Gene Therapy (2003) 10, 134–140 D 2003 Nature Publishing Group All rights reserved 0929-1903/03 $25.00 www.nature.com/cgt

Fas -expressing tumors induce tumor-specific protective immunity in the inoculated hosts but vaccination with the apoptotic tumors suppresses antitumor immunity Yuji Tada,1,2 Jiyang O-Wang,1 Akihiko Wada,1,2 Yuichi Takiguchi,2 Koichiro Tatsumi,2 Takayuki Kuriyama,2 Shigeru Sakiyama,1 and Masatoshi Tagawa1 1Division of Pathology, Chiba Cancer Center Research Institute, Chuo-ku, Chiba, Japan; and 2Department of Respirology, Graduate School of Medicine, Chiba University, Chuo-ku, Chiba, Japan.

The interaction between Fas and (FasL) is involved in the apoptotic death of a number of cells including . Forced expression of FasL in tumors can induce of infiltrating Fas-positive T cells; accordingly, tumors can survive in the milieu of systemic immune responses. However, FasL-expressing murine lung carcinoma (A11) and melanoma (B16) cells did not develop subcutaneous tumors and FasL-expressing A11 (A11/FasL) cells produced few spontaneous lung metastatic foci in syngeneic mice. The mice that rejected A11/FasL cells were resistant to subsequent challenge of parent A11 but not irrelevant B16 cells. Vaccination of mice with UV-treated A11/FasL, but not UV-treated A11 cells, however, augmented the growth rate of A11 but not B16 tumors, both of which were subsequently inoculated. The number of lung metastatic foci of A11 cells was also increased in the mice that received UV-treated A11/FasL but not UV-treated A11 cells. Intraperitoneal injection of UV-treated A11/FasL cells resulted in the production of larger amounts of immunosuppressive TGF-b in peritoneal exudate than that of UV-treated A11 cells. Expression of the CD80 costimulatory molecule in tissues where UV-treated A11/FasL cells were inoculated was lower than the expression at an untreated A11/FasL–injected site. Our results indicated that apoptotic FasL-expressing tumor cells could impair host immune responses against the tumors, in contrast to potent antitumor immunity generated by viable FasL-expressing tumors. Cancer Gene Therapy (2003) 10, 134–140 doi:10.1038/sj.cgt.7700545 Keywords: Fas ligand; protective immunity; apoptosis; immune suppression; TGF-b

as is a transmembrane belonging to the apoptosis of Fas-positive activated T cells that infiltrated Ftumor necrosis factor family and activates an into the tissues.7,8 Ectopic expression of FasL in tumors can, apoptotic signal pathway upon the ligation with its therefore, be an escape mechanism of tumors to evade host physiological ligand (Fas ligand, FasL).1,2 The Fas/FasL immune responses.9–11 However, forced expression of the system has been implicated in clonal deletion of auto- FasL gene in tumors induced regression of the tumors, which reactive lymphocytes and in elimination of activated lym- was postulated to be due to -mediated inflamma- phocytes to down-regulate immune responses. The system tory reactions.12 –14 The immunological significance of FasL thereby plays a crucial role in the maintenance of immune expression in tumors thereby remains controversial.15,16 .3 Loss of Fas expression or mutation of the Local production of can modulate the effects of FasL gene in experimental animal models generated lym- expressed FasL; TGF-b inhibited activation of neutrophils phoproliferative disorders associated with autoimmune and decreased FasL-mediated antitumor activity.17 responses.4,5 FasL is also involved in one of the effector In this study, we established murine lung carcinoma mechanisms through which cytotoxic T lymphocytes or (A11) and melanoma (B16) cells expressing FasL and natural killer cells fulfill their killing function toward target examined whether an antitumor effect or immunological cells.6 tolerance to tumor cells could be observed in the inoculated The immune-privileged sites such as testis and the mice. We found that the FasL expressed in tumors produced anterior chamber of the eyes, where systemic immunity a bipolar effect: induction or suppression of antitumor was poorly induced, expressed FasL locally and induced immunity, depending on the viability status of the tumors.

Received September 19, 2002. Materials and methods Address correspondence and reprint requests to: Dr Masatoshi Tagawa, Mice and cells Division of Pathology, Chiba Cancer Center Research Institute, 666-2 18 Nitona, Chuo-ku, Chiba 260-8717, Japan. A11 cells, established from Lewis lung carcinoma, and E-mail: [email protected] B16 melanoma cells were cultured in Dulbecco’s modified Apoptotic FasL-expressing tumors suppresses immunity Y Tada et al 135 Eagle’s medium supplemented with 10% heat-inactivated fetal calf serum. Syngeneic C57BL/6 mice (6- to 8-week- old females) were purchased from Japan SLC (Hamamatsu, Japan).

Establishment of FasL-expressing cells Cells were transfected with the full-length murine FasL cDNA1 or vector pMKITneo DNA with a lipofectin reagent (Life Technologies, Gaithersburg, MD). G418 (Life Tech- nologies) resistant clones were selected and examined for their expression of surface FasL with flow cytometry.

B-cell apoptosis Spleen cells of C57BL/6 mice were incubated with culture supernatants from a myeloma cell line producing fused soluble CD40 ligand (CD4OL)/CD8 protein19 for 2 days. They were added onto adherent tumor cells and cultured for

Figure 2 Growth of subcutaneous tumors in syngeneic mice. Mice were injected with (A) parent A11, vector DNA–tranfected A11 (A11/Vect) or A11/FasL cells, or (B) parent B16, vector DNA– transfected B16 (B16/Vect) or B16/FasL cells (n=7). Standard error bars are also shown.

12 hours. The spleen cells were then stained with fluorescein isothiocyanate–conjugated monoclonal antimouse B220 (PharMingen, San Diego, CA) and 1 g/mL pro- pidium iodide (PI) for 20 minutes in the presence of 0.1% (wt/vol) sodium azide. The stained cells were analyzed with FACScan (Becton Dickinson, San Jose, CA) through the CellQuest software (Becton Dickinson). Anti-Fas antibody (Jo-2, 1 ng/mL; PharMingen) was used as a positive control to induce apoptosis of Fas-positive B cells. Animal studies Parent or transfected cells (2Â105 ) were subcutaneously Figure 1 Cytotoxic activity of FasL-expressing tumor cells. CD40L- stimulated spleen cells were incubated with A11, A11/FasL, B16, or inoculated into syngeneic mice. Tumor volumes were calcu- lated according to the following formula: 1/2Âlength B16/FasL cells and were examined for B-cell apoptosis (B220- 2 positive and PI-positive fraction). Treatment with apoptosis-inducing (width) . For spontaneous lung , parent and 5 anti-Fas antibody ( Jo- 2) killed spleen B cells. Percent cell transfected cells (2Â10 ) were injected subcutaneously into populations of each fraction are also shown. the abdominal flank of mice. The lungs were fixed with

Cancer Gene Therapy Apoptotic FasL-expressing tumors suppresses immunity Y Tada et al 136 Bouin’s solution and the number of metastatic nodules was of respective cDNA was performed for 25 (b-actin) or 30 counted on day 30.20 Statistical analysis was performed by cycles (CD4, CD8a, CD40, and CD80) with the following one-way analysis of variance (ANOVA). primers and conditions: for -actin , forward (50 -ATGGATGACGATATCGCT-30 ) and reverse 0 0 Vaccination (5 -ATGAGGTAGTCTGTCAGGT-3 ) primers, and 5 sec- onds at 958C for denaturation/10 seconds at 548C for primer Cells were washed with phosphate-buffered saline and were annealing/70 seconds at 728C for primer extension; for the exposed to 200-J (A11 and A11/FasL cells) or 600-J (B16 CD4 gene expression, forward (50 -GAAGGGGAA- and B16/FasL cells) ultraviolet (UV) radiation. They 0 0 5 TCAGCAGAACT-3 ) and reverse (5 -CTTCTCTGCCT- (2Â10 ) were then subcutaneously inoculated into synge- TCCACATCA- 30 ) primers; for the CD8 gene, forward neic mice. More than 95% of the cells became apoptotic (50 -CACCCGAACTCCGAATCTTT-30 ) and reverse (50 - under the experimental condition. To prepare necrotic cells, 0 5 AT CACAGGCGAAGTCCAATC-3 ) primers; for the cells (2Â10 ) were frozen and immediately thawed once, CD40 gene expression, forward (50 -TGATTTGTGCC- and then subcutaneously inoculated into mice. One cycle of AGCCAGGAA-30 ) and reverse (50 -CCCGAAAATGGTG- the freeze/thaw procedure did not disrupt cells into ATGAGGA-30 ) primers; for the CD80 gene expression, fragments but kept membrane configuration intact with 0 0 21 forward (5 -TATTGCTGCCTTGCCGTTAC-3 ) and reverse increased membrane permeability. Mice were immunized (50 -TAATGGTGTGGTTGCGAGTC-30 ) primers, and 5 se- with UV-treated or freeze/thaw–treated cells and then were conds at 958C/10 seconds at 568C/60 seconds at 728C. subcutaneously inoculated with parent cells 14 days later.

Measurement of TGF- Results UV-treated (200-J) or untreated A11 cells (1Â106) were Establishment of FasL-expressing cells inoculated into the peritoneal cavity of syngeneic mice. A11 and B16 cells were transfected with FasL cDNA and Phosphate-buffered saline (5 mL) was injected and two clones that expressed the highest level of FasL on cell aspirated sequentially. The aliquots of the peritoneal exudate surface (A11/FasL and B16/FasL, respectively) were were tested for their concentrations of TGF-b with enzyme- selected. Their ability to kill Fas-positive cells was tested linked immunosorbent assay (R&D Systems, Minneapolis, with cocultured spleen B cells that were stimulated with MN). soluble CD40L (Fig 1). A11/FasL and B16/FasL cells, but not their respective parent cells, killed B220-positive B cells, Reverse transcription polymerase chain reaction (RT-PCR) which came to express Fas molecules upon CD40L A11/FasL cells (1Â107 ), either exposed to 200-J UV or stimulation, implying that membrane-bound FasL on A11 unexposed, were subcutaneously injected into syngeneic or B16 cells was able to induce cell death through the Fas/ mice and, 24 hours later, RNA was extracted from the tissues FasL interaction. Cell proliferation rates in vitro of A11/ at the inoculation sites. First-strand cDNA was synthesized FasL and B16/FasL cells were not different from those of with random primers and amplification of an equal amount respective parent cells (data not shown). Although A11, but

Figure 3 Induction of tumor-specific protective immunity. A11 (A)or B16 cells (B)were inoculated into naive or A11/FasL–rejected mice (n=6). Standard error bars are also shown.

Cancer Gene Therapy Apoptotic FasL-expressing tumors suppresses immunity Y Tada et al 137 not B16, cells were positive for Fas, expression of FasL on A11 did not induce apoptotic cell death. The expression level of class I molecules of the major histocompatibility complexes remained unchanged after the forced expression of FasL (data not shown).

Decreased tumorigenicity of FasL-expressing tumor cells We subcutaneously inoculated FasL-expressing cells or their parent cells into syngeneic mice. The mice that received parent or vector DNA-transfected cells developed tumors, whereas those injected with A11/FasL or B16/ FasL cells did not (Fig 2, A and B). We then inoculated a tumorigenic dose of A11 or B16 cells into the mice that had rejected A11/FasL cells. Half of the mice did not develop A11 tumors and the rest of the mice formed small masses, which did not grow further (tumor volumes developed in naive versus A11/FasL–rejected mice: P<.01, day 24) (Fig 3A). In contrast, all the mice that had rejected A11/ FasL cells developed B16 tumors and the tumor growth was not different from that of B16 tumors in naive mice (P=.52, day 24) (Fig 3B). Tumor-specific protective immunity was, therefore, induced in the mice that rejected A11/FasL cells. A11 cells produced spontaneous lung metastatic foci when they were subcutaneously inoculated in the flank. However, the number of lung foci was significantly decreased when A11/FasL cells were inoculated (Table 1). Expression of FasL in tumors also inhibited spontaneous metastasis of the tumors.

Vaccination effects with FasL-expressing tumor cells As inoculation of A11/FasL cells induced systemic immune responses, we examined whether antitumor effects could be produced by the vaccination of syngeneic mice with A11/ FasL cells. Treatment of parent or FasL-expressing cells with UV did not affect their cytotoxic activity to Fas- positive B cells (data not shown). Mice were immunized with A11 or A11/FasL cells, either UV- or freeze/thaw– treated, and then were inoculated with A11 cells 14 days Figure 4 A: Growth of A11 tumors developed in untreated or vaccinated mice that received UV- or freeze/thaw (F/T)–treated A11 or A11/FasL cells. B: Growth of B16 tumors in untreated or vaccinated mice that received UV-treated B16, B16/FasL, or A11/ Table 1 Spontaneous lung metastasis produced by A11 or A11/ FasL cells. Standard error bars are also shown. FasL cells in naive or vaccinated mice with UV-treated cells

Number of lung Inoculated cells foci on day 30 later. Vaccination of mice with A11 cells, either UV- or Vaccination (2Â105 ) (average±SE) freeze/thaw–treated, retarded tumor growth of A11 cells None A11/parent 2, 7, 10, 34, 51, 58*yz that were subsequently challenged, compared with the (27±9.8) growth of A11 tumors in untreated mice (UV, P=.071; None A11/FasL 0, 0, 0, 0, 0, 1* freeze/thaw, P=.074, day 24) (Fig 4A). However, immu- (0.17±0.17) nization with UV-treated A11/FasL cells significantly en- UV-treated A11/parent 3, 4, 7, 11, 24, 35yx hanced the growth of A11 tumors compared with the growth A11/parent (14±5.2) in untreated mice (P<.01, day 21) or in the mice immu- UV-treated A11/parent 33, 41, 71, 122, 129, 181zx nized with UV-treated A11 cells (P<.01, day 24) (Fig 4A). A11/FasL (96.2±23.5) When mice were immunized with freeze/thaw–treated *P=.021. A11/FasL cells, the growth of A11 tumors remained the yP=.27. same as that in untreated mice. The growth was slightly zP=.022. greater than that in the mice immunized with freeze/thaw– xP<.01. treated A11 cells (P=.082, day 24) but was suppressed

Cancer Gene Therapy Apoptotic FasL-expressing tumors suppresses immunity Y Tada et al 138 compared with that in the mice immunized with UV-treated A11/FasL cells (P=.051, day 21). We also examined the vaccination-induced enhanced tumor growth using B16/FasL cells. Syngeneic mice were immunized with UV-treated B16 or B16/FasL cells and then subsequently inoculated with B16 cells. Immunization with UV-treated B16/FasL cells significantly enhanced the growth of B16 tumors compared with that in untreated and immunized mice with UV-treated B16 cells (P=.020 and P<.01, respectively, day 24) (Fig 4B). In contrast, immunization with UV-treated B16 or A11/FasL cells did not affect the tumor growth of B16 cells subsequently inoculated. Effect of the vaccination on spontaneous lung metastasis was examined by immunization with UV-treated A11 or A11/FasL cells followed by subcutaneous inoculation of A11 cells (Table 1). Immunization with UV-treated A11 cells did not influence the number of lung foci compared with that of the foci developed in untreated mice. However, the focus number was significantly increased in the mice immunized with UV-treated A11/FasL cells.

Intraperitoneal concentrations of TGF- by inoculating FasL-expressing cells Figure 6 Expression of cell surface molecules in the tissues where We sequentially measured the concentrations of TGF-b in UV -treated or untreated A11 / FasL cells were subcutaneously the peritoneal cavity of the mice that received untreated or injected. RT-PCR was performed with RNA that were extracted UV-treated cells (Fig 5). A11 and A11/FasL cells did not 24 hours after the injection. produce detectable amounts of TGF-b in vitro ( <10 pg/mL) and the concentration of TGF-b in the peritoneal cavity of naive mice was 100±25 (pg/mL±SE). When untreated or hours, P=.015; treated cells–injected, 3 vs 24 hours, UV-treated A11 cells were inoculated, the amount of P<.01). The amount of TGF-b at 24 hours in the mice TGF-b became maximal at 9 hours after the inoculation injected with UV-treated A11/FasL cells was significantly and returned to an uninjected level. The amount at each larger than that in untreated A11/FasL– or A11-injected time was not different between untreated A11 and UV- mice (P<.01). In contrast, the concentrations of TNF-a treated A11-injected mice. Inoculation of A11/FasL cells, were not different between the mice injected with UV- untreated or UV-treated, increased the amount of TGF-b treated A11 cells and those with UV-treated A11/FasL cells after the inoculation (untreated cells–injected, 3 vs 24 (data not shown).

Expression of CD80 at tumor injection sites We compared the expression of the CD4, CD8a, CD40, and CD80 genes in tissues where UV-treated or untreated A11/ FasL cells were injected (Fig 6). Expression of the CD4, CD8a, and CD40 genes was higher at the injection site of UV-treated A11/FasL cells than that at the untreated A11/ FasL cells–injected site. In contrast, expression of CD80 costimulator molecule was lower at the injection site of UV-treated A11/FasL cells. These data suggest that CD40 + antigen-presenting cells, CD4 + , and CD8 + T cells migrated into the injection site of UV-treated A11/FasL cells better than that of untreated A11/FasL cells; however, dendritic cells (DCs) at the injection site of UV-treated A11/FasL cells were less activated compared with those at A11/FasL cells–injected site.

Figure 5 Concentrations of TGF-b in the peritoneal exudate from the mice that were inoculated with A11 or A11/FasL cells, either UV- treated or untreated ( À ). The concentrations were sequentially Discussion measured after the inoculation. Concentration of TGF-b in the peritoneal cavity in naive mice was 100±25 pg/mL. Standard error In this study, we showed that immunocompetent mice bars are also shown. produced significant antitumor effects on FasL-expressing

Cancer Gene Therapy Apoptotic FasL-expressing tumors suppresses immunity Y Tada et al 139 murine carcinoma cells, and the mice that had rejected FasL- thereby allow DCs to induce peripheral tolerance to putative expressing tumors generated tumor-specific protective im- tumor antigen(s) in the presence of immunosuppressive munity. Although the present study suggested that cytotoxic cytokines.26 On the contrary, exposure of DCs to necrotic T cells specific for FasL-expressing tumors were generated, tumor cells provides requisite maturation signals, which a possible involvement of other cell populations in the FasL- results in the up-regulation of costimulatory molecules and mediated antitumor responses cannot be excluded. Previous in the augmented induction of antigen-specific CD4 + and studies reported that neutrophils-mediated inflammatory CD8 + T cells.21 At the site where UV-treated A11/FasL reactions were primarily responsible for FasL-mediated cells were inoculated, expression of the CD80 costimulatory antitumor effects12,13 and our separate experiments showed molecule was relatively low compared with that of CD40. As that A11/FasL cells were also rejected in –defective CD40 and CD80 are up-regulated upon the maturation of nude mice and that intraperitoneal inoculation of A11/FasL DCs,21 the increased expression of CD40 observed at the cells induced -dominant peritoneal exudation. inoculation site of UV-treated cells could be due to increased However, we observed that mice inoculated with a mixed numbers of DCs and/or B cells. Relatively lower expression population of A11/FasL and B16 cells developed B16 but of the CD80 gene compared with expression of the CD4, not A11/FasL tumors, suggesting that T cells, which CD8a, and CD40 genes suggests that cognate interaction of recognize the specificity of tumors, could be actively in- DCs and T cells was impaired, although T and antigen- volved in FasL-mediated antitumor responses (manuscript presenting cells were migrated into the inoculation site. in preparation). Extravasation of neutrophils and subsequent Inoculation of UV-treated FasL-expressing tumors thereby release of proinflammatory cytokines from neutrophils can suppressed the maturation of DCs. Our separate experiments be an initial step for antitumor responses, but we presume showed that bone marrow–derived DCs could form cluster that prompt migration of DCs into the tumors and subsequent in vitro with Fas-expressing, but not with parent tumor, cells cognate interaction between antigen-loaded DCs and naive (manuscript in preparation). We thereby presume that T cells facilitate the development of T cell–dependent immature DCs interact with FasL-expressing tumors in vivo systemic immunity. but could not be mature partly due to TGF-b and other Although the FasL-expressing tumors induced systemic immunosuppressive cytokines secreted from ; immunity, vaccination effects were not achieved with the consequently, T cell–mediated antitumor immunity is sup- UV-treated FasL-expressing tumors. Immunization of mice pressed. TGF-b, in fact, prevents the maturation of DCs,27 with A11 cells, treated either with UV or freeze/thaw and Chen et al17 demonstrated that secretion of TGF-b from procedure, retarded subsequent growth of A11 tumors, FasL-expressing tumors allowed the tumor growth; other- suggesting that A11 cells were immunogenic to stimulate wise, FasL-expressing tumors were rejected. systemic immunity. The level of the antitumor activity In conclusion, forced expression of the FasL gene in triggered by the immunization was the same irrespective of tumors generated bivalent immune responses; live FasL- the treatment procedure. However, immunization with UV- expressing tumors induced systemic antitumor responses, treated but not freeze/thaw–treated A11/FasL cells whereas apoptotic tumors suppressed antitumor immunity. enhanced the growth of A11 but not B16 tumors. The Expression of FasL in live tumors is a possible thera- enhanced tumor growth was also achieved by the immuni- peutic strategy for cancer but apoptotic cells included in zation with UV-treated B16/FasL cells. Suppression of the preparation can lessen the therapeutic effects. Deple- systemic immunity was confirmed in the experimental lung tion of immunosuppressive cytokines and provision of metastasis system. Administration of apoptotic FasL- maturation signals to DCs can be crucial to shift a pos- expressing tumor cells, therefore, induced a tumor-specific sible FasL-mediated tolerance toward systemic antitumor tolerant state. Melcher et al22 suggested that tumor cell death responses. by nonapoptotic mechanisms was associated with high immunogenicity, whereas apoptotic cells were considerably less immunogenic. Upon apoptotic cell death, macrophages Acknowledgments phagocytose the apoptotic cells and produce immunoregu- latory factors such as IL-10 and TGF-b that skew immunity This work was supported by a grant-in-aid for scientific to suppressive and anti-inflammatory responses; the pro- research from the Japan Society for the Promotion of Science duction of proinflammatory cytokines was suppressed.23,24 and a grant-in-aid for scientific research on priority areas Our study, in fact, demonstrated the increase of TGF-b but from the Minister of Education, Culture, Sports, Science, and not TNF-a in the mice that received apoptotic FasL- Technology of Japan. expressing cells. Although macrophages can play an important role in noninflammatory and nonimmunogenic clearance of cells undergoing apoptosis,25 we have not References histologically detected the migration of macrophages at the inoculation sites. 1. Suda T, Takahashi T, Goldstein P, et al. Molecular cloning and DCs can also contribute to the silencing of immune expression of the Fas ligand, a novel member of the tumor responses. Immature DCs phagocytose apoptotic cells; necrosis factor family. Cell. 1993;75:1169–1178. however, they do not efficiently cross-present antigens or 2. Nagata S. Fas and Fas ligand: a death factor and its receptor. activate cytotoxic T cells unless they receive maturation Adv Immunol. 1994;57:129–144. stimuli such as TNF-a.21 Uptake of apoptotic cells can 3. Alderson MR, Tough TW, Davis-Smith T, et al. Fas ligand

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