- Email: [email protected]
In vivo acquired mechanisms of tumor cells local defense against the host innate immunity effectors: implication in specific antitumor immunity
Immunology Letters 70 (1999) 37 – 42
www.elsevier.com/locate/
In vivo acquired mechanisms of tumor cells local defense against the host innate immunity effectors: implication in specific antitumor immunity Galina Deichman *, Natalya Dyakova, Lidia Kashkina, Valentina Matveeva, Elvira Uvarova Laboratory of Antitumor Immunity, Institute of Carcinogenesis, N.N. Blokchin Cancer Research Center, Russian Academy of Medical Sciences, Moscow, Russia Accepted 22 June 1999
Abstract As shown earlier, the cells transformed in vitro by several different oncogenes, or spontaneously, during in vivo growth in normal hosts would be gradually replaced by the highly-tumorigenic descendants co-expressing high H2O2-catabolizing and S PGE2-releasing activities. Acquisition of (H2OCA 2 +PGE ) phenotype provides the cells with local defense mechanisms against the S host innate immunity effectors. However, it remained unknown, whether the expression of (H2OCA 2 + PGE ) phenotype is implicated in susceptibility of tumor cells expressing tumor-specific transplantation antigens to rejection in immune animals. Here, S with the use of SV40 in vitro transformed parental cells, negative in expression (H2OCA 2 + PGE ) phenotype, and their in vivo selected descendant tumor cell lines expressing this phenotype, we show that: (1) the rates of in vivo selection of the parental SV40 S tumor cells expressing (H2OCA 2 +PGE ) phenotype are the same in normal and SV40-immune animals; (2) in vivo selected SV40 S CA tumor cells expressing (H2O2 +PGE ) phenotype, although they retain specific immunosensitivity, are 100 times less effectively rejected in SV40-immunized animals, as compared with their in vitro SV40-transformed parental cells. Thus, in vivo acquired immunologically non-specific local mechanisms of tumor cells defense against the host innate immunity effectors, significantly decreases the effectiveness of their specific immunorejection. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Host innate and specific antitumor immunity; Immunoresistance; Immunosensitivity; Natural selection; Tumor cells local defence mechanisms; Tumor progression
1. Introduction During the last 20 years the mechanisms of tumor cells in vivo acquired resistance to cytotoxicity of the host innate immunity effectors, such as macrophages (M¥), NK cells, and neutrophils (Nph), and its possible role in tumor progression were widely studied in many laboratories. Briefly, our main finding in this field, was evidence that at least one such type of resistance is connected with the in vivo natural selection of tumor cells possessing two new properties, i.e. high H2O2-catabolizing (H2OCA 2 ) (antioxidant) activity and * Corresponding author. Present address. Institute of Carcinogenesis, Cancer Research Center, Kashirskoye shosse 24, M-478, Moscow, Russia; Fax: + 7-095-3241205. E-mail address: [email protected] (G. Deichman)
the ability to release PGE2 (PGES2 ) immediately upon contact with NK cells, M¥, or Nph. Although these biochemically different cell properties forming S (H2OCA 2 + PGE ) phenotype are usually considered separately, our data showed that they are acquired by tumor cells in vivo in one step; no intermediate cell variants were noticed and both were co-expressed at the S cell clonal level [1,2]. Acquisition of (H2OCA 2 +PGE ) phenotype by tumor cells during in vivo natural selection appeared to be independent from the type of oncogenes initially transformed these cells in vitro (with the exception of v-src) [3]. Examination of tumor cells of different oncogenetic origin in dynamics of in vivo progression demonstrated that acquired expression of S (H2OCA 2 + PGE ) phenotype coincides with their regular 30–200 times increased tumorigenic activity (TGA),
0165-2478/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 7 8 ( 9 9 ) 0 0 1 2 3 - 6
38
G. Deichman et al. / Immunology Letters 70 (1999) 37–42
followed, or not followed, by spontaneous metastasizing activity (SMA). Recently, we have shown that natural selection of tumor cells expressing (H2OCA 2 + PGES) phenotype begins during the latent period of primary viral carcinogenesis and this process may be completed before the primary tumor appearance (Deichman et al., 1999, submitted). Thus, acquisition of S (H2OCA 2 +PGE ) phenotype may be considered as a stable biochemical marker of the relatively early, premetastatic stage of tumor progression [1 – 5]. S Expression of (H2OCA 2 +PGE ) phenotype appears to be responsible for two different local mechanisms of the tumor cells defense against cytotoxic activity (CTA) of the host innate immunity effectors. (1) The acquired H2OCA 2 , mainly based on the high catalase and/or on glutathione peroxidase activities, was shown to defend tumor cells against the damaging effect of H2O2 and some other reactive oxygen species produced by M¥ and Nph, as a part of their CTA [1,2,6 – 11]. (2) The immediate release of PGE2 by the tumor cells in response to the contact interaction with NK cells or M¥, leads to the suppression of CTA of NK cells, thus demonstrating inducible and almost aggressive forms of malignant tumor cells local defense against NK cells [12 – 17]. The inhibition of PGE2 release by the pretreatment of tumor cells with indomethacin is making them susceptible to CTA of NK cells [18,19]. S Acquisition of (H2OCA 2 +PGE ) phenotype by tumor cells during sc growth and natural selection in normal animals was shown for the cells of non-antigenic tumors, as well as for those expressing TSTA (i.e. tumorspecific transplantation antigens); among the latter ones, simian virus 40 (SV40) and bovine adenovirus type 3 (BAV-3) in vitro transformed cells were examined [3]. As shown, TSTA’s of cells in vivo, or in vitro transformed by DNA tumor viruses are inducing specific antitumor immunity, which is manifested in vivo in specific immunogenicity and in immunosensitivity of virus-transformed cells. The long-term growth of the tumors expressing TSTA’s in immunized animals, in contrast to the growth in normal ones, leads to the gradual selection of immunoresistant tumor cell variants [review in [20]]. The recent data demonstrating the non-specific defense mechanisms of in vivo selected tumor cells against the host innate immunity effectors raise a question on S the possible implication of (H2OCA 2 +PGE ) phenotype in reactions of specific antitumor immunity. Therefore, in the present study, with the use of SV40 in vitro transformed cells we examined the possible influence of: (1) specific antitumor immunity on the rates of in vivo selection of SV40 tumor cells expressing (H2OCA 2 + PGES) phenotype, as compared with the rates of their selection in normal animals; and (2) the expression of S (H2OCA 2 +PGE ) phenotype on the effectiveness of in vivo rejection of SV40 tumor cells in normal and immune animals.
2. Materials and methods
2.1. Origin of cell lines Syrian hamster normal embryo cells transformed in vitro by the strain N128 of wild-type SV40 (cell line HE-wtSV40) and their in vivo selected variants were examined before and in dynamics of in vivo natural and immune selection. Each in vivo cycle of selection lasted 55–60 days. The cells isolated from the tumors of individual animals (normal and SV40 immune) were transferred for cultivation in vitro in DMEM containing 5% FCS and antibiotics; after 3–5 in vitro passages (in order to clear tumor cells from the host stromal cells) they were challenged in vitro for S expression of (H2OCA 2 + PGE ) phenotype and in vivo on the TGA, SMA, and on the specific immunosensitivity.
2.2. The le6els of TGA, SMA, and immunosensiti6ity of the SV40 tumor cell lines The levels of TGA, SMA, and immunosensitivity of the SV40 tumor cell lines were determined with the use of in vivo quantitative transplantation tests. Several cell variants selected in vivo, and particularly, the descendant cell lines (HE-wtSV40-2 SC-4 and HE-wtSV40-2SC-5) in parallel with the parental cell line were challenged in transplantation tests, in normal and SV40-immune animals. Immunization of 2 to 3-month-old normal animals was performed by i.p. inoculation of SV40 (106.2 ID50/1.0 ml) 20 days before the sc challenge of virus-free SV40 tumor cells in quantitative transplantation tests [21,22]. In the immunosensitivity examination, quantitative transplantation tests were performed in parallel in groups of 5–6 normal and SV40-immune animals. Four 10-fold differing doses of tumor cells (from 1–2 ×101 to 1–2 × 104) were inoculated sc in a volume of 0.2 ml into groups of five or six animals; 55– 60 days later the animals were sacrificed under anesthetic and the levels of TGA estimated in the log of 50% transplantation dose (TrD50), were calculated for each group of normal and immune animals. The use of quantitative transplantation test permits us to estimate the number of tumor cells eliminated and/or surviving in normal and/or immune animals. The relative immunosensitivity index, i.e. the difference between TGA level estimated in log TrD50 in SV40-immune animals and the TGA level of the same cells challenged in parallel in normal animals, was considered as positive if it was ] 1.0. Values varying from 0.0 to 0.5 suggested the loss, or significant decrease of immunosensitivity of the SV40 tumor cells examined.
G. Deichman et al. / Immunology Letters 70 (1999) 37–42 S 2.3. Determination of (H2O CA 2 + PGE ) phenotype was performed in 6itro in accordance to [1,3]
2.3.1. H2O2 -catabolizing acti6ity H2O2-catabolizing activity (H2OCA 2 ) was determined in ex tempore prepared cell extracts (2.0 mg of protein per 1.0 ml) of different cell strains with the use of luminol-dependent chemiluminescence (LDC); 0.01 M luminol (Serva) was used. Cells were extracted in a phosphate buffer and lysed with Triton X-100 at 4°C. Extracts in a volume of 0.1 ml, added with ex tempore prepared 10 mM H2O2 (0.1 ml) and 0.1 ml of luminol, were immediately placed at 37°C in thermostated Biolumate (Model 9500, Berthold, Germany). Two samples of each cell extract (H2O2-treated), as well as two samples of H2O2 added with phosphate buffer (extractfree control of the rate of H2O2 inactivation), were used for comparative parallel determination of LDC. The LDC of cell extracts was registered every 10 s in darkness until the complete inactivation of H2O2. The LDC activities of H2O2-treated cell extracts were expressed as a % of LDC of parallel H2O2 extract-free control (considered as 100% at each time interval). H2OCA was considered as positive if inactivation of 95% 2 of H2O2 added to cell extract varied between 20 s and 3 min, (for negative cell variants it usually takes 5–20 min, but for some cell extracts it may take more than 40 – 120 min). 2.3.2. PGE2 -releasing acti6ity PGE2-releasing activity (PGES) was determined by the bioassay specially developed for these studies by Kluchareva et al. [19] and confirmed by parallel RIA. Values of PGES in bioassay ]1.7, and their complete inhibition by indomethacin pretreatment of tumor cells represents a reliable and highly-reproducible positive index of PGES.
39
3. Results and discussion
3.1. The comparati6e rates of in 6i6o acquisition of S (H2O CA 2 + PGE ) phenotype by the tumor cells in conditions of natural and immune selection SV40 in vitro transformed Syrian hamster embryo cells of HE-wtSV40 strain were challenged in several cycles of parallel quantitative transplantation tests in normal and SV40-immune animals. After each cycle, 2–4 tumor-cell variants were isolated from individual animals, established in tissue cultures, and examined on S the expression of (H2OCA 2 + PGE ) phenotype, TGA, SMA, and immunosensitivity. The variant cell lines were used for subsequent in vivo selection; this procedure was repeated several times until the expression of S (H2OCA 2 + PGE ) phenotype and of immunoresistance was achieved. As shown earlier, and confirmed in the present study, SV40 tumor cells were not selectable for immunoresistance in normal animals and they retained expression of TSTA and of immunosensitivity in conditions of numerous in vivo passages [reviewed in [20]]. The results presented in Table 1 demonstrated that: S (1) acquisition of (H2OCA 2 + PGE ) phenotype was achieved in normal and SV40-immune animals at approximately the same time, between the second and the third in vivo cycles of tumor cells selection, i.e. independently of the immune status of animals; (2) the gradual increase of TGA levels and of the rate of acquisition of SMA were the same in normal and in SV40-immune animals, although the number of metastatic nodules in immune animals was, as a rule, lower than in normal ones (data not shown); (3) the rates of in vivo selection of tumor cells for specific immunoresistance, as comS pared with acquisition of (H2OCA 2 + PGE ) phenotype, was delayed, and in the majority of cases was achieved between the third and the fourth cycles of in vivo selection in immune animals. Thus, specific antitumor
Table 1 S The comparative rates of in vivo natural and immune selection of SV40 in vitro transformed cells and acquisition of [H2OCA 2 +PGE ] phenotype Number of in vivo cycles of selection*
I II III IV
Normal animals
SV40-immune animals
Expression of S [H2OCA 2 +PGE ] phenotype***
TGA in log TrD50
0/1** 0/3 3/3 n.d.
3.5 1.7 1.2 0.8
9 9 9 9
0.3 0.3 0.2 0.2
Number of immunoresistant cell variants ****
Expression of S [H2OCA 2 +PGE ] phenotype***
TGA in log TrD50
0/1** 0/3 0/3 0/3
0/1** 0/3 4/4 3/3
4.7 3.7 2.2 1.1
9 9 9 9
0.4 0.2 0.4 0.1
Number of immunoresistant cell variants**** 0/1** 0/3 1/4 2/3
* Each cycle of in vivo selection of tumor cells lasted 55–60 days of sc growth. ** Numerator: the number of tumor cell variants expressing the given property; denominator: the number of tumor cell variants examined. *** Expression of [H2OCA + PGES] phenotype was determined in accordance to Materials and methods. 2 **** Immunosensitivity index of tumor cells represents the difference in individual values of TGA determined (in log TrD50) in SV40-immune animals and normal animals; the values B0.5 suggested the loss of immunosensitivity, i.e. of acquired immunoresistance of cell line.
40
G. Deichman et al. / Immunology Letters 70 (1999) 37–42
immunity does not influence the rate of in vivo natural S selection of tumor cells expressing (H2OCA 2 +PGE ) phenotype and the highly-increased TGA. S 3.2. The acquired expression of (H2O CA 2 + PGE ) phenotype and the effecti6eness of in 6i6o rejection of SV40 tumor cells in normal and in immune animals S The possible influence of (H2OCA 2 +PGE ) phenotype acquired in vivo by TSTA-expressing SV40 tumor cells, on their local susceptibility to rejection by the innate host effectors and in conditions of specific antitumor immunity was studied. For this purpose, the parental HE-wt SV40 cells [immunosensitive and negative in S expression of (H2OCA 2 +PGE ) phenotype] and two of their direct-descendant variant cell lines, [immunosensiS tive and expressing (H2OCA 2 +PGE ) phenotype] were challenged in vivo in parallel quantitative transplantation tests in groups of five to six normal and SV40-immunized animals. The data presented in Table 2 demonstrate that parental SV40 in vitro transformed cells are 1 – 2 orders more efficiently eliminated in normal hosts, as compared with in vivo selected descendant cell lines. The latter two lines manifested significantly increased levels of the resistance to in vivo elimination and correspondingly, the increase of TGA level in normal animals ( B1.2–1.7, vs 3.1– 3.5 logs of TrD50 of the parental cell line). As expected, the parental tumor cells were eliminated in immunized animals significantly more effectively. It may be assumed that contribution of innate host effectors in elimination of the parental SV40-transformed cells is the same in normal and in immune animals. In this case, the real number of tumor cells rejected non-specifically, (near to 103) is almost 100 times lower than the total number of tumor cells rejected in immune animals. However, this situation dramatically changed in the case of descendant tumor cell lines growing in immunized animals. Both SV40-descendant tumor cell lines, although still retaining a high level of immunosensitivity (the same as the parental cells), were 100 times less effectively eliminated in SV40-immune animals, as compared with the parental cell line (Table 2). While the total number of the parental tumor cells eliminated in the immune animals is near to 105, the number of tumor cells of the descendant cell lines eliminated by the immune host is 100 times lower (102 – 103). The decreased effectiveness of specific immunorejection of the descendant tumor cell lines in immune animals correlates with the decreased effectiveness of their nonspecific elimination in normal animals. These unexpected findings suggest that acquired exS pression of (H2OCA 2 +PGE ) phenotype may defend antigenic tumor cells (directly or indirectly) not only
against the innate, but also against the adaptive host immunity effectors. However, the mechanisms of the local interactions of these effectors at the site of antigenic tumor growth are unknown. In the light of recent progress in the understanding of the complex mechanisms of interaction between the effectors of innate and adaptive immunity in the host defence against bacterial infections [23–25], some elements of the same mechanisms may determine the antitumor host defense reactions. Earlier, the dominating role of the host innate effectors in the antitumor defense reactions and in in vivo tumor progression were emphasized [26]. In this case, the problem concerns not only the mechanisms of recognition of autologous tumor cells as ‘non-self’ by innate host effectors and of the subsequent activation of the host T helper and killer cells. The in vivo acquired ability of tumor cells to defend themselves locally against the host innate immunity effectors makes this situation even more complex. For instance, in the interaction of NK cells and PGE2-releasing tumor cells they would exchange their roles: the former are becoming the targets, while the latter ones acquire ability to suppress NK CTA. It may be suggested that local suppression of innate host effectors by the tumor S cells expressing (H2OCA 2 + PGE ) phenotype possibly leads to altered interactions between these two branches of the host defense system and correspondingly, to the decrease of the effectiveness of specific immunorejection of antigenic tumor cells. Earlier, we demonstrated that systemic suppression of the host innate immunity was opening the way for single sc transplanted tumor cells (antigenic, as well as non-antigenic) to ‘sneak through’ the host natural defense barriers and form tumor nodules. Moreover, the systemic suppression of the host innate immunity almost completely suppressed the possibility to induce specific (SV40) antitumor immunity in such animals [27]. Now, as shown in Table 2, the comparison of in vivo transplantability in normal and immune hosts of the SV40 parental in vitro transformed cells and their in vivo selected direct descendants, demonstrate that not only systemic, but also local suppression of the host innate immunity effectors may significantly decrease the real numbers of immunosensitive tumor cells rejectable in immune animals. However, the local suppression of innate host effectors, possibly leading to their altered antigen-presenting function, is not followed by the systemic T cells anergy, or exhaustion, at least at the beginning of tumor growth [20]. Independent (parallel) in vivo mobilization of some other tumor cells defense and/or escape mechanisms, (e.g. FAS–FASL interactions [28]) are not excluded. In general, the data presented are related to one of the main unresolved questions of tumor immunology, i.e. why the effectiveness of specific immunotherapy of antigenic tumors is, as a rule, lower, than expected
Table 2 S Expression of [H2OCA 2 +PGE ] phenotype, tumorigenicity, and effectiveness of in vivo SV40 tumor cells rejection in normal and SV40-immune animals
HE-wtSV40 parental
HE-wtSV40-2SC-4 HE-wtSV40-2SC-5
Number of in vivo selection cycles
0 0 0 0 3 3 3 3
S Expression of [H2OCA 2 +PGE ] (+) or (−)
− − − − + + + +
Animals
Normal Immune (Exp.1) Normal Immune (Exp. 2) Normal Immune Normal Immune
Number of cells transplanted 101
102
103
104
0/5** 0/5 0/6 0/6 2/5 0/6 5/5 2/5
0/5 0/5 0/6 0/6 5/5 2/6 5/5 3/5
5/5 0/5 3/6 0/6 5/5 4/6 5/5 4/5
5/5 2/5 5/6 1/6 5/5 4/6 5/5 5/5
TGA in logTrD50
ISI***
3.1 ]4.6 3.5 ]4.9 1.7 3.3 B1.2 2.4
]1.5 ]1.5 ]1.4 ]1.4 1.6 1.6 ]1.2 ]1.2
* In vitro SV40-transformed parental cells and two lines of their direct in vivo selected descendants were in vivo challenged in parallel sc quantitative transplantation tests in the groups of five to six normal and SV40-immunized animals (see Materials and methods). ** Numerator: number of tumor takes; Denominator: number of animals inoculated sc with the given number of tumor cells. *** Immunosensitivity indices (see Materials and methods).
G. Deichman et al. / Immunology Letters 70 (1999) 37–42
Cell lines*
41
42
G. Deichman et al. / Immunology Letters 70 (1999) 37–42
[29 – 34]? In addition to the several established ways of tumor cells escaping from the host control, the acquired local mechanisms of defense against the host innate immunity effectors seems to be the basic and underestimated factor. The constructive evaluation of the situation, directly related to the in vivo tumor progression, suggests that, either specific immunization against the antigenic tumors is to be performed very early when the tumor cells are still naive, i.e. defenseless, or it should be carried out in combination with the suppression of S (H2OCA 2 +PGE ) phenotype. The use of the latter approach is especially expedient in the cases of spontaneous non- or low-antigenic tumors expressing S (H2OCA 2 +PGE ) phenotype, as it may recover their in vivo susceptibility to the host innate immunity control.
Acknowledgements We greatly appreciate Professor Garri Abelev and Professor Anatole Altstein for the critical reading and discussion of the manuscript. This work was supported by grant No. 99-04-48358 of the Russian Fund for Fundamental Investigations.
References [1] G.I. Deichman, T.E. Kluchareva, V.A. Matveeva, N.E. Kushlinsky, L.S. Bassalyk, E.L. Vendrov, Int. J. Cancer 44 (1989) 904. [2] G.I. Deichman, H.A. Kashleva, T.E. Kluchareva, V.A. Matveeva, Int. J. Cancer 44 (1989) 908. [3] G.I. Deichman, V.A. Matveeva, L.M. Kashkina, N.A. Dyakova, E.N. Uvarova, M.A. Nikiforov, et al., Int. J. Cancer 75 (1998) 277. [4] G.I. Deichman, L.M. Kashkina, T.E. Kluchareva, V.A. Matveeva, E.N. Uvarova, L.G. Burdelya, Adv. Exp. Med. Biol. 400A (1997) 473. [5] G.I. Deichman, L.M. Kashkina, O.A. Misenina, E.G. Gorojanskaya, M.A. Nikiforov, A.V. Gudkov, et al., Int. J. Cancer 66 (1996) 747.
.
[6] C.F. Nathan, S.C. Silverstein, L.H. Brukner, Z.A. Cohn, J. Exp. Med. 149 (1979) 100. [7] C.F. Nathan, B.A. Arrick, H.W. Murray, N.M. De Santis, Z.A. Cohn, J. Exp. Med. 153 (1981) 766. [8] D.O. Adams, C.F. Nathan, Immunol. Today 4 (1983) 166. [9] G.I. Deichman, E.L. Vendrov, Int. J. Cancer 37 (1986) 401. [10] N.A. Lavnikova, L.G. Burdelya, (In Russian), Bull. Exp. Biol. Med. 109 (1990) 954. [11] L.G. Burdelya, Neoplasma 44 (1) (1997) 31. [12] M. Droller, M. Schneider, P. Perlman, Cell Immunol. 39 (1979) 165. [13] M.J. Brunda, R.B. Herberman, H.T. Holden, J. Immunol. 124 (1980) 2682. [14] A.M. Fulton, Int. J. Cancer 33 (2) (1984) 375. [15] R. Young, S. Knies, J. Natl. Cancer Inst. 72 (4) (1984) 919. [16] T.E. Kluchareva, V.A. Matveeva, L.C. Bassalyk, N.E. Kushlinsky, (In Russian), Bull. Exp. Biol. Med 2 (1988) 204. [17] T.E. Kluchareva, V.A. Matveeva, L.M. Kashkina, N.E. Kushlinsky, L.C. Bassalyk, M.O. Prilutskaya, (In Russian), Exp. Oncology 11 (1989) 53. [18] T.E. Kluchareva, V.A. Matveeva, E.N. Uvarova, (In Russian), Bull. Exp. Biol. Med. 9 (1990) 308. [19] T.E. Kluchareva, V.A. Matveeva, N.E. Kushlinsky, Immunol. Lett. 33 (1992) 239. [20] G.I. Deichman, Adv. Cancer Res. 12 (1969) 101. [21] H.O. Sjo¨gren, I. Hellstro¨m, G. Klein, Exp. Cell Res. 23 (1961) 204. [22] K. Habel, Proc. Soc. Exp. Biol. Med. 106 (1961) 722. [23] R. Medzhitov, C.A. Janeway, Curr. Opin. Immunol. 9 (1997) 4. [24] R. Medzhikov, C.A. Janeway, Cell 91 (1997) 295. [25] A. Bandelac, D.T. Fearon, Curr. Opin. Immunol. 9 (1997) 1. [26] G.I. Deichman, Cancer Surveys 7 (1988) 675. [27] G.I. Deichman, T.E. Kluchareva, L.M. Kashkina, V.A. Natveena, et al., Int. J. Cancer 23 (1979) 571. [28] M. Hanhe, D. Rimoldi, M. Schro¨ter, P. Romero, M. Schreier, L.E. French, et al., Science 274 (1996) 1363. [29] G. Klein, in: R.T. Smith, M. Landy (Eds.), Immunology of the Tumor-Host Relationship, Academic Press, New York/London, 1975, p. 201. [30] T. Boon, J.-C. Cerottini, B. Van den Eynde, P. van der Bruggen, A. Van Pel, Ann. Rev. Immunol. 12 (1994) 337. [31] E. Kedar, E. Klein, Adv. Cancer Res. 59 (1992) 245. [32] E. Klein, A. Mantovani, Curr. Opin. Immunol. 5 (1993) 714. [33] H. Schreiber, in: W.E. Paul (Ed.), Fundamental Immunology, Third Edition, Raven Press, New York, 1993, p. 1143. [34] M. Wick, P. Dubey, H. Koeppen, C.T. Siegel, P.E. Fields, L. Chen, et al., J. Exp. Med. 186 (1997) 229.
www.elsevier.com/locate/
In vivo acquired mechanisms of tumor cells local defense against the host innate immunity effectors: implication in specific antitumor immunity Galina Deichman *, Natalya Dyakova, Lidia Kashkina, Valentina Matveeva, Elvira Uvarova Laboratory of Antitumor Immunity, Institute of Carcinogenesis, N.N. Blokchin Cancer Research Center, Russian Academy of Medical Sciences, Moscow, Russia Accepted 22 June 1999
Abstract As shown earlier, the cells transformed in vitro by several different oncogenes, or spontaneously, during in vivo growth in normal hosts would be gradually replaced by the highly-tumorigenic descendants co-expressing high H2O2-catabolizing and S PGE2-releasing activities. Acquisition of (H2OCA 2 +PGE ) phenotype provides the cells with local defense mechanisms against the S host innate immunity effectors. However, it remained unknown, whether the expression of (H2OCA 2 + PGE ) phenotype is implicated in susceptibility of tumor cells expressing tumor-specific transplantation antigens to rejection in immune animals. Here, S with the use of SV40 in vitro transformed parental cells, negative in expression (H2OCA 2 + PGE ) phenotype, and their in vivo selected descendant tumor cell lines expressing this phenotype, we show that: (1) the rates of in vivo selection of the parental SV40 S tumor cells expressing (H2OCA 2 +PGE ) phenotype are the same in normal and SV40-immune animals; (2) in vivo selected SV40 S CA tumor cells expressing (H2O2 +PGE ) phenotype, although they retain specific immunosensitivity, are 100 times less effectively rejected in SV40-immunized animals, as compared with their in vitro SV40-transformed parental cells. Thus, in vivo acquired immunologically non-specific local mechanisms of tumor cells defense against the host innate immunity effectors, significantly decreases the effectiveness of their specific immunorejection. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Host innate and specific antitumor immunity; Immunoresistance; Immunosensitivity; Natural selection; Tumor cells local defence mechanisms; Tumor progression
1. Introduction During the last 20 years the mechanisms of tumor cells in vivo acquired resistance to cytotoxicity of the host innate immunity effectors, such as macrophages (M¥), NK cells, and neutrophils (Nph), and its possible role in tumor progression were widely studied in many laboratories. Briefly, our main finding in this field, was evidence that at least one such type of resistance is connected with the in vivo natural selection of tumor cells possessing two new properties, i.e. high H2O2-catabolizing (H2OCA 2 ) (antioxidant) activity and * Corresponding author. Present address. Institute of Carcinogenesis, Cancer Research Center, Kashirskoye shosse 24, M-478, Moscow, Russia; Fax: + 7-095-3241205. E-mail address: [email protected] (G. Deichman)
the ability to release PGE2 (PGES2 ) immediately upon contact with NK cells, M¥, or Nph. Although these biochemically different cell properties forming S (H2OCA 2 + PGE ) phenotype are usually considered separately, our data showed that they are acquired by tumor cells in vivo in one step; no intermediate cell variants were noticed and both were co-expressed at the S cell clonal level [1,2]. Acquisition of (H2OCA 2 +PGE ) phenotype by tumor cells during in vivo natural selection appeared to be independent from the type of oncogenes initially transformed these cells in vitro (with the exception of v-src) [3]. Examination of tumor cells of different oncogenetic origin in dynamics of in vivo progression demonstrated that acquired expression of S (H2OCA 2 + PGE ) phenotype coincides with their regular 30–200 times increased tumorigenic activity (TGA),
0165-2478/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 7 8 ( 9 9 ) 0 0 1 2 3 - 6
38
G. Deichman et al. / Immunology Letters 70 (1999) 37–42
followed, or not followed, by spontaneous metastasizing activity (SMA). Recently, we have shown that natural selection of tumor cells expressing (H2OCA 2 + PGES) phenotype begins during the latent period of primary viral carcinogenesis and this process may be completed before the primary tumor appearance (Deichman et al., 1999, submitted). Thus, acquisition of S (H2OCA 2 +PGE ) phenotype may be considered as a stable biochemical marker of the relatively early, premetastatic stage of tumor progression [1 – 5]. S Expression of (H2OCA 2 +PGE ) phenotype appears to be responsible for two different local mechanisms of the tumor cells defense against cytotoxic activity (CTA) of the host innate immunity effectors. (1) The acquired H2OCA 2 , mainly based on the high catalase and/or on glutathione peroxidase activities, was shown to defend tumor cells against the damaging effect of H2O2 and some other reactive oxygen species produced by M¥ and Nph, as a part of their CTA [1,2,6 – 11]. (2) The immediate release of PGE2 by the tumor cells in response to the contact interaction with NK cells or M¥, leads to the suppression of CTA of NK cells, thus demonstrating inducible and almost aggressive forms of malignant tumor cells local defense against NK cells [12 – 17]. The inhibition of PGE2 release by the pretreatment of tumor cells with indomethacin is making them susceptible to CTA of NK cells [18,19]. S Acquisition of (H2OCA 2 +PGE ) phenotype by tumor cells during sc growth and natural selection in normal animals was shown for the cells of non-antigenic tumors, as well as for those expressing TSTA (i.e. tumorspecific transplantation antigens); among the latter ones, simian virus 40 (SV40) and bovine adenovirus type 3 (BAV-3) in vitro transformed cells were examined [3]. As shown, TSTA’s of cells in vivo, or in vitro transformed by DNA tumor viruses are inducing specific antitumor immunity, which is manifested in vivo in specific immunogenicity and in immunosensitivity of virus-transformed cells. The long-term growth of the tumors expressing TSTA’s in immunized animals, in contrast to the growth in normal ones, leads to the gradual selection of immunoresistant tumor cell variants [review in [20]]. The recent data demonstrating the non-specific defense mechanisms of in vivo selected tumor cells against the host innate immunity effectors raise a question on S the possible implication of (H2OCA 2 +PGE ) phenotype in reactions of specific antitumor immunity. Therefore, in the present study, with the use of SV40 in vitro transformed cells we examined the possible influence of: (1) specific antitumor immunity on the rates of in vivo selection of SV40 tumor cells expressing (H2OCA 2 + PGES) phenotype, as compared with the rates of their selection in normal animals; and (2) the expression of S (H2OCA 2 +PGE ) phenotype on the effectiveness of in vivo rejection of SV40 tumor cells in normal and immune animals.
2. Materials and methods
2.1. Origin of cell lines Syrian hamster normal embryo cells transformed in vitro by the strain N128 of wild-type SV40 (cell line HE-wtSV40) and their in vivo selected variants were examined before and in dynamics of in vivo natural and immune selection. Each in vivo cycle of selection lasted 55–60 days. The cells isolated from the tumors of individual animals (normal and SV40 immune) were transferred for cultivation in vitro in DMEM containing 5% FCS and antibiotics; after 3–5 in vitro passages (in order to clear tumor cells from the host stromal cells) they were challenged in vitro for S expression of (H2OCA 2 + PGE ) phenotype and in vivo on the TGA, SMA, and on the specific immunosensitivity.
2.2. The le6els of TGA, SMA, and immunosensiti6ity of the SV40 tumor cell lines The levels of TGA, SMA, and immunosensitivity of the SV40 tumor cell lines were determined with the use of in vivo quantitative transplantation tests. Several cell variants selected in vivo, and particularly, the descendant cell lines (HE-wtSV40-2 SC-4 and HE-wtSV40-2SC-5) in parallel with the parental cell line were challenged in transplantation tests, in normal and SV40-immune animals. Immunization of 2 to 3-month-old normal animals was performed by i.p. inoculation of SV40 (106.2 ID50/1.0 ml) 20 days before the sc challenge of virus-free SV40 tumor cells in quantitative transplantation tests [21,22]. In the immunosensitivity examination, quantitative transplantation tests were performed in parallel in groups of 5–6 normal and SV40-immune animals. Four 10-fold differing doses of tumor cells (from 1–2 ×101 to 1–2 × 104) were inoculated sc in a volume of 0.2 ml into groups of five or six animals; 55– 60 days later the animals were sacrificed under anesthetic and the levels of TGA estimated in the log of 50% transplantation dose (TrD50), were calculated for each group of normal and immune animals. The use of quantitative transplantation test permits us to estimate the number of tumor cells eliminated and/or surviving in normal and/or immune animals. The relative immunosensitivity index, i.e. the difference between TGA level estimated in log TrD50 in SV40-immune animals and the TGA level of the same cells challenged in parallel in normal animals, was considered as positive if it was ] 1.0. Values varying from 0.0 to 0.5 suggested the loss, or significant decrease of immunosensitivity of the SV40 tumor cells examined.
G. Deichman et al. / Immunology Letters 70 (1999) 37–42 S 2.3. Determination of (H2O CA 2 + PGE ) phenotype was performed in 6itro in accordance to [1,3]
2.3.1. H2O2 -catabolizing acti6ity H2O2-catabolizing activity (H2OCA 2 ) was determined in ex tempore prepared cell extracts (2.0 mg of protein per 1.0 ml) of different cell strains with the use of luminol-dependent chemiluminescence (LDC); 0.01 M luminol (Serva) was used. Cells were extracted in a phosphate buffer and lysed with Triton X-100 at 4°C. Extracts in a volume of 0.1 ml, added with ex tempore prepared 10 mM H2O2 (0.1 ml) and 0.1 ml of luminol, were immediately placed at 37°C in thermostated Biolumate (Model 9500, Berthold, Germany). Two samples of each cell extract (H2O2-treated), as well as two samples of H2O2 added with phosphate buffer (extractfree control of the rate of H2O2 inactivation), were used for comparative parallel determination of LDC. The LDC of cell extracts was registered every 10 s in darkness until the complete inactivation of H2O2. The LDC activities of H2O2-treated cell extracts were expressed as a % of LDC of parallel H2O2 extract-free control (considered as 100% at each time interval). H2OCA was considered as positive if inactivation of 95% 2 of H2O2 added to cell extract varied between 20 s and 3 min, (for negative cell variants it usually takes 5–20 min, but for some cell extracts it may take more than 40 – 120 min). 2.3.2. PGE2 -releasing acti6ity PGE2-releasing activity (PGES) was determined by the bioassay specially developed for these studies by Kluchareva et al. [19] and confirmed by parallel RIA. Values of PGES in bioassay ]1.7, and their complete inhibition by indomethacin pretreatment of tumor cells represents a reliable and highly-reproducible positive index of PGES.
39
3. Results and discussion
3.1. The comparati6e rates of in 6i6o acquisition of S (H2O CA 2 + PGE ) phenotype by the tumor cells in conditions of natural and immune selection SV40 in vitro transformed Syrian hamster embryo cells of HE-wtSV40 strain were challenged in several cycles of parallel quantitative transplantation tests in normal and SV40-immune animals. After each cycle, 2–4 tumor-cell variants were isolated from individual animals, established in tissue cultures, and examined on S the expression of (H2OCA 2 + PGE ) phenotype, TGA, SMA, and immunosensitivity. The variant cell lines were used for subsequent in vivo selection; this procedure was repeated several times until the expression of S (H2OCA 2 + PGE ) phenotype and of immunoresistance was achieved. As shown earlier, and confirmed in the present study, SV40 tumor cells were not selectable for immunoresistance in normal animals and they retained expression of TSTA and of immunosensitivity in conditions of numerous in vivo passages [reviewed in [20]]. The results presented in Table 1 demonstrated that: S (1) acquisition of (H2OCA 2 + PGE ) phenotype was achieved in normal and SV40-immune animals at approximately the same time, between the second and the third in vivo cycles of tumor cells selection, i.e. independently of the immune status of animals; (2) the gradual increase of TGA levels and of the rate of acquisition of SMA were the same in normal and in SV40-immune animals, although the number of metastatic nodules in immune animals was, as a rule, lower than in normal ones (data not shown); (3) the rates of in vivo selection of tumor cells for specific immunoresistance, as comS pared with acquisition of (H2OCA 2 + PGE ) phenotype, was delayed, and in the majority of cases was achieved between the third and the fourth cycles of in vivo selection in immune animals. Thus, specific antitumor
Table 1 S The comparative rates of in vivo natural and immune selection of SV40 in vitro transformed cells and acquisition of [H2OCA 2 +PGE ] phenotype Number of in vivo cycles of selection*
I II III IV
Normal animals
SV40-immune animals
Expression of S [H2OCA 2 +PGE ] phenotype***
TGA in log TrD50
0/1** 0/3 3/3 n.d.
3.5 1.7 1.2 0.8
9 9 9 9
0.3 0.3 0.2 0.2
Number of immunoresistant cell variants ****
Expression of S [H2OCA 2 +PGE ] phenotype***
TGA in log TrD50
0/1** 0/3 0/3 0/3
0/1** 0/3 4/4 3/3
4.7 3.7 2.2 1.1
9 9 9 9
0.4 0.2 0.4 0.1
Number of immunoresistant cell variants**** 0/1** 0/3 1/4 2/3
* Each cycle of in vivo selection of tumor cells lasted 55–60 days of sc growth. ** Numerator: the number of tumor cell variants expressing the given property; denominator: the number of tumor cell variants examined. *** Expression of [H2OCA + PGES] phenotype was determined in accordance to Materials and methods. 2 **** Immunosensitivity index of tumor cells represents the difference in individual values of TGA determined (in log TrD50) in SV40-immune animals and normal animals; the values B0.5 suggested the loss of immunosensitivity, i.e. of acquired immunoresistance of cell line.
40
G. Deichman et al. / Immunology Letters 70 (1999) 37–42
immunity does not influence the rate of in vivo natural S selection of tumor cells expressing (H2OCA 2 +PGE ) phenotype and the highly-increased TGA. S 3.2. The acquired expression of (H2O CA 2 + PGE ) phenotype and the effecti6eness of in 6i6o rejection of SV40 tumor cells in normal and in immune animals S The possible influence of (H2OCA 2 +PGE ) phenotype acquired in vivo by TSTA-expressing SV40 tumor cells, on their local susceptibility to rejection by the innate host effectors and in conditions of specific antitumor immunity was studied. For this purpose, the parental HE-wt SV40 cells [immunosensitive and negative in S expression of (H2OCA 2 +PGE ) phenotype] and two of their direct-descendant variant cell lines, [immunosensiS tive and expressing (H2OCA 2 +PGE ) phenotype] were challenged in vivo in parallel quantitative transplantation tests in groups of five to six normal and SV40-immunized animals. The data presented in Table 2 demonstrate that parental SV40 in vitro transformed cells are 1 – 2 orders more efficiently eliminated in normal hosts, as compared with in vivo selected descendant cell lines. The latter two lines manifested significantly increased levels of the resistance to in vivo elimination and correspondingly, the increase of TGA level in normal animals ( B1.2–1.7, vs 3.1– 3.5 logs of TrD50 of the parental cell line). As expected, the parental tumor cells were eliminated in immunized animals significantly more effectively. It may be assumed that contribution of innate host effectors in elimination of the parental SV40-transformed cells is the same in normal and in immune animals. In this case, the real number of tumor cells rejected non-specifically, (near to 103) is almost 100 times lower than the total number of tumor cells rejected in immune animals. However, this situation dramatically changed in the case of descendant tumor cell lines growing in immunized animals. Both SV40-descendant tumor cell lines, although still retaining a high level of immunosensitivity (the same as the parental cells), were 100 times less effectively eliminated in SV40-immune animals, as compared with the parental cell line (Table 2). While the total number of the parental tumor cells eliminated in the immune animals is near to 105, the number of tumor cells of the descendant cell lines eliminated by the immune host is 100 times lower (102 – 103). The decreased effectiveness of specific immunorejection of the descendant tumor cell lines in immune animals correlates with the decreased effectiveness of their nonspecific elimination in normal animals. These unexpected findings suggest that acquired exS pression of (H2OCA 2 +PGE ) phenotype may defend antigenic tumor cells (directly or indirectly) not only
against the innate, but also against the adaptive host immunity effectors. However, the mechanisms of the local interactions of these effectors at the site of antigenic tumor growth are unknown. In the light of recent progress in the understanding of the complex mechanisms of interaction between the effectors of innate and adaptive immunity in the host defence against bacterial infections [23–25], some elements of the same mechanisms may determine the antitumor host defense reactions. Earlier, the dominating role of the host innate effectors in the antitumor defense reactions and in in vivo tumor progression were emphasized [26]. In this case, the problem concerns not only the mechanisms of recognition of autologous tumor cells as ‘non-self’ by innate host effectors and of the subsequent activation of the host T helper and killer cells. The in vivo acquired ability of tumor cells to defend themselves locally against the host innate immunity effectors makes this situation even more complex. For instance, in the interaction of NK cells and PGE2-releasing tumor cells they would exchange their roles: the former are becoming the targets, while the latter ones acquire ability to suppress NK CTA. It may be suggested that local suppression of innate host effectors by the tumor S cells expressing (H2OCA 2 + PGE ) phenotype possibly leads to altered interactions between these two branches of the host defense system and correspondingly, to the decrease of the effectiveness of specific immunorejection of antigenic tumor cells. Earlier, we demonstrated that systemic suppression of the host innate immunity was opening the way for single sc transplanted tumor cells (antigenic, as well as non-antigenic) to ‘sneak through’ the host natural defense barriers and form tumor nodules. Moreover, the systemic suppression of the host innate immunity almost completely suppressed the possibility to induce specific (SV40) antitumor immunity in such animals [27]. Now, as shown in Table 2, the comparison of in vivo transplantability in normal and immune hosts of the SV40 parental in vitro transformed cells and their in vivo selected direct descendants, demonstrate that not only systemic, but also local suppression of the host innate immunity effectors may significantly decrease the real numbers of immunosensitive tumor cells rejectable in immune animals. However, the local suppression of innate host effectors, possibly leading to their altered antigen-presenting function, is not followed by the systemic T cells anergy, or exhaustion, at least at the beginning of tumor growth [20]. Independent (parallel) in vivo mobilization of some other tumor cells defense and/or escape mechanisms, (e.g. FAS–FASL interactions [28]) are not excluded. In general, the data presented are related to one of the main unresolved questions of tumor immunology, i.e. why the effectiveness of specific immunotherapy of antigenic tumors is, as a rule, lower, than expected
Table 2 S Expression of [H2OCA 2 +PGE ] phenotype, tumorigenicity, and effectiveness of in vivo SV40 tumor cells rejection in normal and SV40-immune animals
HE-wtSV40 parental
HE-wtSV40-2SC-4 HE-wtSV40-2SC-5
Number of in vivo selection cycles
0 0 0 0 3 3 3 3
S Expression of [H2OCA 2 +PGE ] (+) or (−)
− − − − + + + +
Animals
Normal Immune (Exp.1) Normal Immune (Exp. 2) Normal Immune Normal Immune
Number of cells transplanted 101
102
103
104
0/5** 0/5 0/6 0/6 2/5 0/6 5/5 2/5
0/5 0/5 0/6 0/6 5/5 2/6 5/5 3/5
5/5 0/5 3/6 0/6 5/5 4/6 5/5 4/5
5/5 2/5 5/6 1/6 5/5 4/6 5/5 5/5
TGA in logTrD50
ISI***
3.1 ]4.6 3.5 ]4.9 1.7 3.3 B1.2 2.4
]1.5 ]1.5 ]1.4 ]1.4 1.6 1.6 ]1.2 ]1.2
* In vitro SV40-transformed parental cells and two lines of their direct in vivo selected descendants were in vivo challenged in parallel sc quantitative transplantation tests in the groups of five to six normal and SV40-immunized animals (see Materials and methods). ** Numerator: number of tumor takes; Denominator: number of animals inoculated sc with the given number of tumor cells. *** Immunosensitivity indices (see Materials and methods).
G. Deichman et al. / Immunology Letters 70 (1999) 37–42
Cell lines*
41
42
G. Deichman et al. / Immunology Letters 70 (1999) 37–42
[29 – 34]? In addition to the several established ways of tumor cells escaping from the host control, the acquired local mechanisms of defense against the host innate immunity effectors seems to be the basic and underestimated factor. The constructive evaluation of the situation, directly related to the in vivo tumor progression, suggests that, either specific immunization against the antigenic tumors is to be performed very early when the tumor cells are still naive, i.e. defenseless, or it should be carried out in combination with the suppression of S (H2OCA 2 +PGE ) phenotype. The use of the latter approach is especially expedient in the cases of spontaneous non- or low-antigenic tumors expressing S (H2OCA 2 +PGE ) phenotype, as it may recover their in vivo susceptibility to the host innate immunity control.
Acknowledgements We greatly appreciate Professor Garri Abelev and Professor Anatole Altstein for the critical reading and discussion of the manuscript. This work was supported by grant No. 99-04-48358 of the Russian Fund for Fundamental Investigations.
References [1] G.I. Deichman, T.E. Kluchareva, V.A. Matveeva, N.E. Kushlinsky, L.S. Bassalyk, E.L. Vendrov, Int. J. Cancer 44 (1989) 904. [2] G.I. Deichman, H.A. Kashleva, T.E. Kluchareva, V.A. Matveeva, Int. J. Cancer 44 (1989) 908. [3] G.I. Deichman, V.A. Matveeva, L.M. Kashkina, N.A. Dyakova, E.N. Uvarova, M.A. Nikiforov, et al., Int. J. Cancer 75 (1998) 277. [4] G.I. Deichman, L.M. Kashkina, T.E. Kluchareva, V.A. Matveeva, E.N. Uvarova, L.G. Burdelya, Adv. Exp. Med. Biol. 400A (1997) 473. [5] G.I. Deichman, L.M. Kashkina, O.A. Misenina, E.G. Gorojanskaya, M.A. Nikiforov, A.V. Gudkov, et al., Int. J. Cancer 66 (1996) 747.
.
[6] C.F. Nathan, S.C. Silverstein, L.H. Brukner, Z.A. Cohn, J. Exp. Med. 149 (1979) 100. [7] C.F. Nathan, B.A. Arrick, H.W. Murray, N.M. De Santis, Z.A. Cohn, J. Exp. Med. 153 (1981) 766. [8] D.O. Adams, C.F. Nathan, Immunol. Today 4 (1983) 166. [9] G.I. Deichman, E.L. Vendrov, Int. J. Cancer 37 (1986) 401. [10] N.A. Lavnikova, L.G. Burdelya, (In Russian), Bull. Exp. Biol. Med. 109 (1990) 954. [11] L.G. Burdelya, Neoplasma 44 (1) (1997) 31. [12] M. Droller, M. Schneider, P. Perlman, Cell Immunol. 39 (1979) 165. [13] M.J. Brunda, R.B. Herberman, H.T. Holden, J. Immunol. 124 (1980) 2682. [14] A.M. Fulton, Int. J. Cancer 33 (2) (1984) 375. [15] R. Young, S. Knies, J. Natl. Cancer Inst. 72 (4) (1984) 919. [16] T.E. Kluchareva, V.A. Matveeva, L.C. Bassalyk, N.E. Kushlinsky, (In Russian), Bull. Exp. Biol. Med 2 (1988) 204. [17] T.E. Kluchareva, V.A. Matveeva, L.M. Kashkina, N.E. Kushlinsky, L.C. Bassalyk, M.O. Prilutskaya, (In Russian), Exp. Oncology 11 (1989) 53. [18] T.E. Kluchareva, V.A. Matveeva, E.N. Uvarova, (In Russian), Bull. Exp. Biol. Med. 9 (1990) 308. [19] T.E. Kluchareva, V.A. Matveeva, N.E. Kushlinsky, Immunol. Lett. 33 (1992) 239. [20] G.I. Deichman, Adv. Cancer Res. 12 (1969) 101. [21] H.O. Sjo¨gren, I. Hellstro¨m, G. Klein, Exp. Cell Res. 23 (1961) 204. [22] K. Habel, Proc. Soc. Exp. Biol. Med. 106 (1961) 722. [23] R. Medzhitov, C.A. Janeway, Curr. Opin. Immunol. 9 (1997) 4. [24] R. Medzhikov, C.A. Janeway, Cell 91 (1997) 295. [25] A. Bandelac, D.T. Fearon, Curr. Opin. Immunol. 9 (1997) 1. [26] G.I. Deichman, Cancer Surveys 7 (1988) 675. [27] G.I. Deichman, T.E. Kluchareva, L.M. Kashkina, V.A. Natveena, et al., Int. J. Cancer 23 (1979) 571. [28] M. Hanhe, D. Rimoldi, M. Schro¨ter, P. Romero, M. Schreier, L.E. French, et al., Science 274 (1996) 1363. [29] G. Klein, in: R.T. Smith, M. Landy (Eds.), Immunology of the Tumor-Host Relationship, Academic Press, New York/London, 1975, p. 201. [30] T. Boon, J.-C. Cerottini, B. Van den Eynde, P. van der Bruggen, A. Van Pel, Ann. Rev. Immunol. 12 (1994) 337. [31] E. Kedar, E. Klein, Adv. Cancer Res. 59 (1992) 245. [32] E. Klein, A. Mantovani, Curr. Opin. Immunol. 5 (1993) 714. [33] H. Schreiber, in: W.E. Paul (Ed.), Fundamental Immunology, Third Edition, Raven Press, New York, 1993, p. 1143. [34] M. Wick, P. Dubey, H. Koeppen, C.T. Siegel, P.E. Fields, L. Chen, et al., J. Exp. Med. 186 (1997) 229.