Augmentation of specific immune response against syngeneic SV40-induced tumor-associated antigens by splenectomy

CELLULAR

IMMUNOLOGY

A&230-236

(1979)

Augmentation of Specific Immune SV40-Induced Tumor-Associated

Response Antigens

against Syngeneic by Splenectomy

MOSHE GLASER Department

of Biochemistry, The George Washington 2300 Eye Street, N. W., Washington, Received

November

University Medical D.C. 20037

Center,

28. 1978

Splenectomy before immunization of mice with syngeneic SV40-transformed cells markedly augmented the specific cell-mediated immune response against the corresponding tumorassociated antigens as measured by an in vitro Yr-release assay and an in vivo tumor-cell neutralization assay. This augmentation was not dependent on the time interval between splenectomy and antigen immunization. By performing reconstitution experiments, it was found that thymus-derived cells in spleens of normal syngeneic mice abolished the splenectomy-induced augmentation of immune response. It is inferred that the resident population which normally operates in spleen-intact mice to suppress the specific immune response against SV40-induced tumor-associated antigens is T-cell.

INTRODUCTION The spleen plays an important role in the regulation of immune responses. Some types of cell-mediated immune reactions can be augmented by splenectomy, for example, (1) delayed-type hypersensitivity to sheep erythrocytes, (2) proliferative response to concanavalin A, (3) resistance to Listeriu monocytogenes, and (4) allograft rejection. Recently various investigators have shown great interest in the role of the spleen in immune responses to tumors (5-7). Two main theories exist for the explanation of the regulatory effects of the spleen on the immune response. The first one favors the idea that splenectomy decreases the production of serum blocking factors, especially enhancing antibodies and/or antigen-antibody complexes which can protect the neoplasm from immune rejection (8, 9). The second one favors the concept of removal of suppressor T-cells by splenectomy (10). Information on the feedback mechanisms exerted by the spleen on cell-mediated immune responses to tumor antigens may provide a clue to understanding why immune surveillance fails to destroy some tumors; in addition, it may provide information on general biologic characteristics of the immune system. There have been extensive studies on immune response to SV40-induced syngeneic tumor in our laboratory (1 l- 13). This tumor has been shown to possess tumor-associated antigens which immunize specifically against the growth of the tumor. This immunity can be transferred by T-lymphocytes from immune animals in the tumorcell neutralization assay (14). Cytotoxic T-cells have also been shown in the spleens of animals immune to SV40-transformed cells by the use of a Wr-release assay 230 0008-8749/79/070230-07$02.00/O Copyright All rights

0 1979 by Academic Press, of reproduction in any form

Inc. reserved.

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COMMUNICATIONS

231

(14). In a recent series of experiments, we developed a technique for in vitro sensitization of lymphocytes from mice immune against SV40-induced tumor-associated antigens, plus the corresponding antigens. The 51Cr-release assay employed to measure the activity of these lymphocytes (15) was shown to give good correlation to the in vivo tumor-cell neutralization assay (16) employing a transplantable SV40-induced sarcoma. The present experiments were conducted to investigate the possible control mechanisms responsible for the regulation of cellular processes involved in the immune response against a syngeneic SV40-induced tumor. Evidence is presented there that prior splenectomy augments the cell-mediated immunity, as measured by the two assays mentioned, against tumor-associated antigens of SV40-induced syngeneic tumor, apparently by removal of suppressor T-cells. MATERIALS

AND METHODS

Mice. Two- to three-month-old BALB/c, C57BL/6, and CBF, (BALB/c x C57BL/6)F, female mice were obtained from the NIH breeding colony. Tumors and cell lines. The mksA-Tu5 tumor line was developed by transformation of BALB/c kidney cell line with SV40 (17). This line has been maintained in this laboratory for several years in tissue culture as adherent cells. An ascites form of mksA was developed by intraperitoneal (ip) passage of 1 x IO6 cells in BALB/c mice. The mksA ascites (ASC) grow as a solid tumor when injected subcutaneously (SC) and have a TD5* of 1 x lo2 cells. Both the tissue culture and the ascites forms of mksA tumor express the SV40 tumor antigen (T) and SV40 tumor-associated transplantation antigen (TATA) but are free of infectious virus (18). These cells are poor targets in in vitro 51Cr-release assay (19). C57SV cells (SV40-transformed C57BL/6 mouse embryo fibroblasts) (20) are tissue cultureadherent cells obtained from B. B. Knowles, Wistar Institute, Philadelphia. These cells are good targets for in vitro 51Cr-release assay but do not grow in vivo (14,20). C57SV and mksA cells cross-react both in vivo and in vitro (14). RBL-5, a Rauscher virus-induced leukemia, was maintained in C57BL/6 mice by weekly ip passage of 1 x 10” cells. These cells were adapted to grow in tissue culture cells in suspension in our laboratory. The TD,, of these cells when inoculated SC is 1 x lo4 cells. MSB is an adherent tissue culture cell line derived from an M-MuSV-induced tumor in C57BL/6 mice (21). The cells cross-react with RBL-5 cells and are good targets for the in vitro 51Cr-release assay (21). Immunization. CBF, mice were inoculated ip with 20 x lo6 C57SV cells. Preparation und purification of spleen cell populations. Spleens were removed from normal CBF, mice and single cell suspensions were prepared in RPM1 1640 medium supplemented with 100 pg/ml streptomycin, 100 units/ml penicillin, and 2 mM glutamine (all reagents from Grand Island Biological Co., Grand Island, N.Y .). For removal of T-cells, AKR anti-Thy 1.2 C3H antibody was obtained from H. Holden, NCI, Bethesda, Maryland. The preparation of this antiserum and the treatment of lymphoid cells were performed according to the technique described in detail in Herberman et al. (22). This antiserum lysed more than 97% of C3H thymocytes and 35-40% of C3H spleen cells as assessed by trypan blue dye exclusion. The T-cell response of spleen cells to PHA was abolished after treatment, whereas the B-cell response to LPS was left intact. For removal of B-cells

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and macrophages, the technique employing nylon wool columns, described by Julius et al. (23) was used. About 30% of the starting spleen cell population was recovered in the nonadherent fraction (T enriched); of this 2-4% (as compared to 40-45% in the unfractionated population) were surface immunoglobulin-positive cells. For removal of phagocytic cells, the technique employing iron and magnet described elsewhere (24) was used. This technique removed about 30% of the total cell population and eliminated most phagocytic cells (0.3% remained after treatment in contrast to 5% before treatment) as assessed by latex particle ingestion. In vitro sensitization. Pooled lymph nodes were removed from CBF, immune mice and 5 x lo6 lymph node cells were cultured with 5000 R X-irradiated 3 x lo4 C57SV cells or 1 x lo4 MSB cells in a 2-ml volume of RPM1 1.640 medium supplemented with 25 x 10e3 h4 Hepes solution (Microbiological Associates, Bethesda, Md.), 5 x lob5 M 2-mercaptoethanol (Schwartz-Mann Division, Becton Dickinson, Orangeburg, N.Y.), and 5% fetal bovine serum (Grand Island Biological Co.) in 24-well culture plates (16 mm, Linbro Chemical Co., New Haven, Conn.) at 37°C in a humidified incubator (5% COZ, 95% air) for 5 days. 51Cr-Release assay. The effector cells (the nonadherent cells) were removed from the Linbro wells by gentle pipetting after in vitro sensitization, washed once, and resuspended in RPM1 1640 medium containing 10% fetal bovine serum. The cytotoxicity assay was performed in the wells of flat-bottom microtiter plates (Cooke Laboratory Products, Div. Dynatech Laboratories, Inc., Alexandria, Va.). Tissue target cells were trypsinized and plated in each well at 1 x lo4 cells with 5 &i Nas1Cr04 (New England Nuclear, Boston, Mass.) in 0.2 ml RPM1 1640 medium containing 5% fetal bovine serum. After 18 hr at 37°C in a CO2 incubator, the target cells were washed and effector cells were added in a 0.2-ml volume. After 6 hr incubation at 37°C in a CO, incubator, the plates were centrifuged at 300g for 5 min and 0.1 ml of the supernatant was removed for counting. The target cells were lysed with 1% sodium dodecyl sulfate (SDS) to measure the total releasable 51Cr in each well. Results are calculated from the mean of triplicate samples and expressed as: Percentage

specific lysis cpm 51Cr released by in vitro sensitized cells - cpm 51Cr released by unsensitized cells = x 100. cpm 51Cr released by target cells in 1% SDS

Representative experiments are shown. Each experiment was repeated 3-4 times wth similar results. Tumor-cell neutralization assay. Pooled CBF, lymph node cells were washed twice and resuspended in RPM1 1640 medium. The cells were mixed with mksA ascites cells or RBL-5 tissue culture cells, incubated for 45 min at 37°C and injected SC in O.l-ml aliquots. The ratios of effector to tumor cells using 1 x lo5 mksA and 5 x lo5 RBL-5 cells are specified in the results. These doses of tumor cells produced tumors in all mice injected with tumor cells mixed with nonimmune lymph node cells. Tumor growth was monitored until death of the animals. Lymph node cells from six to eight donors were used for each assay. Representative experiments are shown. Each experiment was repeated three to four times with similar results.

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Splenectomy. Splenectomy was performed in CBF, mice anesthetized with ether. The peritoneal cavity was opened by a small incision under the left costal margin, and the spleen was mobilized outside the abdominal cavity. After the splenic venules were ligated with silk, the spleen was removed, and peritoneum was closed with silk, and the muscle and skin were closed with metal clamps. Sham-operated mice underwent the same procedure except that their spleens were not removed. Splenectomy was usually performed 2 weeks before immunization unless otherwise indicated. RESULTS Effect of splenectomy on immune response to SV40-induced tumor-associated antigens. CBF, mice were splenectomized or sham-splenectomized or left unoperated and 2 weeks later they were inoculated ip with 20 x lo6 C57SV cells. Twenty days thereafter the animals were sacrificed and their pooled lymph node cells were either incubated in vitro for five days with X-irradiated C57SV-sensitizing cells and then tested in a 6-hr 51Cr-release assay against C57SV or MSB target cells, or were incubated for 45 min with mksA ascites or RBL-5 tissue culture cells and injected SC into normal CBF, mice. The results of these experiments are shown in Table 1. Marked augmentation of in vitro cell-mediated cytotoxicity against C57SV target cells but not against MSB cells was observed in all attacker: target cell ratios employed by using lymph node cells from splenectomized mice as opposed to the control groups. By employing the tumor-cell neutralization assay marked augmentation of effector-cell activity against mksA cells but not against RBL-5 cells was seen in splenectomized animals as opposed to the control animals. For example, whereas complete protection against mksA growth was observed with 1OO:l effector:tumor cell ratio in the control group, a ratio as low as 25:l effector:tumor cell was necessary to get similar effects with lymph node cells from splenectomized mice. Both these results implied that splenectomy of mice before tumor-cell immunization augmented cell-mediated immune response against SV40-induced tumor-associated antigens. Effect of splenectomy at different intervals before immunization on immune response against SV40-induced tumor-associated antigens. CBF, mice were splenectomized or sham-splenectomized at different intervals before ip inoculation of TABLE

1

Effect of Splenectomy on Cell-Mediated Cytotoxicity against and Tumor-Cell of SV40-Induced Tumor-Associated antigens Cytotoxicity

(o/o) against

Neutralization

c57sv Treatment

50: 1(1

MSB

25: 1

12:l

6:l

Neutralization

mksA

50: 1

25: 1

1lxl:l”

RBL-5

50: 1

2S:l

12: I

100: I

50: 1

None Sham

45

28

12

4

6

4

1116’

11116

14116

14116

14116

13/lh

splenectomy Splenectomy

48 69

30 58

16 41

7 19

5 8

3 5

3/16 l/16

12116 l/16

13/16 3116

13116 12116

12116 13/16

12116 14116

” Attackwtarget b Effectwtumor r No. of dead

cell ratio. cell ratio. animals/no. of total

animals.

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20 x lo6 C57SV cells. Twenty days after immunization the in vitro sensitization and the in vivo tumor-cell neutralization assays were performed with pooled lymph node cells of these animals. The results in Table 2 demonstrate that an enhanced immune response was already detected 1 week after splenectomy, and this effect was similar even at 12 weeks after splenectomy. These results implied that under these experimental conditions the time of splenectomy before immunization is not critical in order to get an enhanced immune response against SV40-induced tumor-associated antigens. Abrogation of augmented immune response against SV40-induced tumor-associated antigens by transfer of normal spleen cells. CBF, mice were splenectomized and 2 weeks later were inoculated iv with 60 x lo6 various spleen-cell populations from normal CBF, mice and ip with 20 x lo6 C57SV cells. The in vitro sensitization and in vivo tumor-cell neutralization assays were performed 20 days later with pooled lymph node cells of these animals. Table 3 demonstrates that when the splenectomized mice were repopulated with normal spleen cells either unfractionated, complement-treated, nonadherent nylon-column passed, or iron- and magnet-treated, the abrogation of splenectomy-induced augmentation of cell-mediated immune response was observed. However, when the mice were inoculated with spleen cells treated with anti-Thy 1.2 and complement-augmented immune response was still observed similar to the response in nonrepopulated mice. These results implied that thymus-derived lymphocytes in normal spleens suppressed the in vivo cell-mediated immune response against SV40-induced tumor-associated antigens. DISCUSSION The results of the present study demonstrate that splenectomy before immunization of mice with syngeneic SV40-transformed cells augmented the specific TABLE

2

Effect of Splenectomy at Different Intervals before Immunization on Cell-Mediated Cytotoxicity against and Tumor-Cell Neutralization of SV40-Induced Tumor-Associated Antigens

Treatment

Weeks before immunization

Cytotoxicity against C57SV (%I

Neutralization of mskA (dead/total)

14”

13/16*

Sham splenectomy

1 2 4 8 12

16 13 17 14 12

14116 12/16 13116 15/16 14116

Splenectomy

1 2 4 8 12

38 42 40 43 39

l/16 2116 4116 3/16 4116

a Attacker: target cell ratio = 12: 1. b Effector:tumor cell ratio = 25: 1.

235

SHORT COMMUNICATIONS TABLE Abrogation of Augmented Cell-Mediated SV40-Induced Tumor-Associated Reconstitution with spleen cells None Unfractionated Nylon wool column Iron and magnet Anti-Thy 1.2 serum and complement Complement u Attacker:target * Effector:tumor

3

Cytotoxicity against and Tumor-Cell Neutralization Antigens by Transfer of Normal Spleen Cells Cytotoxicity against C57SV (%)

of

Neutralization of mskA (dead/total)

45” 12 14 10

1116” 12116 14/16 13/16

42 15

3116 14/16

cell ratio = 12: 1. cell ratio = 25: 1.

immune response against the corresponding tumor-associated antigens as measured by in vitro 51Cr-release assay and in vivo tumor-cell neutralization assay. These two tests of cell-mediated immune responses to syngeneic tumors were shown to provide good correlation in the in vivo resistance against tumor growth (15, 16). The augmentation effect of splenectomy was abolished by the transfer of thymusderived lymphocytes from normal syngeneic spleen cells; B-cells and phagocytic cells do not seem to be involved in this type of suppression. The demonstration that splenectomy augmented immune responses against syngeneic tumors is not new. Chang and Turk found that splenectomized BALB/c mice were protected from death caused by the growth of methylcholanthrene-induced ascites tumor (6). Similarly augmentation of immune response against syngeneic mm-me melanoma (Cloudman S 91) in splenectomized DBAR mice was reported by Norlund and Gershon (25). These results are also in accord with those showing augmentation of (1) delayed-type hypersensitivity reactions against sheep erythrocytes, (2) proliferative response of lymphocytes to concanavalin A, (3) resistance to Listeriu monocytogenes, and (4) allograft rejection. The new aspect of our study using a syngeneic tumor system is that we clarified one of the causes for such an effect as being suppressor thymus-derived lymphocytes present in normal spleens which suppress in vivo the cell-mediated immune response. This was shown by removal of the spleen which resulted in an augmented immune response and by reconstitution of splenectomized mice with normal thymus-derived spleen cells which resulted in abolishment of the augmented immune response. Whether these suppressor T-cells act directly or through factors released by them to suppress immune responses as was suggested for blocking factors (8,9) is not yet resolved at present. Whatever the mechanism might be it is obvious from this and other studies that prior splenectomy shifts the balance of aggressive/suppressive forces in the direction of the former, thus augmenting immune responses and suggesting that in the normal situation regulatory forces are brought into play which operate to suppress immune responses against a variety of antigens. Finally, although many reports dealt with the augmentation effect of splenectomy performed before the establishment of tumor, the clinical implication derived from those and from the present

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study is that splenectomy after the introduction of a tumor may protect the host against death from progressive tumor growth. Such experiments will be the subject of a future communication. ACKNOWLEDGMENTS I wish to thank Dr. L. W. Law for his interest and support during this work and Ms. Barbara Smith for helping with the preparation of this manuscript.

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