Murine mammary adenocarcinoma cells transfected with p53 ... - Nature

Cancer Gene Therapy (2005) 12, 427–437 All rights reserved 0929-1903/05 $30.00

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Murine mammary adenocarcinoma cells transfected with p53 and/or Flt3L induce antitumor immune responses Hongxun Sang,1,2 Vladimir M Pisarev,2,3 Jennifer Chavez,2 Simon Robinson,2 Yajun Guo,3 Lori Hatcher,2 Corey Munger,2 Cathy B Talmadge,2 Joyce C Solheim,2,3,4 Rakesh K Singh,2 and James E Talmadge2,3 1

International Joint Cancer Institute of Shanghai and Institute of Orthopaedics, Xijing Hospital, Xi’an, 710032, PR China; 2Laboratory of Transplantation Immunology, Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA; 3Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska, USA; and 4Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, USA. Transfection of tumors with tumor-associated antigens (Ags) or cytokines can increase immunogenicity and slow down tumor growth. However, the effect of cotransfection with genes that encode a tumor-associated Ag, such as the tumor suppressor gene p53, and a cytokine has been rarely investigated. We report that transfection of 4T1 mammary tumor cells (p53-null) with the dendritic cell (DC) growth factor, fms-like tyrosine kinase 3 ligand (Flt3L), significantly delayed their growth in vivo, resulting in the rejection of 100% of the tumors formed by injection of tumor cells cotransfected with Flt3L and p53. Immunization with irradiated 4T1 cells transfected with Flt3L induced DC infiltration of the immunization site and significantly increased the antitumor T-cell responses. Further, immunization with irradiated 4T1 cells cotransfected with p53 and Flt3L significantly increased p53-specific immune responses, as compared to vaccination with 4T1 cells transfected with either Flt3L or p53 alone. These responses included increased activity against clone 66 (Cl-66), a sister tumor to 4T1 with high murine mutant p53 expression levels. Challenge with Cl66 revealed that immunization with irradiated 4T1-Flt3L-p53 cells significantly slowed growth, prolonged survival, and resulted in complete remissions. Further, immunization with irradiated 4T1-Flt3L also slowed Cl-66 growth, although to a lesser extent than 4T1-Flt3L-p53. We suggest that immunization with DCs transfected with the Flt3L transgene and a tumor Ag may potentially heighten T-cell responses and therapeutic activity. Cancer Gene Therapy (2005) 12, 427–437. doi:10.1038/sj.cgt.7700809 Published online 28 January 2005 Keywords: vaccine; Flt3L; p53; mammary tumor; dendritic cell

he tumor suppressor gene p53 is mutated and T subsequently overexpressed in 50% of all human malignancies and in 69% of women with breast cancer 1,2

who have four or more positive lymph nodes (LNs).3 Although p53 missense mutations are prevalent, the frequency of a given mutation at a specific codon is low.4 Further, p53 mutations may occur at multiple sites and be heterogeneous within a patient; therefore, a primary tumor and its metastases can express different p53 mutations.5,6 Thus, mutation-specific, p53-based vaccine therapy is impractical. The majority of p53 mutations affect the half-life of the p53 protein in tumor cells,7 resulting in p53 accumulation. This allows processing and presentation of wild-type (wt) p53 epitopes that are not presented by normal cells,6,8 Received August 3, 2004.

Address correspondence and reprint requests to: Dr James E Talmadge, PhD, Department of Pathology and Microbiology, 987660 Nebraska Medical Center, Omaha, NE 68198-7660, USA. E-mail: [email protected]

such that a broad T-cell response against p53 can be induced.6,9 These results suggest that wt p53 sequences can be used for specific immunotherapy by making the entire p53 sequence available as an antigen (Ag). Indeed, human cytotoxic T lymphocytes (CTLs) against wt p53 have been shown to be cytotoxic to tumor cells, but not to normal cells, and to be naturally induced in some patients.6,9 Further, CTLs generated against wt p53 can recognize multiple p53 epitopes, which potentially increases their effectiveness against p53-overexpressing tumors.6,8 Recent studies indicate that administration of dendritic cells (DCs) transfected with the wt p53 gene10–12 or pulsed with p53 peptides,13 or vaccination with an idiotypic p5314 or p53-Pox virus vaccine15 can stimulate a CTL response with antitumor activity, thereby supporting p53 as a potentially useful tumor-associated antigen (TAA). The immune system is tolerant towards many TAAs, making a vaccine adjuvant critical to the induction of a strong cellular immune response against TAAs. Flt3L is a pluripotent, hematopoietic growth factor that can expand

Tumor vaccine with Flt3L and p53 transgenes H Sang et al

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DCs within lymphoid and parenchymal tissue16,17 and also increase type 1 T-cell responses.18,19 The expansion of DCs is important because of their unique ability to efficiently internalize, process, and present Ags, as well as stimulate naı¨ ve and memory T cells.6,12,20 Adjuvant activity by Flt3L has been observed with plasmid Flt3L,21–27 Flt3L protein,18,19,28–34 Flt3L-transfected tumor cells,35–38 and vaccines composed of a plasmid linking an Ag and the receptor-binding domain of Flt3L.39–41 Clinical studies using recombinant Flt3L have also shown adjuvant activity.42–44 Here, we report a novel tumor vaccine strategy whereby a vaccine composed of irradiated 4T1 cells cotransfected with p53 and Flt3L induced a p53-specific T-cell response and nonspecific response to the tumor. Immunization with 4T1 cells expressing Flt3L, or p53 and Flt3L, also induced in vivo protection against challenge with a sister tumor (clone 66 (Cl-66)) that overexpresses p53. This suggests that immunization resulted in immunity to p53 and 4T1 Ags. These observations suggest that a therapy protocol utilizing tumor cells transfected with a TAA (p53) and Flt3L may be a promising strategy for the treatment of tumors.

Materials and methods

Plasmid DNA preparation A human Flt3L plasmid (pNGVL3-hFlex) was obtained from The National Gene Vector Laboratory at the University of Michigan. This plasmid encodes a secreted form of human Flt3L that is 72% identical to murine Flt3L and functions normally in mice, as well as a kanamycin resistance marker. The human p53 plasmid pSec-Tag2/B-p53 was constructed by cloning a mutant human p53 cDNA into the Sec-Tag2/B vector with a Zeocin resistance gene (Invitrogen Corp., Carlsbad, CA). This vector places the gene under the control of the cytomegalovirus immediate early gene promoter.45 The p53 cDNA sequence used in our construct has a mutation at codon 175,45 resulting in an Arg-to-His amino-acid substitution (gift from Dr B Vogelstein, Johns Hopkins Oncology Center, Baltimore, MD). The presence of the p53 coding region in the pSecp53 plasmid was confirmed by DNA sequencing. The p53 sequence is highly conserved and is 75% identical between humans and mouse (87% identical, if conservative substitutions are included). Following the manufacturer’s instructions, the plasmid was transformed into competent DH5a Escherichia coli (Invitrogen Corp., Carlsbad, CA), and plasmid DNA was purified using EndoFree Plasmid Giga kits (Qiagen, Valencia, CA).

Preparation of transfected tumor cells The mammary adenocarcinoma 4T1 is a murine metastatic mammary tumor cell line that is p53 null46,47 (gift from Dr F Miller, Michigan Cancer Foundation, Detroit, MI). Cells (2 105 4T1 cells) were prepared in a 60-mm culture dish for transfection using DMRIE-C Reagent

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(Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. The 4T1 transfected cell lines included: 4T1-pSec (vector control), 4T1-pSec-p53 (4T1-p53), 4T1pNGVL3-hFlex-pSec (4T1-Flt3L), and 4T1-pNGVLhFlex-pSec-p53 (4T1-P53-Flt3L). The transfected cells were selected and cloned in the presence of 800 mg/ml Zeocin (Invitrogen Corp., Carlsbad, CA), and were subsequently maintained in culture media containing 200 mg/ml Zeocin.

RT-PCR To verify the successful transfection of the 4T1 cells, reverse transcription-polymerase chain reaction (RTPCR) for human p53 and Flt3L was undertaken by a method similar to that previously reported.48,49 Briefly, RNA was extracted from 1 106 cells using Trizol (Invitrogen Corp., Carlsbad, CA) and RT-PCR was performed with the AmpliTaq Gold PCR KIT (Roche Inc., Foster City, CA). The specific PCR assay primers used for Flt3L were 50 -ACAACCTATCTCCTCCTGCT G-30 and 50 -GGCACATTTGGTGACAAAGTG-30 , which flank a 307 bp sequence. The p53-specific primers used were 50 -CCTTCCCAGAAAACCTACCA-30 and 50 TCATAGGGCACCACCACACT-30 , which flank a 371 bp sequence. Amplification of a GAPDH sequence was performed as a positive control. After 35 cycles of amplification, aliquots of the PCR reactions were analyzed by electrophoresis in a 2% agarose gel containing 0.5 mg/ml ethidium bromide.

Assessment of Flt3L and p53 protein production To verify the secretion of Flt3L protein, transfected cells were cultured and the supernatant collected for analysis. Flt3L levels were measured with an enzyme-linked immunosorbent assay (ELISA) using anti-human Flt3L antibodies (Abs) (R&D Systems Inc., Minneapolis, MN). The expression of p53 protein in 4T1-p53 and 4T1-p53Flt3L cells was confirmed by Western blot analysis using Ab-7 (Oncogene, Cambridge, MA).

In vitro tumor growth rate assessment The in vitro growth rate of the transfected cells was determined with 1000 cells added to each well of a 96-well plate in triplicate and incubated for 1, 3, or 5 days. The cells were stained with 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl-tetrazolium bromide (MTT) on days 1, 3, and 5 and the absorption at 570 nm was determined with a microtiter plate reader (Bio-Tek Instruments, Inc., Winooski, VT).

Immunization protocol The transfected cells were used as a vaccine following irradiation (50 Gy) in a cesium irradiator. For the challenge study after vaccination, both 4T1 and Cl-66 cells, which are cells from the same parent tumor, were used.50 The Cl-66 tumor cells express high levels of mutant murine p53,51,52 while the 4T1 cells are p53 null.46,47 Female BALB/cAnNCrlBR mice, 6–8 weeks of


age, were purchased from Charles River (Wilmington, MA) and, following a 2-week acclimatization, were immunized subcutaneously (s.c.) with 2 105 transfected or nontransfected 4T1 cells. The negative controls were injected with phosphate-buffered saline (PBS). The mice were boosted with the same number of irradiated cells on days 7, 14, and 21. At 14 days after the last immunization, the mice were challenged with the injection of Cl-66 tumor cells (100,000 cells/injection) into the mammary fat pad or their spleen cells were analyzed to determine their immune responses to p53.

counts per minute (CPM). In order to assess p53-specific proliferation, control culture proliferation was subtracted from each group and mean stimulation indices were calculated.19

ELISpot assay

At 2 weeks after the last immunization, 4 mg of p53 peptides (2 mg each of P15 and P16) in a volume of 30 ml were injected into the pinnas with PBS as the negative control. At 24 hours after challenge, the test and control ears were measured with a No. 7300 caliper (Mitutoyo, South Plainfield, NJ). The degree of edema was defined as the difference in the ear thickness between the control ear and the test ear.

Responder spleen cells from each immunized or control mouse, before or after IVS, were cocultured at 2 105 or 0.5 105 cells/well in 96-well culture plates with or without p53 nonapeptides (20 mg/ml final concentration each peptide), or with normal spleen cells only (control) or medium only (control), as previously described.19 Normal spleen cells plus phorbol 12-myristate 13-acetate (50 ng/ml final concentration) were used as the positive control. The capture Abs were Ab R4-6A2 (antiinterferon-gamma (IFN-g)) or 11B11 (anti-interleukin-4 (IL-4)), (BD-Pharmingen, San Diego, CA), and cytokinesecreting cells were visualized using 1 mg/ml biotinylated secondary Ab to IFN-g or IL-4 (Jackson Immuno Research Laboratories, Inc., West Grove, PA) followed by coincubation with alkaline phosphatase-conjugated avidin (Extravidin, Sigma, St Louis, MO) and Western Blue Substrate (Promega, Madison, WI). The number of spot-forming cells was enumerated by an automatic ELISpot reading system, the Axioplan 2 Imaging System with KS ELISpot software (Carl Zeiss Vesion, MunchenHallbergmous, Germany). The total number of spots was determined and the frequency of cytokine-producing cells was calculated as an average number of spot-forming cells after subtraction of the number of spots from wells containing spleen cell cultures in the absence of the p53 nonamers.

In vitro stimulation (IVS) of splenocytes

In vivo tumor growth and challenge assessment

Synthesis of p53 peptides Based on our previous studies,19 we used human p53 and P16 nonamers P15 (340MFRELNEAL348) 18 26 ( TFSDLWKLL ), identical (P15) or similar (P16) to murine p53-derived peptides. These peptides were predicted to bind to the H-2Kd groove using the algorithm developed by Parker et al.53 The peptides were synthesized by Quality Controlled Biochemicals, Inc. (Hopkinton, MA) using f-moc chemistry and purified by highperformance liquid chromatography (HPLC).

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Spleen cells were harvested and 5 10 cells from each immunized and control mouse were separately stimulated for 5 days in six-well plates with 1 107 irradiated normal BALB/cAnNCrlBr splenocytes, preincubated for 2 hours with both p53 nonamer peptides (20 mg/ml) in Click’s media (Invitrogen, Carlsbad, CA) supplemented with 0.5% normal mouse serum (NMS)-Click’s, 20 mg/ml gentamycin, 4 mM L-glutamine, and 5% rat T-Stim without concanavalin A (Collaborative Biomedical Products, Bedford, MA). Following a 5-day IVS, the cells were harvested and used in T-cell assays.

Mice (8 weeks old) were injected in the mammary fat pad with 1 105 4T1 cells, with or without p53 and/or Flt3L transgenes (groups: 4T1; 4T1-pSec; 4T1-p53; 4T1-Flt3L; and 4T1-Flt3L-p53). Twice a week, the tumor volumes were monitored in two perpendicular dimensions with calipers. The tumor volume was calculated using the formula for a prolated sphere (width2 length/2) and the tumor size was reported as the mean tumor volume. In the tumor challenge study, mice were challenged with 1 105 Cl-66 cells in the mammary fat pads 1 week after the last immunization. Tumor growth was then measured twice a week.

Lymphocyte proliferation Irradiated (50 Gy) normal spleen cells (40,000) in 0.5% NMS-Click’s media were added to each well of a 96-well plate with 20,000 irradiated 4T1-p53 cells or 50 mg/ml of each P15 and P16 peptide as stimulator or with medium alone. Splenocytes from the immunized or control mice, before or after IVS, were plated at 0.2 106 cells/well in 0.5% NMS-Click’s Eagle’s-Hank’s amino acid (EHAA) media and then the cocultures were incubated for 5 days. 3 H-thymidine was added at 1 mCi/well 16–18 hours before cell harvest. Cultures were harvested and 3H-thymidine incorporation was quantified by scintillation counting as

Immunohistochemistry (IHC) for DC accumulation Irradiated (50 Gy) 4T1 tumor cells, with or without Flt3L and p53 transgenes, were injected at 2 105 cells per injection site. The mice were killed 10 days later and the injection sites, together with the surrounding tissue (8–10 mm in diameter), were harvested and embedded in Tissue-Teks OCT (Bakura Finetec, Inc. Torrance, CA) and then frozen at 801C. Tissue sections 5–10 mm thick were fixed with cold acetone, endogenous peroxidase was blocked with hydrogen peroxide, and nonspecific binding was reduced with 10% normal serum. Primary Abs

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included rat anti-mouse DEC-205 (NLDC-145, Serotec Inc., Raleigh, NC) and rat anti-mouse follicular DC (FDC-M1, BD-PharMingen, San Diego, CA). The slides were incubated overnight at 41C, rinsed with PBS and incubated with a biotinylated secondary Ab (biotinylated anti-rat IgG, H þ L, Vector, Inc., Burlingame, CA). The slides were rinsed again and incubated with Vectastains ABC (Vector, Inc.) for 30 minutes before staining with 3,30 -diaminobenzidine (DAB) substrate (Vector, Inc.). The sections were counterstained with hematoxylin and viewed by light microscope.

Statistical analysis SPSS for Windows (SPSS, Inc., Chicago, IL) was used with the Student’s t-test (two-tail) to compare mean values. The repeated-measures test was undertaken with SAS for the tumor growth curves. A P-value r.05 was considered significant.

Figure 2 MTT cell proliferation assessment of transfected and nontransfected 4T1 cells. Proliferation of 4T1 cells following transfection with p53 and/or Flt3L was assessed using an MTT proliferation assay as described in Materials and methods. There were no significant differences in in vitro proliferation between transfected or nontransfected cells.

Results

Transfection of 4T1 cells with Flt3L and p53 transgenes The expression of human Flt3L and p53 mRNA by the transfected 4T1 cell lines was determined by RT-PCR (Fig 1). Secretion of human Flt3L by the transfected 4T1Flt3L and 4T1-Flt3L-p53 cells was confirmed by ELISA, and similar levels were found to be secreted by the two cell lines (71.86 mean76.63 and 61.1575.49 ng/106cells/24 hours, respectively). These Flt3L levels were significantly higher than the background levels observed with 4T1 or

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Tumor growth and survival of mice injected with p53- and/or Flt3L-transfected murine mammary adenocarcinoma cells The growth rates of the transfected 4T1 cell lines were assessed in mice injected orthotopically with 1 105 cells into a posterior, ventral mammary fat pad. The tumor volume was monitored twice a week. The results of these studies are shown in Figure 3a. Tumor growth was significantly slower in the mice injected with 4T1-Flt3L or 4T1-Flt3L-p53 tumor cells as compared to the other three groups (using repeated-measures statistical analysis). In addition, all of the mice injected with 4T1, 4T1-pSec, or 4T1-p53 died within 43–55 days post challenge. In contrast, the mice injected with 4T1-Flt3L or 4T1Flt3L-p53 had significantly increased survival (median of 51 and 58 days, respectively) compared to 4T1 and 4T1-pSec control tumors, with 17% of the 4T1-Flt3L-p53 tumors regressing (Fig 3b).

p53

Figure 1 RT-PCR analysis of human hFlt3L and p53 transgene expression. RT-PCR was performed on 4T1 cells transfected with hFlt3L- and p53-specific primers as described in Materials and methods. Lanes: (M) 100 bp DNA marker, (1) 4T1-pSec, (2) 4T1-FL, (3) 4T1-FL-p53, (4) 4T1-p53, and (5) 4T1-FL-p53. Upper panel: lanes 1–5 contain PCR products from GAPDH amplification. Bottom panel: lanes 2 and 3 contain PCR products from the Flt3L amplification and lanes 4 and 5 contain PCR products from the p53 amplification.

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4T1-p53 cells (0.1770.07 ng/106cells/24 hours). The expression of p53 protein in 4T1 cells transfected with p53, or p53 and Flt3L, was confirmed by Western blot analysis (data not shown). Regardless of Flt3L secretion or p53 expression, the MTT assay determined that the in vitro growth rates (Fig 2) of the transfected and nontransfected 4T1 cells were similar.

DTH response to p53 Ag nonamers in mice after immunization Transfected 4T1 tumor variants, following irradiation, were used as vaccines to assess their immunogenicity. As part of these studies, the DTH responses to p53 nonamer challange were analyzed following immunization. Significantly increased pinna edema was observed in the groups vaccinated with 4T1-p53 and 4T1-Flt3L-p53, as


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Figure 4 DTH responses to p53 peptides following immunizations. Mice were immunized with 4T1 tumor cells, with or without transfected Flt3L and p53 transgenes. At 2 weeks after the final immunization, mice were challenged with 4 mg of p53 nonapeptides (2 mg each P15 and P16 peptide) in the right pinna (and with PBS in the left pinna as a control). After 24 hours, the thicknesses of test and control ears were measured and the values from the control ears subtracted. Results are shown as the mean7SEM differences in ear thicknesses between the test and control ears. *Po.05 vs. PBS group, @Po.05 vs. 4T1-pSec immunized group. This study is representative of one of four studies; N ¼ 4/cohort.

Figure 3 Tumor growth following injection of p53 and/or Flt3L transfected 4T1 tumor cells. Mice were injected s.c. in the mammary fat pad with 1 105 nonirradiated 4T1 cells transfected with p53 and/or Flt3L. The tumor sizes were monitored by measurement of the tumors in two perpendicular dimensions and expressed as mean tumor volumes7SEM (a). The survival duration is shown in (b). *Po.05 vs. PBS group, @Po.05 vs. 4T1-pSec immunized group. This study is representative of one of three such studies. N ¼ 6/cohort.

compared to 4T1- and 4T1-pSec-injected mice (Fig 4). In contrast, the 4T1-Flt3L cohort did not have a significantly increased DTH reaction as compared to control mice. However, there were no differences in the DTH responses induced by 4T1-p53 as compared to 4T1-Flt3L-p53.

In vitro proliferation of lymphocytes in response to p53 Ag In order to study p53-specific lymphocyte proliferation, splenocytes from immunized mice were tested for their lymphoproliferative response against p53 nonamers or irradiated 4T1-p53 cells as stimulators. The splenocytes from mice vaccinated with 4T1-Flt3L-p53 or 4T1-p53 showed significantly higher T-cell proliferative responses to p53 nonamers as compared to splenocytes from

4T1-Flt3L or control (4T1-pSec or PBS) mice (Fig 5a). This difference was not observed in cells cultured in the absence of the p53 nonamers. In contrast to the results with p53 nonamers, when irradiated 4T1-p53 cells were used as the stimulators, only the splenocytes from the 4T1-Flt3L-p53-immunized mice demonstrated a significantly increased proliferative response (Fig 5b). In other studies where splenocytes were stimulated in vitro for 5 days in the presence of p53 nonamers, the lymphoproliferative responses to p53 nonamers and irradiated 4T1p53 cells were similar to that observed without an IVS (data not shown).

Frequency of Ag-specific IFN-g- or IL-4-secreting cells as determined by ELISpot The nature of the p53-specific response (type 1 or type 2) was assessed with ELISpot assays, which revealed a significantly higher frequency of both IFN-g- and IL-4secreting cells (Fig 6) in the mice immunized with 4T1Flt3L-p53, 4T1-Flt3L, or 4T1-p53 cells, as compared to PBS-injected control animals. Further, the frequency of IFN-g-secreting cells was significantly increased in 4T1Flt3L-p53-immunized mice as compared to mice immunized with 4T1-pSec or 4T1-p53 cells (Fig 6a). Compared to the PBS control, there was no difference in the frequency of IFN-g-secreting cells between the cohorts immunized with 4T1-pSec, 4T1-p53, or 4T1-Flt3L.

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Figure 5 In vitro lymphocyte proliferation response to p53 nonamers or 4T1-p53 cells. The graph shows Ag-specific proliferation to p53specific nonamers expressed as the mean stimulation index (SI)7SEM calculated after the subtraction of background counts (a). In another set of experiments, irradiated 4T1-p53 cells were used as stimulators (b). In both studies, no IVS were undertaken in the studies shown and the mean SI was calculated after the subtraction of background counts. Significantly different (Po.05) values were obtained vs. cohorts immunized with: *PBS alone, @4T1-pSec, #4T1p53 and $4T1-Flt3L. This study is representative of one of three studies; N ¼ 4/cohort.

However, the frequency of IL-4-secreting cells in spleens from 4T1-Flt3L-p53-, 4T1-Flt3L-, or 4T1-p53-immunized mice, while significantly increased compared to the PBS control mice, was similar within the mice cohorts immunized with the various transfected 4T1 cell lines (Fig 6b). In control studies where lymphocytes were cultured without peptides, no increase in the epitopespecific response was observed (results not shown). These results suggest that vaccination with 4T1 cells transfected with p53 or Flt3L transgenes could increase the frequency of both IFN-g- and IL-4-secreting cells. However, the response by IFN-g-secreting cells predominated, and the maximum response was obtained using 4T1 cells transfected with both p53 and Flt3L.

Immune protection of mice vaccinated with irradiated, transfected 4T1 tumor cells Mice immunized three times with each of the different irradiated, transfected tumor cells were challenged 1 week

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Figure 6 Frequency of IFN-g- and IL-4-secreting cells in the splenocytes following immunization with transfected 4T1 tumor cell vaccine. Mice were immunized with irradiated 4T1 cells transfected with human Flt3L and/or p53 transgenes. At 2 weeks after the final immunization, mice were killed and, without an IVS, splenocytes were stimulated in the presence of p53 peptides (P15 and P16). After 48 hours, the frequencies of IFN-g- and IL-4-secreting cells were determined by ELISpot assay and reported as mean7SEM. Significance (Po.05) vs. the following groups: *PBS alone, @4T1pSec, and #4T1-p53. This study is representative of one of three studies; N ¼ 4/cohort.

after the last immunization by orthotopic injection with 1 105 Cl-66 tumor cells, and revealed different levels of protection from tumor challenge. As shown in Figure 7, mice immunized with PBS (negative control) developed rapidly growing Cl-66 tumors, resulting in death of all the animals within 50 days. The tumor growth in the 4T1pSec and 4T1-p53 immunized mice was significantly slower (P ¼ .013 and .001, respectively). In contrast, 50 or 100% of the mice immunized with irradiated 4T1Flt3L or 4T1-Flt3L-p53, respectively, had complete tumor regression. The surviving mice, as well as agematched control mice, were again challenged with Cl-66 on day 120. In the secondary challenge study, the control mice developed tumors, while the immunized mice did not (data not shown). Thus, immunization using irradiated 4T1 tumor cells transfected with Flt3L or with Flt3L and p53 transgenes enhanced immunity to Cl-66.


Injection of irradiated, Flt3L-secreting 4T1 cells induces local DC accumulation To gain insight into the mechanism of adjuvant activity by Flt3L, we investigated regional DC recruitment by IHC using Abs for both interdigitating DCs (exhibiting the DEC205 marker) and follicular DCs (bearing the

FDC marker). As shown in Figure 8, after orthotopic injection of irradiated 4T1-Flt3L (Fig 8a) or 4T1-Flt3Lp53 (Fig 8b) cells, numerous FDC þ cells were observed in the draining LNs and surrounding the injected irradiated tumor cells. Staining with Abs to DEC205 demonstrated similar DC accumulation and infiltration, mainly in the areas surrounding the LNs after 4T1-Flt3L-p53 injection (Fig 8c). In contrast, few DCs were seen to infiltrate the injection sites of mice receiving 4T1-pSec cells (Fig 8d). Injection of 4T1 or 4T1-p53 tumor cells also evoked only scant DC infiltration (data not shown). Discussion

Figure 7 Tumor growth in vaccinated mice challenged with Cl-66 tumor cells. At 2 weeks after the last immunization, mice were challenged at a s.c. site contralateral to the vaccine site with 1 105 Cl-66 cells in a mammary fat pad. Tumor growth was measured as described in Materials and methods and is shown in the figure. This study is representative of one of two studies; N ¼ 5/cohort. Significant differences (Pr.05) vs. the following groups: *PBS alone, @4T1-pSec, and #4T1-Flt3L.

The injection of Flt3L-expressing tumor cells has been shown to inhibit tumor growth and protect against subsequent challenge.35,54 Tumor cells engineered to secrete cytokines (granulocyte macrophage colony-stimulating factor (GM-CSF) or Flt3L) can stimulate an antitumor immune response35,54–56 and, in some instances, have been shown to induce DC accumulation at the vaccine site.54,57 In this report, we demonstrate a novel tumor vaccine-adjuvant approach that uses the concomitant delivery of p53 and Flt3L transgenes. We transfected the p53 null, murine mammary adenocarcinoma 4T1 with human Flt3L, p53 or both transgenes, and found that transfection with both Flt3L and p53 enhanced the vaccine response to p53-expressing tumors, as indicated by an increased Ag-specific T-cell response in vitro and protection against tumor challenge in vivo.

Figure 8 IHC staining of DCs accumulated in the vaccinated sites. Regional DC recruitment was detected by IHC ( 100 magnification) using Abs for both interdigitating DCs (DEC-205) and FDCs at the vaccination site. Data was observed 10 days after the s.c. injection of irradiated 4T1FL (a) or 4T1-FL-p53 (b) cells by staining with Abs to FDC. Staining with Abs to DEC-205 demonstrated similar DC accumulation and infiltration (c). In contrast, few positively staining DCs were seen in mice receiving 4T1-pSec cells (d).

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Vaccination with tumor cells transfected with Flt3L was able to induce a non-p53-specific antitumor immunity. This was biologically relevant as mice immunized with 4T1-Flt3L were partially protected against challenge with Cl-66 (Fig 7) and 4T1 cells (results not shown) as compared to PBS-injected control mice, although total protection was not achieved. The findings that vaccination with 4T1-p53-Flt3L induced immune responses to p53 and was partially protective against 4T1 suggested that heightened immunity to 4T1 tumor Ags was also induced. This protection could also be explained by Flt3L activation of NK cells activation as described earlier.58–60 It is noted, however, that flow cytometric analysis did not reveal an increase in NK cells (5.370.34 106/control spleen vs. 6.870.6 106/4T1Flt3L spleen) or their activation (results not shown) and that tumor-specific proliferative responses were observed. Further, Flt3L activity was found to induce DC infiltration into the perivaccination site. Delayed tumor growth and, in some immune assays, the induction of an anti-4T1 immune response were also observed with 4T1 cells transfected with the vector alone (pSec). This suggests that cells transfected with the empty vector induced an inflammatory response, but to a lesser extent than tumor cells transfected with the same vector expressing the Flt3L transgene. The immune response induced by transfection with the empty plasmid vector presumably was associated with the expression of plasmid proteins or insertional mutagenesis, as has previously been reported.39,61 In our studies, we observed a DTH response to p53 peptides in mice immunized with irradiated 4T1 tumor cells transfected with p53 alone, or with p53 and a Flt3L transgene. However, the DTH response did not differ between the groups immunized with 4T1-p53 and 4T1Flt3L-p53. This may be due to the induction of a maximal DTH response by 4T1-p53, as observed previously.19 Spleen cells from mice immunized with 4T1-p53 or 4T1Flt3L-p53 had a significant increase in proliferation relative to spleen cells from control mice and mice immunized with 4T1-pSec or 4T1-Flt3L. However, the proliferation to p53 peptides was not significantly different between spleen cells from either the 4T1-p53 or 4T1-Flt3L-p53 vaccinated groups. In contrast, significantly greater lymphocyte proliferation in response to 4T1-p53 cells was observed with spleen cells from the 4T1Flt3L-p53 immunized mice, as compared to all other cohorts, including mice immunized with 4T1-p53. This suggests that maximum p53-specific immune augmentation was induced by immunization with tumor cells expressing both p53 and Flt3L transgenes. The specificity of the T-cell proliferative response was confirmed by the observation of a four-fold higher proliferation response in the presence of p53 peptide-pulsed autologous spleen cells after a 5-day IVS (results not shown). We suggest that this difference in response to 4T1-p53 by the spleen cells from mice immunized with 4T1-p53 vs. 4T1-Flt3L-p53 may be associated with Flt3L adjuvant activity. We report that mice immunized with irradiated 4T1Flt3L or 4T1-Flt3L-p53 tumor cell vaccines had increased

Cancer Gene Therapy

protection to tumor challenge. This enhancement may partly be due to the local recruitment of DCs to the vaccine site, which could then present Ag to T cells and elicit an Ag-specific antitumor immune response. Interestingly, FDC þ and DEC-205 þ DCs were both observed to infiltrate the vaccine site, indicating that they may both be involved. Immunization with 4T1 tumor cells transfected with p53 or Flt3L transgenes resulted in significant augmentation of p53-specific, type 1 IFN-g-secreting cells. The increase in the frequency of IFN-g þ - and IL-4 þ -secreting cells from the mice immunized with 4T1-Flt3L may reflect the immune-augmenting activity of Flt3L that we have reported previously.62 In addition, the frequency of IFNg-secreting cells was significantly increased in the 4T1Flt3L-p53 immunized mice as compared to cohorts vaccinated with PBS, 4T1-pSec, or 4T1-p53 cells. In contrast, the frequency of IL-4-secreting cells in vaccinated mice was only significantly increased as compared to the PBS control group, but with no differences among the cohorts immunized with the different transfected 4T1 variants. This suggests that vaccination with 4T1 cells overexpressing either p53 or Flt3L transgenes could increase both type 1 and type 2 T-cell responses to p53; however, a higher increase in the type 1 response was induced by vaccination with tumor cells transfected with both p53 and Flt3L. These results are similar to those of Westermann et al,26 who reported that transfection of DCs with Flt3L resulted in an increased type 1 and type 2 responses. There are several cytokines reported to activate DCs in vitro, including GM-CSF, tumor necrosis factor, and CD40L,20 although little is known concerning the signals necessary for DC activation in vivo. However, Flt3L can mobilize and expand DC numbers in vivo within lymphoid and parenchymal organs.16,17 Further, injection of Flt3L prior to vaccination increases type 1 T-cell responses.19,31 Our results suggest that Flt3L-producing tumor cells are able to recruit DCs locally to the injection areas. This may lead to an increase in primary T helper 1 cytokine responses to a tumor vaccine and induction of resistance to tumor challenge. This increase in activity may also be partly due to cytokines secreted by the irradiated tumor cells in addition to Flt3L, or due to the danger signals associated with irradiation-induced apoptosis, all of which can provide in situ adjuvant activity. In our study, mice immunized with irradiated 4T1 tumor cells showed reactivity against a tumor cell line (Cl-66) that overexpresses murine p53, even though human p53 was used for transfection of the immunizing tumor cells, confirming an immune crossreaction between human and murine p53.63 It has recently been demonstrated that vaccination based on xenogeneic homologous molecules may induce significant autoimmunity against a tumor as a crossreaction.64,65 Most importantly, it appears that a cellular vaccine with a known Ag and nonidentified TAAs induces increased protection against another tumor, albeit a closely related one, that expresses the known Ag (p53). We conclude that an irradiated tumor cell vaccine transfected with both p53 and Flt3L


significantly improves immunity as compared to a tumor vaccine containing either transgene alone. In a prior study,19 we reported that the injection of Flt3L for 10 days prior to immune priming with an Advp53 vaccine or boosting with a plasmid-p53 construct significantly increased the vaccine response. These studies extend our previous observations by demonstrating that transfection of tumor cells with p53 and Flt3L resulted in an increased T-cell response to p53. These results suggest that tumor cell expression of Flt3L may result in the induction of a T-cell response with therapeutic or prophylactic activity. At present, we are extending these observations with studies examining the therapeutic potential of intralesional injections of Adv-Flt3L as a more clinically relevant therapeutic and vaccine adjuvant strategy. In summary, we report that a novel vaccine composed of tumor cells transfected with human p53 and Flt3L transgenes can induce a marked antitumor response. In association with the immune response, DC recruitment to the vaccine site occurred, which may benefit Ag presentation and the induction of long-lasting immunity. The p53specific and undefined anti-TAA responses induced by this approach were greater than those associated with a corresponding cellular vaccine expressing either transgene alone, as shown by p53-specific IFN-g ELISpot, proliferation to p53-transfected 4T1 cells, and protection against mammary adenocarcinoma challenge.

Acknowledgments

We thank Michelle Varney, Aihua Li, and Matthew Backora for their assistance with the experiments and Richard Murcek, Lisa Chudomelka, Tina Winekauf, and Kirsten Stites for assisting with the preparation of the manuscript. This research was supported by the Nebraska Research Initiative Programs in Gene Therapy (JET) and in Molecular Therapeutics (JET, RKS, and JCS), by an LB595/Cattlemen’s Ball Grant (JCS), and by an International Exchange Award from the National Science Foundation and by the Department of Health, People’s Republic of China (HS).

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