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ICANCER RESEARCH 31, 864—867,June 1971) Phytohemagglutinin Unresponsiveness in Mouse Spleen Cells Induced by Methylcholanthrene Sarcomas' William H. Adler,2 Tomoo Takiguchi, and Richard T. Smith3 Tumor Biology Unit, Department ofPathology, University of Florida, CollegeofMedicine, Gainesville,Florida 32601 SUMMARY The composition of spleen cell populations and the response of spleen cells in vitro to phytohemagglutinin (PHA) were studied in mice bearing methyicholanthrene-induced sarcomas and mice immunized with membrane antigen. With the use of discontinuous albumin density gradients to separate spleen cell suspensions, it was found that tumor-bearing mice and immunized mice had a shift in their spleen cell population, with a depletion of the dense, small lymphocyte population and a majority representation of a less dense, large mononuclear cell population. The PHA reactivity of the spleen cells from the tumor-bearing and immunized mice was less than normal. This finding correlated with the shift in the spleen cell population because the less dense cell types respond minimally to PHA. In mice in which the tumors were excised, the PHA response of the whole spleen cell population returned to normal. Our findings with the tumor-bearing mice suggest that a poor response to PHA may not be indicative of an immune deficiency but rather may demonstrate the possibility that the mice are undergoing an active immune response to their tumors. In human cancer patients, a poor peripheral lymphocyte response to PHA and an inability to demonstrate delayed hypersensitivity reactions have been interpreted as indication of an immune deficiency. However, it could be speculated that these findings in these patients may also suggest the presence of an active immune response to their tumors, rather than an immune deficiency. At present, speculation provides a different working hypothesis evaluation of the immune status of cancer patients. this for the INTRODUCTION Several recent lines of investigation have suggested that cell-mediated immunity is impaired in most individuals carrying malignant tumors (13). Several parameters have been I Supported in part by American Cancer Society Grants ACS-T-463 and hN-62-G, Florida Division of the American Cancer Society Grant F-7-OUF, National Institute of Child Health and Human Development Grant HD-00384, National Institute of General Medical Sciences Grant GM-01996, and Training Grant AI-00401 in cellular immunobiohogy from the National Institute of Allergyand Infectious Diseases. 2 Present address: Fort Detrick, Md. 21701. 3To whom requests for reprints should be addressed, at Department of Pathology, College of Medicine, University of Florida, Gainesville, Fla. 32601. Received January 864 8, 197l;accepted February 23, 1971. measured, including lowered absolute lymphocyte levels (12), lessened lymphocyte responses to mitogenic stimulation (5, 16), and decreased capacity to be sensitized by chemical agents such as 2,4-dinitrochlorobenzene (3) or 2 ,4-dinitrofluorobenzene (8). Moreover, the association of immunological deficiency with neoplastic disorders has been most clear in those individuals who have abnormal or deficient function of their thymus-dependent lymphoid system (6). It has been tempting to link these observations causally in general support of a hypothesis that immune surveillance has a major role in prevention of cancer through the thymus-de pendent, cell-mediated immune mechanism (15). For exploration of the mechanisms underlying decreased manifestations of cell-mediated immunity in tumor-carrying animals, in vitro lymphocyte stimulation by PHA4 was assayed in the spleen cells from methylcholanthrene sarcoma-bearing mice, and the results were compared with the PHA response in the spleen cells of animals immunized with a nonneoplastic membrane antigenic stimulus, either sheep RBC or allogeneic cells. The results suggest that decreased PHA reactivity found in the spleen cell population of tumor-bearing animals may be related to immunological commitment rather than immunological deficiency. MATERIALS AND METHODS A/J and CBA strains of mice used in these experiments were originally purchased from R. B. Jackson Laboratories and have been maintained for 3 years in our laboratory through subsequent inbreeding. Tumor production was accomplished by injections of 0.1 mg methylcholanthrene in 0.1 ml olive oil i.m. into female mice. The tumors that arose 6 to 12 weeks later were serially passed in female A/J mice. The 3 A/J methylcholanthrene sarcomas used in these experiments were designated MA-2, MA-6, and MA-7. Tumors were not used after a 5th mouse passage. The tumor was passed by giving recipient syngeneic mice injections of 2 X 106 dispersed tumor cells s.c. A palpable tumor was easily demonstrated 2 weeks after inoculation, and the mice were used for the experiments 4 weeks after the tumors appeared. Tumor removal was accomplished by surgical excision when the tumor measured 1 cm in diameter. The culture method used has been reported in detail previously (2). The same serum donor *as used for all experiments reported here. PHA-stimulated (10 p1/tube) cultures consisted of 15 X 106 mononuclear spleen cells in 3 4The abbreviation used is: PHA, phytohemagglutinin. CANCER RESEARCH VOL.31 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1971 American Association for Cancer Research. Methykholanthrene density gradients. The layers of the fractionated cells were designated: Layer A, between the 10 and 23% albumin solutions; Layer B, between the 23 and 26% albumin; Layer C, between 26 and 29% albumin; and Layer D, the most dense, small lymphocyte population, between 29 and 35% albumin solutions. The pellet was not used for culture or considered in the enumeration of the separated populations. Each layer is represented as the proportion of cells in that layer in relation to the total cells recovered in all 4 layers. As previously described, cells found in each layer were relatively homogeneous with regard to density in that between 73 to 95% would migrate to the same layer when subjected to a 2nd identical gradient fractionation (1). ml culture medium (RPM! 1640 with 5% heat-inactivated human serum). PHA-stimulated and control cultures were maintained for 48 hr. Tritiated thymidine (1 pCi, Schwarz/Mann, Orangeburg, N. Y., 1.9 Ci/mmole, I pCi/2 p1) was added to each culture during the final 20 hr. At the end of this time, the cultures were processed as described previously (2). The amount of activity in the 5% trichloroacetic acid precipitates was determined by liquid scintillation counting. The results are expressed as means of 5 replicate cultures. Individual variation of cultures from the mean was within 10%. Immunization was accomplished by injection of 0.1 ml of a 50% washed sheep RBC suspension i.v. The 2nd and 3rd immunizing injections were given at 10-day intervals. A/i mice immunized with alloantigen received 0.1 ml of a CBA spleen cell suspension containing 1 X 106 leukocytes. The method of Raidt et a!. ( 11), modified as previously described ( 1), was used for separation of spleen cells on discontinuous albumin RESULTS Gradient of the @,.oma.2 Id 71 Distribution of Spleen Cells in a mononuclear cell population of the normal mouse spleen (1). The density distribution of spleen cells was compared between mice carrying methylcholanthrene-induced sarcomas, and normal mice. As shown in Chart 1, a shift occurred in the proportion of cells of various density. In the tumor-bearing animals, there was a greater proportion of the less dense, larger cell subpopulations. The small, dense lymphocytes were markedly depleted. The PHA Stimulation of Spleen Cells from Tumor-bearing Mice. The A and B layer cells from normal mouse spleens are affected minimally by PHA and have high background thymidine-3 H incorporation levels (1). Since cells of these density characteristics are dominant in tumor-bearing mice, it was of interest to determine their relative responses to PHA stimulation. The results shown in Table 1 are representative of the findings in groups of mice carrying the 3 different methylcholanthrene-induced sarcomas. Spleen cells from these animals have a markedly decreased responsiveness to PHA stimulation. In the mice carrying the MA-2 sarcoma, a normal degree of responsiveness to PHA returned after the tumor had 14,41 A Density Tumor-bearing Mice. A relatively dense, small lymphocyte population, designated Layers C and D, constitutes a majority normal @—oma..7 6( Tumor Effect on Spleen Cells D LAYERS Chart 1. Spleen cells from A/J mice, either normal or ones carrying methylchohanthrene-induced sarcomas of syngeneic different origin, designated MA-2, MA-6, and MA-i, were separated by discontinuous albumin density gradient fractionation. The resultant profile is expressed in terms of percentage of cells found in each layer of the gradient as compared to the total number of cells recovered in all the layers. Layer A is the least dense population; Layer D is the most dense. Table 1 Comparison of the PHA response ofA/J spleen cells from normal and methylcholanthrene-induced tumor-bearing mice and of micefrom which the tumor hasbeen excised. In these experiments, 15 X 106 spleen cells were cultured in 3 ml of medium, with or without 10 @tl PHA, for 48 hr. Tritiated thymidine was added for the final 20 hr of culture. Results are expressed as the mean cpm of 5 replicate samples with less than 10% individual variation. Each of the groups in these series of experiments involved the use of pooled spleen cells obtained from the spleensof at least 4 mice.Thymidine-3 H incorporation (mean cpm/culture) incorporationaNormal Experimental groups A/J A/J A/J A/J a Mean A/J mice with mice with mice with mice with cpm in MA-6 tumorsb MA-i tumors@@ MA-2 tumorsb MA-2 tumors excisedc the b Mice in this group PHA-stimulated had a greater cultures than Unstimulated PHA-stimulated 4,872 44,332 17,704 20,693 5,626 151,023 42,931 63,951 41,326 143,846 divided 1-cm diameter by the mean transplanted, cpm in the syngeneic, Increment of incorporation 146,951 (—)1401 46,211 20,633 138,220 control Ratio of 31.8 1 3.6 1.9 25.4 cultures. methylcholanthrene-induced sarcoma. c Thesemice had carried a greater than 1-cmdiameter MA-2 sarcoma;the tumor wasexcised 1 month before testing. JUNE 1971 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1971 American Association for Cancer Research. 865 w.H.Adler,i: Takiguchi, andR.T.Smith been excised. Thymidine incorporation in unstimulated cultures of spleen cells from mice carrying the tumors was higher than in the control groups of mice or the groups from which the tumor had been excised. This correlated well with the observation that the spleen cell population shifted toward dominant representation Methylcholanthrene Tumors. The results of experiments with tumor-bearing mice were strikingly similar to those found previously in animals immunized with sheep RBC or allogeneic spleen cells (1). In both instances, a shift in spleen cell subpopulation representation occurred, as demonstrated in Chart 2. In mice thus immunized, the mitogenic stimulation of either sheep RBC stroma or of allogeneic cells in mixed leukocyte cell culture was most effective in the B layer cells ( 1). Raidt et al. ( 11) also found that the B layer contained a high proportion of antibody-forming cells. a sheep RBC-immunized and ailogeneic cell-immunized mice was examined. of A and B layer types of cells. Comparison of Effects of Immunization with the Effects of Bearing PHA Reactivity of Spleen Cells from Either Sheep RBC- or Allogeneic Cell-immunized Mice. Since the spleens of the immunized mice contain more B layer cells and less small, dense lymphocytes, the PHA reactivity of the spleen cells from ___normal G@O imraunizad Mice that had received either I or 3 immunizing injections of sheep RBC i.v. showed changes both in degree of background unstimulated thymidine incorporation and stimulation by PHA (Table 2). This change was less pronounced in the group immunized with allogeneic cells. As in tumor-bearing mice, a shift in spleen cell population toward greater representation of B layer cells was associated with higher background incorporation of tritiated thymidine and impaired reactivity to PHA. DISCUSSION In both human and animal hosts, cell-mediated immunity and antibody with specificity toward tumor-unique antigens have been demonstrated in many systems. Moreover, the immune state of the host appears to have a role in determining the outcome of the host-tumor interactions. Experimental tumor rejection appears to be mediated most effectively by immune cells, although whether through cell-mediated or antibody secretion mechanisms is not resolved (1 5). Hellström et al. (7) found evidence that a major component of the A I C D LAYERS Chart 2. Spleen cells from CBA mice, normal and immunized with sheep RBC, were separated by discontinuous albumin density gradient fractionation. The profile is expressed as the percentage of cells found in each layer of the gradient as compared to the total number of cells recovered in all layers. Layer A is the least dense population; Layer D is the most dense. immune response to a tumor, a blocking factor, presumably antibody, can be associated with faster progress of the tumor to the detriment of the host. This antibody-like factor can inhibit the in vitro tumor recognition response of host lymphocytes. Our present studies demonstrate that the PHA reactivity of mouse spleen cells is markedly affected by the state of the immune mechanisms of the animals. Whether the animal was marshalling an immune response to sheep RBC or allogeneic cells or putatively to a methylcholanthrene tumor, similar changes occur. The PHA-reactive cell subpopulation of the spleen, mainly small lymphocytes, decrease in their proportional representation in the spleen, and the total Table 2 Comparison of the PHA response ofA/J and CBA spleen cells from normal and sheep RBC- or alloantigen-immunized mice In these experiments, 15 X 106 spleen cells were cultured in 3 ml of medium, with or without 10 i.@h PHA, for 48 hr. Tritiated thymidine was added for the final 20 hr of culture. Results are expressed as the mean cpm of 5 replicate samples with less than 10% individual variation. Each of the groups in these series of experiments involved the use of pooled spleen cells obtained from the spleens of at least 4 mice.Thymidine-3 H incorporation (mean cpm/culture) incorporationaNormal Experimental groups A/J A/J receiving 1 injection of sheep RBCb A/J receiving 1 injection of CBA spleen cellsb NormalCBA CBA receiving 1 injection of sheep RBCb CBA receiving 3 injections ofsheep RBCb Unstimulated i,468 50,004 32,415 7,612 26,201 24,626 PHA-stimulated 139,388 173,143 291,5 14 180,013 113,889 71,274 Increment of incorporation Ratio of 131,920 123,139 259,099 172,401 87,688 46,648 a Mean cpm in the PHA-stimulated cultures divided by the mean cpm in the control cultures. b Immunized mice received 0.1 ml of a 50% suspension of sheep RBC i.v. or 1 X 106 CBA spleen 18.7 3.5 9.0 24 4.3 2.9 cells i.v., once or at 10-day intervals. The mice were sacrificed 2 days after the last injection, and the in vitro response to PHA was assayed. 866 CANCER RESEARCH VOL.31 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1971 American Association for Cancer Research. Methyicholanthrene reactivity of the spleen cells to PHA stimulation decreases. The cells involved in a tumor-specific, sheep RBC, or allogeneic cell immune response may be antibody-forming cells or lymphocytes dividing in the presence of membrane antigens as occurs in a purified protein derivative-driven clone of Bacillus Calmette-Guerin-sensitized cells (9). These stimulated cell populations may produce, by expansion, an actual space occupying lesion that crowds out other potentially responsive cells. On the other hand, there could be a functional lesion, possibly produced by the stimulated lymphocytes, which may secrete inhibitors in vivo analogous to the inhibitors that are known to be elaborated in vitro by lymphoblasts (14). A relatively rapid return to a normal response in an animal in which the tumor was removed lends support to this idea. Recent experiments of Ming and Ming (10) show quantitative cellular changes in the spleens of tumor-bearing mice. Their studies were interpreted to suggest that a major shift in such spleens was toward antibody-producing cells or their precursors. These findings permit an alternative interpretation of the PHA unresponsiveness of peripheral lymphocytes from cancer patients and the inability to sensitize these patients with chemical compounds. Instead of reflecting a state of immunological deficiency in any absolute terms, the failure to sensitize tumor patients may reflect evidence of an intensive immune response, especially an antibody response, to his tumor. Hellström's data and previous studies of the phenomenon of enhancement (4) suggest that this might be detrimental to the host and for this reason might correlate with a relatively poor prognosis. As a working hypothesis, it could be suggested that the inability to sensitize a patient to obtain a delayed hypersensitive response or a poor peripheral lymphocyte response to PHA in vitro may indicate a high degree of immune commitment to the tumor rather than an immune deficiency state. This hypothesis would suggest a different approach in the evaluation of the immune status of a cancer patient. REFERENCES 1. Adler, W. H., Peavy, D., and Smith, R. T. The Effects of PHA, PPD, Allogeneic Cells and Sheep Erythrocytes on Albumin Gradient Fractionated Mouse Spleen Cell Populations. Cell Immunol., 1.78—91, 1970. 2. Adler, W. H., Takiguchi, T., Marsh, B., and Smith, R. T. Cellular Recognition by Mouse Lymphocytes in Vitro. I. Definition of a New Technique and Results of Stimulation by Phytohemagghutinin and Specific Antigens. J. Expth. Med., 131: 1049—1078, 1970. 3. Eilber, F. R., and Morton, D. L. Impaired Immunologic Reactivity and Recurrence following Cancer Surgery. Cancer, 25: 362—367, 1970. 4. Flexner, S., and Jobling, J. W. On the Promoting Influence of Heated Tumor Emulsions on Tumor Growth. Proc. Soc. Exptl. Biol. Med., 4: 156—157,1907. 5. Garrioch, D. B., Good, R. A., and Gatti, R. A. Lymphocyte Response to PHA in Patients with Non-Lymphoid Tumors. Lancet, 1:618,1970. 6. Good, R. A., In: R. T. Smith, and M. Landy (eds.), Immune Surveillance, pp. 442-443. New York: Academic Press, Inc., 1970. i. Helhström,I., Hellström, K., and Sjögren, H. Serum Mediated Inhibition of Cellular Immunity to Methyhcholanthrene-induced Murine Sarcomas. Cell Immunoh., 1. 18—30,1970. 8. Levin, A. G., McDonough, E. F., Jr., Miller, D. G., and Southam, C. M. Delayed Hypersensitivity Response to DNFB in Sick and Healthy Ann. N. Y. Acad. Sci. U. S., 120: 400—409, 10. Ming, S. C., and Ming, R. L. Neutralizing Effect of Spleen Cells on I 1. 12. 13. 15. We acknowledge the technical assistance of Mrs. Karen Sarbeck and the help of Mrs. Charlotte Adler in preparation of the manuscript. Persons. 1964. 9. Marshall, W. B., Valentine, F. T., and Lawrence, H. S. Cellular Immunity in Vitro, Clonal Proliferation of Antigen Stimulated Lymphocytes. J. Expth. Med., 130: 32i—344, 1969. 14. ACKNOWLEDGMENTS Tumor Effect on Spleen Cells 16. Transplanted Methylcholanthrene-induced Sarcomas. Scientific Proceedings of the American Association of Pathologists and Bacteriologists. Am. J. Pathol., 59: 90-A, 1970. Raidt, D. J., Mishell, R. I., and Dutton, R. W. Cellular Events in the Immune Response. J. Expth. Med., 128. 681—698, 1968. Riesco, A. Five-Year Cancer Cure: Relation to Total Amount of Peripheral Lymphocytes and Neutrophils. Cancer, 25: 135—140, 1970. Smith, R. T., and Adler, W. H. Human Tumor Immunity. New EngI. J. Med., 282: 1320, 1970. Smith, R. T., Bausher, J. A. C., and Adler, W. H. Studies of an Inhibitor of DNA Synthesis and a Nonspecific Mitogen Elaborated by Human Lymphoblasts. Am. J. Pathol., 60. 495—504, 1970. Smith, R. T., and Landy, M. (eds.). Immune Surveillance. New York: Academic Press, Inc., 1970. Stjernswärd, J., Clifford, P., and Svedmyr, E. General and Tumor-distinctive Cellular Immunological Reactivity. In. D. Burkett and D. Wright (eds.), Burkitt's Lymphoma, pp. 164—iii. Edinburgh and London: E & S Livingston, 1970. JUNE 1971 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1971 American Association for Cancer Research. 867 Phytohemagglutinin Unresponsiveness in Mouse Spleen Cells Induced by Methylcholanthrene Sarcomas William H. Adler, Tomoo Takiguchi and Richard T. Smith Cancer Res 1971;31:864-867. 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