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Original Article |
ludewigb{at}pathol.unizh.ch
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Key Words: dendritic cells • immunotherapy • autoimmunity • tumor immunity • vaccination
Dendritic cells (DCs) are potent immunostimulatory cells facilitating antigen transport to lymphoid tissues and efficient stimulation of T cells 1213. It has thus been suggested that targeting immunogenic tumor antigens to DCs may represent an efficient means for induction of antitumor immunity 14. Indeed, DC vaccination against experimental murine model tumors has given encouraging results 151617181920. However, to be effective, DC vaccination usually had to be initiated before or within a few days after tumor cell transfer. Although in some of these studies tumor antigens not exclusively expressed by the tumor had been used 181920, autoimmune side-effects had not been observed. This lack of autoimmunity may be due to the fact that only low CTL activities present for a relatively short time were sufficient to control and clear these early and small tumors without or before causing clinically apparent autoimmune disease. In contrast, clinically established tumors seem to require a strong and sustained CTL response to guarantee complete remission of more sizeable tumors 21. Although strong antitumor CTLs directed against a unique and truly tumor-specific antigen may cure the tumor without causing autoimmunity, this may be a serious problem for CTL responses against antigens shared between tumor cells and nonneoplastic cells. In the latter case, it is important to evaluate the optimal conditions for induction of tumor-specific CTLs by antitumor vaccination and whether a therapeutic window exists between induction of tumor immunity and autoimmunity against so far immunologically ignored self-antigens.
Preparation and Peptide Pulse of DCs.
Transplantation of Tumor Pieces and Monitoring of Tumor Growth.
Cytotoxicity Assays.
Cell Rejection Assay.
Measurement of Blood Glucose.
Immunohistology.
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A large number of tumor antigens has been identified, and antitumor responses against these antigens can be elicited in mice or humans 1234. Tumor antigens most suitable for immunotherapy are, ideally, the strictly tumor-specific antigens that are shared by different tumors or mutated proteins expressed uniquely by tumor cells 2. However, many tumor antigens that are shared with other tissues are recognized by tumor-specific CTLs, such as the melanocyte differentiation antigen tyrosinase-related protein 2 (TRP-2; references 5 and 6) or the antigen p53 7, which is specifically overexpressed in tumors. Therefore, for certain tumor antigens, an efficient immune response may potentially be harmful, because immunity against self-antigens may cause autoimmunity. Indeed, efficient tumor control in human patients may be associated with autoimmune phenomena, e.g., antimelanoma immunity that sometimes correlates with vitiligo 8, certain gynecological tumors that appear to induce paraneoplastic cerebellar degeneration 9, or Wegener's granulomatosis, which is often associated with renal cell carcinoma 10. It is therefore important to assess the potential sequelae of immunity against tumor antigens that are also expressed by normal cells of peripheral organs, i.e., antigens that are located strictly outside of the immune system and are shared between tumors and normal host tissues. It has been shown that such antigens are largely ignored by T cells but can be reacted against if sufficient antigen reaches secondary lymphoid organs during a sufficient time period 11.
Materials and Methods
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Mice and Viruses.
Mice were obtained from the Institut für Labortierkunde (University of Zürich, Switzerland). Transgenic mice expressing lymphocytic (L)CMV-GP (glycoprotein) under the control of the rat insulin promotor (RIP-GP mice; reference 22), transgenic mice expressing the LCMV GP33 epitope in all tissues (H8 mice; reference 23), and SM-LacZ mice expressing β-galactosidase under the control of the SM22
promotor (2126nlz; reference 24) have been previously described. Experiments were carried out with age- (8–16 wk) and sex-matched animals. The β-galactosidase–recombinant vaccinia virus (VV-LacZ) was provided by Dr. R. Drillien (Université Louis Pasteur, Strasbourg, France) and grown and plaqued on BSC-40 cells.
Generation of DCs from bone marrow cultures of H8 and C57BL/6 mice has been described 25. C57BL/6 DCs were pulsed with β-galactosidase peptide β-gal 497–504 (reference 26) at a concentration of 10–6 M for 60 min at 37°C. Cells were washed three times with BSS and injected intravenously in a volume of 0.5 ml of BSS.
The MC-GP cell line 27 and the EL4-LacZ cell line 26 were described previously. Tumor cells in single-cell suspensions were injected subcutaneously into the flanks of T cell–immunodeficient mice (H-2b RAG-1–/–). Growing tumor pieces were dissected into small tumor pieces of 2 x 2 x 2 mm containing 
2–5 x 106 tumor cells and transplanted into the flanks of naive recipient mice. Tumor size was assessed twice per week, and the animals were killed when the tumor volume reached 7 cm3. Tumor volume was calculated by the formula V = 
x abc/6, where a, b, and c are the orthogonal diameters. Tumor cells were checked before transplantation and at the end of each experiment for tumor antigen expression.
Spleen cells (4 x 106 per well) from primed mice were restimulated for 5 d in 24-well tissue culture plates with 2 x 106 β-gal 497–504-pulsed, irradiated (3,000 rads) spleen cells in IMDM supplemented with 10% FCS, penicillin/streptomycin, and 0.001 M 2-ME. Restimulated spleen effector cells from one well were resuspended in 1 ml of MEM/2% FCS, and threefold serial dilutions were made (indicated as dilution of culture). EL-4 cells were pulsed with β-gal 497–504 (10–6 M; 1.5 h at 37°C) and used in a standard 5-h 51Cr-release assay. Unlabeled EL-4 cells served as controls.
To monitor DC-induced CTL activity directly in vivo, naive or SM-LacZ mice primed with 2 x 105 β-gal 497–504-pulsed DCs on days 0 and 2 were adoptively transfused with β-gal 497–504-pulsed splenocytes on day 8. β-gal 497–504-pulsed splenocytes were labeled with a high intensity of CSFE (5- and 6-carboxyfluorescein diacetate succinimidyl ester; Molecular Probes, Inc.) fluorescence, whereas unpulsed control cells were labeled with a low intensity of CSFE fluorescence. 4 x 107 cells from each preparation were transferred intravenously into naive or DC-primed mice, and the percentage of CSFE-positive cells in PBL was determined by FACS® analysis after 24 h. Erythrocytes were lysed with FACS lysis solution (Becton Dickinson), and the cell suspensions were analyzed on a FACScanTM flow cytometer (Becton Dickinson).
The glucose concentration in blood obtained from a tail vein was measured using an ELITE hemoglucometer (Bayer AG). Mice were considered diabetic with values >14 mM at two consecutive measurements.
Freshly removed organs were immersed in HBSS and snap-frozen in liquid nitrogen or fixed in 4% buffered formalin and subsequently embedded in paraplast. Frozen tissue sections were cut in a cryostat and fixed in acetone for 10 min. Sections were incubated with anti–mouse mAb against CD8+ cells (YTS169.4.2; reference 28), followed by goat anti–rat Ig (Caltag Labs.) and alkaline phosphatase–labeled donkey anti–goat Ig (Jackson ImmunoResearch Labs.). Endothelial cells and smooth muscle cells were stained on deparaffinized sections using rabbit anti–von Willebrand factor (DAKO A/S) or anti–smooth muscle actin (clone 1A4; Sigma Chemical Co.), respectively. Alkaline phosphatase was visualized using AS-BI phosphate/new fuchsin. Sections were counterstained with hemalum.
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Development of Autoimmune Diabetes as a Consequence of Curative Tumor Immunity.
We have previously shown that both the strength of the initial activation and the maintenance of CTL activity by repetitive DC immunization are important parameters for the induction of an autoimmune disease via DCs 29. Correspondingly, a single immunization with DCs is usually not sufficient to cure an established and peripherally growing tumor; maintenance of CTL activity by repetitive DC immunization is required to control and to eventually cure such a tumor 21. In a first set of experiments, we evaluated the consequences of an antitumor response against a model tumor antigen that is also expressed on strictly peripheral pancreatic β islet cells. RIP-GP mice express the GP of LCMV exclusively in pancreatic β islet cells, where the neo–self-antigen is immunologically ignored unless mice are infected with LCMV 22 or immunized with GP-expressing DCs 29 to induce an appropriate anti–LCMV-GP–specific immune response in secondary lymphoid organs. LCMV-GP was also transfected into fibrosarcoma cell line MC57 (MC-GP; references 21 and 27). Small pieces of MC-GP tumors (2 x 2 x 2 mm) were implanted subcutaneously into RIP-GP mice, and therapeutic treatment was started when successful tumor growth could be determined by palpation (tumor diameter 
5 mm, usually around day 14 after transplantation). Normal C57BL/6 mice do not generate an anti–LCMV-GP response after transplantation of MC-GP tumor pieces because tumor cells do not reach lymphoid organs 21. Similarly, in RIP-GP mice, MC-GP tumor pieces grew without inducing a specific immune response, and the mice remained normoglycemic (Fig. 1 A). When treatment of mice was started 14 d after transplantation with repetitive injection of 2 x 105 DCs constitutively expressing the immunodominant CTL epitope of LCMV-GP (H8-DC), the treatment also led to efficient tumor control in RIP-GP mice (Fig. 1 B). However, these recipients rapidly developed diabetes with an early onset and a severe disease outcome (30% of the mice died before day 21; Fig. 1 B), comparable to or slightly more severe than that of RIP-GP mice without MC-GP tumors treated with the same protocol (10% of the mice died before day 21; Fig. 1C).
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Taken together, these results show that previously ignored self-antigens, such as LCMV-GP in RIP-GP mice or β-galactosidase in SM-LacZ mice, may serve as target antigens for vaccination using antigen-expressing DCs. However, potentially harmful autoimmune responses against these self-antigens are induced by DC treatment when a strong and sustained CTL activity is necessary to eliminate the tumor. Depending on the tumor size and the correspondingly needed prolonged maintenance of antitumor/antiself CTL activity, severe and even lethal autoimmunity may develop before tumor growth is controlled.
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Immunological Ignorance as an Avenue for Therapeutic Tumor Immunotherapy.
It is conceivable that therapeutic tumor immunotherapy is most promising when tumor antigen–specific T cells of high avidity have not been centrally or peripherally deleted, i.e., the antigen has stayed outside of lymphatic tissues and therefore has been immunologically ignored. It appears that immunological ignorance of antigens strictly expressed in peripheral tissues is a function of antigen distribution and expression level. For example, in transgenic model situations with low levels of antigen expression exclusively in pancreatic β islets 2230 or solely in cells of the cardiovascular system, this report shows that the antigens are immunologically ignored. In contrast, high levels of self-antigen expression leads either to central 3132 or peripheral tolerance 3334. Similarly, tumor antigens can be either immunologically ignored when expressed at low levels in the periphery 213536 or induce a CTL immune response when they are expressed abundantly and reach local secondary lymphoid organs 37. It has been suggested that presentation of such tumor antigens by bone marrow–derived APCs in secondary lymphoid organs may lead to tumor-specific T cell unresponsiveness, particularly in the CD4+ T cell compartment 3839. In a study with patients with metastatic melanoma, Lee et al. 40 found that in 1 out of 11 patients, tumor-specific CD8+ T cells might also become unresponsive. From these examples, it is not clear whether such tumor-specific CD8+ and/or CD4+ T cell unresponsiveness is the general rule that leads to a failure of tumor immunity or rather the exception.
We favor the view that central and/or peripheral tolerance mechanisms usually do not exist against peripheral tumor antigens of sarcomas and carcinomas and that the majority of tumor and self-antigens are immunologically ignored. This is supported by the findings that high frequencies of tumor-specific CTLs exist in both tumor patients and healthy individuals 4142 and that antitumor CTLs may be of high avidity 43. Most importantly, the potent (unimpaired) function of antitumor T cells can be observed in melanoma patients suffering from vitiligo, where tumor-specific T cells mediate autoimmune depigmentation 8. It is therefore important for tumor immunotherapy to use as targets antigens that have been previously immunologically ignored. However, immunization with, for example, DCs efficiently presenting so far immunologically ignored shared tumor antigens will cause rejection of tumors but will also lead to autoimmunity. The two experimental systems used in this report model such a shared natural tumor situation: immune surveillance against potentially immunogenic tumor antigens fails because the tumor antigens are immunologically ignored for too long. Consequently, neither autoimmunity nor an antitumor response was induced. DC priming controlled the tumor and elicited a severe autoimmune response against pancreatic β-islet cells or cells of the cardiovascular system that was life threatening. Of course, immunotherapy with antigens derived from less essential organs may cause transient and less severe disease.
Implications for the use of DCs in Antitumor Immunotherapy.
Some experimental murine tumors express target antigens restricted solely to the tumor and can therefore be efficiently cured without inducing autoimmune phenomena using, for example, DCs 151617. In cases in which non–tumor-restricted antigens are used, autoimmune sequelae are probably not observed when the requirements for immune activity are rather low and tumor elimination is achieved before autoimmune symptoms appear 181920. This probably depends largely on the relative size of the tumor versus the size and importance of the autoimmune target, as well as on the relative precursor and effector T cell frequencies. Furthermore, it is possible that tumor cells may provide certain factors compensating for the lack of healthy tissue that is destroyed during curative antitumor treatment (e.g., in the model study of Speiser et al. [36], where insulin-producing insulinoma cells compensate for the destruction of pancreatic β-islet cells during antitumor treatment).
Under certain experimental transgenic conditions, high-avidity CTLs reactive for both tumor and self-antigens may be deleted. The remaining appropriately activated low-avidity CTLs may provide protection against subsequent tumor challenges but may not suffice to provoke autoimmunity 44. Alternatively, it is possible that CD4+ T cells alone mediate tumor immunity. For example, it has been shown in a transgenic mouse model that CD4+ T cell–mediated antitumor immune responses do not lead to obvious autoimmune sequelae when a retroviral tumor antigen is expressed in normal lymphoid tissues 45. However, it is questionable whether CD4+ T cell–mediated antitumor immune responses alone may be sufficient to cure rapidly growing tumors in the periphery via adoptive immunotherapy.
As stated, in situations where self- and tumor-reactive, high-avidity CTLs are not centrally or peripherally deleted and the antigens are immunologically ignored, such as in RIP-GP or SM-LacZ mice, strong antitumor responses that are also directed against self-antigens will eventually cause autoimmunity. This has also been observed in nontransgenic situations when mice were immunized with the melanoma antigen gp75/tyrosinase-related protein 1, leading to mainly T helper–mediated protection against tumor challenge and also to mild depigmentation 4647. Similarly, in clinical studies, induction of autoimmune depigmentation has been associated with a good prognosis for melanoma patients, indicative of successful control of the tumor by the immune system 8. Recently, it has been shown that paraneoplastic cerebellar degeneration (PCD) is probably mediated by CTLs specific for tumor antigens present on gynecological tumors and in neuronal tissue 48. Interestingly, PCD-associated gynecological tumors have been documented to regress with the onset of autoimmune neurological disease 9. The clinical consequences of autoimmune reactions against normal melanocytes causing vitiligo during melanoma treatment or a strong autoimmune reaction against organs where the loss of function can be substituted, e.g., with insulin for damage of pancreatic β islet cells, are acceptable during immunotherapeutic antitumor treatment. In contrast, other autoimmune diseases, such as reactions against cells in the cardiovascular system or neuronal tissue, may impose limitations on antitumor therapy with DCs using antigens also expressed by cells in vital organs.
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Acknowledgments |
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This work was supported by the Swiss National Science Foundation, the Deutsche Forschungsgemeinschaft (to B. Ludewig), and the Kanton Zürich.
Submitted: 25 October 1999
Revised: 28 December 1999
Accepted: 7 January 2000
Abbreviations used in this paper: DCs, dendritic cells; GP, glycoprotein; RIP, rat insulin promotor.
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| TABLE OF CONTENTS |
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) or without (
) peptide β-gal 497–504 were used in vitro as targets in a 51Cr-release assay. Effector cells were β-gal 497–504-restimulated spleen cells from C57BL/6 mice infected with 2 x 106 pfu VV-LacZ. (B) 2 x 107 EL4-LacZ tumor cells were injected subcutaneously into the flanks of C57BL/6 mice. 8 d later, splenocytes were restimulated in vitro using irradiated, β-gal 497–504-pulsed spleen cells for 5 d, and the CTL activity was determined in a 51Cr-release assay on β-gal 497–504-labeled EL4 target cells (closed symbols) or on EL4 cells without peptide (open symbols). (C) Small pieces of EL4-LacZ tumors were implanted subcutaneously in the flanks of SM-LacZ mice on day 1, and tumor growth was assessed at the indicated time points. (D) SM-LacZ mice received small EL4-LacZ tumor pieces as in C and were treated from day 0 repetitively by intravenous injection with 3–5 x 105 β-gal 497–504-pulsed DCs. Arrows, day of injection. Numbers indicate the proportion of tumor rejection (or tumor growth) in tumors tested. Results from one of two comparable experiments are shown.