Effects of Murine Tumors Upon Delayed Hypersensitivity to Dinitrochlorobenzene. 11. Transitory Delayed Hypersensitivity in Tumor-Bearing Mice 1 J. Milburn Jessup 2,
3. 4
and Max H. Cohen
2
ABSTRACT-A model for the study of tumcr-tnduced nonspecific suppression of delayed hypersensitivity was developed in BALBtc mice bearing syngeneic transplantable tumors. Twelve or more days after tumor inoculation, tumor·bearing and normal mice were given injections of 2 mg dinitrochlorobenzene (DNCB) dissolved in dimethyl sulfoxide (DMSO). One hind footpad was challenged 7 or 10 days later with 0.05 mg DNCB in 50% DMSO. Footpads were measured 24 hours later with a micrometer. Mice whose tumors spontaneously regressed had normal footpad responses to DNCB, whereas mice bearing progressively growing tumors had suppressed footpad responses when these mice were challenged 10 days after DNCB sensitization. When challenged 7 days after DNCB sensitization, tumor-bearing mice had positive footpad responses. Because tumor burdens that varied trom 1 to 16% of body weight were equally suppressive, the suppression of tootpad response seen 10 days after DNCB sensitization was not associated with advancing tumor burden. In other experiments, sera and spleen cells were passively transferred from tumor bearers to normal recipients who were simultaneously sensitized to DNCB. Spleen cells could inhibit sensitization to DNCB if they were harvested from tumor bearers 13-24 days after tumor lnlttation. lf collected more than 25 days after tumor inoculation, spleen cells lost this inhibitory activity. If harvested 25 or more days after tumor initiation, sera similarly inhibited sensitization to DNCB In normal recipients. The results indicated that delayed hypersensitivity was Inltiated in tumor-bearlnq mice but that the expression of delayed hypersensitivlty could be inhibited by cellular and humoral factors.-J Natl Cancer Inst 59: 12211226,1977.
Cancer patients who can be sensitized to simple chemieals such as DNCB may have a better prognosis or response to therapy than patients who cannot be sensitized (1). Recent reports also indicate that patients with advanced, disseminated malignant diseases may be anergie to DNCB or other non-tumor-associated antigens whereas patients with minimal, localized cancer may (2) or may not (3-.5) be anergic. Identification of the factors that enable some cancer patients to be sensitized to non-tumor-associated antigens might result in a better appreciation of the mechanisms by which tumors immunosuppress their hosts and might suggest better approaches to immunotherapy. Studies of the primary cell-mediated immunity responses to non-tumor-associated antigens in tumor-bearing animals have generally dealt with the ability of the host to reject skin allografts. Linder (6) demonstrated tumor-associated immunosuppression by showing that mice bearing primary 3methylcholanthrene-induced sarcomas could reject strongly antigenic skin allografts but would accept skin grafts with only weak non-H-2-antigenic differences. Mice bearing advanced spontaneous mammary adenocarcinomas also accepted weakly antigenie skin grafts. Matsuyama and Nakamura (7) similarly showed that a transplantahle murine fibrosarcoma could suppress a tumor-bearing animal's ability to reject an unrelated tumor allograft. McCarthy (8) demonstrated that mice hearing the Ehrlich ascites carVOL. 59, NO. 4, OCTOBER 1977
1221
cinoma subcutaneously, but not intraperitoneally, could reject skin allografts or xenografts. The data from these experiments suggest that tumor-hearing animals have an inhibition of immunity to weak transplantation antigens, but they may be primarily sensitized to strong transplantation antigens. We previously described an assay in which mice were sensitized subcutaneously with 2 mg DNCB and challenged 10 days later with 0.05 mg DNCB in a footpad (9). The footpad swelling that resulted measured delayed hypersensitivity to DNCB and could be passively transferred to normal mice by immune lymphoid cells but not by sera. When DNCB sensitization was performed 10 days after inoculation of 10 6 syngeneic lymphoma or fibrosarcoma cells, subsequent DNCB footpad challenge resulted in depressed or absent footpad reactivity to DNCB. However, nonspecific inflarnmatory responses were intact in tumor-bearing mice, since tumor-bearing and tumor-free animals developed equivalent inflammatory reactions at the DNCB challenge site. The purpose of this report was to study the time course of DNCB anergy in tumor-bearing mice and its relationship to lymphoid cells and sera ofthese animals.
MATERIALS AND METHODS Anz·maL5.-BALB/c mice, 2-3 months old, were obtained from the Animal Production Unit, National Institutes of Health. Housed in cages in groups of 10-25, they were fed water and chow ad libitum and weighed between 16 and 20 g when used in an experiment. Tumors. - RLO"I was obtained from Dr. T. Aoki of the Viral Leukemia and Lymphoma Branch, National Cancer Institute. This radiation-induced lymphoma of BALB/c male mice possesses the X.l antigen (10, 11). RLO"l is immunogenic for BALB/c mice and spontaneously regresses in about a third of untreated mice. It kills its host by metastasizing to lungs, liver, and spleen. Meth A is a 3-methylcholanthrene-induced fibrosarcoma, which was obtained from Dr. R. Cassell at the Sloan-Ketter-
ABBREVIATIONS USED: DNCB = dinitrochlorobenzene; DMSO = dimethyl sulfoxide; % FPS ::::: percent footpad swelling; HBSS = Hanks' balanced salt solution: TW ITBW = tumor weighr/rotal body weight.
I
Received November 11, 1976; accepted April 11 , 1977.
2 Surgery Branch, National Cancer Institute, National Institutes of
Health, Public Health Service, V.S. Department ofHealth, Education, and Welfare, Bethesda, Md. 20014. 3 Currently a visiting scientist at the NCI Frederick Cancer Research Center, P.O. BoxB, Frederick, Md. 21701. 4 A ddress reprint requests to Dr. Jessup at the Frederick Cancer Research Center.
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NATL CANCER INST
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JESSUP AND COHEN
ing Institute, Rye, New York (12). It is immunogenic in syngeneic BALB/c fernales and will also spontaneously regress in one-third to two-thirds ofinoculated mice. Both tumors were carried by serial passage of ascites in BALB/mice of the sex of origin. DNCB sensitization and challenge. - The method of DNCB sensitization was asdescribed previously (9). Mice were inoculated sc with 2 mg DNCB crystals (Eastman Kodak Co., Rochester, N.Y.) dissolved in 0.10 ml DMSO Q. T. Baker Chemical Co., Phillipsburg, N.J.) in one flank. Under light ether anesthesia, mice were inoculated 7 or 10 days later with 0.05 ml of 0.1 % DNCB in 50% DMSO in the plantar aspect of a hind footpad; 24 hours later, both hind footpads were measured with a micrometer (MG/National Co., New York, N.Y.). We then ca1culated % FPS by the formula: %FPS= thickness ofinjected footpad - thickness ofuninjected footpad xl00. thickness of uninjected footpad
Preparation ofspleen cells.-From 3 to 6 mice were killed by cervical dislocation, and their left flanks were shaved and prepared with 70% ethanol. Spleens were removed aseptically, pooled, and finely minced in cold HBSS in a petri dish. After clumps were allowed to settle, 20 ml of the supernatant was removed by aspiration and layered over 10 ml of lymphocyte separation medium (LSM; Litton Bionetics, Inc., Kensington, Md.) in 50-mI plastic centrifuge tubes. These tubes were then centrifuged at 3,000 rpm for 5 minutes at room temperature. Cells collected at the interface were removed by aspiration, washed three times in cold HBSS, and then counted. Cell viability was determined by trypan blue dye exc1usion. Collected cells were at least 90% viable. Serum collection. - Tumor-bearing or normal mice under light ether anesthesia were bled from the retro-orbital plexus at various times after tumor inoculation as already stated. The sera were pooled, allowed to clot for an hour at room temperature, centrifuged at 3,000 rpm for 20 minutes, and then kept at 4 0 C for several hours until use. Experimental design. -Groups of mice were inoculated sc in one flank with 106 RLO"I or Meth A cells in 0.10 ml of cold HBSS. Littermates were not given injections. After 12-30 days, tumor-bearing mice and littermates were sensitized to DNCB in the opposite flank. Although mice inoculated with tumor might have tumors that regress spontaneously, all mice developed small papules at the site of tumor inoculation. Therefore, when DNCB sensitization was performed within 15 days of tumor inoculation, we could not predict which mice would have a tumor that would undergo spontaneous regression. Seven or 10 days after DNCB sensitization, mice were challenged in the hind footpad, ipsilateral to the prior DNCB sensitization. Both hind footpads were then measured 24 hours later with a micrometer. Tumor areas were determined in tumor- bearing mice by measurement with a caliper of the two largest perpendicular diameters and ca1culation of the product. Determination of tumor area in this way was a good approximation of tumor burden,since regression analysis of tumor area versus the ratio TW/TBW yielded the regression line TW/TBW (%) = 0.064 X tumor area (cms) - 0.036 with
J
NATL CANCER INST
a coefficient of correlation 0.93, which was significant at P
3. 4
and Max H. Cohen
2
ABSTRACT-A model for the study of tumcr-tnduced nonspecific suppression of delayed hypersensitivity was developed in BALBtc mice bearing syngeneic transplantable tumors. Twelve or more days after tumor inoculation, tumor·bearing and normal mice were given injections of 2 mg dinitrochlorobenzene (DNCB) dissolved in dimethyl sulfoxide (DMSO). One hind footpad was challenged 7 or 10 days later with 0.05 mg DNCB in 50% DMSO. Footpads were measured 24 hours later with a micrometer. Mice whose tumors spontaneously regressed had normal footpad responses to DNCB, whereas mice bearing progressively growing tumors had suppressed footpad responses when these mice were challenged 10 days after DNCB sensitization. When challenged 7 days after DNCB sensitization, tumor-bearing mice had positive footpad responses. Because tumor burdens that varied trom 1 to 16% of body weight were equally suppressive, the suppression of tootpad response seen 10 days after DNCB sensitization was not associated with advancing tumor burden. In other experiments, sera and spleen cells were passively transferred from tumor bearers to normal recipients who were simultaneously sensitized to DNCB. Spleen cells could inhibit sensitization to DNCB if they were harvested from tumor bearers 13-24 days after tumor lnlttation. lf collected more than 25 days after tumor inoculation, spleen cells lost this inhibitory activity. If harvested 25 or more days after tumor initiation, sera similarly inhibited sensitization to DNCB In normal recipients. The results indicated that delayed hypersensitivity was Inltiated in tumor-bearlnq mice but that the expression of delayed hypersensitivlty could be inhibited by cellular and humoral factors.-J Natl Cancer Inst 59: 12211226,1977.
Cancer patients who can be sensitized to simple chemieals such as DNCB may have a better prognosis or response to therapy than patients who cannot be sensitized (1). Recent reports also indicate that patients with advanced, disseminated malignant diseases may be anergie to DNCB or other non-tumor-associated antigens whereas patients with minimal, localized cancer may (2) or may not (3-.5) be anergic. Identification of the factors that enable some cancer patients to be sensitized to non-tumor-associated antigens might result in a better appreciation of the mechanisms by which tumors immunosuppress their hosts and might suggest better approaches to immunotherapy. Studies of the primary cell-mediated immunity responses to non-tumor-associated antigens in tumor-bearing animals have generally dealt with the ability of the host to reject skin allografts. Linder (6) demonstrated tumor-associated immunosuppression by showing that mice bearing primary 3methylcholanthrene-induced sarcomas could reject strongly antigenic skin allografts but would accept skin grafts with only weak non-H-2-antigenic differences. Mice bearing advanced spontaneous mammary adenocarcinomas also accepted weakly antigenie skin grafts. Matsuyama and Nakamura (7) similarly showed that a transplantahle murine fibrosarcoma could suppress a tumor-bearing animal's ability to reject an unrelated tumor allograft. McCarthy (8) demonstrated that mice hearing the Ehrlich ascites carVOL. 59, NO. 4, OCTOBER 1977
1221
cinoma subcutaneously, but not intraperitoneally, could reject skin allografts or xenografts. The data from these experiments suggest that tumor-hearing animals have an inhibition of immunity to weak transplantation antigens, but they may be primarily sensitized to strong transplantation antigens. We previously described an assay in which mice were sensitized subcutaneously with 2 mg DNCB and challenged 10 days later with 0.05 mg DNCB in a footpad (9). The footpad swelling that resulted measured delayed hypersensitivity to DNCB and could be passively transferred to normal mice by immune lymphoid cells but not by sera. When DNCB sensitization was performed 10 days after inoculation of 10 6 syngeneic lymphoma or fibrosarcoma cells, subsequent DNCB footpad challenge resulted in depressed or absent footpad reactivity to DNCB. However, nonspecific inflarnmatory responses were intact in tumor-bearing mice, since tumor-bearing and tumor-free animals developed equivalent inflammatory reactions at the DNCB challenge site. The purpose of this report was to study the time course of DNCB anergy in tumor-bearing mice and its relationship to lymphoid cells and sera ofthese animals.
MATERIALS AND METHODS Anz·maL5.-BALB/c mice, 2-3 months old, were obtained from the Animal Production Unit, National Institutes of Health. Housed in cages in groups of 10-25, they were fed water and chow ad libitum and weighed between 16 and 20 g when used in an experiment. Tumors. - RLO"I was obtained from Dr. T. Aoki of the Viral Leukemia and Lymphoma Branch, National Cancer Institute. This radiation-induced lymphoma of BALB/c male mice possesses the X.l antigen (10, 11). RLO"l is immunogenic for BALB/c mice and spontaneously regresses in about a third of untreated mice. It kills its host by metastasizing to lungs, liver, and spleen. Meth A is a 3-methylcholanthrene-induced fibrosarcoma, which was obtained from Dr. R. Cassell at the Sloan-Ketter-
ABBREVIATIONS USED: DNCB = dinitrochlorobenzene; DMSO = dimethyl sulfoxide; % FPS ::::: percent footpad swelling; HBSS = Hanks' balanced salt solution: TW ITBW = tumor weighr/rotal body weight.
I
Received November 11, 1976; accepted April 11 , 1977.
2 Surgery Branch, National Cancer Institute, National Institutes of
Health, Public Health Service, V.S. Department ofHealth, Education, and Welfare, Bethesda, Md. 20014. 3 Currently a visiting scientist at the NCI Frederick Cancer Research Center, P.O. BoxB, Frederick, Md. 21701. 4 A ddress reprint requests to Dr. Jessup at the Frederick Cancer Research Center.
J
NATL CANCER INST
1222
JESSUP AND COHEN
ing Institute, Rye, New York (12). It is immunogenic in syngeneic BALB/c fernales and will also spontaneously regress in one-third to two-thirds ofinoculated mice. Both tumors were carried by serial passage of ascites in BALB/mice of the sex of origin. DNCB sensitization and challenge. - The method of DNCB sensitization was asdescribed previously (9). Mice were inoculated sc with 2 mg DNCB crystals (Eastman Kodak Co., Rochester, N.Y.) dissolved in 0.10 ml DMSO Q. T. Baker Chemical Co., Phillipsburg, N.J.) in one flank. Under light ether anesthesia, mice were inoculated 7 or 10 days later with 0.05 ml of 0.1 % DNCB in 50% DMSO in the plantar aspect of a hind footpad; 24 hours later, both hind footpads were measured with a micrometer (MG/National Co., New York, N.Y.). We then ca1culated % FPS by the formula: %FPS= thickness ofinjected footpad - thickness ofuninjected footpad xl00. thickness of uninjected footpad
Preparation ofspleen cells.-From 3 to 6 mice were killed by cervical dislocation, and their left flanks were shaved and prepared with 70% ethanol. Spleens were removed aseptically, pooled, and finely minced in cold HBSS in a petri dish. After clumps were allowed to settle, 20 ml of the supernatant was removed by aspiration and layered over 10 ml of lymphocyte separation medium (LSM; Litton Bionetics, Inc., Kensington, Md.) in 50-mI plastic centrifuge tubes. These tubes were then centrifuged at 3,000 rpm for 5 minutes at room temperature. Cells collected at the interface were removed by aspiration, washed three times in cold HBSS, and then counted. Cell viability was determined by trypan blue dye exc1usion. Collected cells were at least 90% viable. Serum collection. - Tumor-bearing or normal mice under light ether anesthesia were bled from the retro-orbital plexus at various times after tumor inoculation as already stated. The sera were pooled, allowed to clot for an hour at room temperature, centrifuged at 3,000 rpm for 20 minutes, and then kept at 4 0 C for several hours until use. Experimental design. -Groups of mice were inoculated sc in one flank with 106 RLO"I or Meth A cells in 0.10 ml of cold HBSS. Littermates were not given injections. After 12-30 days, tumor-bearing mice and littermates were sensitized to DNCB in the opposite flank. Although mice inoculated with tumor might have tumors that regress spontaneously, all mice developed small papules at the site of tumor inoculation. Therefore, when DNCB sensitization was performed within 15 days of tumor inoculation, we could not predict which mice would have a tumor that would undergo spontaneous regression. Seven or 10 days after DNCB sensitization, mice were challenged in the hind footpad, ipsilateral to the prior DNCB sensitization. Both hind footpads were then measured 24 hours later with a micrometer. Tumor areas were determined in tumor- bearing mice by measurement with a caliper of the two largest perpendicular diameters and ca1culation of the product. Determination of tumor area in this way was a good approximation of tumor burden,since regression analysis of tumor area versus the ratio TW/TBW yielded the regression line TW/TBW (%) = 0.064 X tumor area (cms) - 0.036 with
J
NATL CANCER INST
a coefficient of correlation 0.93, which was significant at P
