[ RESEARCH 37, 320-322, January 1977]

Brief Communication

Differences in the Responsiveness of Splenic, , and Peripheral Lymphoid Cells to Tumor Membrane Extracts1

JamesG.Krueger,RosalindA.Segal,andRexC.Moyer TheThormanCancerResearchLaboratory,TrinityUniversity,SanAntonio, Texas78284

SUMMARY measure the response by [3H]thymidine incorporation. Max imal stimulation is achieved with as little as 1 ng of protein. The blastogenic response of normal , lymph node, A close correlation between the blastogenic response to and peripheral blood lymphoid cells to tumor-associated membrane fragments and killed tumor cells has been antigens (TAA) derived from two syngeneic C57BL/6J tu shown (12). mors was measured by [3H]thymidine incorporation. Pe While a great deal of information is available on the im nipheral blood cells were responsive to TAA from B16 mela mune response to tumor cells, little information is available noma and from BW10232 mammary carcinoma at both a on the distribution of responsive cells throughout an ani high (10_i to 10@ mg/mI) and a low (10@ to 10@ mg/mI) mal. Current studies of afferent or efferent immune re concentration of antigen. While peripheral blood cells al sponses to tumor cells frequently use lymphoid cells from 1 ways responded to TAA, spleen cells and lymph node cells area or “lymphoidcompartment,―e.g., lymph nodes, the did not. When spleen and lymph node cells did respond, spleen, or peripheral blood, without attempting to correlate they sometimes responded at different concentrations of the responses from the other lymphoid compartments. TAA than did the peripheral blood cells. Spleen cells gener This study compares the blastogenic response of lymph ally responded to “low―concentrationsof TAA, while lymph oid cells from the spleen, lymph nodes, and peripheral node cells responded to “high―concentrationsof TAA. blood to membrane fragments from 2 syngeneic munine tu These data suggest two subpopulations of lymphoid cells mors. Data suggest that 2 subpopulations of lymphoid cells capable of response to TAA. Spleen cells from mice bearing may be responsive to tumor antigens and that lymphoid the BW10232 mammary carcinoma became responsive to cells from different areas do not always show similar re BW10232 TAA at low concentrations of antigen. Lymph sponses. node cells became responsive at high concentrations of BW10232 antigen. The response of both subpopulations to MATERIALS AND METHODS BW10232 TAA was amplified in peripheral blood cells. Spleen cells were 30 times more responsive in the tumor Mice. Fourteen- to 16-week-old female C57BL/6J mice, bearer than in normal animals, while lymph node cells were obtained from The Jackson Laboratory, Bar Harbor, Maine, only 3 times more responsive. It is shown that lymphoid were used for all experiments. cells taken from different areas or “lymphoidcompart Tumors.TwosyngeneicC57BL/6Jtumorswereobtained ments―do not always show similar responses and should from The Jackson Laboratory in s.c. transplant: BW10232 not be considered equivalent in evaluating immune ne mammary carcinoma and B16 melanoma. Tumors were sponses to tumor cells. maintained in s.c. transplant and were transplanted by stan dardtechniques(5). INTRODUCTION Preparation of Membrane Extracts. Membrane extracts An immunological response to syngeneic tumor cells has rich in TAA were prepared by the method of Poon and been well established (3, 6, 8, 13). Lymphoid cells respond Cauchi (12) using low salt extraction. Protein content was to syngeneic tumor cells in vitro by blastogenic transforma estimated by the Folin method (9). tion (1) and subsequent cytotoxicity (2). It has been demon LymphoidCellCulturewithTAA. Peripheralbloodcells, strated that lymphoid cell division is necessary for the pro spleen cells, or lymph node cells (from the inguinal, superfi duction of cytotoxic T-cells (10). Thus, blastogenic transfor cial cervical, brachial, and axillary lymph nodes) were cul mation probably represents the initial cellular immune ne tuned in Roswell Park Memorial Institute Medium 1640 con sponse to tumor cells. A sensitive method for evaluating taming penicillin (100 IU) and streptomycin (100 @g/ml). blastogenic response to tumor cells is to stimulate lymphoid Peripheral blood cells were cultured directly from whole cells with small membrane fragments containing TAA2 and blood (4, 7). One million spleen cells or lymph node cells were added to 1.0 ml of medium containing antibiotics and I This study was supported in part by Grant R-A-44 from the A. W. Morn supplemented with 15% fetal calf serum (same lot for all son Trust. 2 The abbreviation used is: TAA, tumor-associated-antigen. experiments). All cultures were set up in triplicate. TAA was Received August 30, 1976; accepted October 28, 1976. added in concentrations ranging from 10_i to 10@ mg pro

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teinper ml medium. Cultureswere incubatedat37°ina TAA; 1 peak was at 10@ mg protein per ml, and the other humidified atmosphere of CO2and for a total of 72 hr. A was at 5 x 10-s mg protein per ml (Chart 2A). The B16 72-hr culture produced maximal stimulation of lymphoid melanoma TAA induced a bimodal response in spleen cells, cells by TAA. During the final 24 hr of growth, the cells were with 1 peak at 10@ mg protein per ml and another at 10@ labeled with 0.5 @Ciof[5-methyl-3H]thymidine (2.0 Ci/103 mg protein per ml (Chart 2B). Lymph node cells had a uni mole). Cells were extracted on glass fiber filters and treated modal response to the B16 anfigen, in the range of 10_i to with 6% perchlonic acid. Acid insoluble counts were deter 5 x 10@ mg protein per ml (Chart 2C). mined by liquid scintillation counting (4, 7). LymphoidCellsfromMicewithBW10232MammaryCar cinomas. The response of lymphoid cells from animals bearing the BW10232 mammary tumor for 10 days (host RESULTS survival is approximately 20 days) was measured as de scnibed for lymphoid cells from healthy animals. At this Response of Lymphoid Cells from Normal Animals. Pe point in tumor growth, the lymphoid cell response to nipheral blood cells from healthy C57BL/6J mice showed BW10232 TAA was greater in tumor-bearing animals than in blastogenic responses to TAA from both syngeneic C57BL/ normal animals. Peripheral cell response was amplified in 6J tumors (Charts 1A and 2,4). Peripheral blood cells were both peaks of response to BW10232 TAA, although the the only normal cells to respond to BW10232 antigen response at the lower antigen concentration (5 x 10_6mgi (Charts 1,B and C). Peripheral blood cells exhibited a bimo ml) was greater (Chart 1A). The response of spleen cells to dal response to BW10232 TAA. Peaks in response were BW10232 antigen was approximately 30 times higher in the observed at 10_2and 5 x 10_6mg protein per ml. Peripheral tumor bearer than in normal mice. The responding cells blood cells showed a bimodal response to B16 melanoma were sensitive mainly to the lower antigen concentration of 5 x 10@ mg protein per ml (Chart 1B). Lymph node cells 16 A were responsive only at the higher antigen concentration of C 214 5 x 10@ mg protein pen ml (Chart 1C). 0 &12 C 12 0 @1o ,.‘ a) bE

N .E ______

.@,@,‘ là —' 102 i0@ iO—@i0@:_5'10_6 1@y-7 10― ;E , i t I ‘—1 @ mg. Protein/mi 10W' 10.2 10@ 10@ 10@ 10.6 iø-@ . mg Protein/mi @35 0 B C @3O 0

g25 0 0 @E20 a 0 @ .@15 — C ‘C @0 \ ‘0 a) @- x C = a. E U -C _i&i 102 iO@ 10 10 10@6 10@ 10' mg Protein/mi. =

C C 0 0

@20 C @ 0 20 a. 0 C - o15 a @ -,U x 0 C ;— 10 a. a) @ U C @- 0. @ E o• u:! 5 E >‘ -C = iô-' 102 i03 iO'@ i@-@ 10.6 iO@ 10' 1-&― 1&2' @3i@-@ iô-@ ió.6 mg Protein/mi = mg Protein/mi Chart 1. Blastogenic response of lymphoid cells to BW10232 mammary tumorTAA. The lymphoid cells are from (A) peripheral blood, (B) the spleen, Chart 2. Blastogenic response of lymphoid cells to B16 melanoma TAA. and (C) lymph nodes. •,lymphoid cells from normal mice; 0, control The Iymphoid cells are from (A) peripheral blood, (B) the spleen, and (C) response, i.e. , no TAA in culture; A, lymphoid cells from mice bearing the lymph nodes. •,lymphoidcells from normal mice; 0, control response, i.e., BW10232 tumor for 10 days; & control response. no TM in culture.

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DISCUSSION tumors using either spleen cells, lymph node cells, on pe nipheral blood cells, are often observed in the literature. We The present study suggests the existence of 2 peripheral suggest that the difference in the response of cells from lymphoid cell subpopulations responsive to membrane separate lymphoid compartments may clarify interpretation fragments, i.e., TAA, from syngeneic C57BL/6J tumors. of some of these studies. It might be helpful in future stud One subpopulation appears to be sensitive to “high―con ies to locate the compartment of responsive cells, since the centrations of antigen, while another appears to be sensi location may differ with each tumor, and to compare the tive to “low―concentrations.The idea of 2 subpopulations responses of cells from several compartments during the of lymphoid cells responsive to TAA is further supported by progress of anti-tumor immunity. the response to B16 melanoma TAA. The spleen cell ne sponseto B16TAAis chiefly at 10@mg/mI, but a minority of REFERENCES cells respond at 10@mg/mI. The lymph node response is to high concentrations of antigen only. It would seem difficult 1. Anderson, A. J., McBride, C. M., and Hersh, E. M. Blasto to explain the difference between the spleen and lymph genic Responses to Cultured Allogeneic Tumor Cells in Vitro. Cancer node responses if only 1 population of cells was present. Aes., 32: 988-992, 1972. 2. Cerottini, J-C., and Brunner, K. T. Cell-Mediated Cytotoxicity, Allograft In healthy C57BL/6J mice, cells responsive to BW10232 Rejection, and Tumor Immunity. Advan. Immunol., 18: 67—132,1974. mammary carcinoma TAA are found only in the peripheral 3. Haughton, G., and Amos, D. B. of Carcinogenesis. Cancer Aes., 28: 1839-1848, 1968. blood. However, in mice carrying the mammary tumor, the 4. Heiniger, H.-J., Taylor, B. A., Hands, E. J., and Meier, H. Heritability of lymph node cells become responsive to high concentra the Phytohemagglutinin Responsiveness of and Its Ada tions of antigen, arid spleen cells become responsive to low tionship to Laukemogenesis. Cancer Res., 35: 825-831 , 1975. 5. Kaliss, N. Transplanted Tumors. Jackson Laboratory Notes, No. 410, concentrations of antigen. In peripheral blood of mice with 1972. BW10232 mammary tumors, the responsiveness of both 6. Klein, G. Experimental Studies in Tumor Immunology. Federation Proc., 28: 1739-1753, 1969. subpopulations is intensified. These results indicate that 7, Krueger, J. G., and Moyer, A. C. A Serum Inhibitor of Cell Mediated lymphoid cells from different compartments do not always Immunological Functions in Healthy Mice and Possible Correlations with show similar responses to a TAA and that characteristic C-Type Viral Expression and Spontaneous Neoplasia. In: H. E. Nieburgs (ed), Proceedings of the Third International Symposium on Detection responses to a TAA can change when animals carry tumors. and Prevention of Cancer. New York: Marcel Dekker, in press. The difference in responsiveness of cells from separate 8. Law, L. W. Studiesof the Significanceof TumorAntigensin the Induc tion and Repression of Neoplastic Diseases. Cancer Res., 29: 1-21 , 1969. lymphoid compartments and the existence of 2 subpopula 9. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein tions of lymphoid cells is substantiated by work with tumors Measurement with the Folin Phenol Reagent. J. Biol. Chem., 193: 265- from other mouse strains and in other animals (Refs. 7 and 275,1951. 10. Lundak, A. L., Granger, F. A., and Kaplan, M. S. T-Lymphocyte Cell 11; J. G. Krueger, R. A. Segal, and A. C. Moyer, unpub Division: A Requirement for the Tumor Specific Immune Response in lished observations). Vitro. In: H. E. Nieburgs (ed), Proceedings of the Third International The immunological nature of the 2 lymphoid subpopula Symposium on Detection and Prevention of Cancer. New York: Marcel Dekker, in press. tions sensitive to tumor cell membrane fragments is not 11. Oppenheim,J.J., Wolstencroft, A. A., and Gell, P. G. H. Delayed Hyper known . While it is thought that the cells responsive to tumor sensitivity in the Guinea-Pig to a Protein-Hapten Conjugate and Its Ada tionship to in Vitro TransfOrmation of Lymph Node, Spleen, , and antigens are T-cells (2), the responsive populations could Peripheral Blood Lymphocytes. Immunology, 12: 89-102, 1967. be B-cells, , , or some other 12. Poon, S. P. and Cauchi, M. N. Blastogenic Response of Lymphoid Cells lymphoid cells. Further investigation will be necessary to to Tumor Cells or Tumor Membrane Extracts in Wtro. Cancer Aes. , 33: 2677-2682, 1973. clarify this point. 13. SjOgren, H. 0. Transplantation Methods as a Tool for Detection of Contradictory data on immune response to many different Tumor-specific Antigens. Progr. Exptl. Tumor Aes., 6: 289—322,1965.

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James G. Krueger, Rosalind A. Segal and Rex C. Moyer

Cancer Res 1977;37:320-322.

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