Cancer
Immunity, Vol. 2, p. 4 (16 April 2002) Submitted:
26 March 2002. Accepted: 26 March 2002.
Contributed by: PK Srivastava
Immunization against a dominant tumor antigen abrogates immunogenicity of the tumor
Amira Makki1, Gunnar Weidt1, Nathalie E. Blachere1*, Leo Lefrançois2, and Pramod K. Srivastava1,2
1Center for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut School of Medicine, Farmington, CT 2Division of Immunology, Department of Medicine, University of Connecticut School of Medicine, Farmington, CT *Present address: Rockefeller University, 1230 York Avenue, New York, NY 10021
Keywords:
mice, immunization, cytotoxic T Lymphocytes, immunodominant epitopes, tumor immunity
Abstract
To study the role of subdominant epitopes in tumor rejection we have used EL4 tumor cells and their ovalbumin (OVA)-transfected counterpart E.G7. Immunization of mice with irradiated EL4 cells conferred protection against challenge with EL4 and E.G7. Surprisingly, immunization with irradiated E.G7 cells did not protect against a subsequent challenge with EL4 or E.G7. Growth of E.G7 tumors in E.G7 immunized mice was not due to loss of expression of OVA or MHC I by the tumor cells in vivo. Adoptive transfer of OVA-specific transgenic T cells, immunization of mice with native or heat-denatured OVA or infection with a recombinant virus expressing OVA also failed to induce rejection of E.G7 tumors. Lack of immunogenicity of the OVA-expressing tumor could not be overcome by combination of a CD40 activating antibody with immunization against E.G7 or OVA. Our results suggest that immunization against subdominant epitopes is more effective than vaccination against dominant epitopes.
Introduction
T cells play an important role in tumor rejection. Demonstration in cancer patients of a cytotoxic T lymphocyte response against a number of molecules and the identification of CTL epitopes on tumors and normal cells have encouraged the belief that tumors can be controlled by the immunization with such epitopes. Regardless, tumors have been generally observed to grow progressively even in the presence of a T cell response (1, 2, 3, 4, 5).
One mechanism by which tumor cells escape the anti-tumor immune response is the down-regulation, mutation or loss of tumor antigens (6, 7, 8). Immunization with complex antigens elicits CTLs only against a small number of dominant epitopes out of a broad range of potential epitopes. CTL responses against subdominant epitopes are weak or not measurable, but can be evoked if a vaccine misses the dominant epitopes (9, 10, 11). For an optimal anti-tumor response it might be useful to induce CTLs against subdominant epitopes for two reasons: (a) A combined vaccination against more than one epitope, including subdominant epitopes, could prevent the outgrowth of epitope loss variants of the tumor cells, and (b) many of the human tumor 'antigens' characterized thus far are dominant 'antigens' and have also been detected in normal cells (12, 13). Tolerance to these self-molecules is likely to exist. Subdominant epitopes may not elicit such tolerance (14, 15).
In this backdrop, we have explored the role of subdominant epitopes in tumor rejection. As model tumors, we chose the EL4 thymoma and its OVA-transfected counterpart E.G7 (16). OVA is a well-characterized antigen with two defined dominant CTL epitopes: ova257-264 (17) and ova176-183 (18). The parental tumor line EL4 presents one dominant epitope that has not yet been sequenced (15). However, in E.G7 cells OVA-epitopes are dominant over the endogenous EL4 epitopes (16, 19, 20, 21). Previous studies have shown that rejection of EL4 (15) and E.G7 (19, 22) is mediated by CD8+ T cells.
Results
E.G7 cells induce OVA-specific CTL but can not protect against E.G7 tumor growth
We examined the hierarchy of T cell epitopes in E.G7 cells by a CTL assay. C57BL/6 mice were immunized with irradiated E.G7 cells as described in Materials and Methods, and splenocytes from the immunized mice were stimulated in vitro with the dominant OVA epitope 257-264. In CTL assays, the mixed tumor lymphocyte cultures showed lytic activity against E.G7, but not against EL4 tumor cells. Vaccination with EL4 cells induced CTLs against both EL4 and E.G7 (Fig. 1A). We deduced that the OVA epitope SIINFEKL is dominant over the endogenous EL4 epitopes, as measured in vitro. In studies in vivo, immunization with EL4 cells conferred protection against EL4 and against E.G7. However, immunization with irradiated E.G7 cells did not protect against a subsequent challenge with EL4 nor E.G7 (Fig. 1B). This was surprisingly, as vaccination with E.G7 was observed to elicit OVA-specific CTLs as shown in Fig.1A.
Immunization with E.G7 cells induces OVA-specific CTLs but does not protect against E.G7 tumor growth. (A) C57BL/6 mice were immunized with irradiated E.G7 or EL4 cells. Seven days later, spleens of immunized mice were stimulated in vitro with ova257-264 or irradiated EL4 cells for 5 days. Lytic activity against E.G7 and EL4 was measured by 51Cr release assay. (B) C57BL/6 mice were immunized with irradiated E.G7 cells, EL4 cells or buffer. One week later they were challenged with live E.G7 or EL4 cells s.c. and tumor growth was measured. Each line denotes the kinetics of tumor growth in a single mouse.
Lack of immunogenicity of E.G7 is not due to antigen loss
We isolated cells from tumors growing in E.G7-immunized and E.G7-challenged mice and used them without further cultivation as targets in a CTL assay. Cells derived from five of six tumors were recognized by OVA-specific CTLs to the same extent as ova257-264 peptide-pulsed cells from the same tumor (Fig. 2 A, C-F). Thus, loss of expression of OVA or MHC class I does not explain the growth of E.G7 tumors in E.G7 immunized mice.
E.G7 tumor growth is not due to antigen loss. C57BL/6 mice were immunized with irradiated E.G7 cells. One week later, they were challenged with 2.5 x 106 live E.G7 cells. At a tumor diameter of more than 20 mm, tumor cells from individual mice (6 mice, designated A-F) were isolated and used as targets in a 51Cr release assay with or without sensitization by ova257-264. Spleen cells from VSV-OVA infected mice stimulated in vitro with ova257-264 for 5 days were used as effector cells.
One tumor whose cells could not be killed by OVA-specific CTLs had lost the expression of the ova257-264 epitope, but not MHC class I, as it could be lysed after being pulsed with the SIINFEKL epitope (Fig. 2B). From this tumor we established a cell line "E.G7 OVA LOSS". Vaccination of C57BL/6 mice with irradiated E.G7 OVA LOSS cells was observed to confer protection against a subsequent challenge with live E.G7 cells (Fig. 3A). This observation was similar to the results obtained by immunization with EL4. Thus, the failure of E.G7 cells to elicit a protective immune response against itself could be attributed to an immune response against the dominant epitope of OVA.
Immunization with the tumor line "E.G7 OVA LOSS", but not OVA protein, protects mice against E.G7 tumor growth. C57BL/6 mice were immunized twice, one week apart, with irradiated "E.G7 OVA LOSS" cells (A), native OVA (B), heat-denatured OVA (C), irradiated E.G7 cells (D) or buffer (E). Seven days later, they were challenged with live E.G7 cells and tumor growth was measured. Each line denotes the kinetics of tumor growth in a single mouse.
Adoptive transfer of OVA-specific T cells and immunization with OVA fail to induce protective immunity against E.G7
We adoptively transferred naive and in vitro stimulated spleen cells from the transgenic mouse line OT-1 that expresses a T cell receptor specific for ova257-264 in the context of H-2Kb molecules (23, 24) into normal C57BL/6 mice. Recipient mice could not reject a subsequent challenge with live E.G7 cells (Fig. 4A and C) although the in vitro stimulated OT-1 spleen cells demonstrated high cytolytic activity in a CTL assay (data not shown).
Transfer of OVA-specific spleen cells from OT-1 mice does not prevent E.G7 tumor growth. Naive (A) or in vitro stimulated (C) OT-1 spleen cells were adoptively transferred into naive C57BL/6 mice. Two days later, mice were challenged with live E.G7 cells. Control mice received the same number of naive (B) or in vitro stimulated spleen cells from normal C57BL/6 mice (D) and were also challenged with live E.G7 cells. Each line denotes the kinetics of tumor growth in a single mouse.
To investigate the fate of OVA-specific T cells in vivo we adoptively transferred CD45.1-positive OT-1 spleen cells into normal C57BL/6 mice (CD45.1-negative). The mice were then challenged with buffer or E.G7 or EL4 cells or were immunized with 5 mg OVA protein i.p. The spleens and draining and non-draining lymph nodes of recipient mice were examined by cytofluorometry. Six days after tumor challenge, spleens and lymph nodes of mice challenged with EL4 or E.G7 cells had the same numbers of CD45.1+ CD8+ cells as the control mice that had received buffer after adoptive transfer (data not shown). In contrast, we observed expansion of OT-1 cells in mice immunized with OVA (1.76% CD45.1+CD8+ lymph node cells 6 days after OVA injection compared to 0.26% in control mice), as described earlier (25).
We tested the ability of immunization with OVA protein to elicit resistance to E.G7 and CTLs against SIINFEKL/Kb. Mice were immunized with native or heat-denatured OVA or infected with recombinant vesicular stomatitis virus-expressing OVA (VSV-OVA) (25) and tested for CTL activity. All forms of antigen elicited potent CTL response to SIINFEKL/Kb (Fig. 5A). Nevertheless, all groups of OVA-immunized mice failed to reject the E.G7 tumor (Fig. 3B and 3C, 6A). Surprisingly, mice infected with wild type VSV rejected the E.G7 tumor (Fig. 6B). Thus, the introduction of the OVA antigen into the vaccine in fact abrogated the protective unspecific immune response conferred by VSV infection in this set of experiments.
Induction of OVA-specific cytolytic activity by heat-denatured and native OVA protein, E.G7 cells and VSV-OVA viral infection. (A) C57BL/6 mice were immunized with irradiated E.G7 cells, heat-denatured or native OVA or infected with VSV-OVA. One week after the last immunization or after viral infection, spleen cells were stimulated in vitro with ova257-264 for 5 days and the secondary lytic activity against E.G7 was tested by 51Cr release assay. Lytic activity against EL4 at the E:T ratio shown was <10%. (B) C57BL/6 mice were immunized with irradiated E.G7 cells or infected i.v. with VSV-OVA. One week later, primary lytic activity of spleen cells against E.G7 was measured. Lytic activity against EL4 at the E:T ratio shown was <2%.
Infection with an OVA-expressing virus induces tolerance against E.G7. C57BL/6 mice were injected i.v. with VSV-OVA (A), VSV (B) or buffer (C). Two weeks later, mice were challenged with live E.G7 cells and tumor growth was monitored. Each line denotes the kinetics of tumor growth in a single mouse.
Vaccination of mice with OVA in combination with anti-CD40 antibodies has been shown to induce memory CTLs against OVA (26). However, combination of immunization with irradiated E.G7 or heat-denatured OVA with injection of a CD40-activating antibody also failed to elicit rejection of E.G7 tumors (Table 1).
Combination of immunization against OVA with CD40-activation does not protect against E.G7 tumor growth
Discussion
The experiments presented here indicate that the presence of the dominant antigen OVA in a complex tumor vaccine abrogates an otherwise protective immune response against the OVA-expressing tumor E.G7. Irradiated EL4 cells can protect against the growth of EL4 tumors and the OVA-expressing cell line E.G7. However, if mice are immunized with E.G7 cells, they can not reject EL4 or E.G7 tumors. Our results are consistent with other studies that show failure of irradiated E.G7 cells to protect against a subsequent E.G7 challenge (4, 19). As also shown by other groups, immunization with free ova257-264 peptide in incomplete Freund's adjuvant or PBS (19, 27) and with native OVA (28) fails to protect from E.G7 tumor growth. Previous studies in other systems have also shown protection from tumor growth by CTLs against subdominant epitopes (15, 29, 30) or viral infection (31, 32).
In contrast, protection against E.G7 tumors was observed after i.p. injection of OVA by Fenton et al. (33) and el-Shami et al. described rejection of E.G7, but not of EL4, tumors following immunization with ova257-264 peptide-pulsed RMA-S.B7 cells (21). Interestingly, the initial CTL response in the latter study was confined to the dominant OVA epitope 257-264; however, after rejection of E.G7, epitope spreading of the CTL response to two other OVA epitopes and to endogenous EL4 epitopes occurred, and was presumably responsible for rejection. Our results are thus consistent with the latter study.
Rejection of E.G7 tumors has also been achieved by immunization with dendritic cells pulsed with ova257-264 peptide or OVA (19) or with dendritic cells infected with an OVA-expressing adenovirus (34). Surprisingly, direct infection of mice with this virus could not induce rejection of a subsequent E.G7 challenge, although it elicited a strong CTL response (34). Our results that vaccination against OVA can not protect from E.G7 tumors, despite the induction of an effective OVA-specific CTL response, are consistent with that experience.
Several mechanisms have been described to explain the failure of the immune system to induce an effective anti-tumor response. Tumor cells can escape the immune response by down-regulation of their MHC expression or by in vivo selection of tumor cells that have lost the expression of the target antigen (35, 36, 37). In our system, the majority of tumors in E.G7 challenged mice were recognized by OVA-specific CTLs in a CTL assay, suggesting that growth of E.G7 tumors after E.G7 immunization can not be explained by antigen loss of the tumor cells.
Tolerance can be due to ignorance of the antigen (38, 39) or to anergy if the antigen is not presented with a sufficient co-stimulatory signal (40, 41). Deletion or suppression of activated CTLs can occur (42, 43, 44, 45, 46, 47) as well as migration of CTLs away from the tumor site (4). The precursor frequency of CTLs can be too low, so that the T cells loose the competition with the growing tumor, or the tumor cells could produce humoral factors that locally block cytotoxicity (reviewed in 1). We exclude the possibility that the OVA antigen is ignored by the immune system since we observe effective priming of OVA-specific CTLs after immunization with irradiated E.G7 cells or with OVA protein and after infection with VSV-OVA. Moreover, the CTL response against OVA after immunization with irradiated E.G7 cells dominates the response to endogenous EL4 epitopes. The failure to induce a CTL response against the subdominant EL4 epitopes better explains the growth of EL4 tumor after immunization with E.G7 in our study.
The adoptive transfer of high numbers of naive or in vitro stimulated OVA-specific T cells into normal C57BL/6 mice was not able to protect against E.G7 tumor growth, suggesting that the failure of the immune system to control E.G7 tumor growth is not a quantitative problem of OVA-specific precursor T cells. Although injection of native OVA was able to trigger vigorous expansion of OVA-specific CD8+ T cells in vivo, neither native nor heat-denatured OVA could protect mice from E.G7 tumor growth. To sum up these data, effective priming of CTLs as measured by CTL assays in vitro does not correspond with protection against tumor growth in vivo.
Why do CTLs against epitopes that are subdominant in OVA or E.G7 protect against OVA-expressing tumors whereas dominant CTLs do not? A possible explanation is a transient activation of dominant CTLs after antigen contact followed by a down-regulation of this response due to suppressor cells or deletion of these CTLs (42, 43, 44, 45, 46, 47). Failure to maintain an anti-tumor CTL response induced by viral infection despite the presence of antigenic tumor cells has been previously described (48). These mechanisms of down-regulation may be operative for the stronger, and not for the weaker, response against subdominant epitopes.
The results presented here indicate that vaccination against a dominant antigen, by itself or in context with tumor cells or viral infection, may not necessarily confer tumor protection, and may indeed obstruct generation of effective and protective immune response to other antigens. Immunization against subdominant epitopes may circumvent this hurdle. They also indicate that CTL response is not a surrogate for protective tumor immunity. Some of these points have been argued previously (49).
Abbreviations
OVA, ovalbumin; VSV, vesicular stomatitis virus
Acknowledgements
This work was supported by NIH grant CA84479 to P.K.S. and by a sponsored research agreement with Antigenics Inc. in which one of us (P.K.S.) has a significant financial interest.
References
1. Wojtowicz-Praga S. Reversal of tumor-induced immunosuppression: a new approach to cancer therapy. J Immunother 1997; 20: 165-77. (PMID: 9181454)
2. Marincola FM. The multiple ways to tumor tolerance. J Immunother 1997; 20: 178-79. (PMID: 9181455)
3. Rowley DA, Stach RM. A first or dominant immunization. I. Suppression of simultaneous cytolytic T cell responses to unrelated alloantigens. J Exp Med 1993; 178: 835-40. (PMID: 8338500)
4. Shrikant P, Mescher MF. Control of syngeneic tumor growth by activation of CD8+ T cells: efficacy is limited by migration away from the site and induction of nonresponsiveness. J Immunology 1999; 162: 2858-66. (PMID: 10072534)
5. Biddison WE, Palmer JC. Development of tumor cell resistance to syngeneic cell-mediated cytotoxicity during growth of ascitic mastocytoma P815Y. Proc Natl Acad Sci U S A 1977; 74: 329-33. (PMID: 402006)
6. Urban JL, Burton RC, Holland JM, Kripke ML, Schreiber H. Mechanisms of syngeneic tumor rejection. Susceptibility of host-selected progressor variants to various immunological effector cells. J Exp Med 1982; 155: 557. (PMID: 6977009)
7. Meeker T, Lowder J, Cleary ML, Stewart S, Warnke R, Sklar J, Levy R. Emergence of idiotype variants during treatment of B-cell lymphoma with anti-idiotype antibodies. New Engl J Med 1985; 312: 1658. (PMID: 3923352)
8. Flyer DC, Pretell J, Campbell AE, Liao WS, Tevethia MJ, Taylor JM. Biology of simian virus 40 (SV40) transplantation antigen (TrAg). X. Tumorigenic potential of mouse cells transformed by SV40 in high responder C57BL/6 mice and correlation with the persistence of SV40 TrAg, early proteins and sequences. Virology 1983; 131: 207-20. (PMID: 6316651)
9. Vitiello A, Yuan L, Chestnut RW, Sidney J, Southwood S, Farness P, Jackson MR, Peterson PA, Sette A. Immunodominance analysis of CTL responses to influenza PR8 virus reveals two new dominant and subdominant Kb-restricted epitopes. J Immunology 1996; 157: 5555-62. (PMID: 8955206)
10. Mylin LM, Bonneau RH, Lippolis JD, Tevethia SS. Hierarchy among multiple H-2b-restricted cytotoxic T-lymphocyte epitopes within simian virus 40 T antigen. J Virol 1995; 69: 6665-6677. (PMID: 7474076)
11. Sercarz EE, Lehmann PV, Ametani A, Benichou G, Miller A, Moudgil K. Dominance and crypticity of T cell antigenic determinants [review]. Ann Rev Immunol 1993; 11: 729-66. (PMID: 7682817)
12. Boon T, Cerottini JC, Van den Eynde B, van der Bruggen P, Van Pel A. Tumor antigens recognized by T lymphocytes [review]. Ann Rev Immunol 1994; 12: 337-65. (PMID: 8011285)
13. Chen YT, Old LJ. Cancer-testis antigens: targets for cancer immunotherapy. Cancer J Sci Am 1999; 5: 16-7. (PMID: 10188055)
14. Schoenberger SP, Sercarz EE. Harnessing self-reactivity in cancer immunotherapy. Semin Immunol 1996; 8: 303-309. (PMID: 8956459)
15. Johnston JV, Malacko AR, Mizuno MT, McGowan P, Hellstrom I, Hellstrom KE, Marquardt H, Chen L. B7-CD28 costimulation unveils the hierarchy of tumor epitopes recognized by major histocompatibility complex class I-restricted CD8+ cytolytic T lymphocytes. J Exp Med 1996; 183: 791-800. (PMID: 8642283)
16. Moore MW, Carbone FR, Bevan MJ. Introduction of soluble protein into the class I pathway of antigen processing and presentation. Cell 1988; 54: 777-785. (PMID: 3261634)
17. Rotzschke O, Falk K, Stevanovic S, Jung G, Walden P, Rammensee HG. Exact prediction of a natural T Cell epitope. Eur J Immunol 1991; 21: 2891-94 (PMID: 1718764)
18. Lipford GB, Hoffman M, Wagner H, Heeg K. Primary in vivo responses to OVA. Probing the predictive value of the Kb binding motif. J Immunol 1993; 150: 1212-22. (PMID: 7679422)
19. Porgador A, Snyder D, Gilboa E. Induction of antitumor immunity using bone marrow-generated dendritic cells. J Immunol 1996; 156: 2918-26. (PMID: 8609412)
20. Martinez-Kinader B, Lipford GB, Wagner H, Heeg K. Sensitization of MHC class I-restricted T cells to exogenous proteins: evidence for an alternative class I-restricted antigen presentation pathway. Immunology 1995; 86: 287-295. (PMID: 7490131)
21. el-Shami K, Tirosh B, Bar-Haim E, Carmon L, Vadai E, Fridkin M, Feldman M, Eisenbach L. MHC class I-restricted epitope spreading in the context of tumor rejection following vaccination with a single immunodominant CTL epitope. Eur J Immunol 1999; 29: 3295-3301. (PMID: 10540341)
22. Kim TS, Chung SW, Hwang SY. Augmentation of antitumor immunity by genetically engineered fibroblast cells to express both B7.1 and interleukin-7. Vaccine 2000; 18: 2886-94. (PMID: 10812232)
23. Hogquist KA, Jameson SC, Heath WR, Howard JL, Bevan MJ, Carbone FR. T cell receptor antagonist peptides induce positive selection. Cell 1994; 76: 17-27. (PMID: 8287475)
24. Kim SK, Reed DS, Heath WR, Carbone F, LeFrancois L. Activation and migration of CD8 T cells in the intestinal mucosa. J Immunol 1997; 159: 4295-4306. (PMID: 9379025)
25. Kim SK, Reed DS, Olson S, Schnell MJ, Rose JK, Morton PA, LeFrancois L. Generation of mucosal cytotoxic T cells against soluble protein by tissue-specific environmental and costimulatory signals. Proc Natl Acad Sci U S A 1998; 95: 10814-10819. (PMID: 9724787)
26. Lefrancois L, Altman JD, Williams K, Olson S. Soluble antigen and CD40 triggering are sufficient to induce primary and memory cytotoxic T cells. J Immunol 2000; 164: 725-32. (PMID: 10623816)
27. Minev BR, McFarland BJ, Spiess PJ, Rosenberg SA, Restifo NP. Insertion signal sequence fused to minimal peptides elicits specific CD8+ T-cell responses and prolongs survival of thymoma-bearing mice. Cancer Research 1994; 54: 4155-61. (PMID: 7518351)
28. Zhou F, Rouse BT, Huang L. Prolonged survival of thymoma-bearing mice after vaccination with a soluble protein antigen entrapped in liposomes: a model study. Cancer Research 1992; 52: 6287-91. (PMID: 1384958)
29. Newmaster RS, Mylin LM, Fu TM, Tevethia SS. Role of a subdominant H-2Kd-restricted SV40 tumor antigen cytotoxic T lymphocyte epitope in tumor rejection. Virology 1998; 244: 427-41. (PMID: 9601511)
30. Feltkamp MC, Vreugdenhil GR, Vierboom MP, Ras E, van der Burg SH, ter Schegget J, Melief CJ, Kast WM. Cytotoxic T lymphocytes raised against a subdominant epitope offered as a synthetic peptide eradicate human papillomavirus type 16-induced tumors. Eur J Immunol 1995; 25: 2638-42. (PMID: 7589138)
31. van der Most RG, Murali-Krishna K, Whitton JL, Oseroff C, Alexander J, Southwood S, Sidney J, Chesnut RW, Sette A, Ahmed R. Identification of Db- and Kb-restricted subdominant cytotoxic T-cell responses in lymphocytic choriomeningitis virus-infected mice. Virology 1998; 240: 158-67. (PMID: 9448700)
32. Weidt G, Utermohlen O, Heukeshoven J, Lehmann-Grube F, Deppert W. Relationship among immunodominance of single CD8+ T cell epitopes, virus load, and kinetics of primary antiviral CTL response. J Immunol 1998; 160: 2923-31. (PMID: 9510196)
33. Fenton RG, Taub DD, Kwak LW, Smith MR, Longo DL. Cytotoxic T-cell response and in vivo protection against tumor cells harboring activated ras proto-oncogenes. J Natl Cancer Inst 1993; 85: 1294-02. (PMID: 8340941)
34. Brossart P, Goldrath AW, Butz EA, Martin S, Bevan MJ. Virus-mediated delivery of antigenic epitopes into dendritic cells as a means to induce CTL. J Immunol 1997; 158: 3270-76. (PMID: 9120283)
35. Ferrone S, Marincola FM. Loss of HLA class I antigens by melanoma cells: molecular mechanisms, functional significance and clinical relevance [review]. Immunology Today 1995; 16: 487. (PMID: 7576053)
36. Marincola FM, Hijazi YM, Fetsch P, Salgaller ML, Rivoltini L, Cormier J, Simonis TB, Duray PH, Herlyn M, Kawakami Y, Rosenberg SA. Analysis of expression of the melanoma-associated antigens MART-1 and gp100 in metastatic melanoma cell lines and in in situ lesions. J Immunother Emphasis Tumor Immunol 1996; 19: 192-205. (PMID: 8811494)
37. Chen Y, Stockert E, Tsang S, Coplan KA, Old LJ. Immunophenotyping of Melanomas for Tyrosinase: Implications for Vaccine Development. Proc Natl Acad Sci U S A 1995; 92: 8125-8129. (PMID: 7667256)
38. Ochsenbeing AF, Klenerman P, Karrer U, Ludewig B, Pericin M, Hengartner H, Zinkernagel RM. Immune surveillance against a solid tumor fails because of immunological ignorance. Proc Natl Acad Sci U S A 1999; 96: 2233-8. (PMID: 10051624)
39. Hermans IF, Daish A, Yang J, Ritchie DS, Ronchese F. Antigen expressed on tumor cells fails to elicit an immune response, even in the presence of increased numbers of tumor-specific cytotoxic T lymphocyte precursors. Cancer Res 1998; 58: 3909-17. (PMID: 9731502)
40. Mueller DL, Jenkins MK, Schwartz RH. Clonal expansion versus functional clonal inactivation: a costimulatory signalling pathway determines the outcome of T cell antigen receptor occupancy [review]. Ann Rev Imm 1989; 7: 445. (PMID: 2653373)
41. Staveley-O'Carroll K, Sotomayor E, Montgomery J, Borrello I, Hwang L, Fein S, Pardoll D, Levitsky H. Induction of antigen-specific T cell anergy: An early event in the course of tumor progression. Proc Natl Acad Sci U S A 1998; 95: 1178-1183. (PMID: 9448305)
42. Moskophidis D, Lechner F, Pircher H, Zinkernagel RM. Virus persistence in acutely infected immunocompetent mice by exhaustion of antiviral cytotoxic effector T cells. Nature 1993; 362: 758-61. (PMID: 846928)
43. Kurts C, Kosaka H, Carbone FR, Miller JF, Heath WR. Class I-restricted cross-presentation of exogenous self-antigens leads to deletion of autoreactive CD8(+) T cells. J Exp Med 1997; 186: 239-45. (PMID: 9221753)
44. North RJ. Cyclophosphamide-facilitated adoptive immunotherapy of an established tumor depends on elimination of tumor-induced suppressor T cells. J Exp Med 1982; 155: 1063-74. (PMID: 6460831)
45. Benacerraf B, Germain RN. A single major pathway of T-lymphocyte interactions in antigen-specific immune suppression. Scand J Immunol 1981; 13: 1-10. (PMID: 6972088)
46. Groux H, O'Garra A, Bigler M, Rouleau M, Antonenko S, de Vries JE, Roncarolo MG. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 1997; 389: 737-742. (PMID: 9338786)
47. Bronte V, Wang M, Overwijk WW, Surman DR, Pericle F, Rosenberg SA, Restifo NP. Apoptotic death of CD8+ T lymphocytes after immunization: induction of a suppressive population of Mac-1+/Gr-1+ cells. J Immunol 1998; 161: 5313-20. (PMID: 9820504)
48. Speiser DE, Miranda R, Zakarian A, Bachmann MF, McKall-Faienza K, Odermatt B, Hanahan D, Zinkernagel RM, Ohashi PS. Self antigens expressed by solid tumors Do not efficiently stimulate naive or activated T cells: implications for immunotherapy. J Exp Med 1997; 186: 645-53. (PMID: 9271580)
49. Srivastava PK. Do human cancers express shared protective antigens? or the necessity of remembrance of things past [review]. Semin Immunol 1996; 8: 295-302. (PMID: 8956458)
50. Nikolic-Zugic J, Bevan MJ. Role of self-peptides in positively selecting the T-cell repertoire. Nature 1990; 344: 65-7. (PMID: 2304556)
51. Kearney ER, Pape KA, Loh DY, Jenkins MK. Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo. Immunity 1994; 1: 327-39. (PMID: 788941)
Materials
and methods
Mice and cell lines
C57BL/6 and bm1 mice (50) were purchased from The Jackson Laboratory (Bar Harbor, ME). C57BL/6-Ly-5.2 mice were obtained from Charles River (Wilmington, MA) through the National Cancer Institute animal program. The OT-1 mouse line (23) was generously provided by W. R. Heath (WEHI, Parkville, Australia) and F. Carbone (Monash Medical School, Prahran, Victoria, Australia) and was maintained as a C57BL/6-Ly-5.2 line.
EL4 cells are a thymoma of C57BL/6 origin. The E.G7 cell line was derived from EL4 by transfection with chicken OVA cDNA (16). Cells were maintained in RPMI 1640 without glutamine (Gibco, USA) supplemented with 5% fetal calf serum, 1% L-glutamine, 1% penicillin/streptomycin, 1% sodium pyruvate and 1% non-essential amino acids (all from Gibco, USA). Media for E.G7 cells also contained 400 µg/ml G418 (Gibco, USA).
The cell line "E.G7 OVA LOSS" was established from tumors growing in mice immunized and subsequently challenged with E.G7 cells. C57BL/6 mice were vaccinated and challenged with E.G7 cells as described below. Tumors from these mice were isolated 18 days later and a single cell suspension was prepared. Cells were grown in complete medium without G418 as described above.
Immunization, virus infection and tumor challenge
For the immunization of mice, tumor cells (EL4, E.G7 or E.G7 OVA LOSS) were irradiated with 7500 rad and washed three times. 2 x 107 irradiated tumor cells were injected twice s.c., with a one-week interval. For the two simultaneous subcutaneous immunizations with E.G7 on one flank and EL4 on the other, we used 1 x 107 cells of each line.
Immunization with OVA was performed with 5 mg of native OVA (Grade VI, Sigma) injected i.p. or 1 mg of heat-denatured (boiled for 5 min) OVA administered by s.c. injection. For CTL induction we injected 1 mg native or heat-denatured OVA s.c.
For virus infection, 1 x 106 PFU of VSV or VSV-OVA were injected i.v. The production of VSV-OVA has been described previously (25). One week after the last immunization or two weeks after virus infection mice were challenged s.c. with 2.5 x 106 EL4 or E.G7 cells, respectively.
Measurement of cytolytic activity
Mice were splenectomized one week after the last immunization or after viral infection. 8 x 106 spleen cells from immunized mice were cultured in 24-well plates as a mixed lymphocyte tumor culture with 8 x 104 irradiated (7500 rad) tumor cells. Alternatively, spleen cells of immunized mice were stimulated in vitro with 10-6 M ova257-264 peptide. After 5 days, cytolytic activity of spleen cells was measured in a 51Cr release assay.
To test the expression of OVA by tumors growing in E.G7 immunized and E.G7 challenged mice, we used spleen cells from VSV-OVA infected mice stimulated in vitro with 10-6 M ova257-264 peptide for 5 days. A single cell suspension was prepared from tumor tissue of mice immunized and challenged with E.G7, and cells were immediately used as targets in a 51Cr release assay. As a positive control, we used these tumor cells pulsed with 10-6 M ova257-264.
Adoptive transfer and analysis by flow cytometry
This method was adapted from Kearney et al. (51). Spleen cells from OVA-T cell receptor-transgenic mice OT-1 (Ly5.2-positive) were injected through the tail vein into normal C57BL/6 mice (Ly-5.2-negative) either directly or following 2 days of in vitro stimulation with OVA. For in vitro stimulation, OT-1 spleen cells were incubated with 2.5 mg/ml OVA in a 24-well plate (1 x 106 cells/well). After 2 days in culture, cells were washed three times and injected i.v. (4 x 106 stimulated or 5 x 106 naive OT-1 spleen cells per recipient mouse). Two days after adoptive transfer, recipient mice were challenged with 2.5 x 106 live E.G7 or EL4 tumor cells or injected with 5 mg OVA i.p. As a control, we adoptively transferred naive or in vitro stimulated spleen cells from normal C57BL/6 mice. On days 2 and 7 of in vitro culture, the cytolytic activity of OT-1 spleen cells was measured in a 51Cr release assay.
Six days after tumor cell challenge, spleen, draining and non-draining lymph nodes were collected, homogenized, filtered, washed in PBS and incubated with Fc Block (CD16/CD32- antibody, PharMingen, USA) at 4°C for 10 min followed by antibody incubation at 4°C for 45 min. Cells were washed three times in PBS and fluorescence intensity was measured by FACScan (Becton Dickinson, USA). Transferred OT-1 cells were identified as CD45.1 (Ly-5.2)+ cells. Directly conjugated antibodies CD45.1-PE and CD8-FITC were purchased from PharMingen, USA.
CD40 activation
C57BL/6 mice were immunized with 2 x 107 irradiated E.G7 cells s.c. or 1 mg heat-denatured OVA (boiled for 5 min) s.c. on day 0. 100 µg of the purified CD40-activating antibody FGK45 in 200 µl PBS or a rat IgG2a control antibody (PharMingen, USA) were given i.v. on days 0, 1 and 2. The antibody FGK45 was generously provided by S. P. Schoenberger (Division of Immune Regulation, La Jolla Institute for Allergy and Immunology, San Diego, CA, USA). Two weeks later mice were challenged with 2.5 x 106 live E.G7 cells and tumor growth was measured.
Contact
Address
correspondence to:
Pramod K. Srivastava
Center for Immunotherapy
MC1601
University of Connecticut School of Medicine
Farmington, CT 06030-1601
USA
E-mail:
