Antitumor Immunity in Strain 2 Guinea Pigs Immunized With Potassium Chloride Extracts of L 2 C Tumor Cells 1,2

Transcription

1 Antitumor Immunity in Strain 2 Guinea Pigs Immunized With Potassium Chloride Extracts of L 2 C Tumor Cells 1,2 Donald P. Braun, 3 James C. D. Hengst, 3 Margalit B. Mokyr,3 and Sheldon Dray 3,4 ABSTRACT-Extracts of L 2 C tumor cells stimulated In vitro production of macrophage migration inhibitory factor (MIF) in peritoneal exudate cells from guinea pigs immunized with L 2 C tumor cells. Guinea pigs immunized with extracts of L 2 C tumor cells that were active In vitro (in the MIF assay) were completely resistant to challenge with viable tumor cells given 2 weeks later. Furthermore, guinea pigs Immunized with extracts of L 2 C tumor cells within 1 hour after challenge with viable L 2 C tumor cells survived substantially longer than did nonimmunized controls. The immunoprotective and immunotherapeutic effects seen in guinea pigs given Injections of viable L 2 C tumor cells were obtained with extracts of L 2C tumor cells but not with extracts of another guinea pig tumor (line 10 hepatoma) or with extracts of normal guinea pig lymphoid cells.-j Natl Cancer Inst 60: , Leukemia L 2 C arose spontaneously in a female strain 2 guinea pig at the National Institutes of Health and has been maintained since 1954 by serial passage in inbred strain 2 guinea pigs. The leukemia was originally described by Congdon and Lorenz (1), and the leukemia cells were shown to possess surface immunoglobulins and complement receptors characteristic of B-lymphocytes (2). L 2 C leukemia exhibits a striking hematologic and pathologic similarity to acute lymphoblastic leukemia in man (1, 3, 4) and has be~n used to evaluate the potency of clinically active drugs (5). The presence of TAT A on L 2 C cells was demonstrated by immunoprotection experiments in which animals were immunized with sublethal tumor innocula (6, 7) or inactivated tumor cells (8, 9). Further analysis of the T AT A of L 2 C cells would be useful for understanding the nature of immunity induced by these tumors. One approach to this problem is to extract these antigens and to assess them for their biologic activity. In this study, the biologic activity of KCl extracts of L 2 C cells was evaluated in vitro by the MIF assay and in vivo by immunoprotection tests. MATERIALS AND METHODS Animals.-Inbred female strain 2 guinea pigs were obtained from the National Institutes of Health (Bethesda, Md.) and used to establish a breeding colony. All experiments were performed with age-matched, female animals weeks old. Tumors.-Guinea pig leukemia [Eli Nadel's (EN) L 2 C strain] was generously supplied by Dr. I. Green, National Institutes of Health, and maintained by serial intradermal transplantation. Animals were inoculated with 2 X 10 7 viable L 2 C cells; 2 weeks later, animals with white blood cell counts greater than 100,000/mm 3 were exsanguinated by cardiac puncture. The blood was drawn into heparinized syringes, and the leukocyte-rich plasma was obtained by gelatin sedimentation of red blood cells (8). These cells were washed three times in Hanks' balanced salt solution prior to their use. The ascites form of Ln-lO hepatoma cells (passages 10-15), originally induced by the administration of diethylnitrosamine in the drinking water of strain 2 guinea pigs (10), was also employed in these experiments. Extraction of tumor cells. - Washed L 2 C or Ln-lO tumor cells were suspended in 18 ml of chilled 0.15 M phosphate-buffered saline (ph 7.2) per 1 ml of packed tumor cells. Tumor antigens were extracted with 3 M KCI by the method of Reisfeld and Kahan (11). Particulate material was sedimented by ultracentrifugation at 131,000Xg for 2 hours. Supernatants containing extracted tumor antigens were dialyzed for 16 hours against 100 volumes of saline, and precipitates that formed during dialysis were removed by centrifugation at 48,000Xg for 1 hour. The supernatants were concentrated in a vacuum dialysis apparatus and sterilized by passage through a 0.22-JL Millipore filter (Millipore Corp., Bedford, Mass.). Antigenic preparations obtained by this procedure were designated as tumor extracts. The protein concentration was determined by the method of Lowry et al. (12) and was used as an index to estimate the relative quantity of antigen in different extract preparations. Immunization to L 2 C leukemia.-strain 2 guinea pigs were immunized by injection of 25X 10 6 viable L 2 C cells emulsified in CFA (Difco Laboratories, Detroit, Mich.) in the back footpads. Animals were challenged intradermally 3 weeks later with 2 X 10 5 viable L 2 C cells, and hosts that survived tumor challenge were used for experimentation 2-6 weeks later. The procedure used for immunization of strain 2 guinea pigs to Ln-l0 hepatoma was previously described (13). ABBREVIATIO USED: TAT A = tumor-associated transplantation an tigens; MIF = macrophage migration inhibitory factor; Ln-IO = line 10; CF A = complete Freund's adjuvant; PEC = peritoneal exudate cells; MMI = mean migration index. 1 Received August 23,1977; accepted November 7,1977. 'Supported in part by Public Health Service grant CAl824l from the National Cancer Institute. 3 Department of Microbiology, University of Illinois at the Medical Center, Chicago, Ill We acknowledge the expert technical assistance of Katherine Siessmann whose efforts were invaluable in the performance of this work, as were the editorial contributions of Linda Grossman. VOL. 60, NO.4, APRIL J NATL CANCER IT

2 900 BRAUN, HENGST, MOKYR, AND DRAY In vitro testing of tumor extract activity. -The capillary tube MIF assay was utilized to assess the biologic activity of tumor extract preparations. PEC were induced in normal, Ln-lO-immune, or L 2 C-immune animals by the injection of 30 ml of light mineral oil 72 hours before collection, as described in (14). Separate pools of tumor extracts were diluted and tested for their migration inhibitory effects on normal and tumor-immune PEC in the MIF assay. The raw values of cell migration for antigen-treated and antigen-untreated cells were utilized to calculate an MMI according to the method of Jureziz et al. (15). MMI's of60% or less were considered to represent positive migration inhibition. In vivo testing of tumor extract activity.-the in vivo biologic activity of tumor cell extracts was evaluated by one of two ways. 1) Guinea pigs were immunized by a single sc injection (1 ml) of mg extracted L 2 C, Ln- 10, or normal guinea pig tissue proteins emulsified in an equal volume of CFA. Two weeks following the immunization, animals were challenged with 5X 10 4 viable L 2 C cells given intradermally on the same side or on the contralateral side as the immunizing injection. 2) Guinea pigs were given intradermal injections of 5 X 10 4 viable L 2 C cells followed within 1 hour by the injection of mg extracted L 2 C or Ln-lO tumor proteins emulsified in an equal volume of CFA. Guinea pigs received a total of 2 ml of the tumor protein in adjuvant emulsion divided into eight separate injection sites, including both front and rear footpads and both inguinal and axillary node areas. Survival time was monitored to determine the effectiveness of treatment with tumor proteins in CFA as compared with CFA alone. The significance of the differences in survival times was analyzed by Student's t-test for 2 means. Probability values of less than 0.05 were considered significant. RESULTS Survival Times of Strain 2 Guinea Pigs Inoculated With Graded Doses of EN-L 2 C Leukemia Cells Groups of age-matched strain 2 guinea pigs (6-8 animals/group) were given intradermal injections of graded doses of viable L 2 C tumor cells. The animals were monitored three times weekly to determine a) the interval of tumor growth necessary to achieve a nodule of2 mm in diameter (i.e., the latency period) and b) the survival times of each dose group. When animals received 1 x 10 2 viable L 2 C cells, the latency period was 25 days and the mean survival time was 40.3±3.2 days (table 1). A dose of 1 x 10 4 viable tumor cells achieved 100% lethality (table 1). When animals received doses of L 2 C cells of 1 x 10 5 or greater, the survival time of the animals decreased by about 2 days with each 10glO increase in dose of tumor cells. Excision of L 2 C Nodules To determine whether excision of leukemia nodules alters the survival time of tumor-bearing guinea pigs, groups of animals were inoculated with various doses of J NATL CANCER IT L 2 C tumor cells and the resulting nodules were surgically excised. These experiments were performed in one of two ways: 1) From animals that had received graded doses of L 2 C cells ( ), nodules were excised surgically when they reached 7 mm in diameter; or 2) from animals that had received the same dose of tumor cells (3 x 10 4 ), nodules were surgically excised on various days after tumor challenge. When guinea pigs that had received the graded doses ( ) of L 2 C cells were treated by surgical excision of their 7-mm leukemia nodules, the survival time of the tumor-bearing host was not altered (data not shown). However, when guinea pigs receiving 3 X 10 4 leukemia cells were treated by surgical excision of the inoculated area of skin on day 0 or day 3 (prior to the appearance of palpable tumor nodules), the survival time of the host was significantly prolonged (table 2). Excision of the injection site on day 0 (30 min after the injection of tumor cells) led to the apparent cure of all the animals (survival times of >90 days), whereas excision of the injection site on day 3 prolonged survival in some but not all animals (33% of the animals were cured, 27% survived 48.7±6.6 days, and 40% died at the same time as the controls from which injection sites and nodules were not excised). Excision of the injection site on day 5 did not alter survival time. Thus L 2 C leukemia cells metastasized within 5 days post tumor challenge, and surgical excision of the tumor only within the first 3 days of growth prolonged the survival of the host. TABLE l.-8urvival time of strain 2 guinea pigs inoculated with graded doses of EN-L 2C leukemia cells Dose ofen-loc lxlob lxl0 7 lxl0 lxl0' 5xl0' Ix 10' 1 x10 3 lx10 2 Leukemia incidence" 6/6 7/7 7/B 6/B Latency period,b days IB ± B± ± ± ± ± ±1. 7 c 40.3±3.2c "No. of guinea pigs with tumors/total No. of guinea pigs inoculated. b Time required for tumors to achieve a diameter of 2 mm. c Mean survival time was calculated only for tumor-bearing TABLE 2.-Effect of excision of injected site on the survival time of guinea pigs inoculated with 3 xl O' LoG leukemia cells Excision performed Leukemia inci- on day dence" 0 0/5 > / ±5.3 b 5 14/ ± ± /4 21.2± ±2.1 No excision 10/ ±1.6 "No. of guinea pigs with tumors/total No. of guinea pigs inoculated. b Mean survival time was determined only for tumor-bearing VOL. 60, No.4, APRIL 1978

3 ANTITUMOR IMMUNITY TO L2C TUMOR CELLS 901 MIF Testing of L 2 C Tumor Antigen Extracts PEC from L 2 C-immune animals showed significant inhibition of migration in the presence of L 2 C tumor extracts (P<O.OOl for 2 mg L 2 C tumor extracts/ml; table 3). The same extracts tested at the same concentration of protein did not inhibit nor,nal cell migration. Results were similar with five different extracts of L 2 C tumor cells. The L 2 C-immune PEe were inhibited by L 2 C tumor antigen preparations but not by Ln-10 hepatoma antigen preparations (table 3). Similarly, Ln-10-immune PEC were inhibited by Ln-1O antigen preparations (P<O.OOI for 1 mg Ln-1O tumor extracts/ml; table 3) but not by the L 2 C antigen preparations. Increasing the dose of either tumor antigen preparation to 4 mg/ml did not significantly inhibit migration when the antigens were tested on cells sensitized to the other tumor (MMI's>90% in all experiments). Survival of Guinea Pigs Immunized With L 2 C Tumor Antigen Extracts 14 Days Before Challenge With L 2 C Tumor Cells Animals (5/group) were given single sc injections of either CFA alone or mg of extracted L 2 C tumor proteins emulsified in CFA; they were challenged 2 weeks later with 5x 10 4 viable L 2 C cells. Injection of either CF A alone or 0.1 mg of extracted L 2 C tumor protein in CF A did not extend the survival times of these animals as compared to the controls (table 4). Some protective effect was demonstrated in animals given injections of 0.5 mg L 2 C tumor proteins in CFA. Complete protection against L 2 C challenge was afforded in 39 of 40 guinea pigs pretreated with either 1 or 3 mg of extracted L 2 C tumor protein in CFA (tables 4, 5). This level of protection was in the range of that provided by immunization with 25x 10 6 viable L 2 C cells in CF A. Furthermore, positive delayed-type hypersensitivity skin test reactions were observed hours after tumor cell challenge. The immunity elicited with L 2 C tumor cell extracts required CF A, since animals that received up to 10 mg of L 2 C tumor proteins not emulsified in CF A did not survive L 2 C challenge better than untreated controls (table 4). The immunoprotective activity of the L 2 C tumor extracts seen in the preceding experiments was investigated further to determine whether Ln-IO extracts (that TABLE 5.-Survival time of guinea pigs immunized with extracts of L 2 G tumor cells, Ln-lO tumor cells, or normal lymphoid cells and challenged 14 days later with 5 xlo' L 2 G tumor cells Dose of soluble extract, mg Animals immunized with No. of survivors/total No. inoculated Survival 0/5 18.2±0.2 GFAonly 0/5 18.2±0.2 Normal extract" 1.0 0/5 18.2±0.2 Normal extract" 3.0 0/5 18.4±0.2 Ln-10 extract 1.0 0/5 17.6±0.9 Ln-10 extract 3.0 0/5 18.0±0 L 2 C extract 1.0 >90 L.C extract 3.0 >90 a An extract of spleen cells and blood cells from normal guinea pigs. TABLE 3.-MMI's of PEG from normal, L 2 G-immune, or Ln-lO-immune guinea pigs after incubation of PEG with KGl-extracted L 2G or Ln-lO tumor antigens MMI's" of PEC incubated with: Guinea pigs immunized with L 2 C extracted proteins Ln-10 extracted proteins L 2 C leukemia cells Ln-10 hepatoma cells a Values are expressed as means±se. 1 mg/ml 138.4± ± ±28 2 mg/ml 122.8± ± ±30 1 mg/ml 108.7± ±6 2 mg/ml 102.1± ±11 TABLE 4.-Survival time of guinea pigs immunized with L 2 G tumor extracts either in GFA or in saline 14 days prior to inoculation with 5xlO' L 2 G tumor cells Animals immunized with CFA only 0.1 mg L 2 C extract in CFA 0.5 mg L 2 C extract in CFA 1.0 mg L 2 C extract in CFA 3.0 mg L 2 C extract in CFA 3.0 mg L 2 C extract in saline 10.0 mg L 2 C extract in saline Expt No. 1b 0/5 (18-24) 0/5 (21) 0/5 (18-22) 4/5 (21) 0/5 (17-27) 0/5 (19-27) Fraction of animals surviving the leukemia challenge a Expt No. 2b 0/5 (19-24) 0/5 (21) 0/5 (20-24) 2/5 (21) 4/5 (21) a No. of survivors/no. of animals inoculated. Numbers in parentheses indicate ranges of survival times in days. b Tumor cells were injected in the same side in which the antigen was administered. C Tumor cells were. injected in the side contralateral to the antigen injection site. Expt No.3' 0/5 (19-24) 0/5 (21-25) 4/5 (23) VOL. 60, NO.4, APRIL 1978 J NATL CANCER IT

4 902 BRAUN, HENGST, MOKYR, AND DRAY were positive in MIF and skin test assays) or an extract of normal guinea pig lymphoid. cells could immunize against a challenge with L 2 C tumor cells. Guinea pigs (5/ group) were given injections of 1 or 3 mg of protein extracted from Ln-lO hepatoma, L 2 C leukemia, or normal guinea pig lymphoid cells in CFA; they were challenged 2 weeks later with 5 X 10 4 L 2 C tumor cells (table 5). Survival was not prolonged in guinea pigs pretreated with either normal lymphoid cell proteins (1 or 3 mg in CFA) or Ln-lO tumor cell proteins (1 or 3 mg in CFA). In contrast, preinjection of 1 or 3 mg of L 2 C proteins com pletely prevented tumor growth in L 2 C-challenged Survival of Guinea Pigs Immunized With L 2C Tumor Antigen Extracts on the Same Day as the Challenge With L 2 C Tumor Cells Since extracts of L 2 C tumor cells had immunoprotective effects, the immunotherapeutic effects of these extracts were investigated. Animals were given intradermal injections of 5x 10 4 viable L 2 C cells and then 1 hour later were given injections of mg extracted L 2 C proteins in CF A. The tumor antigen preparation in CF A was administered into eight separate injection sites including both front and rear footpads and both axillary and inguinal node areas. Two separate preparations of L 2 C antigen were used for these experiments (table 6). Survival was substantially prolonged in animals that received extracted L 2 C tumor proteins in C FA ( mg, expt No.1, table 6; 5.5 mg, expt No.2, table 6). In contrast, treatment of strain 2 guinea pigs with extracted Ln-lO tumor proteins ( mg in CFA) on the same day as the L 2 C tumor challenge did not prolong survival (table 7). DISCUSSION The present study demonstrates that KCI extracts of L 2 C tumor cells can stimulate in vitro MIF production in PEC from guinea pigs immunized with L 2 C tumor cells. L 2 C extracts, which were active in vitro, were also active in vivo, since guinea pigs immunized with L 2 C tumor extracts were completely resistant to a lethal challenge with L 2 C tumor cells given 2 weeks later. Furthermore, immunization of guinea pigs within 1 hour after challenge with L 2 C tumor cells effectively prolonged the survival of the hosts. The immunoprotective activity of extracted tumor antigens is the most relevant criterion for their functional integrity. To protect an animal against challenge with a lethal dose of tumor cells, the extract must be immunogenic and capable of eliciting the type and level of immunity needed to handle the existing tumor burden. Our KCI extracts of L 2 C tumor cells meet this criterion, since they could elicit immunoprotective activity against a subsequent challenge with a lethal dose of L 2 C tumor cells. These findings are in accord with a number of reports that have illustrated the capacity of KCI extracts of tumor cells to induce specific tumor resistance in syngeneic recipients (16-19). The degree of tumor antigen-induced protection that has been reported, however, is variable presumably due to differences in: a) the immunogenicity of tumors, b) the means by which tumor antigens are administered to the host, i.e., dose, route, adjuvant, and injection schedules, c) the stability of tumor antigens to extraction, and d) the susceptibility of tumors to immune-mediated rejection. Preliminary reports by Hu et al. (20) indicate that KCI extracts of L 2 C tumor cells administered in adjuvant to strain 2 guinea pigs completely protect against a subsequent challenge with L 2 C. These antigen-immunized animals exhibit positive delayed-type hypersensitivity skin reactions upon challenge with viable tumor cells. Our data are in accord with these preliminary reports in that immunoprotection was induced by extracted L 2 C antigens in adjuvant and that positive delayed-type hypersensitivity skin reactions were elicited following challenge with L 2 C tumor cells. In addition, we have demonstrated that adjuvant-administered antigen preparations capable of inducing immunoprotection also can TABLE 7.-8urvival time of guinea pigs immunized with extracts of L 2 G tumor cells or Ln-IO tumor cells 1 hour after challenge with 5 xl O' L 2G tumor cells Animals immunized with" CFAonly 1.1 mg Ln-lO extract in CFA 2.75 mg Ln-10 extract in CFA 5.5 mg Ln-10 extract in CFA 5.5 mg L 2 C extract in CFA 18.2± ± ± ± ± ±5.1 " No. of guinea pigs used was 5/group. b Probability values were calculated relative to CF A alone by Student's t-test for 2 means. =not significant. <0.01 TABLE 6.-8urvival time of guinea pigs immunized with L 2 G tumor extracts (emulsified in GFA) 1 hour after challenge with 5 xl O' L 2G tumor cells Expt No. I" Expt No.2" Animals immunized with days, pb days, mean±se mean±se pb 21.3±0.5 (7) 22.0±1.0 (3) CFAonly 28.8±3.6 (6) 24.2±1.7 (5) 0.55 mg L 2 C extract in CFA 26.0±3.4 (6) 1.1 mg L 2 C extract in CF A 41.6±2.6 (5) < ±3.4 (5) 2.75 mg L 2 C extract in CFA 39.7±5.8 (6) < ±2.3 (5) 5.5 mg L 2 C extract in CFA 42.4±6.1 (5) < ±7.9 (5) <0.001 "Numbers in parentheses indicate No. of guinea pigs used. b Probability values were calculated relative to CFA alone by Student's t-test for 2 means. =not significant. J NATL CANCER IT VOL. 60, NO.4, APRIL 1978

5 ANTITUMOR IMMUNITY TO L2C TUMOR CELLS 903 elicit MIF production in PEC from tumor-immune Thus the MIF test might be a useful method of screening extracts for their in vivo potency. Guinea pigs immunized with L 2 C tumor extracts exhibited positive delayed-type hypersensitivity skin reactions hours after challenge with viable L 2 C tumor cells. This indicates that the response engendered by administration of soluble L 2 C tumor extracts has a component of cell-mediated immunity. Studies by Law et at. (19), Kahan et at. (21), and Mokyr et at. (22), which demonstrate that lymphoid cells from animals treated with soluble tumor antigens can retard tumor cell outgrowth in Winn assays, are consistent with the notion that KCI-extracted tumor antigens induce cellmediated responses in the host. In addition, reports showing that the immunity induced by L 2 C cells in CF A can be adoptively transferred with cells but not with sera from tumor-immune animals emphasize the cellular nature of the immunity to L 2 C tumor cells (8, 23). Our studies with extracted L 2 C tumor antigens demonstrate that CF A is needed to elicit a potent antitumor response in tumor-challenged These observations are in accord with those of Ellman and Green (24) who directly compared the tumor resistance of guinea pigs when immunized with irradiated tumor cells either in saline or in adjuvant. Animals immunized with cells in CF A were protected against challenge with greater numbers of viable L 2 C cells and were protected against challenge in shorter intervals post immunization as compared to hosts immunized with cells in saline. Our study extends the observations of Ellman and Green by demonstrating the superiority of adjuvant-administered extracts of L 2 C tumors over saline-administered materials. Administration of L 2 C tumor antigens in saline did not extend the survival of tumor-challenged Furthermore, the degree of pr'otection obtained by immunization with adjuvant-administered L 2 C tumor proteins was in the range of that obtained by immunization with 25 X 10 6 viable cells in C FA and also in the range reported by Ellman and Green with 50x 10 6 irradiated cells in CF A (24). Tumor antigen extracts have been reported to be immunoprotective (16-19). Here we show the immunotherapeutic effect of L 2 C tumor extracts. Thus L 2 C antigens administered in CF A prolonged the survival of guinea pigs given injections of tumor cells 1 hour earlier. These data imply that the immunization with tumor antigens elicited both a rapid development of tumor immunity and a substantial level of immunity, since significant extensions of survival were obtained in treated animals challenged with a tumor known to be highly metastatic and rapidly proliferative. Biologically active tumor cell extracts may be applicable to the therapy of human neoplastic disease. Thus the administration of tumor extracts to patients during remission periods when the tumor burden is minimal may impair the viability of the remaining neoplastic cells. Furthermore, the solubilization and purification of the active principals in KCl extracts may afford insight into the nature of host immunity toward tumor growth. REFERENCES (1) CONGDON CC, LORENZ E: Leukemia in guinea pigs. Am j Pat hoi 30: , 1954 (2) SHEVACH EM, ELLMAN L, DAVIE jm, et al: L 2 C guinea pig lymphatic leukemia: A "B" cell leukemia. Blood 39:1-12,1972 (3) GROSS L, DREYFUSS Y, EHRENREICH T, et al: Experimental studies on leukemia in guinea pigs. Acta Haematol (Basel) 43: ,1970 (4) OPLER SR: Animal model of viral oncogenesis. Nature 215:184, 1967 (5) PEARSON jw, PERK K, CHIRIGOS MA, et al: Drug therapy against a transplantable guinea pig leukemia. Cancer Res 35: , 1975 (6) GROSS L: Specific, active, intradermal immunization against leukemia in guinea pigs. Acta Haematol (Basel) 44: 1-10, 1970 (7) GROSS L, FELDMAN DG, EHRENREICH T, et al: Comparative studies of subcutaneous and intradermal leukemic tumors in guinea pigs. Cancer Res 33: ,1973 (8) ELLMAN L, GREEN I: Guinea pig leukemia: Immunoprotection and immunotherapy. Cancer 28: ,1971 (9) MURPHY SG, LAUFMAN H, LoBUGLIO AF: Immune response to L 2 C guinea pig leukemia-specific antigens. j Nat! Cancer Inst 58: ,1977 (10) RAPP Hj, CHURCHILL WH jr, KRONMAN BS, et al: Antigenicity of a new diethylnitrosamine-induced transplantable guinea pig hepatoma: Pathology and formation of ascites variant. j Nat! Cancer Inst 41:1-11,1968 (11) REISFELD RA, KAHAN BD: Biologic and chemical characterization of human histocompatibility antigens. Fed Proc 29:2034, 1970 (12) LOWRY OH, ROSEBROUGH Nj, FARR AL, et al: Protein measurement with the Folin phenol reagent. j BioI Chern 193: , 1951 (13) ZBAR B, BERTEIN I, TANAKA T, et al: Tumor immunity produced by the intradermal inoculation of living tumor cells and Mycobacterium bovis (strain BCG). Science 170: , 1970 (14) PAQUE RE, KNISKERN Pj, DRAY S, et al: In vitro studies with "transfer factor": Transfer of the cell-migration inhibition correlate of delayed hypersensitivity in humans with celllysates from humans sensitized to histoplasmin, coccidioidin, or PPD. j ImmunolI03: , 1969 (15) jureziz RE, THOR DE, DRAY S: Transfer of the delayed hypersensitivity skin reaction in the guinea pig using RNA-treated lymphoid cells. j Immunol 105: , 1970 (16) MELTZER MS, LEONARD Ej, HARDY AS, et al: Protective tumor immunity induced by potassium chloride extracts of guinea pig hepatomas. j Nat! Cancer Inst 54: , 1975 (17) PELLIS NR, TOM BH, KAHAN BD: Tumor-specific and allospecific immunogenicity of soluble extracts from chemically induced murine sarcomas. j Immunol 113: , 1974 (18) PELLIS NR, KAHAN BD: Specific tumor immunity induced with soluble materials: Restricted range of antigen dose and of challenge tumor load for immunoprotection. j Immunol 115: ,1975 (19) LAW LW, HENRIKSEN 0, APELLA E: Tumour rejection properties of solubilized TSTA from an SV40-induced neoplasm. Nature 257: , 1975 (20) Hu CP, SCHWARTZ BD, GREEN I: Immunogenic properties of solubilized tumor associated transplantation antigens (TAT A) of guinea pig L 2 C leukemia. Fed Proc 36:1260, 1977 (21) KAHAN BD, TOM BH, MOKYR MB, et al: Purification of tumorspecific antigens: An overview of the relevance to human colon carcinoma. Cancer 36: , 1975 (22) MOKYR MB, KAHAN BD, PELLIS NR: Kinetics and characterization of soluble antigen-induced tumor resistance. Fed Proc 36:1205,1977 (23) GROSS L: Studies on the nature of acquired immunity against leukemia in guinea pigs. Acta Haematol (Basel) 45: , 1971 (24) ELLMAN L, GREEN 1: The role of complete Freund's adjuvant in the immunoprotection of L 2 C guinea pig leukemia. Proc Soc Exp BioI Med 138: , 1971 VOL. 60, NO.4, APRIL 1978 j NATL CANCER IT