Immune competence and immunosuppressive factors in splenectomized tumor-bearing mice

CELLULARIMMUNOLOGY28, 15-21 (1977)

Immune Competence and Immunosuppressive Factors in Splenectomized Tumor-Bearing Mice 1 RICI~ARD B.

WHITNEY, BARBARA L. POPE, AND JULIA G. LEVY

Department of Microbiology, University of British Columbia, Vancouver, British Coh~mbia, Canada VdT 1V/5 Received June 25, 1976 Splenectomy prior to tumor cell inoculation had no effect on subsequent tumor growth rate. The immunosuppressive serum factor of tumor bearers, which inhibits lymphocyte proliferation, developed to usual levels in splenectomized tumor bearers. Suppressor cells normally found in spleen but not lymph nodes of large-tumor bearers were still not observed in lymph nodes of splenectomized tumor bearers. Spleen cells of sham-tumor bearers had suppressed mitogen responses. Lymph node cells were not suppressed in either sham-tumor bearers or splenectomized tumor bearers. Tumorspecific immunity was absent in both spleen and lymph node cells of sham-operated mice with large tumors and in lymph nodes of splenectomized mice with large tumors. INTRODUCTION Numerous studies have shown that the spleen may be very influential in regulating the immune response to tumors. Whether it is beneficial or detrimental to the tumor-bearing host remains to be clarified. For example, splenectomy of the tumor host has in some cases been found to inhibit tumor growth (1, 2) and to reduce tumor metastases (3). However, in another study tumor growth was not affected (4) while in yet another both positive and negative effects on growth were observed depending on the dose of tumor cells injected (5). Additionally, the spleen has been found to regulate lymphocyte trapping in the lymph nodes draining tumor grafts (6), and sera from splenectomized tumor bearers were less effective in blocking lymphocyte-mediated tumor cell destruction in vitro than were sera from untreated tumor bearers (7). It is established now that immunosuppression is often a common feature of progressive tumor growth in both nonhuman animals and humans (8-12). Previous studies in our laboratory have shown that the decrease in lymphocyte competence was concomitant with the appearance of an immunosuppressive serum factor which inhibited lymphocyte proliferation (13-15). Additionally, mice with tumors developed suppressor cells in their spleens which inhibited mitogen responses of normal lymphocytes just as the serum factor did (16). Suppressor cells were not detected in tumor-bearer lymph nodes, however. 1 This work was supported by National Cancer Institute of Canada grants to Julia G. Levy and R. B. Whitney. 15 Copyright © 1977 by AcademicPress, Inc. All rights of reproductionin any form reserved.

ISSN 0008-8749

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WHITNEY, POPE AND LEVY

The purposes of the present study were to determine the effects of splenectomy on tumor growth and on the induction of the immunosuppressive serum factor described above using a methylcholanthrene-induced sarcoma system. It was also of interest to determine whether tumor-specific and general competence of lymph node cells of tumor bearers was altered by splenectomy and whether suppressor cells, normally present only in spleens of tumor bearers (16) devloped in lymph nodes in the absence of the spleen. MATERIALS AND METHODS

Mice. Female DBA/2J mice, aged 2-4 months, obtained from the Jackson Laboratory were used throughout. Tumor. A methylcholanthrene-induced sarcoma syngeneic to DBA/2J mice obtained from the Jackson Laboratory and maintained in vivo and in vitro for 4 years in our laboratory was used. Culture techniques have been described previously (12, 13). All tumors were larger than 3.0 g when used. Splenectomy. Spleens were removed under Nembutal anesthesia. For shams, incisions were made, and the spleen was handled with forceps before closure. Tumor cells were injected 3 weeks after surgery. Collection and preparation of serum. Mice were exsanguinated from the heart under ether anesthesia. The blood was clotted overnight at 4°C after which the serum was separated, inactivated at 56°C for 30 min, and stored at - 2 0 ° C . Fractionation and assay of mouse serum. Serum taken from splenectomized and sham-operated tumor bearing and normal mice was fractionated on Sephadex G-150 in order to obtain the immunoglobulin (Ig)-containing fraction. The Ig peak was precipitated with 40% saturated (NH4)2SO4 and reconstituted to its original volume in physiological saline as previously described (17). The immunosuppressive activity of the Ig fraction was assessed by titrating various concentrations of the material in culture with Con A-stimulated normal DBA/2J splenocytes as previously described (17). The albumin fractions were also collected, concentrated, and assayed for inhibitory activity. In no instance was this material found to be inhibitory to Con A-stimulated cells. Culture system for mitogen stimulation. These methods have been described in detail previously (12, 13). Briefly, spleen or lymph node cells were cultured in 0.2 ml of RPMI-1640 medium in microtiter plates with either 5% fetal calf serum (FCS) in tests for competence or suppressor cells or 2% FCS in tests on mouse serum fractions. The medium also contained gentamicin at 50 ~g/ml. Mitogens were added in 0.05-ml volumes at final concentrations of 4 ~,g/ml for concanavalin A (Con A) or 25 ~g/ml for lipopolysaccharide (LPS). One microcurie of [aH]thymidine was added to each culture after 48 hr and the cultures were harvested 18 hr later. V/inn test. This method has been described previously (12, 18). Briefly, 6 × 106 lymphoid cells were mixed with 2 × 104 tumor cells and incubated in vitro for 1 hr. The combination was then injected subcutaneously into normal recipients who were followed for tumor development for 6 weeks after injection. Data presentation. Stimulation results were expressed as mean counts per minute (cpm) of three or four replicate cultures. Standard deviations were always less than 10~. Statistical significance was determined using a Students' t test.

IMMUNE STATUS OF SPLENECTOMIZED TUMOR BEARERS

17

RESULTS Splenectomy 3 weeks prior to tumor cell inoculation had no effect on tumor growth. Tumors appeared after the same latent period and subsequently grew at the same rate in sham-treated and splenectomized mice (Fig. 1). There was no significant difference between the groups at any time. As in previous studies (12, 16), both Con A and LPS responses of spleen cells were markedly decreased in nonsplentcomized animals which, in this case, were the sham-tumor bearers (Table 1). However, there was no difference between lymph node Con A responses of normal and tumor-bearing mice in the absence or presence of the spleen. Also, there was no difference between the controls and splenectomized tumor-bearer lymph node Con A responses. The L P S responses of both types of tumor bearers were actually enhanced. Unstimulated spleen and lymph node cells of tumor bearers incorporated more [~H]thymidine than did normal cells. However, since treatment with anti-O serum and complement abolishes the Con A response but does not affect the unstimulated incorporation (18), it seems best to disregard these unstimulated levels when considering whether the level of mitogen stimulation is suppressed. Tumor-specific immunity usually develops shortly after initial tumor cell inoculation (10, 12, 19). However, after a certain point of tumor growth it disappears (10, 12, 19). Using the Winn test, an adoptive transfer method, it was found that tumor-specific immunity was completely lacking in the spleen and lymph nodes of sham-splenectomized normal and large-tumor-bearing mice (Table 2) as previously 2,C

1..'

u

0.5

0

i

a

n

I

7

14

21

28

TIME

35

{DAYS)

FIG. 1. Tumor growth in splenectomized and sham-splenectomized mice. ( • spleneetomized ; ( O O ) , splenectomized.

• ), Sham

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WI~ITNEY, POPE AND LEVY

TABLE 1 General Lymphocyte Competence in Sham-Treated and Splenectomized Mice Assessed by Responsiveness to Mitogens Lymphoid cell typea

Surgicaltreatment

a[H]thymidine incorporation (cpm)b None

Con A

LPS

Spleen (N) Spleen (T)

Sham Sham

12,200 17,300

253,000 94,000

81,100 16,600

Lymph node (N) Lymph node (T)

Sham Sham

3,200 12,700

247,000 269,000

47,900 80,300

Lymph node (N) Lymph node (T)

Splenectomy Splenectomy

3,740 17,500

231,000 284,000

71,000 93,300

a Spleen or lymph node cells, 8 X 105,from normal (N) or tumor-bearing (T) mice were tested. bThe mean counts per minute of triplicate cultures are given. observed (12). Splenectomized normal or large-tumor-bearing mice also had no demonstrable anti-tumor activity in their lymph nodes. Spleen cells from sham-operated tumor bearers inhibited both the Con A and L P S responses of normal spleen or lymph node cells (Table 3) as previously reported (16). Also as observed before, lymph node cells of sham-treated tumor bearers were not in the least suppressive. Further, splenectomy did not promote the development of suppressor cells in tumor bearer lymph nodes. Serum from normal and tumor-bearing mice was collected and separated on Sephadex G-150 into a high molecular weight (Ig-containing fraction) and a lower molecular weight fraction. Immunosuppressive activity has been shown to separate in the high molecular weight fraction (17). Table 4 shows that highly suppressive activity was present in the high molecular weight fraction of sera of both sham-treated and splenectomized tumor bearers. As has been found before, no suppressive activity was detected in the low molecular weight fraction of each. The serum concentration for optimum suppression was given. As with normal whole serum (13, 14), high concentrations of the normal serum fraction also will inhibit stimulation. No explanation can be given at present for the considerably higher stimulation observed in the presence of splenectomized normal serum relative to sham-treated normal serum. DISCUSSION Previous studies have shown that mice bearing the transplantable methylcholanthrene-induced sarcoma used herein, as well as many other tumors, produce a soluble serum factor which seems to be an immunoglobulin (17; unpublished observations) and which acts by inhibiting lymphocyte proliferation (15; unpublished observations). Additionally, these same tumor bearers develop a population of suppressor cells in their spleens but not their lymph nodes which are also capable of inhibiting the proliferation of normal lymphocytes when cultured together (16). Since these factors could be responsible, at least in part, for the decline of immune competence which so frequently accompanies progressive tumor growth (812), and since some reports (1-3) have shown that splenectomy can to some de-

IMMUNE

19

S T A T U S OF SPLENECTO2cIIZED T U M O R BEARERS

gree reduce tumor growth, it was important to determine if removal of the spleen could affect the development of suppressor factors and cells and, consequently, the immune competence of the tumor host. The results showed that splenectomized mice with large tumors had just as high levels of the serum inhibitor as did the sham-operated control tumor bearers. Thus, it can be concluded that the splenic environment was not the sole requirement for production of the serum inhibitor. It obviously could be produced elsewhere. This was in contrast to another report by Hellstr6m et al. where it was found that splenectomized tumor bearers had less tumor-specific blocking factor in their serum than did nonsplenectomized mice (7). In sham-operated tumor bearers suppressor cells for mitogen responses were found in the spleen but not the lymph nodes as previously reported (16). It was thought that in the absence of the spleen production of suppressor cells might be taken over by the lymph nodes. However, no suppressor activity was detected in splenectomized tumor-bearer lymph nodes. It was still possible that some suppressor cells could be present but were not detected because they were below a critical level needed for suppression. Additional studies using varying densities of I-Iypaque-Ficoll might permit detection of some lymph node suppressor cells of density different than the splenic suppressor cells. However, the present results indicated that there was no dramatic change in the normally negative suppressor properties of lymph node cells in splenctomized tumor bearers. Mice with large tumors have depressed responses to both T and B mitogens, and the tumor-specific immunity which develops during early tumor growth is no longer detectable (12). Splenectomy did not affect these conditions either positively or negatively. A major source of suppressor cells, the spleen, was removed. However, since the suppressive serum factor was still present in high concentration, the results were not unusual. Furthermore, since the suppression of the immune response during tumor growth was not altered by splenectomy, the lack of any effect on actual tumor growth was likewise not unusual. The observations reported herein together with those of previous studies (1317) raise some important questions concerning the nature and source of the serum inhibitor and the splenic suppressor cells and, ultimately, their relationship to actual tumor growth. TABLE 2 Tests for Tumor-Specific Immunity in Splenectomized or Sham-Treated Normal and Tumor-Bearing Mice Lymphoid cell type"

Surgical treatment

Tumor frequencyb

Spleen (N) Spleen (T)

Sham Sham

10/10 10/10

Lymph node (N) Lymph node (T)

Sham Sham

10/10 10/10

Lymph node (N) Lymph node (T)

Splenectomy Splenectomy

10/10 12/12

Spleen or lymph node cells, 6 X 106, from normal (N) or tumor-bearing (T) mice were mixed with 2 X 104 tumor cells and injected together subcutaneously. bThe frequency of tumor development in recipients after 6 weeks is given.

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WHITNEY, POPE AND LEVY TABLE 3 Effect of Tumor Bearer Lymphoid Cells on Mitogen Stimulation Normal Lymphoid Cells

Composition of culture a

[*H-]thymidine incorporation (cmp) b

Surgical treatment of 3 X 105 population

None

Con A

LPS

5 X 105 3 X 10 5 Ns Ns Ns Ns

Ns Ts T1 T1

Sham Sham Sham Splenectomy

12,200 24,400 27,200 28,200

263,000-137,000 ( - 4 6 ) 232,000 ( - 8 ) 265,000 (-I-5)

81,100-33,500 ( - 5 9 ) 91,900 (-t-13) 101,000(-}-25)

N1 N1 N1 N1

N1 Ts TI TI

Sham Sham Sham Splenectomy

3,200 24,600 7,280 10,800

247,000-160,000 ( - 3 5 ) 256,000 (+4) 256,000 (-t-4)

47,900-38,300 ( - 2 0 ) 66,800 (+39) 72,800 (-t-52)

a Cultures consisted of 5 X 10~normal spleen (Ns) or lymph node (N1) cells plus 3 X 105 normal or tumor-bearer (Ts or T1) spleen or lymph node cells. b Results are given as mean counts per minute of triplicate cultures followed by percentage of control level. Consider first the serum inhibitor which all evidence indicates is at Ieast in p a r t an i m m u n o g l o b u l i n ( 1 7 ; unpublished o b s e r v a t i o n s ) , thus, i m p l y i n g it to be a l y m p h oid cell product. T h e data here showed that it is p r o d u c e d as efficiently in the absence as in the presence of the spleen. Also, w o r k in p r o g r e s s indicates the serum factor a p p e a r s earlier in the course of t u m o r g r o w t h than do the splenic s u p p r e s s o r cells, and it d i s a p p e a r s m o r e r a p i d l y after surgical t u m o r resection ( u n p u b l i s h e d o b s e r v a t i o n s ) . Therefore, it seems unlikely that the splenic s u p p r e s s o r cells are the p r o d u c e r s of the serum inhibitor. N o r m a l l y , the spleen m a y be a source of the inhibitor but clearly it is not an essential source. T h e observations s u p p o r t the possibility that the inhibition of general i m m u n e competence d u r i n g t u m o r g r o w t h is a two-cell t y p e process. T h e soluble inhibitor for e x a m p l e m i g h t be a T-cell p r o d u c t which w o u l d interact with and activate another cell, possibly a maerophage, for suppression. Since m a c r o p h a g e s are plentiful in spleen but not l y m p h nodes, TABLE 4 Effect of the Immunoglobulin Fraction from Splenectomized and Sham-Operated Normal and Tumor-Bearing Mice on the Uptake of [-3H~Thymidine in Con A-Stimulated Cultures of Normal Mouse Spleen Cells Source of immunoglobulin fraction ~

Surgical treatment

Protein added per culture (rag)

[3H]thymidine incorporation (cpm) b Con A

N T N T None

Sham Sham Splenectomy Splenectomy Splenectomy

0.06 0.06 0.05 0.05 0.05

20,700 1,830 (91) 114,000 9,540 (92) 44,600

a Serum was taken from normal (N) or tumor-bearing (T) mice which had been splenectomized or sham operated. b Results are expressed as mean counts per minute of triplicate cultures followed by percentage of inhibition,

IMMUNE STATUS OF SPLENECTOMIZED TUi~iOR BEARERS

21

this could explain the strong suppression by tumor-bearer spleen cells and the lack of suppression by lymph node cells from the same animal. Other studies have indicated that the suppressor spleen cells may be maerophages (16, 20). The lack of inhibition of mitogen responsiveness in lymph nodes in animals which have severely suppressed spleen mitogen responses would then be due to the absence or low numbers of the activated suppressor macrophages. This, however, raises a question regarding the observed suppression of tumor-specific immune responses in lymph node cells of tumor-bearing animals. A potential explanation could be that different suppressive mechanisms are involved. In several recent studies tumor-bearer thymus cells suppressed tumor-specific immunity (21, 22) while, in one case, not affecting mitogen responses. The ultimate question concerns how these factors and cells affect the outcome of tumor growth. It may well be that the nonspecific serum inhibitor and splenic suppressor cells do not directly influence tumor growth but are important in that they reduce immunologic competence toward general infection which is one of the major immediate causes of death in cancer patients (23). Thus, they may be critical elements in determining whether or not a patient will survive after primary tumor therapy. If his general immune competence is normal, his survival chances may be improved. Indeed, a number of studies has now shown that prognosis is much better in patients with strong immune responses at the time of treatment than in those who are severely immunosuppressed (24, 25). Experiments in progress should help to clarify some of these points. REFERENCES 1. Old, L. J., Clarke, D. A., Benacerraf, B., and Stockert, E., Ezperientia 18, 335, 1962. 2. Ferret, J. F., Transplantation 6, 160, 1968. 3. Milas, L., and Mujagie, H., Int. Y. Cancer 11, 186, 1973. 4. Bard, D. S., and Pilch, Y. H., Cancer Res. 29, 1125, 1969. 5. Nordlund, J. J., and Gershon, R. K., J. Immunol. 114, 1486, 1975. 6. Fightlin, R. S., Lytton, B., and Gershon, R. K., Y. Immunol. 115, 345, 1975. 7. Hellstr6m, I., Hellstr6m, K. E., and SjiSgren, H. 0., Cell. Immunol. 1, 18, 1970. 8. Golub, S. H., O'Connell, T. X., and Morton, D L., Cancer Res. 34, 1833, 1974. 9. Adler, W. H., Takiguchi, T., and Smith, R. T., Cancer Res. 31,864, 1971. 10. Deckers, P. J., Davis, R. C., Parker, G A., and Mannick, J. A. Cancer Res. 33, 33, 1973. 11. Hellstr/Sm, I., Warner, G. A., Hellstr/~m, K. E., and Sj/Sgren, H. O., Int. J. Cancer 11, 280, 1973. 12. Whitney, R. B., Levy, J. G., and Smith, A. G., Y. Nat. Cancer lust. 53, 111, 1974. 13. Whitney, R. B., and Levy, J. G., Eur. J. Cancer 10, 739, 1974. 14. Whitney, R. B., and Levy, J. G., J. Nat. Cancer Inst. 54, 733, 1975. 15. Whitney, R. B., and Levy, J. G., ./. Nat. Cancer Inst. 55, 1447, 1975. 16. Pope, B. L., Whitney, P~. B., Levy, J. G., and Kilburn, D. G., J. ImmunoI. 116, 1342, 1976. 17. Levy, J G., Smith, A. G., Whitney, R. B., McMaster, R., and Kilburn, D. G., Immunology 30, 565, 1976. 18. Whitney, R. B., Levy, J. G., and Smith, A. G., Brit. d. Cancer 31, 157, 1975. 19. Le Francois, D., Youn, J. K., Belehradek, J, and Barski, G., J. Nat. Cancer Inst. 46, 981, 1971. 20. Kirchner, H., Chused, T. M., I-Ierberman, ]~. B., Holden, H. T., and Laurin, D. H., Y. Exp. Med. 139, 1473, 1974 21. Fujimoto, S., Greene, M. I., and Sehon, A. H., J. Immunol. 116, 791, 1976. 22. Takei, F., Levy, J. G., and Kilburn, D. G., J. Immunol. 116, 288, 1976. 23. Inagaki, J., Rodriguez, V., and Bodey, G. P., Cancer 33, 568, 1974. 24. Krant, l'Vf.J., Manskopf, G., Brandrup, C. S., and Madoff, M. A., Cancer 21, 623, 1968. 25. Eilber, F. R., Nizze, J. A, and ~Iorton~ D. L., Ca~,cer 35~ 660~ 1975.