue culture medium at a concentration of 4 X 107/ml. Portions of 0.25 ml containing. 107 cells were then dropped onto MCA-A monolayers and assayed for immune cytolysis by the method described. As controls, cell suspensions were made from the spleens of normal, untreated strain-2 guinea pigs, and 0.25 ml droplets were applied to MCA-A monolayers in an identical fashion. Mter 48 hours of incubation, the spleen cell droplets were eluted and the monolayers inspected for plaques. Results are shown in table 1. As table 1 indicates, only 3 areas of lysis were formed by normal strain-2 spleen cells in 45 tests. However, 13 plaques in 14 tests were formed by spleen cell suspensions prepared from the spleens of strain-2 guinea pigs that had been immunized with MCA-A. Statistically significant, these observations indicated that this in vitro cytolytic assay, used as a measure of lymphoid cell immunoreactivity against transplantation antigens by Granger and Weiser (25), Moller (26), and' Roseneau and Moon (27), was sufficiently sensitive to demonstrate immunity to tumorspecific transplantation antigens in the MCA-A guinea pig tumor-host system. In these and all subsequent experiments, the addition of phytohemagglutinin (PHA) to the medium before the application of the spleen cell droplets did riot affect the results. Spleen cells from normal, untreated strain-2 guinea pigs preincubated' with RNA extracted VOL: 45, NO.3, SEPTEMBER 1970

4 546 RAMMING AND PILCH TABLE I.-Plaques formed on monolayers of strain-2 guinea pig MCA-A at 48 hours by spleen cells Number of plaques formed Spleen cell source Total No. of tests PHA * in medium No PHA in medium Total Strain-2 guinea pigs immunized with MCA-A Untreated strain-2 guinea pigs 6/6 0/15 7/8 3/30 13/14t 3/45 Phytohemagglutlnln. tp<o.ol hy x' with Yates' correction when compared to untreated straln-2 guinea pigs. from the spleens of pigs immunized with MCA-A (MCA-A immune RNA) were applied to monolayers of MCA-A, and the results are shown in table 2. In 36 of 40 tests, strain-2 spleen cells, which had been incubated with MCA-A-immune RNA, formed zones of immune cytolysis or plaques. When normal strain-2 spleen cells were incubated with RNA extracted from the spleens of untreated strain-2 guinea pigs who had never been exposed to MCA-A, plaques were formed in only 10 of 49 tests. This result indicates that RNA extracted from strain-2 guinea pig spleens did not nonspecifically stimulate spleen cells to mediate a cytolytic reaction. It also demonstrated that any RNA remaining as a contaminant after the RNA-incubated spleen cells were washed was not in itself cytolytic. Intact RNA appeared essential for the reaction, since spleen cells incubated with RNA extracted from pigs immunized with MCA-A and then degraded by treatment with ribonuclease did not mediate immune cytolysis. It was necessary to establish that the activity of the RNA preparations was not due simply to contamination of these RNA extracts with tumorspecific transplantation antigens. Therefore, preparations known to be rich in solubilized tumorspecific transplantation antigens were prepared by two methods. Holmes et at., who prepared extracts of MCA-A tissue, showed them to contain tumorspecific transplantation antigens (21). These extracts were immunogenic in strain-2 guinea pigs and could elicit delayed cutaneous hypersensitivity reactions in vivo. When strain-2 spleen cells, incubated under the usual circumstances with high concentrations of these soluble antigen preparations but without RNA, were applied to MCA-A monolayers, no plaques were formed (table 3). To determine the specificity of the reaction, strain-2 spleen cells incubated with MCA-Aimmune RNA were applied to monolayers of a different strain-2 tumor. A 3-methylcholanthreneinduced osteogenic sarcoma (MCA-25) induced and carried in strain-2 guinea pigs had been shown by Holmes et at. (21) to contain tumor-specific transplantation antigens which did not cross-react with MCA-A. Droplets of strain-2 spleen cells that had been incubated with MCA-A-immune RNA were applied to monolayers of MCA-25 TABLE 2.-Plaques formed on monolayers of strain-2 guinea pig MCA-A at 48 hours by spleen cells incubated with RNA Groups Number of plaques formed Total No. of tests PHA * in medium No PHA in medium Total Strain-2 spleen cells incubated with RNA extracted from the lymphoid organs of strain-2 guinea pigs: Immunized with MCA-A Immunized with MCA-A (ribonuclease) t Unimmunized N orroal, untreated strain-2 spleen cells (no RNA) 7/7 0/2 4/17 0/15 29/33 3/13 6/32 3/30 36/40t 3/15 10/49 3/45 Phytohemagglutinin. tp<o.01 hy x' with Yates' correction when compared to every other group. tthese RNA preparations were treated with ribonuclease before Incubation with spleen cells. JOURNAL OF THE NATIONAL CANCER INSTITUTE

5 TRANSFER OF TUMOR-SPECIFIC IMMUNITY WITH RNA 547 TABLE 3.-Plaques formed on monolayers of strain-2 guinea pig MCA-A at 48 hours by strain-2 spleen cells incubated with soluble antigen extracts of MCA-A Groups PHA* in media Number of plaques formed Total No. of tests No PHA in media Total Antigen preparation: Holmes' method Oettgen's method Normal, untreated strain-2 spleen cells (no soluble antigens) 0/2 0/2 0/15 0/10 0/12 3/30 0/12 0/14 3/45 Phytohemagglutinin. in the usual manner. The results are shown in table 4. Cells incubated with MCA-A-immune RNA, which had induced zones of lysis in 90% of MCA-A monolayers on which they had been applied, formed plaques in less than 30% of the tests on MCA-25 monolayers. (This difference is significant at P<O.Ol.) In addition, this degree of plaque formation did not differ significantly from the nonspecific lysis of MCA-A monolayers caused by cells incubated with RNA from unimmunized animals. Hence the zones of lysis in MCA-25 monolayers induced by spleen cells incubated with "MCAimmune" RNA perhaps were due to nonspecific factors. Spleen cells incubated with MCA-Aimmune RNA appeared to react specifically only with MCA-A tumor cells. DISCUSSION As Granger and Weiser (25), Moller (26), and Roseneau and Moon (27) have shown, lymphoid cells from animals immunized to histocompatibility antigens can cause discrete areas of lysis in monolayers of target cells containing these antigens. Our studies applied this technique of immune cytolysis to the detection of immunity to tumorspecific transplantation antigens. Spleen cells from strain-2 guinea pigs immunized with MCA-A caused lysis of MCA-A target cells in tissue culture. The capacity of lymphoid cells from immunized animals to mediate immunity to tumor-specific transplantation antigens of chemically induced tumors is now a well-established fact and has been studied in several tumor-host systems (28-35). Our observations further support these findings. Wilson and Wecker (36) and Bondevik and Mannick (37) showed that nonimmune lymphoid cells, incubated with RNA extracted from the lymphoid organs of animals immunized to strong histocompatibility antigens, cause specific immune cytolysis of target cells in vitro. Alexander et al. (38, 39) reported inhibition in the growth of sarcomas of rats given RNA extracts prepared from the lymphocytes of immunized sheep. The data we presented here suggest that RNA extracted from the spleens of syngeneic animals immunized to tumor-specific antigens confers upon previously normal, nonimmune spleen cells a specific immunoreactivity to tumor-specific antigens, as TABLE 4.-Plaques formed on monolayers of strain-2 guinea pig tumors at 48 hours by spleen cells incubated with RNA Groups N umber of plaques formed Total No. of tests MCA-A monolayers MCA-25 monolayers Strain-2 spleen cells incubated with RNA extracted from the lymphoid organs of strain-2 guinea pigs: Immunized with MCA-A Unimmunized 36/40 10/49 8/27* 0/3 P<O.Ol by x' with Yates' correction when compared with identical cells dropped on MCA-A monolayers. No significant difierences, P>O.80, when compared with cells incubated with RNA from unimmunized guinea pigs dropped on MCA-A monolayers.. VOL. 45, NO.3, SEPTEMBER 1970

6 548 RAMMING AND PILCH evidenced by the ability of these cells to mediate specific immune cytolysis of tumor cells in vitro. This reaction was not PHA dependent. Normal spleen cells not incubated with RNA, or spleen cells incubated with RNA extracted from the spleens of animals not previously exposed to MCA-A, did not produce lysis of MCA-A target cells. The immunoreactivity mediated by RNA appeared directed against the tumor-specific antigens of MCA-A only. When spleen cells incubated with "MCA-immune" RNA were dropped on monolayers of MCA-25; a sarcoma known not to cross-react with MCA-A, the degree of lysis observed was significantly less than that observed on MCA-A monolayers and was not significantly different from that observed with spleen cells preincubated with RNA from unimmunized animals. Previous work from this laboratory (18, 19) demonstrated that the development of murine tumor isografts could be inhibited by injection of spleen cells incubated with heterologous "immune RNA," i.e., RNA extracted from the lymph nodes and spleens of guinea pigs immunized with the murine tumor to be treated. RNA from nodes and spleens of guinea pigs immunized with normal mouse tissues did not induce a significant response. Thus the tumor specificity of this type of immune RNA could only be inferred. The completely syngeneic guinea pig system herein described, however, eliminates the difficulties of interpretation inherent in the xenogeneic mouse-guinea pig system. Strain-2 guinea pigs immunized with strain-2 tumor recognize as foreign only tumorspecific antigens. Since RNA from such animals can induce specific immune cytolysis of syngeneic tumor target cells, the mediation of an immune response to tumor-specific antigens by "tumorimmune" RNA is confirmed. We believe this is the first report of such a phenomenon. One experimental system has shown that, within RNA-rich preparations extracted from peritoneal macrophages exposed in vitro to T 2 phage, exists a class of RNA which does not appear to contain antigen and which can induce previously normal lymph node cells to produce IgM antibody in vitro (40). This may be a type of true informational RNA. Although incorporation of informational RNA during incubation may be solely responsible for the Conversion of normal spleen cells to immunoreactive cells (41-43), Gottlieb (44-46), Friedman and co-workers (47-49), Haurowitz (50), and Askonas and Rhodes (51) have submitted convincing evidence that there is persistence of small amounts of antigen bound to RNA extracted from immune lymphoid cells and that the immunologic activity of such an RNA preparation is due to RNA-antigen complexes. Such RNA-antigen complexes may sensitize lymphoid cells much more efficiently than antigen not complexed with RNA, as Askonas and Rhodes (51) demonstrated with the antigen hemocyanin. These authors showed that antigen-containing RNA preparations were at least 20 times more immunogenic than simple antigen alone. Friedman (48) and Fishman and Adler (40) called such RNAantigen complexes of greatly increased immunogenicity "super antigens." The role of RNA in these complexes may be analogous to that of the carrier protein in a hapten-protein conjugate, in which the carrier protein, although necessary for immunogenicity, does not alter the antigenic specificity of the haptenic moiety but, rather, has a nonspecific adjuvant function (52, 53). Just as a given protein may be a carrier for many different haptens and a specific protein is not required for each hapten, so a single RNA or group of RNA's may nonspecifically complex with many antigens to form super antigens of vastly increased immunogenicity. This would obviate the necessity of postulating the existence of specific RNA's for each different antigen, as would be required if these responses were mediated by distinct informational RNA's specific for each antigen. The RNA preparations we used contained some protein, which might represent contamination of the RNA by tumor-specific antigens. However, spleen cells incubated with extracts of MCA-A known to be rich in such antigens, and containing times the amount of protein found in the RNA preparations, could not mediate immune cytolysis of MCA-A cells; therefore, the responses observed were probably not due to simple contamination of the RNA preparations with tumorspecific antigens. MCA-A-immune RNA did not mediate immune cytolysis of MCA-25 cells, another 3-methylcholanthrene-induced strain-2 guinea pig sarcoma known not to cross-react antigenically with MCA- JOURNAL OF THE NATIONAL CANCER INSTITUTE

7 TRANSFER OF TUMOR-SPECIFIC IMMUNITY WITH RNA 549 A. This observation supports the concept that the immune cytolysis mediated by RNA from guinea pigs immunized with MCA-A was directed specifically against the tumor-specific antigens of MCA-A. Finally, the finding that treatment of the RNA extracts with ribonuclease inactivated them demonstrates that intact RNA is necessary for the activity of these preparations. The isolation and characterization of immunologically active complexes of RNA with tumor-specific antigens (a "super tumor antigen"), if indeed they exist within these preparations, remain to be accomplished. REFERENCES (I) FISHMAN M: Anitbody formation in vitro. J Exp Med 114: , 1961 (2) FISHMAN M, HAMMERSTROM RA, BOND VP: In vitro transfer of macrophage RNA to lymph node cells. Nature (London) 198: , 1963 (3) MICHELAZZI L, NOVELLI A, BALDINI 1, et al: Induction of antibody response in 1ympohid cells in vivo and in vitro by RNA-immuno-carrier from immune serum. Experientia 21 : , 1965 (4) FRIEDMAN H: ACQ.uisition of antibody plaque forming activity by normal mouse spleen cells treated in vitro with RNA extracted from immune donor spleens. Biochem Biophys Res Comm 17: , 1964 (5) ---: Appearance of antibacterial agglutinins in X-irradiated rabbits receiving immune spleen nucleoproteins. Experientia 19 : , 1963 (6) ---: Transfer of agglutinin formation by immune lymph node cell fractions. Fed Proc 18:567, 1959 (7) FISHMAN M, ADLER FL: Antibody formation initiated in vitro. II. Antibody synthesis in X-irradiated recipients of diffusion chambers containing nucleic acids derived from macrophages incubated with antigen. J Exp Med 117: , 1963 (8) PINCHUCK P, FISHMAN M, ADLER FL, et al: Antibody formation: Initiation in "nonresponder" mice by macrophage synthetic polypeptide RNA. Science 160: , 1968 (9) COHEN EP, NEWCOMB RW, CROSBY LK: Conversion of nonimmune spleen cells to antibody-forming cclls by RNA: Strain specificity of the response. J Immun 95: , 1965 (10) COHEN EP, PARKS JJ: Antibody production by nonimmune spleen cells incubated with RNA from immunized mice. Science 144: , 1964 (11) FRIEDMAN H: Antibody plaque formation by normal mouse spleen cell cultures exposed in vitro to RNA from immune mice. Science 146: , 1964 (12) THOR DE: Delayed hypersensitivity in man: A correlate in vitro and transfer by an RNA extract. Science 157: , 1967 (13) ---: Human delayed hypersensitivity: An in vitro correlate and transfer by an RNA extract. Fed Proc 27:16-20, 1968 (14) JUREZIZ RE, THOR DE, DRAY S: Transfer with RNA extracts of the cell migration inhibition correlate of delayed hypersensitivity in the guinea pig. J lmmun 101 : , 1968 (I5) THOR DE, DRAY S: The cell-migration-inhibition correlate of delayed hypersensitivity. Convertion of human nonsensitive lymph node cells to sensitive cells with an RNA extract. J Immun 101 : , 1968 (16) MANNICK JA, EGDAHL RH: Transfer of heightened, immunity to skin homografts by lymphoid RNA. J Clin Invest 43: , 1964 (17) SABBADINI E, SEHON AH: Acceleration of allograft rejection induced by RNA from sensitized donors. Int Arch Allerg 32:55-63, 1967 (18) RAMMING KP, PILCH YH: Ribonucleic acid mediated inhibition and enhancement of tumor growth in mice. Surg!,'orum 20: , 1969 (19) ---: Mediation of immunity to tumor isografts in mice by heterologous ribonucleic acid. Science 168: , 1970 (20) MORTON DL, GOLDMAN L, WOOD DA: Tumorspecific antigenicity of methylcholanthrene (MCA) and dibenzanthracene (DBA) induced sarcomas of guinea pigs. Fed Proc 24:684, 1965 (21) HOLMES EC, KAHAN BD, MORTON DL: Soluble tumor-specific transplantation antigens from methy1- cholanthrene-induced guinea pig sarcomas. Cancer 25: , 1970 (22) RAMMING KP, PILCH YH: Transfer of transplantation immunity by ribonucleic acid. Transplantation 7: , 1969 (23) LOWRY OH, ROSEBROUGH NJ, FARR AL, et al: Protein measurement with the folin phenol reagent. J BioI Chem 193: , 1951 (24) OETTGEN' HF, OLD LJ, McLEAN LP, et al: Delayed hypersensitivity and transplantation immunity elicited by soluble antigens of chemically induced tumors induced in inbred guinea pigs. Nature (London) 220: , 1968 (25) GRANGER GA, WEISER RS: Homograft target cells: Specific destruction in vitro by contact interaction with immune macrophages. Science 145: , 1964 (26) MOLLER E: Contact-induced cytotoxicity by lymphoid cells containing foreign isoantigens. Science 147: , 1965 (27) ROSENAU W, MOON HD: Lysis of homologous cells by sensitized lymphocytes in tissue culture. J Nat Cancer Inst 27: , 1961 (28) MITCHISON NA, DUBE OL: Studies on the im~nological response to foreign tumor transplants in the mouse; role of lymph node cells in conferring immunity by adoptive transfer. J Exp Med 102: ,1955 VOL. 45, NO.3, SEPTEMBER 1970

8 550 RAMMING AND PILCH (29) KLEIN E, SjOGREN HO: Humoral and cellular factors in homograft and isograft immunity against sarcoma cells. Cancer Res 20: , 1960 (30) OLD L], BOYSE EH, CLARKE DA, et al: Antigeneic properties of chemically induced tumors. Ann NY Acad Sci 101 :80-106, 1962 (31) YOSHIDA TO, SOUTHAM CM: Attempts to find cell associated immune reactions against autochthonous tumors. ] ap] Exp Med 33 : , 1963 (32) BARD DS, PILCH YH: The role of the spleen in the immunity to a chemically induced sarcoma in C3H mice. Cancer Res 29: , 1969 (33) BARD DS, HAMMOND WG, PILCH YH: The role of regional lymph nodes in the immunity to a chemically induced sarcoma in C3H mice. Cancer Res 29: , 1969 (34) DELORME EJ, ALEXANDER P: Treatment of primary fibrosarcoma in the rat with immune lymphocytes. Lancet 2: ,1964 (35) JEEJEEBHOY HF, RABBAT AG: Inhibition of the growth of mouse polyoma tumors by lymph node fragments from specifically immunized rats. Cancer Res 29: , 1969 (36) WILSON DB, WECKER EE: Quantitative studies on the behavior of sensitized lymphoid cells in vitro. Ill. Conversion of "normal" lymphoid cells to an immunologically active status with RNA derived from isologous lymphoid tissues of specifically immunized rats.] Immun 97: , 1966 (37) BONDEVIK H, MANNICK ]A: RNA-mediated transfer of lymphocyte VB. target cell activity. Proc Soc Exp BioI Med 129: , 1968 (38) ALEXANDER P, DELORME EJ, HAMILTON LD, et al: Effect of nucleic acids from immune lymphocytes on rat sarcomata. Nature (London) 213: , 1967 (39) ---: Stimulation of anti-tumor activity of the host with RNA from immune lymphocytes. In Nucleic Acids in Immunology (Plescia OJ, Braun W, eds.). New York, Springer-Verlag, 1968, pp (40) FISHMAN M, ADLER FL: The role of macrophage RNA in the immune response. Sympos Quant BioI 32: , 1967 (41) CAMBELL DH, GARVEY js: Nature of retained antigen and its role in immune mechanisms. Advances Immun 3: ,1963 (42) ADLER FL, FISHMAN M, DRAY S: Antibody formation initiated in vitro. III. Antibody formation and allotypic specificity directed by ribonucleic acid from peritoneal exudate cells. J Immun 97: , 1966 (43) BELL C, DRAY S: Conversion of non-immune spleen cells by ribonucleic acid of lymphoid cells from an immunized rabbit to produce IGM antibody of foreign light chain allotype. j Immun 103: , 1969 (44) GOTTLIEB AA: The antigen-rna complex of macrophages. In Nucleic Acids in Immunology (Plescia OJ, Braun W, eds.). New York, Springer-Verlag, 1968, pp (45) ---: Studies on the binding of soluble antigens to a unique ribonucleoprotein fraction of macrophage cells. Biochemistry (Wash) 8: ,1969 (46) GOTTLIEB AA, GLISlN VR, DOTY P: Studies on macrophage RNA involved in antibody production. Proc Nat Acad Sci USA 57: , 1967 (47) FRIEDMAN H: Immune responsiveness and unresponsiveness to Shigella: The role of antigen containing RNA extract~. Bact Proc 38:1966 (48) ---: The nature of immunogenic RNA-antigen complexes in immune and tolerant mice. In Nucleic Acids in Immunology (Plescia OJ, Braun W, eds.). New York, Springer-Verlag, 1968, pp (49) FRIEDMAN H, STAVITSKY AB, SOLOMON jm: Induction in vitr9 of antibodies to phage T2: Antigens in the RNA extract employed. Science 149: , 1965 (50) HAUROWITZ, F: Antibody formation. Physiol Rev 45:1-47, 1965 (51) ASKONAS BA, RHODES jm: Immunogenicity of antigencontaining ribonucleic acid preparations from macrophages. Nature (London) 205: , 1965 (52) BRAUN W: Influence of nucleic acid degradation products on antibody synthesis. In Molecular and Cellular Basis of Antibody Formation (Sterzl], ed.). New York, Academic Press Inc., 1965, pp (53) MERRITT K, JOHNSON AG: Studies on the adjuvant action of bacterial endotoxins on antibody formation. VI. Enhancement of antibody formation by nucleic acids. J Immun 94 : , 1965

9 1 111"1,11,1,,, 'I /,,, rill " /1111/1111 FIGURE I.-After incubation with RNA spleen cells are dropped through the media onto the MCA-A monolayer and diffuse into circular areas I.(}-2.0 cm in diameter. RAMMING AND PILCH 551

10 FIGURE 2.-Zone of lysis in monolayer of MCA-A caused by spleen cells incubated with RNA extracted from the lymph nodes and spleens of guinea pigs immunized with :MCA-A: Clear plaque corresponds to area previously covered by spleen cells, which have been eluted. 552 RAMMING AND PILCH

11 FIGURE 3.-Intact monolayer of lv[ca-a. Spleen cells incubated with RNA extracted from guinea pigs not immunized with MCA-A had been dropped onto the middle of the monolayer. No zones of lysis are apparent after elution of the spleen cell droplet. RAMMING AND PILCH