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Splenectomy, suppressor cell activity, and survival in tumor bearing rats
JOURNAL
OF SURGICAL
RESEARCH
29, 527-532(1980)
Splenectomy, JOHN D. MEYER,*
Suppressor Cell Activity, in Tumor Bearing Rats
BERTIE
F. ARGYRIS,
PH.D.,?
AND
*Hamilton College. Clinton, New York 13323, and Departments State University of New York, Upsfaie Medical Center, Submitted
for publication
December
and Survival
JOHN
A.
MEYER,
M.D.S*r
of TMicrobiology and Uurgery, Syracuse, New York I3210 5, 1979
Young female rats of the Fischer strain were subjected to splenectomy at 10 weeks of age. Three weeks later, these animals and a comparable group of intact animals were inoculated subcutaneously with 3 x IO6 cells of the syngeneic Ward colon carcinoma. Tumor growth was recorded by serial measurement. Lymph node cells from control, intact tumor-bearing, and asplenic tumorbearing animals were tested for reactivity in mixed lymphocyte culture (MLC) and for ability to suppress the allogeneic mixed lymphocyte reaction. At 3 weeks after tumor inoculation, lymphocyte reactivity in MLC was somewhat depressed in asplenic tumor-bearing hosts but severely depressed in intact hosts (P < 0.001); suppressor activity was demonstrable in intact hosts but not in asplenic hosts (P < 0.001). At 5 weeks, lymphocyte reactivity in MLC was severely depressed in both groups; suppressor cell activity was present in both groups and differences were no longer significant. Median survival of intact tumor-bearing animals was 97 days after inoculation (P < 0.0601 by the log-rank test), and 120 days in asplenic animals (P < 0.001). We conclude that both depression of lymphocyte reactivity and onset of suppressor activity are delayed in asplenic tumor-bearing animals. Host survival was significantly prolonged.
INTRODUCTION Cells capable of suppressing lymphocyte reactivity to alloantigens, or to plant mitogens, have been identified in many tumorbearing animal hosts and in human patients with cancer [2, 3, 9- 11, 221. Suppressor cells of this type, not shown to react with any degree of immunologic specificity, have been thought to be a factor in diminishing immunologic competence of the tumor-bearing host. Whether increasing nonspecific suppressor cell activity correlates to any degree with host survival or prognosis remains an open question. The concept that these cells have any relevance to the tumorhost balance has recently been challenged [9]. MATERIALS
AND METHODS
Animals. Young female rats of the Fischer strain (Charles River Breeding Laboratories, Wilmington, Mass.) were obtained at age 8 L Address reprint requests 750 E. Adams St., Syracuse,
to: Dr. John A. Meyer, N. Y. 13210. 527
weeks and used at 10weeks. The Ward colon carcinoma [20], specific to the Fischer strain, was obtained through the courtesy of Dr. Fred Rosen, Roswell Park Memorial Institute, and was maintained in our laboratory by subcutaneous trocar inoculation every 5 weeks. For the mixed lymphocyte cultures (MLC), we used stimulating cells from the “controlled outbred” strain SD/N f BR (Taconic Farms, Germantown, N. Y.). Animals of this strain were also obtained at 8 weeks of age and used at 10 to 12 weeks. These animals are referred to hereafter as SD/N. Splenectomy. At 10 weeks of age, half the Fischer strain animals were subjected to initial splenectomy. These animals are referred to hereafter as asplenic. Tumor inoculation. Three weeks after splenectomy, all the asplenic and intact animals were anesthetized with chloral hydrate. Cubes of tumor tissue 1.5 mm to a side (approximately 3 X lo6 tumor cells) were implanted subcutaneously by trocar into all 0022-4804/80/120527-06$01.00/O Copyright All rights
Q 1980 by Academic Press, Inc. of reproduction in any form reserved
528
JOURNAL
OF SURGICAL
RESEARCH:
recipient animals, at the Site I of Auerbach et al. 151, immediately caudad to the right shoulder. Tumor
measurement
and host survival.
Subcutaneous tumors were measured in three diameters with a small caliper, to the nearest millimeter, at weekly intervals beginning at 2 weeks after inoculation. The mean of the three measurements was taken as the tumor diameter. Last measurements were made at 7 weeks after inoculation; after this, tumors were beginning to ulcerate through the skin and accurate measurement was not feasible. Cages were inspected daily and the data of death was noted for all animals. Mixed lymphocyte culture and assay for suppressor cells. Our technique of MLC has
been described previously [4], modified slightly for these experiments since spleen cells were not available from the experimental animals. We used lymph node cells as responding and suppressor cells in spite of their expected lesser activity, and spleen from nonsensitized animals as stimulating cells. Assays were done on intact and asplenic tumor-bearing animals at 3 and 5 weeks after tumor inoculation. Animals were quickly sacrificed by CO, narcosis, and submandibular and mediastinal lymph nodes were obtained by aseptic dissection. Spleens were obtained from animals of the stimulator strain SD/N, and also from nonsensitized animals of the Fischer strain to serve as syngeneic controls on the allogenic stimulation. Cultures were carried out with 2 x lo5 nonsensitized Fischer lymph node as responding cells and 2 x lo5 SD/N mitomycintreated spleen as stimulating cells, each culture in a total volume of 0.2 ml. After 72 hr, 1 PCi of [3H]TdR was added, and at 96 hr the cultures were harvested, dried, and counted in a Packard scintillation counter. Cells of the responding strain (Fischer) are designated as A, and cells of the stimulating strain (SD/N) as B. Results are expressed as average cpm + SE of triplicate syngeneic (AA,,J and allogeneic (ABA cultures. Percentage of response is calculated as the ratio
VOL.
29, NO.
6, DECEMBER
of differences AA, cultures
1980
in cpm between AB,
AB, - AA,,, (experimental) AB, - AA,,, (control)
x IO0
and
7
where control cultures are those using nonsensitized A-strain cells and are arbitrarily considered as showing 100% response. For assay of suppressor activity, sensitized lymph node cells treated with mitomycin (SLN,; i.e., lymph node cells from tumor-bearing animals) were added to the MLC in two different cell concentrations, 2.5 and 5 x lo5 cells, keeping the total volume of MLC at 0.2 ml as before. Activity was recorded as percentage of the control cpm of allogeneic stimulation for that culture: (AB, + SLN,) (AB, + NSLN,)
- (AA, - (AA,
+ SLN,) + NSLN,) x 100.
In these experiments, the results reported are those in which 5 x lo5 SLN, or NSLN, cells were added to the MLC. All cultures were done in triplicate, and three animals from each of the asplenic and intact tumorbearing groups were tested for reactivity and suppression at both 3 and 5 weeks after tumor inoculation. Statistical analysis. Lymphocyte reactivity in MLC was recorded as the cpm with allogeneic stimulation minus cpm with syngeneic culture (AB, - AA,,,). Suppressive activity of cells from tumor-bearing animals was recorded as percentage of the control value of allogeneic stimulation for that culture, by the formula noted above. Both of these sets of figures were compared for significance by Student’s t test. Survival of intact and asplenic tumorbearing animals was compared by the logrank test, otherwise known as the MantelHaenszel test for survivorship data [ 13, 161. Since groups can be compared at any point in the follow-up period, comparison was made between groups at the time of median
MEYER,
ARGYRIS,
AND
MEYER:
SUPPRESSOR
survival of intact animals (97 days) and at the time of median survival of asplenic animals (120 days).
ACTIVITY
growth. Twenty-one animals in each group were observed throughout their course, for recording of tumor growth and host survival. All tumors grew after inoculation but a few regressed completely between 4 and 5 weeks; two regressed in intact animals and four in asplenic animals. Measurements on Weeks 5 through 7 include only the nonregressing tumors. Differences in tumor size between groups, though technically significant, were very small in absolute terms and on each occasion represented distinctly less than a week’s growth (Fig. 1). Mixed lymphocyte culture. Lymph node cell reactivity in MLC at 3 weeks is summarized in Table 1. Assays were done on three animals in each of the asplenic and intact groups; cultures were performed on two successive days with experimental animals mixed on each day. No animals showing any sign of tumor regression were used in these assays. Lymphocyte reactivity in asplenic tumor-bearing animals was somewhat depressed below that of controls, but reactivity was severely depressed in intact tumor-bearing animals (P < 0.001). Ability of lymph node cells from tumorbearing animals to suppress mixed lympho-
s
4 AFTER
f
529
SURVIVAL 1
LYMPH NODE CELL RESPONSIVENESS IN MLC INTACT AND ASPLENIC TUMOR-BEARING ANIMALS AT 3 WEEKS AFTER
OF
INOCULATION
Tumor
WEEKS
HOST
TABLE
RESULTS
2
AND
8
I
INOCULATION
FIG. 1. Semilogarithmic plot of mean tumor diameters in intact and asplenic animals at Weeks 2 through 7 after inoculation. Brackets indicate 95% confidence limits (mean c 2 SE)
Donor of responding cells
[3H]TdR incorporation (cpm k SE) ‘%l 14,649 + 2,802 9,231 k 246 3,301 + 489 5,692 k 1,010
Control No. 2 Asplenic No. 2 Asplenic No. 3 Intact No. 3
16,065
Mean cpm of AB, - AA,,, Asplenic animals 10,102 Intact animals 3,356 Mean percentage response Asplenic animals 71 Intact animals 24
Percentage response
AAm
Control No. 1 Asplenic No. I Intact No. 1 Intact No. 2
+ 12,326 + 11,185 + 2,171 +
1
884 622 335 259
973 + 68 683 + 41 265 r 53 349 f 63
100 63 22
+ + k t
100
1494 933 773 476
39
49 33 56 36
78 71 12
+ 475 SE + 635 SE
P < 0.001
f 2.7 SE t 4.7 SE
Pi
0.001
Note. Responding cells: 2 x IO5 Fischer lymph node, as listed. Stimulating cells: 2 x lo5 SD/N spleen, mitomycin treated. Separate cultures were done on successive days, each including a nonsensitized control and both intact and asplenic sensitized (tumor-bearing) animals.
cyte reactivity of nonsensitized animals at 3 weeks is summarized in Table 2. The cultures were again performed on successive days, with mixed groups and with a new control on each day. Cells from asplenic animals showed no suppression of MLR (mean percentage response 114.7 + 8.8 SE), while cells from intact animals showed significant suppression (mean percentage response 55.4 5 1.8, P < 0.001). At 5 weeks after inoculation, the tumors were much larger. Lymphocyte reactivity in MLC was severely depressed in both groups (l-2% response in intact animals, 2-3% in asplenic). Suppression of the allogeneic MLR was prominent in both groups. Cultures with cells added from asplenic animals showed a mean percentage response of 66.5 -+ 5.6; with cells added from intact animals 56.5 ? 1.1; the difference is not significant. Survival. Date of death was recorded for all of the animals, 21 in each experimental
JOURNALOFSURGICALRESEARCH:VOL.29,NO.6,DECEMBER
530
TABLE2 SUPPRESSIONOFALLOGENEIC MLR BY LYMPH CELLSFROMINTACTANDASPLENICTUMOR-BEARING ANIMALS AT 3 WEEKS AFTER INOCULATION
No.
i3H]TdR incorporatiom (cpm f SE)
DOllOr
Control (F-NSLN, added)
NODE
(F-SLN, added)
A&
I
AA,
Asplenic No. I Intact No. I Intact No. 2
28,283 23,487 16,095 14,619
k 2,532 f 813 ? %8 ? 666
1,306 945 864 551
t 231 + 74 + I58 * II4
100 83 56 52
Asplenic No. 2 Asplenic No. 3 Intact No. 3
23,428 27,627 31.763 13,472
2 2,185 f 1,053 2 1,666 -t 446
2,548 2,608 2,419 1,425
+ I61 ? 83 * I65 + 67
100 119 140 57
Mean response as percentage of control for that day’s Asplenic animals 114.7 2 8.8 SE P c 0.001 Intact animal 55.4 ? I.8 SE
culture
No. 2
Norr. Responding cells: 2 x IV Fischer nonsensitized lymph node (F-NSLN). Stimulating cells: 2 x lo’ SD/N spleen, mitomycin treated. Added cells: 5 x ItV Fischer LN. mitomycin treated: nonsensitized (NSLN,) to control cultures; sensitized (SLN,), as indicated, to other cultures. Separate cultures were done on successive days, each including a NS control and both intact and asplenic sensitized animals.
group, which were retained for observation of tumor growth and survival. As noted above, tumors regressed completely in two intact animals and in four asplenic animals; these long survivors did not affect the determination of median survival. Median survival was 97 days in intact animals and 120 days in asplenic animals (Fig. 2). Though the increased life span is only about 24%, the difference in group survival is highly significant when compared by the log-rank test, P < 0.0001 at 97 days and P < 0.001 at 120 days.
1980
viewing the problem, we should try to distinguish initially between cells described as suppressing reactivity of host lymphocytes to mitogens in experimental animals [2,3,9, 1 l] or in human cancer patients [ 10, 211, from those cells demonstrated to specifically suppress host anti-tumor cytotoxicity in animals [8, 181 or in human patients [21]. It is not clear whether these groups of suppressor cells are entirely distinct. The concept that nonspecific suppressor cells reflect diminishing host immunologic reactivity, in particular against its own tumor, has been challenged by Hanna and Kripke [9]. Their study concluded that nonspecific suppressor cells could not be regarded as having a regulatory role in the development of an immune response against syngeneic or autochthonous tumors. The source of suppressor cells in practically all these studies has been the spleen. Suppressor cells can be demonstrated in lymph nodes and thymus as well [ 1,8], and in peripheral blood in human patients [lo, 21, 221. Many studies have noted progressive splenic enlargement and thymic atrophy in the presence of a growing tumor [6,7, 151. A recent study found that concomitant with the generation of nonspecific suppressor cells in melanoma-bearing mice, aggregations of macrophages with lymphocytes in strong physical association could be identified in the spleen [ 171. Fractionation of spleen cell suspensions permitted isolation of supASPLENIC Median 17.0
DISCUSSION
Our data suggest that, in this syngeneic model, preliminary splenectomy delays the generation of nonspecific suppressor cells. After this initial delay, with tumors having grown quite large, suppressor activity was prominent in both asplenic and intact hosts. Nonetheless, survival of asplenic animals was significantly prolonged. No consensus is apparent in the recent literature regarding the significance of suppressor cells in tumor-bearing hosts. In re-
DAYS
AFTER
INOCULATION
FIG. 2. Survival of intact and asplenic animals after tumor inoculation. Value of P by the log-rank test on Day 97,
MEYER, ARGYRIS,
AND MEYER: SUPPRESSOR
pressor activity to the aggregated cells. Splenectomy has been found to diminish thymic atrophy in the presence of progressively growing tumor [ 151, and to diminish the thymic involution of increasing age in non-tumor-bearing animals [ 141. A contrary finding has been reported by Amsbaugh et al. [ 11, that splenectomy 7- 10 days before immunization did not impair the induction or expression of suppressor T-cell activity. However, the animals did not bear tumors, and tumor-associated antigens were not involved. On theoretical grounds, it might appear likely that splenectomy would remove a large bulk of cytotoxic or potentially cytotoxic cells. If this were the case, survival of asplenic animals should have been shortened, while in fact it was significantly prolonged. Actual suppressor cells have been identified variously, as T cells in some studies [3, 12, 181 or as macrophages in others [9, 111. In peripheral blood from human patients, Zembala et al. [22] found suppressor cells to be adherent.Jerrels et al. [lo] found that nonspecific suppressor cells in cancer patients could be adherent or nonadherent, while Yu et al. [21] found that human specific suppressor cells were adherent and expressed the 8 antigen. Cytotoxic cells, on the other hand, possessed receptors for C’3 but showed no surface immunoglobulins. These studies seeking to precisely characterize the suppressor cells in tumor bearing hosts have not completely resolved the problem and have left their exact nature uncertain. We have sought instead to look for functional consequences of suppressor activity. The final question is whether specific or nonspecific suppressor cells bear any relation to host prognosis or survival; very little information is available. A number of studies have found correlation between lymphocyte reactivity and prognosis in cancer patients, but without the demonstration of suppressor cells, these studies may not be pertinent to the present discussion. Yu et al. [21] identified specific suppressor cells in 4
ACTIVITY
AND HOST SURVIVAL
531
of 10 human patients with osteogenic sarcoma, but not in any patient relatives or normal controls. Three of the 4 patients had pulmonary metastases, while the fourth developed metastases soon afterward. Only 1 of the 6 patients not showing specific suppressor cells had metastatic disease. The authors conceded that their experimental methods may have been responsible for failure to demonstrate suppressor cells in more of the patients, since of 13 patients in whom cytotoxicity was not initially demonstrable, it could be demonstrated in 11 after cell fractionation. To our knowledge, only this study [21] suggests a correlation between suppressor cell activity and host prognosis. Our data indicate an association between delayed suppressor activity, admittedly nonspecific, and longer host survival. We have not shown cause and effect, only association. Much additional study of the consequences of suppressor activity seems necessary. REFERENCES 1. Amsbaugh, D. F., Prescott, B., and Baker, P. J. Effect of splenectomy on the expression of regulatory T cell activity. J. Zmmunol. 121: 1483, 1978. 2. Anaclerio, A., Moras, M. L., Conti, G., Mistrello, G., and Spreafico, F. Suppression of mitogen responses and graft-versus-host reaction by splenocytes from mice bearing Lewis lung carconima. .I. Nat/. Cancer Inst. 61: 471, 1978. 3. Argyris, B. F. Activation of suppressor cells by syngeneic tumor transplants in mice. Cancer Res. 38: 1269, 1978. 4. Argyris, B. F., and DeLustro, F. Immunologic unresponsiveness of mouse spleen sensitized to allogeneic tumors. Cell. Zmmunol. 28: 390, 1977. 5. Auerbach, R., Morrissey, L. W., and Sidky, Y. Regional differences in the incidence and growth of mouse tumors following intradermal or subcutaneous inoculation. CancerRes. 38: 1739,1978. 6. Edwards, A. J., Rowland, G. F., Sumner, M. R., and Hurd, C. M. Changes in lympho-reticular tissues during growth of a murine adenocarcinoma. I. Histology and weight of lymph nodes, spleen, and thymus. J. Natl. Cancer Inst. 47: 301, 1971. 7. Efskind, L., Godal, T., Grude, T., Marton, P. F., Mossige, J., and Hoglo, S. Progressive atrophy of the thymus in mice carrying Lewis lung carcinoma grafts. Cancer Zmmunol. Zmmunother. 3: 207, 1978.
532
JOURNAL
OF SURGICAL
RESEARCH:
8. Fujimoto, S., Greene, M. I., and Sehon, A. H. Regulation of the immune response to tumor antigens. I. Immunosuppressor cells in tumor-bearing hosts. II. Nature of immunosuppressor cells in tumor-bearing hosts. J. Immunol. 116: 791, 1976. 9. Hanna, N., and Kripke, M. L. Immunologic significance of nonspecific suppressor cells in spleens of tumor-bearing mice. Cell. Immunol. 43: 293, 1979. 10. Jerrels, T. R., Dean, J. H., Richardson, G. L., McCoy, J. L., and Herberman, R. B. Role of suppressor cells in depression of in vitro lymphoproliferative responses of lung cancer and breast cancer patients. J. Null. Cancer Inst. 61: 1001, 1978. 11. Kirchner, H., Muchmore, A. V., Chused, T. M., Holden, H. T., and Herberman, R. B. Inhibition of the proliferation of lymphoma cells and T lymphocytes by suppressor cells from the spleens of tumor-bearing mice. J. Immunol. 114: 206, 1975. 12. Manor, Y., Treves, A. J., Cohen, I. R., and Feldman, M. Transition from T cell protection to T cell enhancement during tumor growth in an allogeneic host. Transplantation 22: 360, 1976. 13. Mantel, N. Evaluation of survival data and two new rank order statistics arising in its consideration. Cancer Chemother. Rep. 50: 163, 1%6. 14. Meyer, J. A., and Meyer, J. D. Splenectomy and the thymic involution of increasing age. Arch. Surg. 113: 972, 1978.
15. Meyer, J. D., and Meyer, J. A. Prevention by splenectomy of thymus weight depletion in the presence of progressively growing tumor in rats. J. Natl. Cancer Inst. 59: 1023, 1977.
VOL. 29, NO. 6, DECEMBER
1980
16. Peto, R., Pike, M. C., Arm&age, P., Breslow, N. E., Cox, D. R., Howard, S. V., Mantel, N., McPherson, K., Peto, J., and Smith, P. G. Design and analysis of randomized clinical trials requiring prolonged observation of each patient. II. Analysis and examples. Bit. J. Cancer 35: 1, 1977. 17. Stelzer, G. T., Shellhaas, J. L., and Wallace, J. H. Macrophage-lymphocyte interaction and the regulation of the immune response to murine melanoma. Proc. Amer. Assoc. Cancer Res. 20: 12(No. 47), 1979. 18. Takei, F., Levy, J. G., and Kilbum, D. G. In vitro induction of cytotoxicity against syngeneic mastocytoma and its suppression by spleen and thymus cells from tumor-bearing mice. J. Zmmunol. 116: 288, 1976.
19. Takei, F., Levy, J. G., and Kilbum, D. G. Characterization of suppressor cells in mice bearing syngeneic mastocytoma. J. Immunol. 118: 412, 1977.
20. Ward, J. M., Yamamoto, R. S., Weisburger, J. H., and Benjamin, T. Transplantation of chemically induced metastatic mutinous adenocarcinomas of the jejunum and colon in rats. J. Natl. Cancer Inst.
51: 1997, 1973.
21. Yu, A., Watts, H., Jalfe, N., and Parkman, R. Concomitant presence of tumor-specific cytotoxic and inhibitor lymphocytes in patients with osteogenic sarcoma. N. Engl. J. Med. 297: 121, 1977. 22. Zembala, M., Mytar, B., Popiela, T., and Asherson, G. L. Depressed in vitro peripheral blood lymphocyte response to mitogens in cancer patients: The role of suppressor cells. In?. J. Cuncer 19: 605, 1977.
OF SURGICAL
RESEARCH
29, 527-532(1980)
Splenectomy, JOHN D. MEYER,*
Suppressor Cell Activity, in Tumor Bearing Rats
BERTIE
F. ARGYRIS,
PH.D.,?
AND
*Hamilton College. Clinton, New York 13323, and Departments State University of New York, Upsfaie Medical Center, Submitted
for publication
December
and Survival
JOHN
A.
MEYER,
M.D.S*r
of TMicrobiology and Uurgery, Syracuse, New York I3210 5, 1979
Young female rats of the Fischer strain were subjected to splenectomy at 10 weeks of age. Three weeks later, these animals and a comparable group of intact animals were inoculated subcutaneously with 3 x IO6 cells of the syngeneic Ward colon carcinoma. Tumor growth was recorded by serial measurement. Lymph node cells from control, intact tumor-bearing, and asplenic tumorbearing animals were tested for reactivity in mixed lymphocyte culture (MLC) and for ability to suppress the allogeneic mixed lymphocyte reaction. At 3 weeks after tumor inoculation, lymphocyte reactivity in MLC was somewhat depressed in asplenic tumor-bearing hosts but severely depressed in intact hosts (P < 0.001); suppressor activity was demonstrable in intact hosts but not in asplenic hosts (P < 0.001). At 5 weeks, lymphocyte reactivity in MLC was severely depressed in both groups; suppressor cell activity was present in both groups and differences were no longer significant. Median survival of intact tumor-bearing animals was 97 days after inoculation (P < 0.0601 by the log-rank test), and 120 days in asplenic animals (P < 0.001). We conclude that both depression of lymphocyte reactivity and onset of suppressor activity are delayed in asplenic tumor-bearing animals. Host survival was significantly prolonged.
INTRODUCTION Cells capable of suppressing lymphocyte reactivity to alloantigens, or to plant mitogens, have been identified in many tumorbearing animal hosts and in human patients with cancer [2, 3, 9- 11, 221. Suppressor cells of this type, not shown to react with any degree of immunologic specificity, have been thought to be a factor in diminishing immunologic competence of the tumor-bearing host. Whether increasing nonspecific suppressor cell activity correlates to any degree with host survival or prognosis remains an open question. The concept that these cells have any relevance to the tumorhost balance has recently been challenged [9]. MATERIALS
AND METHODS
Animals. Young female rats of the Fischer strain (Charles River Breeding Laboratories, Wilmington, Mass.) were obtained at age 8 L Address reprint requests 750 E. Adams St., Syracuse,
to: Dr. John A. Meyer, N. Y. 13210. 527
weeks and used at 10weeks. The Ward colon carcinoma [20], specific to the Fischer strain, was obtained through the courtesy of Dr. Fred Rosen, Roswell Park Memorial Institute, and was maintained in our laboratory by subcutaneous trocar inoculation every 5 weeks. For the mixed lymphocyte cultures (MLC), we used stimulating cells from the “controlled outbred” strain SD/N f BR (Taconic Farms, Germantown, N. Y.). Animals of this strain were also obtained at 8 weeks of age and used at 10 to 12 weeks. These animals are referred to hereafter as SD/N. Splenectomy. At 10 weeks of age, half the Fischer strain animals were subjected to initial splenectomy. These animals are referred to hereafter as asplenic. Tumor inoculation. Three weeks after splenectomy, all the asplenic and intact animals were anesthetized with chloral hydrate. Cubes of tumor tissue 1.5 mm to a side (approximately 3 X lo6 tumor cells) were implanted subcutaneously by trocar into all 0022-4804/80/120527-06$01.00/O Copyright All rights
Q 1980 by Academic Press, Inc. of reproduction in any form reserved
528
JOURNAL
OF SURGICAL
RESEARCH:
recipient animals, at the Site I of Auerbach et al. 151, immediately caudad to the right shoulder. Tumor
measurement
and host survival.
Subcutaneous tumors were measured in three diameters with a small caliper, to the nearest millimeter, at weekly intervals beginning at 2 weeks after inoculation. The mean of the three measurements was taken as the tumor diameter. Last measurements were made at 7 weeks after inoculation; after this, tumors were beginning to ulcerate through the skin and accurate measurement was not feasible. Cages were inspected daily and the data of death was noted for all animals. Mixed lymphocyte culture and assay for suppressor cells. Our technique of MLC has
been described previously [4], modified slightly for these experiments since spleen cells were not available from the experimental animals. We used lymph node cells as responding and suppressor cells in spite of their expected lesser activity, and spleen from nonsensitized animals as stimulating cells. Assays were done on intact and asplenic tumor-bearing animals at 3 and 5 weeks after tumor inoculation. Animals were quickly sacrificed by CO, narcosis, and submandibular and mediastinal lymph nodes were obtained by aseptic dissection. Spleens were obtained from animals of the stimulator strain SD/N, and also from nonsensitized animals of the Fischer strain to serve as syngeneic controls on the allogenic stimulation. Cultures were carried out with 2 x lo5 nonsensitized Fischer lymph node as responding cells and 2 x lo5 SD/N mitomycintreated spleen as stimulating cells, each culture in a total volume of 0.2 ml. After 72 hr, 1 PCi of [3H]TdR was added, and at 96 hr the cultures were harvested, dried, and counted in a Packard scintillation counter. Cells of the responding strain (Fischer) are designated as A, and cells of the stimulating strain (SD/N) as B. Results are expressed as average cpm + SE of triplicate syngeneic (AA,,J and allogeneic (ABA cultures. Percentage of response is calculated as the ratio
VOL.
29, NO.
6, DECEMBER
of differences AA, cultures
1980
in cpm between AB,
AB, - AA,,, (experimental) AB, - AA,,, (control)
x IO0
and
7
where control cultures are those using nonsensitized A-strain cells and are arbitrarily considered as showing 100% response. For assay of suppressor activity, sensitized lymph node cells treated with mitomycin (SLN,; i.e., lymph node cells from tumor-bearing animals) were added to the MLC in two different cell concentrations, 2.5 and 5 x lo5 cells, keeping the total volume of MLC at 0.2 ml as before. Activity was recorded as percentage of the control cpm of allogeneic stimulation for that culture: (AB, + SLN,) (AB, + NSLN,)
- (AA, - (AA,
+ SLN,) + NSLN,) x 100.
In these experiments, the results reported are those in which 5 x lo5 SLN, or NSLN, cells were added to the MLC. All cultures were done in triplicate, and three animals from each of the asplenic and intact tumorbearing groups were tested for reactivity and suppression at both 3 and 5 weeks after tumor inoculation. Statistical analysis. Lymphocyte reactivity in MLC was recorded as the cpm with allogeneic stimulation minus cpm with syngeneic culture (AB, - AA,,,). Suppressive activity of cells from tumor-bearing animals was recorded as percentage of the control value of allogeneic stimulation for that culture, by the formula noted above. Both of these sets of figures were compared for significance by Student’s t test. Survival of intact and asplenic tumorbearing animals was compared by the logrank test, otherwise known as the MantelHaenszel test for survivorship data [ 13, 161. Since groups can be compared at any point in the follow-up period, comparison was made between groups at the time of median
MEYER,
ARGYRIS,
AND
MEYER:
SUPPRESSOR
survival of intact animals (97 days) and at the time of median survival of asplenic animals (120 days).
ACTIVITY
growth. Twenty-one animals in each group were observed throughout their course, for recording of tumor growth and host survival. All tumors grew after inoculation but a few regressed completely between 4 and 5 weeks; two regressed in intact animals and four in asplenic animals. Measurements on Weeks 5 through 7 include only the nonregressing tumors. Differences in tumor size between groups, though technically significant, were very small in absolute terms and on each occasion represented distinctly less than a week’s growth (Fig. 1). Mixed lymphocyte culture. Lymph node cell reactivity in MLC at 3 weeks is summarized in Table 1. Assays were done on three animals in each of the asplenic and intact groups; cultures were performed on two successive days with experimental animals mixed on each day. No animals showing any sign of tumor regression were used in these assays. Lymphocyte reactivity in asplenic tumor-bearing animals was somewhat depressed below that of controls, but reactivity was severely depressed in intact tumor-bearing animals (P < 0.001). Ability of lymph node cells from tumorbearing animals to suppress mixed lympho-
s
4 AFTER
f
529
SURVIVAL 1
LYMPH NODE CELL RESPONSIVENESS IN MLC INTACT AND ASPLENIC TUMOR-BEARING ANIMALS AT 3 WEEKS AFTER
OF
INOCULATION
Tumor
WEEKS
HOST
TABLE
RESULTS
2
AND
8
I
INOCULATION
FIG. 1. Semilogarithmic plot of mean tumor diameters in intact and asplenic animals at Weeks 2 through 7 after inoculation. Brackets indicate 95% confidence limits (mean c 2 SE)
Donor of responding cells
[3H]TdR incorporation (cpm k SE) ‘%l 14,649 + 2,802 9,231 k 246 3,301 + 489 5,692 k 1,010
Control No. 2 Asplenic No. 2 Asplenic No. 3 Intact No. 3
16,065
Mean cpm of AB, - AA,,, Asplenic animals 10,102 Intact animals 3,356 Mean percentage response Asplenic animals 71 Intact animals 24
Percentage response
AAm
Control No. 1 Asplenic No. I Intact No. 1 Intact No. 2
+ 12,326 + 11,185 + 2,171 +
1
884 622 335 259
973 + 68 683 + 41 265 r 53 349 f 63
100 63 22
+ + k t
100
1494 933 773 476
39
49 33 56 36
78 71 12
+ 475 SE + 635 SE
P < 0.001
f 2.7 SE t 4.7 SE
Pi
0.001
Note. Responding cells: 2 x IO5 Fischer lymph node, as listed. Stimulating cells: 2 x lo5 SD/N spleen, mitomycin treated. Separate cultures were done on successive days, each including a nonsensitized control and both intact and asplenic sensitized (tumor-bearing) animals.
cyte reactivity of nonsensitized animals at 3 weeks is summarized in Table 2. The cultures were again performed on successive days, with mixed groups and with a new control on each day. Cells from asplenic animals showed no suppression of MLR (mean percentage response 114.7 + 8.8 SE), while cells from intact animals showed significant suppression (mean percentage response 55.4 5 1.8, P < 0.001). At 5 weeks after inoculation, the tumors were much larger. Lymphocyte reactivity in MLC was severely depressed in both groups (l-2% response in intact animals, 2-3% in asplenic). Suppression of the allogeneic MLR was prominent in both groups. Cultures with cells added from asplenic animals showed a mean percentage response of 66.5 -+ 5.6; with cells added from intact animals 56.5 ? 1.1; the difference is not significant. Survival. Date of death was recorded for all of the animals, 21 in each experimental
JOURNALOFSURGICALRESEARCH:VOL.29,NO.6,DECEMBER
530
TABLE2 SUPPRESSIONOFALLOGENEIC MLR BY LYMPH CELLSFROMINTACTANDASPLENICTUMOR-BEARING ANIMALS AT 3 WEEKS AFTER INOCULATION
No.
i3H]TdR incorporatiom (cpm f SE)
DOllOr
Control (F-NSLN, added)
NODE
(F-SLN, added)
A&
I
AA,
Asplenic No. I Intact No. I Intact No. 2
28,283 23,487 16,095 14,619
k 2,532 f 813 ? %8 ? 666
1,306 945 864 551
t 231 + 74 + I58 * II4
100 83 56 52
Asplenic No. 2 Asplenic No. 3 Intact No. 3
23,428 27,627 31.763 13,472
2 2,185 f 1,053 2 1,666 -t 446
2,548 2,608 2,419 1,425
+ I61 ? 83 * I65 + 67
100 119 140 57
Mean response as percentage of control for that day’s Asplenic animals 114.7 2 8.8 SE P c 0.001 Intact animal 55.4 ? I.8 SE
culture
No. 2
Norr. Responding cells: 2 x IV Fischer nonsensitized lymph node (F-NSLN). Stimulating cells: 2 x lo’ SD/N spleen, mitomycin treated. Added cells: 5 x ItV Fischer LN. mitomycin treated: nonsensitized (NSLN,) to control cultures; sensitized (SLN,), as indicated, to other cultures. Separate cultures were done on successive days, each including a NS control and both intact and asplenic sensitized animals.
group, which were retained for observation of tumor growth and survival. As noted above, tumors regressed completely in two intact animals and in four asplenic animals; these long survivors did not affect the determination of median survival. Median survival was 97 days in intact animals and 120 days in asplenic animals (Fig. 2). Though the increased life span is only about 24%, the difference in group survival is highly significant when compared by the log-rank test, P < 0.0001 at 97 days and P < 0.001 at 120 days.
1980
viewing the problem, we should try to distinguish initially between cells described as suppressing reactivity of host lymphocytes to mitogens in experimental animals [2,3,9, 1 l] or in human cancer patients [ 10, 211, from those cells demonstrated to specifically suppress host anti-tumor cytotoxicity in animals [8, 181 or in human patients [21]. It is not clear whether these groups of suppressor cells are entirely distinct. The concept that nonspecific suppressor cells reflect diminishing host immunologic reactivity, in particular against its own tumor, has been challenged by Hanna and Kripke [9]. Their study concluded that nonspecific suppressor cells could not be regarded as having a regulatory role in the development of an immune response against syngeneic or autochthonous tumors. The source of suppressor cells in practically all these studies has been the spleen. Suppressor cells can be demonstrated in lymph nodes and thymus as well [ 1,8], and in peripheral blood in human patients [lo, 21, 221. Many studies have noted progressive splenic enlargement and thymic atrophy in the presence of a growing tumor [6,7, 151. A recent study found that concomitant with the generation of nonspecific suppressor cells in melanoma-bearing mice, aggregations of macrophages with lymphocytes in strong physical association could be identified in the spleen [ 171. Fractionation of spleen cell suspensions permitted isolation of supASPLENIC Median 17.0
DISCUSSION
Our data suggest that, in this syngeneic model, preliminary splenectomy delays the generation of nonspecific suppressor cells. After this initial delay, with tumors having grown quite large, suppressor activity was prominent in both asplenic and intact hosts. Nonetheless, survival of asplenic animals was significantly prolonged. No consensus is apparent in the recent literature regarding the significance of suppressor cells in tumor-bearing hosts. In re-
DAYS
AFTER
INOCULATION
FIG. 2. Survival of intact and asplenic animals after tumor inoculation. Value of P by the log-rank test on Day 97,
MEYER, ARGYRIS,
AND MEYER: SUPPRESSOR
pressor activity to the aggregated cells. Splenectomy has been found to diminish thymic atrophy in the presence of progressively growing tumor [ 151, and to diminish the thymic involution of increasing age in non-tumor-bearing animals [ 141. A contrary finding has been reported by Amsbaugh et al. [ 11, that splenectomy 7- 10 days before immunization did not impair the induction or expression of suppressor T-cell activity. However, the animals did not bear tumors, and tumor-associated antigens were not involved. On theoretical grounds, it might appear likely that splenectomy would remove a large bulk of cytotoxic or potentially cytotoxic cells. If this were the case, survival of asplenic animals should have been shortened, while in fact it was significantly prolonged. Actual suppressor cells have been identified variously, as T cells in some studies [3, 12, 181 or as macrophages in others [9, 111. In peripheral blood from human patients, Zembala et al. [22] found suppressor cells to be adherent.Jerrels et al. [lo] found that nonspecific suppressor cells in cancer patients could be adherent or nonadherent, while Yu et al. [21] found that human specific suppressor cells were adherent and expressed the 8 antigen. Cytotoxic cells, on the other hand, possessed receptors for C’3 but showed no surface immunoglobulins. These studies seeking to precisely characterize the suppressor cells in tumor bearing hosts have not completely resolved the problem and have left their exact nature uncertain. We have sought instead to look for functional consequences of suppressor activity. The final question is whether specific or nonspecific suppressor cells bear any relation to host prognosis or survival; very little information is available. A number of studies have found correlation between lymphocyte reactivity and prognosis in cancer patients, but without the demonstration of suppressor cells, these studies may not be pertinent to the present discussion. Yu et al. [21] identified specific suppressor cells in 4
ACTIVITY
AND HOST SURVIVAL
531
of 10 human patients with osteogenic sarcoma, but not in any patient relatives or normal controls. Three of the 4 patients had pulmonary metastases, while the fourth developed metastases soon afterward. Only 1 of the 6 patients not showing specific suppressor cells had metastatic disease. The authors conceded that their experimental methods may have been responsible for failure to demonstrate suppressor cells in more of the patients, since of 13 patients in whom cytotoxicity was not initially demonstrable, it could be demonstrated in 11 after cell fractionation. To our knowledge, only this study [21] suggests a correlation between suppressor cell activity and host prognosis. Our data indicate an association between delayed suppressor activity, admittedly nonspecific, and longer host survival. We have not shown cause and effect, only association. Much additional study of the consequences of suppressor activity seems necessary. REFERENCES 1. Amsbaugh, D. F., Prescott, B., and Baker, P. J. Effect of splenectomy on the expression of regulatory T cell activity. J. Zmmunol. 121: 1483, 1978. 2. Anaclerio, A., Moras, M. L., Conti, G., Mistrello, G., and Spreafico, F. Suppression of mitogen responses and graft-versus-host reaction by splenocytes from mice bearing Lewis lung carconima. .I. Nat/. Cancer Inst. 61: 471, 1978. 3. Argyris, B. F. Activation of suppressor cells by syngeneic tumor transplants in mice. Cancer Res. 38: 1269, 1978. 4. Argyris, B. F., and DeLustro, F. Immunologic unresponsiveness of mouse spleen sensitized to allogeneic tumors. Cell. Zmmunol. 28: 390, 1977. 5. Auerbach, R., Morrissey, L. W., and Sidky, Y. Regional differences in the incidence and growth of mouse tumors following intradermal or subcutaneous inoculation. CancerRes. 38: 1739,1978. 6. Edwards, A. J., Rowland, G. F., Sumner, M. R., and Hurd, C. M. Changes in lympho-reticular tissues during growth of a murine adenocarcinoma. I. Histology and weight of lymph nodes, spleen, and thymus. J. Natl. Cancer Inst. 47: 301, 1971. 7. Efskind, L., Godal, T., Grude, T., Marton, P. F., Mossige, J., and Hoglo, S. Progressive atrophy of the thymus in mice carrying Lewis lung carcinoma grafts. Cancer Zmmunol. Zmmunother. 3: 207, 1978.
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8. Fujimoto, S., Greene, M. I., and Sehon, A. H. Regulation of the immune response to tumor antigens. I. Immunosuppressor cells in tumor-bearing hosts. II. Nature of immunosuppressor cells in tumor-bearing hosts. J. Immunol. 116: 791, 1976. 9. Hanna, N., and Kripke, M. L. Immunologic significance of nonspecific suppressor cells in spleens of tumor-bearing mice. Cell. Immunol. 43: 293, 1979. 10. Jerrels, T. R., Dean, J. H., Richardson, G. L., McCoy, J. L., and Herberman, R. B. Role of suppressor cells in depression of in vitro lymphoproliferative responses of lung cancer and breast cancer patients. J. Null. Cancer Inst. 61: 1001, 1978. 11. Kirchner, H., Muchmore, A. V., Chused, T. M., Holden, H. T., and Herberman, R. B. Inhibition of the proliferation of lymphoma cells and T lymphocytes by suppressor cells from the spleens of tumor-bearing mice. J. Immunol. 114: 206, 1975. 12. Manor, Y., Treves, A. J., Cohen, I. R., and Feldman, M. Transition from T cell protection to T cell enhancement during tumor growth in an allogeneic host. Transplantation 22: 360, 1976. 13. Mantel, N. Evaluation of survival data and two new rank order statistics arising in its consideration. Cancer Chemother. Rep. 50: 163, 1%6. 14. Meyer, J. A., and Meyer, J. D. Splenectomy and the thymic involution of increasing age. Arch. Surg. 113: 972, 1978.
15. Meyer, J. D., and Meyer, J. A. Prevention by splenectomy of thymus weight depletion in the presence of progressively growing tumor in rats. J. Natl. Cancer Inst. 59: 1023, 1977.
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16. Peto, R., Pike, M. C., Arm&age, P., Breslow, N. E., Cox, D. R., Howard, S. V., Mantel, N., McPherson, K., Peto, J., and Smith, P. G. Design and analysis of randomized clinical trials requiring prolonged observation of each patient. II. Analysis and examples. Bit. J. Cancer 35: 1, 1977. 17. Stelzer, G. T., Shellhaas, J. L., and Wallace, J. H. Macrophage-lymphocyte interaction and the regulation of the immune response to murine melanoma. Proc. Amer. Assoc. Cancer Res. 20: 12(No. 47), 1979. 18. Takei, F., Levy, J. G., and Kilbum, D. G. In vitro induction of cytotoxicity against syngeneic mastocytoma and its suppression by spleen and thymus cells from tumor-bearing mice. J. Zmmunol. 116: 288, 1976.
19. Takei, F., Levy, J. G., and Kilbum, D. G. Characterization of suppressor cells in mice bearing syngeneic mastocytoma. J. Immunol. 118: 412, 1977.
20. Ward, J. M., Yamamoto, R. S., Weisburger, J. H., and Benjamin, T. Transplantation of chemically induced metastatic mutinous adenocarcinomas of the jejunum and colon in rats. J. Natl. Cancer Inst.
51: 1997, 1973.
21. Yu, A., Watts, H., Jalfe, N., and Parkman, R. Concomitant presence of tumor-specific cytotoxic and inhibitor lymphocytes in patients with osteogenic sarcoma. N. Engl. J. Med. 297: 121, 1977. 22. Zembala, M., Mytar, B., Popiela, T., and Asherson, G. L. Depressed in vitro peripheral blood lymphocyte response to mitogens in cancer patients: The role of suppressor cells. In?. J. Cuncer 19: 605, 1977.