- Email: [email protected]
Suppressive effect of X-irradiated tumor cell presensitization on the induction of syngeneic tumor immunity
CELLULAR
IMMUNOLOGY
Suppressive
59, 181-186 (1981)
Effect of X-Irradiated Tumor Cell Presensitization the Induction of Syngeneic Tumor Immunity
II. Opposite Effects of Intravenous Administration of TNP-Conjugated Cells on the Development of Antitumor and Anti-TNP-self Cytotoxic Effector Cells HIROMI Immunology
FUJIWARA’
AND GENE
on
Tumor
M. SHEARER
Branch, National Cancer Institute. National Bethesda, Maryland 20205
Institutes
of Health,
Received June 3, 1980: accepted July 30, 1980
A comparison was made of the effects of iv inoculation of trinitrophenyl (TNP)-conjugated syngeneic X5563 tumor cells on the development of anti-TNP-modified self (TNP-self) and anti-X5563 tumor-specific T-cell-mediated cytotoxic responses.Administration of syngeneic TNP-conjugated spleen cells or X-irradiated TNP-conjugated X5563 cells resulted in an appreciable augmentation in the generation of TNP-reactive cytotoxic effector cell activity as measured by subsequent in vitro sensitization with TNP-self. In contrast, iv inoculation with either TNP-modified or unmodified X-irradiated X5563 tumor cells did not prime mice for antitumor cytotoxic responses, but abolished the ability of mice to develop antitumor cytotoxic effector cells even after effective in vivo immunization with tumor cells. The possible mechanisms by which the suppression of antitumor cytotoxic response was induced by conditions which simultaneously induced an enhanced anti-TNP-self cytotoxic response are discussed.
INTRODUCTION A previous study demonstrated that intravenous (iv) administration of trinitrophenyl (TNP)-conjugated syngeneic spleen cells (TNP-self) abolished the ability of mice to develop TNP-contact sensitivity, whereas the identical treatment leads to augmented generation of TNP-reactive cytotoxic effector cell activity as shown by subsequent in vitro sensitization with TNP-self (1). It was also shown in a preceding report (2) that iv inoculation of X-irradiated syngeneic tumor cells can have different immunological effects in two murine tumor systems. For example, in the X5563 plasmacytoma tumor system, anti-tumor-specific cytotoxic effector T cells could be generated by intradermal (id) inoculation of viable tumor cells followed by the tumor resection (immunization procedure) (3). However, iv inoculation of X-irradiated tumor cells did not lead to any significant antitumor immune response, but abolished the ability to develop tumor immunity even when ’ Address correspondenceto Dr. H. Fujiwara, National Cancer Institute, 9000 Rockville Pike, Building 10, Room 4B17, Bethesda, Md. 20205. 181 OOOS-8749/81/050181-06$02,00/O Copyright 0 1981 by Academic Press, Inc. All rights oi reproduction in any form rcscr.4.
182
FUJIWARA
AND SHEARER
followed by the appropriate immunization procedure (2). In contrast, in a MM102 mammary tumor system to which immune response was shown to be exclusively anti-tumor-specific antibody (4, 5), pretreatment with X-irradiated MM102 tumor cells augmented the induction of antitumor immunity (2). Thus, the results of these models indicate that iv presensitization with “modified self” or tumor antigens can result in priming or suppression, depending on the immunological parameter studied. In the present study, we have further investigated the effects of iv administration of cells simultaneously expressing TNP-self and tumor antigens on the generation of each corresponding effector cell population. The results demonstrate that mice inoculated iv with TNP-modified X-irradiated syngeneic X5563 tumor cells exhibited an augmented in vitro anti-TNP-self cytotoxic effector cell response. In contrast, the same inoculated animals failed to develop an antitumor cytotoxic response even when the immunization procedure was performed before in vitro restimulation with tumor antigens. These results illustrate that iv immunization can result in different effects on the same type of immune response (i.e., the generation of cytotoxic effector cells) depending on the target antigens studied. MATERIALS
AND METHODS
Mice. Female C3H/HeN mice were obtained from Charles River Laboratory (Wilmington, Mass.) and used at 7-9 weeks of age. Tumor. The X5563 plasmacytoma was used in this study. It was derived from C3H/He strain (6), and maintained by serial intraperitoneal passages into syngeneic C3H/He mice in the ascitic form. Preparation of TNP-conjugated cells. Spleen or tumor cells were incubated with trinitrobenzene sulfonate (TNBS) at concentrations of 1 or 0.1 mM for 10 min, at 37°C as previously described (7, 8). In vitro sensitization for cytotoxic effector cells and cytotoxicity assay. Spleen cells (5 X 106) were sensitized in vitro to lo6 irradiated (2000 R) TNBS-modified syngeneic spleen cells (TNP-self) or lo5 irradiated ( 10,000 R) unmodified or TNBS-modified X5563 tumor cells and assayed on unmodified or TNBS-modified X5563 tumor target cells, as previously described (7-9). In vivo priming of anti-X5563 tumor speci’c cytotoxic response. C3H/He mice were intradermally (id) inoculated with lo6 viable X5563 tumor cells, followed by surgical resection of the tumor 7 days later (3). RESULTS The results shown in Table 1 confirm that iv administration of TNP-self can result in the enhanced generation of anti-TNP-self cytotoxic response as measured by subsequent in vitro sensitization to TNP-self. C3H/He mice were inoculated iv with 5 X lo7 of 1 mM TNP-self cells. Fourteen days later, spleen cells (5 X 106) from these primed and unprimed mice were sensitized in vitro with lo6 of irradiated (2000 R) 1 mM or 0.1 mM TNP-self cells. Effector cells generated after 5 days of culture were assayed on TNP-conjugated syngeneic X5563 tumor target cells. As shown in Table 1, iv inoculation with TNP-self led to appreciable priming of anti-TNP-self cytotoxic response. We also verify with the results shown in Table 2 that iv presensitization with X-irradiated tumor cells can abolish the ability of syngeneic C3H/He mice to
EFFECTS OF IMMUNIZATION
WITH TNP-TUMOR
183
CELLS ON CML
TABLE 1 Augmented Generation of TNP-Reactive Effector Cell Activity in the Spleen Cells from C3H/He Mice Inoculated iv with TNP-self Percentage specific lysis assayed on targetsb TNBSX5563 Stimulating cells ( 1Oh/well) 1 m&f TNP-spleen cells 0.1 mM TNP-spleen cells
Responding cells” (5 X 106/well)
X5563 2O:l
2O:l
IO:1
5:1
Normal TNP-spleen cellinoculated
-0.3 (1.07)
52.8 (1.04)
35.2 (1.01)
20.3 (1.05)
0.6 (1.02)
74.0 (1.02)
57.2 (1.03)
41.3 (1.05)
Normal TNP-spleen cellinoculated
-0.6 (1.04)
34.2 (1.02)
21.5 (1.03)
12.2 (1.02)
0.2 (1.02)
62.9 (1.02)
46.3 (1.02)
26.2 (1.03)
’ Spleen cells (5 X 106)from normal mice or mice inoculated iv with 5 X 10’ TNP-spleen cells (1 mM TNBS) were sensitized in vitro with irradiated (2000 R) stimulating cells (106). b Effector cell activities generated after 5 days of culture were assayed on unmodified X5563 and TNP-X5563 (10 mM TNBS) plasmacytoma. The percentage specific lysis was expressed as the geometric mean of triplicate cultures and the value in parentheses represents the standard error.
develop antitumor cytotoxic response. C3H/He mice were inoculated three times iv with lo6 of 7000-R X-irradiated X5563 tumor cells at 4-day intervals. Four days after the final inoculation, spleen cells from these injected and uninjected mice were sensitized in vitro to 10’ of irradiated (10,000 R) X5563 tumor cells. No significant antitumor cytotoxic response was detected in spleen cells from either treated or untreated mice (Expt A, Table 2). Furthermore, suppressive effect of TABLE 2 Suppressive Effect of iv Administration of X-Irradiated Tumor Cells on the Development of AntiX5563 Tumor Cytotoxic Response Percentage specific lysis assayed on X5563 cells
In vivo treatment In vitro
Expt
Group
iv presensitization”
Immunization procedureb
sensitization’ with
4O:l
2O:l
IO:1
A
I II
+
-
lo5 X5563 IO5 X5563
8.3 (1.05) 3.8 (1.02)
2.2 (1.03) 0.2 (1.04)
2.2 (1.10) 1.6 (1.06)
B
I II III
+
+ +
lo5 X5563 10’ X5563 10’ X5563
1.2 (1.05) 51.6 (1.01) 2.1 (1.02)
0.3 (1.03) 46.2 (1.00) -0.2 (1.04)
0.5 (1.05) 34.2 (1.01) 1.6 (1.03)
’ Mice were inoculated iv with lo6 of 7000-R X-irradiated X5563 tumor cells three times at 4-day intervals. b Four days after iv presentation, mice were inoculated id with IO6viable tumor cells, followed by the tumor resection 7 days later. ‘Spleen cells (5 X 106) from mice 4 days after iv presentation (Expt A) or 7 days after the tumor resection (Expt B) were sensitized in vitro to lo5 irradiated (10,000 R) X5563 tumor cells.
184
FUJIWARA
AND
SHEARER
presensitization with X-irradiated tumor cells on the subsequent ability of these mice to develop antitumor immune response is shown by the following experiments (Expt B, Table 2). Four days after the final iv injection with X-irradiated tumor cells, mice were inoculated id with lo6 viable X5563 cells followed 7 days later by the surgical resection of the tumor (immunization procedure). Seven days after the tumor resection, spleen cells from either normal control mice (Group I), mice which received the immunization procedure (Group II), or mice which received the immunization procedure after the iv pretreatment with irradiated tumor cell (Group III) were sensitized in vitro to 10’ X5563 tumor cells. As shown under the results in Expt B of Table 2, the immunization procedure led to strong generation of an antitumor cytotoxic response. However, iv administration of tumor cells abolished the ability to develop antitumor cytotoxic response even when followed by the appropriate immunization procedure. Since contrasting results were obtained in separate experiments for antitumor and anti-TNP-self cytotoxic responses after iv pretreatment, a priming study was performed by inoculating mice iv with cells expressing both tumor antigens and TNP-self determinants. C3H/He mice were given three iv inoculations of IO6 Xirradiated (7000 R) X5563 cells modified with 1 mM TNBS at 4-day intervals. Spleen cells from normal mice, or mice inoculated with unmodified or TNBSmodified X5563 cells were sensitized in vitro to TNP-self and effecters were assayed on unmodified or TNBS-modified X5563 cells (Table 3). Detectable and strong priming effects for anti-TNP cytotoxic response were observed in the TNP-X5563 tumor-cell-inoculated group when in vitro stimulation was provided by 1 or 0.1 mM TNP-self, respectively. No significant lysis was observed on unmodified X5563 tumor target cells. An identical iv regimen was studied for its effects on the development of antitumor cytotoxic response (Table 4). When spleen cells from normal mice, or mice inoculated with X5563 or TNP-X5563 cells were sensitized in vitro to X5563 or TNP-conjugated X5563 cells, no significant anti-X5563 tumor cytotoxic activity was detected (Expt A, Table 4). Furthermore, the mice inoculated with X-irradiated TABLE Augmented
3
Generation of TNP-Reactive Effector Cell Activity in the Spleen Cells from C3H/He Mice Inoculated iv with TNP-Modified Syngeneic X5563 Tumor Cells Percentage specific lysis assayed on targets X5563
Stimulating cells (106/well)
Responding cells” (5 X 106/well)
TNBSX5563
4O:l
4O:l
2o:l
lo:1
1 mM TNP-spleen cells
Normal X5563-inoculated TNP-X5563-inoculated
-4.1 (1.01) -1.7 (1.11) 1.0 (1.03)
58.4 (1.04) 59.2 (1.02) 71.3 (1.02)
58.2 (1.03) 57.0 (1.03) 67.8 (1.05)
58.5 (1.02) 47.9 (1.02) 64.5 (1.02)
0.1 mM TNP-spleen cells
Normal X5563-inoculated TNP-X5563-inoculated
-2.9 (1.03) 0.0 (1.03) 0.5 (1.02)
15.2 (1.03) 7.2 (1.02) 62.2 (1.01)
16.2 (1.04) 5.0 (1.07) 57.7 (1.01)
10.5 (1.07) 2.6 (1.00) 52.8 (1.07)
’ Spleen cells from normal mice, or mice inoculated iv with lo6 of 7000-R X-irradiated unmodified or TNBS-modified (1 mM) X5563 cells three times at 4-day intervals were sensitized in vitro to 1 or 0.1 mM TNP-self.
EFFECTS
OF IMMUNIZATION
WITH TABLE
TNP-TUMOR
CELLS
185
ON CML
4
Suppressive Effect of iv Presensitization with X5563 or TNP-X5563 Cells on the Development of Anti-X5563 Tumor Cytotoxic Response In viva treatment
Expt Group A
B
iv presensitization” with
In vitro
Percentage specific lysis assayed on X5563 cells
Immunization procedureb
sensitization with
4O:l
20: 1
1O:l
I
none
-
IO’ X5563 lo5 TNP-X5563
-0.2 (1.03) 0.2 (1.02)
-0.8 (1.07) 1.3 (1.02)
1.4 (1.05) 1.4 (1.03)
II
X5563 cells
-
10’ X5563 10’ TNP-X5563
-1.8 (1.01) -0.8 (1.02)
1.7 (1.03) 1.4 (1.03)
-1.7 (1.08) 1.0 (1.02)
-
lo5 X5563 10’ TNP-X5563
0.4 (1.03) 1.1 (1.03)
0.0 (1.03) -0.2 (1.03)
-0.9 (1.04) -0.2 (1.04)
III
TNP-X5563
cells
I
none
-
lo5 X5563 IO5 TNP-X5563
0.0 (1.05) 1.6 (1.08)
0.4 (1.03) 0.7 (1.07)
0.0 (1.07) 0.8 (1.08)
II
none
+
10’ X5563 lo5 TNP-X5563
64.2 (1.01) 66.9 (1.02)
57.4 (1.03) 64.6 (1.03)
43.8 (1.02) 53.3 (1.00)
III
X5563 cells
+
10’ X5563 10’ TNP-X5563
9.2 (1.03) 9.1 (1.01)
4.3 (1.08) 5.2 (1.02)
1.3 (1.07) 1.4 (1.02)
IV
TNP-X5563
+
lo5 X5563 lo5 TNP-X5563
4.1 (1.01) 2.2 (1.06)
1.1 (1.03) 1.3 (1.02)
0.2 (1.05) 1.4 (1.02)
y Mice were inoculated iv with lo6 of 7000-R X-irradiated X5563 cells three times at 4-day intervals. b See Table 2.
unmodified
or TNBS-modified
(1 mM)
cells lost the ability to generate anti-X5563 tumor cytotoxic response even tumor when followed by the appropriate immunization procedure (Exp B, Table 4), which is consistent with the results of Table 2. DISCUSSION The present study demonstrates that iv inoculation with syngeneic cells expressing both TNP-self and tumor antigens results in priming for the anti-TNP-self cytotoxic response, and in simultaneous suppression of the ability to subsequently prime in vivo for a secondary cytotoxic response to a syngeneic tumor. In our previous work (10) and that of others (1 l), it was demonstrated that in vivo priming to TNP-self was mediated by T cells which include radiosensitive (precursor and/or helper) and radioresistant (helper) populations. Suppressor cells were also shown to be generated by iv inoculation with TNP-self (12). However, the augmented generation of the anti-TNP-self cytotoxic response after iv administration of TNP-self indicates that the overall magnitude of precursors and helpers activated is greater than that of suppressors. Although the induction of helper cells for the tumor antigens has not been examined, such helper cell activity, if any, could not function to trigger the effector cell precursors which recognize the tumor antigens. Furthermore, it should be noted that if anti-TNP-self helper cells could be induced by iv inoculation with TNP-
186
FUJIWARA
AND
SHEARER
X5563 cells, these helper cells also failed to collaborate with anti-tumor-specific precursor population. The discrepancy between anti-TNP-self and antitumor cytotoxic responsescould be accounted for by postulating that the anti-TNP-self and antitumor cytotoxic responses are not equally dependent on helper cells. In this model, anti-TNP-self effector clones would be less dependent on helper cell collaboration. In contrast, anti-tumor-specific effector clones that would be critically dependent on helpers might be affected by a suppressive mechanism(s) before anti-TNP-self or antitumor helper cells were stimulated by iv administration of TNP-tumor cells to interact with effector cell precursors. It has recently been reported that enhanced T-cellmediated immune responses against TATA could be generated by preinducing hapten (TNP)-reactive helper (amplifier) T lymphocytes and by subsequently immunizing with TNP-modified syngeneic tumor cells (13, 14). This approach was based on the postulation of Mitchison (15) that such manipulations might induce augmented tumor immunity. When this TNP-amplifier mechanism was utilized into the present system, it was found that the preinduced TNP-reactive helper cells could prevent the animals from suppression of antitumor immunity induced by iv inoculation of TNP-conjugated syngeneic tumor cells (Fujiwara et al., manuscript in preparation). These observations support the above hypothesis that helper cells play a crucial role in developing cytotoxic T-cell responses against weak antigens such as tumor antigens and that such immunity depends on a delicate balance between help and suppression. Studies are in progress to determine the cellular mechanisms underlying the suppressive effect of iv administration of tumor antigens on the development of antitumor cytotoxic effector cell populations when helper cells are not preinduced. ACKNOWLEDGMENTS The authors are grateful to Drs. Richard Hodes and Howard Dickler for reviewing the manuscript and to the Immunology Branch’s office support staff for preparation of the manuscript.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Fujiwara, H., Levy, R. B., Shearer, G. M., and Terry, W. D., J. Immunol. 123, 423, 1979. Fujiwara, H., Hamaoka, T., and Kitagawa, M., Gunn 69, 793, 1978. Fujiwara, H., Hamaoka, T., Nishino, Y., and Kitagawa, M., Gann 68, 589, 1977. Yamazaki, M., Shinoda, H., and Mizuno, D., Gann 67, 651, 1976. Fujiwara, H., Hamaoka, T., Teshima, K., Aoki, H., and Kitagawa, M., Immunology 31, 239, 1976. Potter, M., Fahey, J. L., and Pilgrim, H. I., Proc. Sot. Exp. Biol. 94, 322, 1957. Shearer, G. M., Eur. J. Immunol. 4, 527, 1974. Shearer, G. M., Rehn, T. G., and Garbarino, C. A., J. Exp. Med. 141, 1348, 1975. Schmitt-Verhulst, A. M., Pettinelli, C. B., Henkart, P. A., Lunney, J. K., and Shearer, G. M., J. Exp. Med. 147, 352, 1978. Fujiwara, H., and Shearer, G. M., J. Immurtol. 124, 1271, 1980. Finberg, R., Greene, M. I., Benacerraf, B., and Burakoff, S. J., J. Immunol. 123, 1205, 1979. Finberg, R., Burakoff, S. J., Benacerraf, B., and Greene, M. I., J. Immunol. 123, 1210, 1979. Hamaoka, T., Fujiwara, H., Teshima, K., Aoki, H., Yamamoto, H., and Kitagawa, M., J. Exp. Med. 149, 185, 1979. Fujiwara, H., Hamaoka, T., Shearer, G. M., Yamamoto, H., and Terry, W. D., J. Immunol. 124, 863, 1980. Mitchison, N. A., 7’ranspZunt. Proc. 2, 92, 1970.
IMMUNOLOGY
Suppressive
59, 181-186 (1981)
Effect of X-Irradiated Tumor Cell Presensitization the Induction of Syngeneic Tumor Immunity
II. Opposite Effects of Intravenous Administration of TNP-Conjugated Cells on the Development of Antitumor and Anti-TNP-self Cytotoxic Effector Cells HIROMI Immunology
FUJIWARA’
AND GENE
on
Tumor
M. SHEARER
Branch, National Cancer Institute. National Bethesda, Maryland 20205
Institutes
of Health,
Received June 3, 1980: accepted July 30, 1980
A comparison was made of the effects of iv inoculation of trinitrophenyl (TNP)-conjugated syngeneic X5563 tumor cells on the development of anti-TNP-modified self (TNP-self) and anti-X5563 tumor-specific T-cell-mediated cytotoxic responses.Administration of syngeneic TNP-conjugated spleen cells or X-irradiated TNP-conjugated X5563 cells resulted in an appreciable augmentation in the generation of TNP-reactive cytotoxic effector cell activity as measured by subsequent in vitro sensitization with TNP-self. In contrast, iv inoculation with either TNP-modified or unmodified X-irradiated X5563 tumor cells did not prime mice for antitumor cytotoxic responses, but abolished the ability of mice to develop antitumor cytotoxic effector cells even after effective in vivo immunization with tumor cells. The possible mechanisms by which the suppression of antitumor cytotoxic response was induced by conditions which simultaneously induced an enhanced anti-TNP-self cytotoxic response are discussed.
INTRODUCTION A previous study demonstrated that intravenous (iv) administration of trinitrophenyl (TNP)-conjugated syngeneic spleen cells (TNP-self) abolished the ability of mice to develop TNP-contact sensitivity, whereas the identical treatment leads to augmented generation of TNP-reactive cytotoxic effector cell activity as shown by subsequent in vitro sensitization with TNP-self (1). It was also shown in a preceding report (2) that iv inoculation of X-irradiated syngeneic tumor cells can have different immunological effects in two murine tumor systems. For example, in the X5563 plasmacytoma tumor system, anti-tumor-specific cytotoxic effector T cells could be generated by intradermal (id) inoculation of viable tumor cells followed by the tumor resection (immunization procedure) (3). However, iv inoculation of X-irradiated tumor cells did not lead to any significant antitumor immune response, but abolished the ability to develop tumor immunity even when ’ Address correspondenceto Dr. H. Fujiwara, National Cancer Institute, 9000 Rockville Pike, Building 10, Room 4B17, Bethesda, Md. 20205. 181 OOOS-8749/81/050181-06$02,00/O Copyright 0 1981 by Academic Press, Inc. All rights oi reproduction in any form rcscr.4.
182
FUJIWARA
AND SHEARER
followed by the appropriate immunization procedure (2). In contrast, in a MM102 mammary tumor system to which immune response was shown to be exclusively anti-tumor-specific antibody (4, 5), pretreatment with X-irradiated MM102 tumor cells augmented the induction of antitumor immunity (2). Thus, the results of these models indicate that iv presensitization with “modified self” or tumor antigens can result in priming or suppression, depending on the immunological parameter studied. In the present study, we have further investigated the effects of iv administration of cells simultaneously expressing TNP-self and tumor antigens on the generation of each corresponding effector cell population. The results demonstrate that mice inoculated iv with TNP-modified X-irradiated syngeneic X5563 tumor cells exhibited an augmented in vitro anti-TNP-self cytotoxic effector cell response. In contrast, the same inoculated animals failed to develop an antitumor cytotoxic response even when the immunization procedure was performed before in vitro restimulation with tumor antigens. These results illustrate that iv immunization can result in different effects on the same type of immune response (i.e., the generation of cytotoxic effector cells) depending on the target antigens studied. MATERIALS
AND METHODS
Mice. Female C3H/HeN mice were obtained from Charles River Laboratory (Wilmington, Mass.) and used at 7-9 weeks of age. Tumor. The X5563 plasmacytoma was used in this study. It was derived from C3H/He strain (6), and maintained by serial intraperitoneal passages into syngeneic C3H/He mice in the ascitic form. Preparation of TNP-conjugated cells. Spleen or tumor cells were incubated with trinitrobenzene sulfonate (TNBS) at concentrations of 1 or 0.1 mM for 10 min, at 37°C as previously described (7, 8). In vitro sensitization for cytotoxic effector cells and cytotoxicity assay. Spleen cells (5 X 106) were sensitized in vitro to lo6 irradiated (2000 R) TNBS-modified syngeneic spleen cells (TNP-self) or lo5 irradiated ( 10,000 R) unmodified or TNBS-modified X5563 tumor cells and assayed on unmodified or TNBS-modified X5563 tumor target cells, as previously described (7-9). In vivo priming of anti-X5563 tumor speci’c cytotoxic response. C3H/He mice were intradermally (id) inoculated with lo6 viable X5563 tumor cells, followed by surgical resection of the tumor 7 days later (3). RESULTS The results shown in Table 1 confirm that iv administration of TNP-self can result in the enhanced generation of anti-TNP-self cytotoxic response as measured by subsequent in vitro sensitization to TNP-self. C3H/He mice were inoculated iv with 5 X lo7 of 1 mM TNP-self cells. Fourteen days later, spleen cells (5 X 106) from these primed and unprimed mice were sensitized in vitro with lo6 of irradiated (2000 R) 1 mM or 0.1 mM TNP-self cells. Effector cells generated after 5 days of culture were assayed on TNP-conjugated syngeneic X5563 tumor target cells. As shown in Table 1, iv inoculation with TNP-self led to appreciable priming of anti-TNP-self cytotoxic response. We also verify with the results shown in Table 2 that iv presensitization with X-irradiated tumor cells can abolish the ability of syngeneic C3H/He mice to
EFFECTS OF IMMUNIZATION
WITH TNP-TUMOR
183
CELLS ON CML
TABLE 1 Augmented Generation of TNP-Reactive Effector Cell Activity in the Spleen Cells from C3H/He Mice Inoculated iv with TNP-self Percentage specific lysis assayed on targetsb TNBSX5563 Stimulating cells ( 1Oh/well) 1 m&f TNP-spleen cells 0.1 mM TNP-spleen cells
Responding cells” (5 X 106/well)
X5563 2O:l
2O:l
IO:1
5:1
Normal TNP-spleen cellinoculated
-0.3 (1.07)
52.8 (1.04)
35.2 (1.01)
20.3 (1.05)
0.6 (1.02)
74.0 (1.02)
57.2 (1.03)
41.3 (1.05)
Normal TNP-spleen cellinoculated
-0.6 (1.04)
34.2 (1.02)
21.5 (1.03)
12.2 (1.02)
0.2 (1.02)
62.9 (1.02)
46.3 (1.02)
26.2 (1.03)
’ Spleen cells (5 X 106)from normal mice or mice inoculated iv with 5 X 10’ TNP-spleen cells (1 mM TNBS) were sensitized in vitro with irradiated (2000 R) stimulating cells (106). b Effector cell activities generated after 5 days of culture were assayed on unmodified X5563 and TNP-X5563 (10 mM TNBS) plasmacytoma. The percentage specific lysis was expressed as the geometric mean of triplicate cultures and the value in parentheses represents the standard error.
develop antitumor cytotoxic response. C3H/He mice were inoculated three times iv with lo6 of 7000-R X-irradiated X5563 tumor cells at 4-day intervals. Four days after the final inoculation, spleen cells from these injected and uninjected mice were sensitized in vitro to 10’ of irradiated (10,000 R) X5563 tumor cells. No significant antitumor cytotoxic response was detected in spleen cells from either treated or untreated mice (Expt A, Table 2). Furthermore, suppressive effect of TABLE 2 Suppressive Effect of iv Administration of X-Irradiated Tumor Cells on the Development of AntiX5563 Tumor Cytotoxic Response Percentage specific lysis assayed on X5563 cells
In vivo treatment In vitro
Expt
Group
iv presensitization”
Immunization procedureb
sensitization’ with
4O:l
2O:l
IO:1
A
I II
+
-
lo5 X5563 IO5 X5563
8.3 (1.05) 3.8 (1.02)
2.2 (1.03) 0.2 (1.04)
2.2 (1.10) 1.6 (1.06)
B
I II III
+
+ +
lo5 X5563 10’ X5563 10’ X5563
1.2 (1.05) 51.6 (1.01) 2.1 (1.02)
0.3 (1.03) 46.2 (1.00) -0.2 (1.04)
0.5 (1.05) 34.2 (1.01) 1.6 (1.03)
’ Mice were inoculated iv with lo6 of 7000-R X-irradiated X5563 tumor cells three times at 4-day intervals. b Four days after iv presentation, mice were inoculated id with IO6viable tumor cells, followed by the tumor resection 7 days later. ‘Spleen cells (5 X 106) from mice 4 days after iv presentation (Expt A) or 7 days after the tumor resection (Expt B) were sensitized in vitro to lo5 irradiated (10,000 R) X5563 tumor cells.
184
FUJIWARA
AND
SHEARER
presensitization with X-irradiated tumor cells on the subsequent ability of these mice to develop antitumor immune response is shown by the following experiments (Expt B, Table 2). Four days after the final iv injection with X-irradiated tumor cells, mice were inoculated id with lo6 viable X5563 cells followed 7 days later by the surgical resection of the tumor (immunization procedure). Seven days after the tumor resection, spleen cells from either normal control mice (Group I), mice which received the immunization procedure (Group II), or mice which received the immunization procedure after the iv pretreatment with irradiated tumor cell (Group III) were sensitized in vitro to 10’ X5563 tumor cells. As shown under the results in Expt B of Table 2, the immunization procedure led to strong generation of an antitumor cytotoxic response. However, iv administration of tumor cells abolished the ability to develop antitumor cytotoxic response even when followed by the appropriate immunization procedure. Since contrasting results were obtained in separate experiments for antitumor and anti-TNP-self cytotoxic responses after iv pretreatment, a priming study was performed by inoculating mice iv with cells expressing both tumor antigens and TNP-self determinants. C3H/He mice were given three iv inoculations of IO6 Xirradiated (7000 R) X5563 cells modified with 1 mM TNBS at 4-day intervals. Spleen cells from normal mice, or mice inoculated with unmodified or TNBSmodified X5563 cells were sensitized in vitro to TNP-self and effecters were assayed on unmodified or TNBS-modified X5563 cells (Table 3). Detectable and strong priming effects for anti-TNP cytotoxic response were observed in the TNP-X5563 tumor-cell-inoculated group when in vitro stimulation was provided by 1 or 0.1 mM TNP-self, respectively. No significant lysis was observed on unmodified X5563 tumor target cells. An identical iv regimen was studied for its effects on the development of antitumor cytotoxic response (Table 4). When spleen cells from normal mice, or mice inoculated with X5563 or TNP-X5563 cells were sensitized in vitro to X5563 or TNP-conjugated X5563 cells, no significant anti-X5563 tumor cytotoxic activity was detected (Expt A, Table 4). Furthermore, the mice inoculated with X-irradiated TABLE Augmented
3
Generation of TNP-Reactive Effector Cell Activity in the Spleen Cells from C3H/He Mice Inoculated iv with TNP-Modified Syngeneic X5563 Tumor Cells Percentage specific lysis assayed on targets X5563
Stimulating cells (106/well)
Responding cells” (5 X 106/well)
TNBSX5563
4O:l
4O:l
2o:l
lo:1
1 mM TNP-spleen cells
Normal X5563-inoculated TNP-X5563-inoculated
-4.1 (1.01) -1.7 (1.11) 1.0 (1.03)
58.4 (1.04) 59.2 (1.02) 71.3 (1.02)
58.2 (1.03) 57.0 (1.03) 67.8 (1.05)
58.5 (1.02) 47.9 (1.02) 64.5 (1.02)
0.1 mM TNP-spleen cells
Normal X5563-inoculated TNP-X5563-inoculated
-2.9 (1.03) 0.0 (1.03) 0.5 (1.02)
15.2 (1.03) 7.2 (1.02) 62.2 (1.01)
16.2 (1.04) 5.0 (1.07) 57.7 (1.01)
10.5 (1.07) 2.6 (1.00) 52.8 (1.07)
’ Spleen cells from normal mice, or mice inoculated iv with lo6 of 7000-R X-irradiated unmodified or TNBS-modified (1 mM) X5563 cells three times at 4-day intervals were sensitized in vitro to 1 or 0.1 mM TNP-self.
EFFECTS
OF IMMUNIZATION
WITH TABLE
TNP-TUMOR
CELLS
185
ON CML
4
Suppressive Effect of iv Presensitization with X5563 or TNP-X5563 Cells on the Development of Anti-X5563 Tumor Cytotoxic Response In viva treatment
Expt Group A
B
iv presensitization” with
In vitro
Percentage specific lysis assayed on X5563 cells
Immunization procedureb
sensitization with
4O:l
20: 1
1O:l
I
none
-
IO’ X5563 lo5 TNP-X5563
-0.2 (1.03) 0.2 (1.02)
-0.8 (1.07) 1.3 (1.02)
1.4 (1.05) 1.4 (1.03)
II
X5563 cells
-
10’ X5563 10’ TNP-X5563
-1.8 (1.01) -0.8 (1.02)
1.7 (1.03) 1.4 (1.03)
-1.7 (1.08) 1.0 (1.02)
-
lo5 X5563 10’ TNP-X5563
0.4 (1.03) 1.1 (1.03)
0.0 (1.03) -0.2 (1.03)
-0.9 (1.04) -0.2 (1.04)
III
TNP-X5563
cells
I
none
-
lo5 X5563 IO5 TNP-X5563
0.0 (1.05) 1.6 (1.08)
0.4 (1.03) 0.7 (1.07)
0.0 (1.07) 0.8 (1.08)
II
none
+
10’ X5563 lo5 TNP-X5563
64.2 (1.01) 66.9 (1.02)
57.4 (1.03) 64.6 (1.03)
43.8 (1.02) 53.3 (1.00)
III
X5563 cells
+
10’ X5563 10’ TNP-X5563
9.2 (1.03) 9.1 (1.01)
4.3 (1.08) 5.2 (1.02)
1.3 (1.07) 1.4 (1.02)
IV
TNP-X5563
+
lo5 X5563 lo5 TNP-X5563
4.1 (1.01) 2.2 (1.06)
1.1 (1.03) 1.3 (1.02)
0.2 (1.05) 1.4 (1.02)
y Mice were inoculated iv with lo6 of 7000-R X-irradiated X5563 cells three times at 4-day intervals. b See Table 2.
unmodified
or TNBS-modified
(1 mM)
cells lost the ability to generate anti-X5563 tumor cytotoxic response even tumor when followed by the appropriate immunization procedure (Exp B, Table 4), which is consistent with the results of Table 2. DISCUSSION The present study demonstrates that iv inoculation with syngeneic cells expressing both TNP-self and tumor antigens results in priming for the anti-TNP-self cytotoxic response, and in simultaneous suppression of the ability to subsequently prime in vivo for a secondary cytotoxic response to a syngeneic tumor. In our previous work (10) and that of others (1 l), it was demonstrated that in vivo priming to TNP-self was mediated by T cells which include radiosensitive (precursor and/or helper) and radioresistant (helper) populations. Suppressor cells were also shown to be generated by iv inoculation with TNP-self (12). However, the augmented generation of the anti-TNP-self cytotoxic response after iv administration of TNP-self indicates that the overall magnitude of precursors and helpers activated is greater than that of suppressors. Although the induction of helper cells for the tumor antigens has not been examined, such helper cell activity, if any, could not function to trigger the effector cell precursors which recognize the tumor antigens. Furthermore, it should be noted that if anti-TNP-self helper cells could be induced by iv inoculation with TNP-
186
FUJIWARA
AND
SHEARER
X5563 cells, these helper cells also failed to collaborate with anti-tumor-specific precursor population. The discrepancy between anti-TNP-self and antitumor cytotoxic responsescould be accounted for by postulating that the anti-TNP-self and antitumor cytotoxic responses are not equally dependent on helper cells. In this model, anti-TNP-self effector clones would be less dependent on helper cell collaboration. In contrast, anti-tumor-specific effector clones that would be critically dependent on helpers might be affected by a suppressive mechanism(s) before anti-TNP-self or antitumor helper cells were stimulated by iv administration of TNP-tumor cells to interact with effector cell precursors. It has recently been reported that enhanced T-cellmediated immune responses against TATA could be generated by preinducing hapten (TNP)-reactive helper (amplifier) T lymphocytes and by subsequently immunizing with TNP-modified syngeneic tumor cells (13, 14). This approach was based on the postulation of Mitchison (15) that such manipulations might induce augmented tumor immunity. When this TNP-amplifier mechanism was utilized into the present system, it was found that the preinduced TNP-reactive helper cells could prevent the animals from suppression of antitumor immunity induced by iv inoculation of TNP-conjugated syngeneic tumor cells (Fujiwara et al., manuscript in preparation). These observations support the above hypothesis that helper cells play a crucial role in developing cytotoxic T-cell responses against weak antigens such as tumor antigens and that such immunity depends on a delicate balance between help and suppression. Studies are in progress to determine the cellular mechanisms underlying the suppressive effect of iv administration of tumor antigens on the development of antitumor cytotoxic effector cell populations when helper cells are not preinduced. ACKNOWLEDGMENTS The authors are grateful to Drs. Richard Hodes and Howard Dickler for reviewing the manuscript and to the Immunology Branch’s office support staff for preparation of the manuscript.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Fujiwara, H., Levy, R. B., Shearer, G. M., and Terry, W. D., J. Immunol. 123, 423, 1979. Fujiwara, H., Hamaoka, T., and Kitagawa, M., Gunn 69, 793, 1978. Fujiwara, H., Hamaoka, T., Nishino, Y., and Kitagawa, M., Gann 68, 589, 1977. Yamazaki, M., Shinoda, H., and Mizuno, D., Gann 67, 651, 1976. Fujiwara, H., Hamaoka, T., Teshima, K., Aoki, H., and Kitagawa, M., Immunology 31, 239, 1976. Potter, M., Fahey, J. L., and Pilgrim, H. I., Proc. Sot. Exp. Biol. 94, 322, 1957. Shearer, G. M., Eur. J. Immunol. 4, 527, 1974. Shearer, G. M., Rehn, T. G., and Garbarino, C. A., J. Exp. Med. 141, 1348, 1975. Schmitt-Verhulst, A. M., Pettinelli, C. B., Henkart, P. A., Lunney, J. K., and Shearer, G. M., J. Exp. Med. 147, 352, 1978. Fujiwara, H., and Shearer, G. M., J. Immurtol. 124, 1271, 1980. Finberg, R., Greene, M. I., Benacerraf, B., and Burakoff, S. J., J. Immunol. 123, 1205, 1979. Finberg, R., Burakoff, S. J., Benacerraf, B., and Greene, M. I., J. Immunol. 123, 1210, 1979. Hamaoka, T., Fujiwara, H., Teshima, K., Aoki, H., Yamamoto, H., and Kitagawa, M., J. Exp. Med. 149, 185, 1979. Fujiwara, H., Hamaoka, T., Shearer, G. M., Yamamoto, H., and Terry, W. D., J. Immunol. 124, 863, 1980. Mitchison, N. A., 7’ranspZunt. Proc. 2, 92, 1970.