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. 2011 Jan 15;60(4):559–573. doi: 10.1007/s00262-010-0955-5

Reduced tumorigenesis of EG7 after interleukin-10 gene transfer and enhanced efficacy in combination with intratumorally injection of adenovirus-mediated lymphotactin and the underlying mechanism

Jianbin Zhang 1, Zhidong Zhou 1,2, Cheng Wang 1, Jiangen Shen 1, Yun Zheng 1, Lihuang Zhang 1, Jianli Wang 1, Dajing Xia 1,✉
PMCID: PMC11029026  PMID: 21240488

Abstract

Although interleukin-10 (IL-10) is commonly regarded as an immunosuppressive cytokine, a wealth of evidence is accumulating that IL-10 also possesses some immunostimulating antitumor properties. Previous studies demonstrated that forced expression of the IL-10 gene in tumor cells could unexpectedly produce antitumor effects. In this study, we explored the tumorigenesis of EG7 cells transduced with IL-10 gene. In vivo, IL-10 gene transfer reduced tumorigenic capacity of EG7 cells and prolonged survival of the EG7 tumor-bearing mice. It was found that the cytotoxicities of cytotoxic T lymphocytes (CTL) and natural killer cells (NK cells) were enhanced. Assessment of the immune status of the animals showed prevalence of a systemic and tumor-specific Th2 response (high levels of IL-4 and IL-10). To improve the therapeutic efficacy, we combined with intratumoral injection of adenovirus-mediated lymphotactin (Ad-Lptn) into the overestablished EG7 tumor model. More significant inhibition of tumor growth were observed in EG7 tumor-bearing mice that received combined treatment with IL-10 and Lptn gene than those of mice treated with IL-10 or Lptn gene alone. The highest NK cells and CTL activity was induced in the combined therapy group, increasing the production of IL-2 and interferon-γ (IFN-γ) significantly but decreasing the expression of immune suppressive cells (CD4+Foxp3+ Treg cells and Gr1+CD11b+ MDSCs). The necrosis of tumor cells was markedly observed in the tumor tissues, accompanying with strongest expression of Mig (monokine induced by interferon-gamma) and IP-10 (interferon-inducible protein 10), weakest expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases-2 (MMP-2). In vivo, depletion analysis demonstrated that CD8+ T cells and NK cells were the predominant effector cell subset responsible for the antitumor effect of IL-10 or Lptn gene. These findings may provide a potential strategy to improve the antitumor efficacy of IL-10 and Lptn.

Keywords: Interleukin-10, Lymphotactin, Natural killer cells, Cytotoxic T lymphocytes, Cytokine gene therapy, Antitumor immunity, Regulatory T cells, Myeloid-derived suppressor cells

Introduction

Cytokine gene transfer to tumor cells has been recently developed as an attractive mechanism of drug delivery in situ without the toxic effects associated with systemic administration.

Although the relationship between IL-10 and cancer has been studied extensively, the ultimate role of IL-10 in tumor biology remains enigmatic. The significance of IL-10 production within the tumor microenvironment, which can be sustained by malignant cells and tumor-infiltrating macrophages (TIM) and lymphocytes, including NK cells and T cells, is debated. IL-10 can favor tumor growth in vitro by stimulating cell proliferation and inhibiting cell apoptosis. High systemic levels of IL-10 correlate with poor survival of some cancer patients.

However, this might reflect just the bulk of disease, but no correlation is reported. Moreover, opposite findings (higher IL-10 with better survival) are observed when the cytokine levels are assessed in tumor samples [1]. Chinese hamster ovary (CHO) cells [2], mammary adenocarcinoma cells [3, 4], melanoma cells [5, 6], CT26 colon carcinoma cells [7], and Burkitt lymphoma cells [8] transfected with IL-10 were less effective at establishing primary tumors and/or metastasis than untransfected cells in syngeneic and SCID mice. Additionally, IL-10 gene transfer to peritoneal mesothelial cells effectively suppressed peritoneal dissemination of gastric cancer cells, which was correlated with transiently high levels of IL-10 in the peritoneal cavity [9]; systemic administration of IL-10 has inhibited tumor metastasis and stimulated antitumor immune responses in various murine models [10]. But the mechanisms behind these antitumor effects are poorly understood, partially due to IL-10 short half life. Thus, we transfected EG7 cells with IL-10 gene and established a cell clone stably expressing IL-10 (500 pg/ml every 106 cells) to maintain a high concentration of IL-10. C57BL/6 mice were inoculated subcutaneously (s.c.) with EG7 cells as a model to observe their tumorigenicity. Our data showed that IL-10 gene transfer to EG7 cells reduced their tumorigenic capacity and prolonged survival of the EG7 tumor-bearing mice.

Lymphotactin (Lptn) is a C chemokine that specifically regulates the migration of T cells and NK cells. Cotransfection of Lptn and IL-2 genes to tumor cellular vaccine induces potent antitumor immunity. In our previous study, we demonstrated that Lptn cotransfection enhances the therapeutic efficacy of dendritic cells genetically modified with melanoma antigen gp100 and also illustrated that intratumoral injection of Ad-Lptn potentiates the antibody-targeted superantigen therapy of cancer. We hypothesized that intratumoral Lptn gene therapy induced local accumulation of T cells and NK cells in the tumor site, which can be activated subsequently by EG7 cells to overcome the occurrence of hyporesponsiveness state and promoted the potent antitumor effects of IL-10. Our study illustrated that the combined treatment with IL-10 and Lptn may elicit more significant antitumor effects through more efficient induction of specific and non-specific antitumor immune responses.

Materials and methods

Animals

Female C57BL/6 mice (H-2 Kb) aged 6–8 weeks were purchased from Joint Ventures Sipper BK Experimental Animal Co. (Shanghai, China) and housed in a specific pathogen-free condition for all experiments.

Cell lines

EG7 cell line is derived from the murine T-cell lymphoma EL-4 transfected with cDNA for OVA and lack of B7-1 expression (kindly provided by Prof. E. Gilboa, Duke University, USA). EG7 cells were transfected with the expression vector IL-10 using a Bio-Rad gene pulser (Bio-Rad Laboratories, Mississauga, Ontario, Canada). The transfected EG7 cells were selected for growth in the medium containing G418 (0.5 mg/ml) and then maintained in the medium containing G418 (0.5 mg/ml). EG7/IL-10 cell clone that stably expressed IL-10 (500 pg/ml every 106 cells). YAC-1, an NK-sensitive lymphoma cell line of A/S (H-2a) origin, EL4, a T-lymphoma cell line of C57BL/6 origin, and 293, a continuous cell line derived from human embryonic kidney were obtained from American Type Culture Collection (ATCC, Manassas, VT, USA) and maintained in RPMI-1640 medium supplemented with penicillin 100 U/ml, streptomycin 100 μg/ml, 2-mercaptoethanol 50 mmol/l, and 10% fetal calf serum. EG7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (FCS, Hyclone, Logan, UT, USA), penicillin (100 U/ml), streptomycin (100 mg/ml), and G418 (0.5 mg/ml), 50 μg/ml gentamycin, and 2 mM glutamine. All culture media were purchased from Gibco-BRL (Gaithersburg, Md., USA), and fetal calf serum was provided by Shanghai Institute of Biological Products (Shanghai, China).

Reagents

Recombinant murine IL-2 was purchased from Pharmigen (San Diego, CA, USA). The IL-2, IL-4, IL-10, and IFN-γ ELISA kits were purchased from R&D System Inc (Minneapolis,MN, USA). G418 was purchased from CalBiochem (Merck, Darmstadt, Germany). Cytotox 96® Non-adioactive Cytotoxicity Assay kit was purchased from Promega (Madison, WI, USA). Trizol Reagent was purchased from Bio Basic (Bio Basic Inc. Canada). RevertAid™ First Strand cDNA Synthesis Kit #K1622 was purchased from Fermentas (Vilnius, Lithuania). Gene Ruler™ 100-bp DNA ladder #SM0241 was purchased from Fermentas (Vilnius, Lithuania). Blend Taq was purchased from Toyobo CO., LTD (Osaka, Japan). dNTPs 10 mM was purchased from Takara Biotechnology (Dalian, China) CO., LTD. The primer of VEGF, MMP-2, IP-10, and Mig was synthesized by Shanghai Biotechnology Company. Rabbit anti-VEGF (Catalog number: BA0407), SABC Immunohistochemistry kit (Product number: SA1022), and DAB kit (Product number: TBD550) were purchased from Boster (Wuhan, China).

Preparation of recombinant adenoviruses

Replication-defective recombinant adenoviruses Ad-LacZ harboring the β-galactosidase gene and Ad-Lptn harboring the murine Lptn gene were constructed from human adenovirus serotype 5 using homologous recombination. The procedures were described previously by us. The expression of these genes was driven by a CAG promoter. The recombinant adenovirus were released from 293 cells by three freezing/thawing cycles and subsequently propagated with 293 cells. The titers of the adenoviral preparations were determined by plaque-forming assay on 293 cells. Briefly, serial tenfold dilutions of adenovirus were added to 24-well plates (Corning, NY, USA), containing confluent 293 cell monolayers. After 48 h of incubation in a humidified atmosphere, the end-point of 50% infectivity was determined according to cytopathic effect. The recombinant adenovirus produced were diluted to a titer of 1010 PFU/ml in phosphate-buffered solution (PBS) and stored at −80°C for experiments.

Tumor formation assay

The C57BL/6 mice were divided into two groups and were injected s.c. into a back limb with a dose of 5 × 105 EG7 cells or EG7/IL-10 cells, respectively. The length and width of tumor mass were measured with calibers every other day after tumor inoculation. Tumor size was expressed as 1/2 (length + width). Three mice of each group were killed 3 weeks after tumor inoculation, and the tumors in the lateral rear leg were extracted and weighted. Splenocytes isolated from the mice were used for cytotoxic assay of NK cells and CTL and cytokine induction and measurement as follows. Seven mice in each group were observed for their survival period. All experiments were performed three times using individual treatment groups of ten mice. Data are representative of three experiments performed.

Immunotherapy of the pre-established tumor model

Three days after s.c. inoculation of 5 × 105 EG7 cells or EG7/IL-10 cells, the tumor-bearing mice were divided into six groups and were injected intratumorally with any of the following preparations: PBS, Ad-LacZ, or Ad-Lptn. Three days after the tumor inoculation, 2 × 109 pfu virus/0.1 ml was injected intratumorally, and a booster of the same dose was given twice at weekly intervals. The length and width of the tumor mass were measured with caliper every other day after tumor inoculation. Tumor size was expressed as 1/2 (length + width). Three tumor-bearing mice of each group were killed 5 days after the last injection of Ad-Lptn, and the tumors in the lateral rear leg were extracted and weighted. Seven mice in each group were observed for their survival period.

Cytotoxic assay of NK and CTL cells

Splenic lymphocytes were isolated from the mice 5 days after the last treatment. The erythrocytes were depleted with 0.83% ammonium chloride, and macrophages were removed by adherence of splenocytes on plastic plates for 2 h. The non-adherent lymphocytes were directly used as NK effector cells. The lymphocytes were cocultured with inactivated EG7 or EG7/IL-10 (treated by 50 μg/ml mitomycin for 30 min) for 7 days in the presence of 20 U/ml recombinant murine IL-2 (San Diego, CA, USA) and then collected as CTL effector cells. The NK-cell activity and CTL activity were determined by lactate dehydrogenase (LDH) release assay with CytoTox 96® Non-Radioactive Cytotoxicity Assay kit (Promega, Madison, WI, USA) Zoll 1998 p. 311. The target cells (YAC-1 cells, EG7 cells or EG7/IL-10) were washed three times with RPMI 1640 medium containing 5% FCS to remove adherent LDH derived from lysed cells. The cell suspension was diluted with RPMI 1640 medium containing 5% FCS to give final concentration of 1 × 105 cells/ml. One hundred microliters target cell suspension and 100 μl different ratios of effector cells were pipetted together into the wells of a round-bottomed microtiter plate. Suspensions containing exclusively effector cells, target cells, or culture medium, respectively, served as controls to estimate the LDH background. The plates were incubated for 4 h in a humidified 5% CO2 atmosphere at 37°C. After incubation, they were centrifuged for 10 min. Then, 100 μl of the supernatant from each well was transferred to the corresponding well of enzymatic assay plate. Fifty microliters reconstituted substrate mix (containing lactate and NAD+) was added to each well. The plate was covered and incubated at room temperature (protected from light). Thirty minutes later, 50 μl stop solution was added to each well. The reaction was measured in an ELISA reader at a wavelength of 490 nm. Calculations were carried out according to the following formula: % of specific lysis = 100 × (experimental−effector spontaneous−target spontaneous)/(target maximum−target spontaneous).

Cytokine induction and measurement of splenocytes

The non-adherent splenocytes derived from mice killed 5 days after the last treatment and the cells were washed three times with PBS. For induction of cytokines, 5 × 106 cells of splenocytes were incubated with inactivated EG7 cells, EG7/IL-10 (treated by 50 μg/ml mitomycin for 30 min) at a 10:1 ratio in a total volume of 1 ml at 37°C, 5% CO2 for 72 h. Culture supernatants were harvested at 24 h (for IL-2 assay), 48 h (for IL-4 and IL-10 assay), and 72 h (for IFN-γ assay) and stored at −20°C for measurement of cytokines using a standard sandwich ELISA technique with corresponding kits purchased from R&D System Inc (Minneapolis, MN, USA).

Determination of intratumoral IP-10, Mig, VEGF, and MMP-2 mRNA expression by semiquantitative RT–PCR

Subcutaneous tumor nodules were taken from killed tumor-bearing mice 5 days after the last treatment. Total RNA was extracted from subcutaneous tumor tissues by Trizol Reagent (Bio Basic Inc. Canada), according to the instructions of the manufacturer. From each sample, 1 μg total RNA was reverse-transcribed (RT) using a RevertAid™ First Strand cDNA Synthesis Kit #K1622 (Fermentas, Vilnius, Lithuania) in a total volume of 20 μl. cDNA as readout of the mRNA was quantitated in a competitive PCR using specific primers for IP-10, Mig, VEGF, and MMP-2. Primers for amplification of IP-10 were 5′-ACCATGAACCCAAGTGCTGCCGTC-3′ (sense) and 5′-GCTTCACTCCAGTTAAGGAGCCCT-3′ (antisense), with an expected PCR product of 311 bp. Primers for Mig amplification were 5′-ACTCAGCTCTGCCATGAAGTCCGC-3′ (sense) and 5′-AAAGGCTGCTGCCA GGGAAGGC-3′ (antisense), with an expected PCR product of 478 bp. Primers for amplification of VEGF were 5′-CTGCTCTCTTGGGTGCACTGG-3′ (sense) and 5′-CACCGCCTT GGCTTGTCACAT-3′ (antisense), with an expected PCR product of VEGF121 (431 bp) and VEGF165 (563 bp). Primers for amplification of MMP-2 were 5′-CACCATCGCCCATCATCAAGT-3′ (sense) and 5′-′TGGATTCGAGAAAAGCGCAGCGG-3′ (antisense), with an expected PCR product of 399 bp. Primers for amplification of β-actin were 5′-TGGAATCCTGTGGCATCCATGAAA C-3′ (sense) and 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′ (antisense), with an expected PCR product of 348 bp.

PCR products of IP-10, Mig, VEGF, MMP-2, and β-actin were visualized by electrophoresis in a 2.5% agarose gel containing 0.5 μg/ml ethidium bromide. Densities of the amplified IP-10, Mig, VEGF, MMP-2, and β-actin were analyzed using the Kodak EDAS120 digital imaging system version 3 (Gibco-BRL).

Immunohistochemical staining of the tumor tissues

Subcutaneous tumor nodules were taken from killed tumor-bearing mice 5 days after the last treatment. The tumor samples were fixed in 10% formalin solution, dehydrated, and embedded in paraffin. Immunohistochemical staining of thin-sliced sections was performed using SABC Immunohistochemistry kit (Product Number: SA1022, Boster, Wuhan, China) and DAB substrate (Product number: TBD550, Boster, Wuhan, China) according to the manufacturer’s instructions. To evaluate angiogenesis, sections were reacted with polyclonal rabbit anti-VEGF antibody (Catalog number: BA0407, Boster, Wuhan, China), the marker for new vessels, and counterstained by hematoxylin. The average number of tumor cells staining with VEGF from five randomly chosen hpf was counted to determine the ratio of VEGF expression.

Histological examination

Subcutaneous tumor nodules were taken from killed tumor-bearing mice 5 days after the last treatment. The tumor samples were fixed in 10% formalin solution, dehydrated, and embedded in paraffin. Thin-sliced sections were staining with hematoxylin and eosin. For the evaluation of tumor necrosis, we assigned the following: − no necrosis; + less than one-third of the tumor size; ++ between one-third and two-thirds of the tumor size; +++ more than two-thirds of the tumor size.

Flow cytometric analysis

Spleen cells were obtained from tumor-bearing mice after last treatment. Splenocytes (1 × 105) were suspended in PBS and was incubated for 30 min at 4°C with anti-CD4 (BD Bioscience) antibody conjugated to fluorescein isothiocyanate (FITC) and anti-Foxp3 (BD Bioscience) antibody conjugated to phycoerythrin-cy5 (PE-cy5). Similarly, splenocytes were incubated for 30 min at 4°C with anti-Gr1 (BD Bioscience) antibody conjugated to phycoerythrin (PE) and anti-CD11b (BD Bioscience) antibody conjugated to phycoerythrin (PE) cyanine5 (Cy5). Subsequent to incubation, the cells were washed twice with PBS and then analyzed by FACScan flow cytometry using CellQuest (Becton–Dickinson, Mountain View, CA) software. The corresponding isotype controls (rat IgG1, IgG2a, and IgG2b) were purchased from eBioscience (San Diego, CA) and BD Bioscience, respectively.

In vivo depletion of immune cell subsets

Mice were challenged with 5 × 105 EG7 cells or EG7/IL-10 cells, respectively. Four days before tumor challenge, the mice started to receive a total of five intraperitoneal injections of ascites (0.1 ml per mouse per injection) from hybridoma-bearing mice at intervals of 3 days. The monoclonal antibodies used were GK1.5 anti-CD4, 2.43 anti-CD8, or PK136 anti-NK1.1 (HB 191; ATCC). Normal rat IgG (Sigma, St Louis, MO, USA) was given as mock control. Depletion of T-cell subsets and NK cells was monitored by flow cytometry, which showed >90% specific depletion in splenocytes.

Statistics

All the experiments were run in triplicate, and the results are means ± SD of triplicate determinations (or representation data from one or two independent experiments). Statistical analysis was performed using Student’s t test and log-rank test (for survival analysis). The difference was considered statistically significant when the P value was less than 0.05.

Results

Interleukin-10 reduced the tumorigenesis of EG7 cells

The genes encoding IL-10 were transduced into EG7 cells, and G418-resistant clones were selected. Normal C57BL/6 mice were inoculated s.c. with a dose of 5 × 105 EG7 or EG7/IL-10 cells. The results (Fig. 1a) showed that the tumorigenesis of the IL-10-transfected EG7 clones was markedly reduced compared with that of wild-type (WT) cell lines (P < 0.05, days 21). Three tumor-bearing mice in each group were killed 3 weeks after the tumor inoculation, and the tumors in the lateral rear leg were extracted and weighted. The results (Fig. 1b) illustrated that the weight of EG7/IL10 tumors were also less than that of EG7 tumors (1.40 vs. 0.61 g, P < 0.05, days 21). Seven tumor-bearing mice left in each group were observed for their survival period. The results (Fig. 1c) showed that EG7/IL10-bearing mice survived much longer than EG7 mice (P < 0.05). Two of seven EG7/IL10-bearing mice were tumor-free and survived more than 90 days. These data suggested that IL-10 gene transfer elicited more potent and specific antitumor effect in vivo.

Fig. 1.

Fig. 1

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Tumorigenicity of EG7 or EG7/IL-10 cells s.c. inoculated into C57BL/6 mice at a dose of 5 × 105 cells. Ten mice (n = 10) each group were analyzed. The length and width of the tumor mass were measured with caliper every other day. Tumor size was expressed as 1/2 (length + width). a Inhibition of tumor growth in EG7 tumors after IL-10 gene transfer. b Tumor weight of each group (days 21). c Survival period of tumor-bearing mice left in each group during 90 days

Increased NK-cell activity after IL-10 gene transfer

Three weeks after the tumor inoculation, the splenocytes isolated from killed tumor-bearing mice of each group were used in cytotoxic assay against YAC-1 cells at effector:target (E:T) ratios at 25:1, 50:1, and 100:1 by LDH release assay. As is shown (Fig. 2a), NK-cell activity of EG7IL-10 tumor-bearing mice increased when compared with that of wild-type mice (39.47 vs. 17.43% P < 0.05). It suggested that the enhanced non-specific immunity might also be involved in the antitumor response.

Fig. 2.

Fig. 2

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Induction higher of NK and CTL activity by IL-10. Normal C57BL/6 mice (n = 10) were inoculated s.c. with a dose of 5 × 105 EG7 or EG7/IL-10 cells. Three mice in each group were killed 3 weeks after the tumor inoculation. a Their splenocytes were isolated and used in cytolytic assays against YAC-1 cells. b Their splenocyte were restimulated in vitro for 7 days with inactivated EG7 cells or EG7/IL-10 cells in the presence of recombinant murine IL-2 (20 U/ml) and assayed for their cytotoxicity against EG7 cells or EG7/IL-10 cells, respectively. Data are representative of three independent experiments

Enhanced specific CTL cytotoxicity induced by IL-10

The splenocytes derived from mice of each group were restimulated in vitro with inactivated EG7 or EG7/IL-10 cells for 7 days in the presence of recombinant murine IL-2 (20 U/ml) and then collected as CTL effector cells. EG7 or EG7/IL-10 cells were used as target cells, respectively. The CTL activity was determined at E:T ratios of 25:1, 50:1, and 100:1 by LDH release assay. Consistent with the protection against the challenge of tumor, the EG7/IL-10 tumor-bearing mice exhibited a specific CTL response which was higher than that of wild-type mice (50.42 vs. 27.53% P < 0.05) (Fig. 2b).

More Th2 type cytokine production after IL-10 gene transfer

Three weeks after the tumor inoculation, we determined the production of IL-2 (for 24 h), IL-4, IL-10 (for 48 h), and IFN-γ (for 72 h) by splenocytes stimulated in vitro with inactivated EG7 or EG7/IL-10 cells at a 10:1 ratio in a total volume of 1 ml at 37°C, 5% CO2. The data (Fig. 3) showed that splenocytes isolated from EG7/IL-10 tumor-bearing mice secreted higher levels of IL-4 and IL-10 and lower levels of IFN-γ when compared with those from wild-type mice (342 vs. 219 pg/ml, 479 vs. 321 pg/ml, 330 vs. 1,157 pg/ml P < 0.05).

Fig. 3.

Fig. 3

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Production of IL-2 (a), IL-4 (c), IL-10 (d), and IFN-γ (b) by lymphocytes derived from tumor-bearing mice. Three mice in each group were killed 3 weeks after the tumor inoculation. 5 × 106 splenocytes were incubated with inactivated EG7 or EG7/IL-10 cells at a 10:1 ratio in a total volume of 1 ml at 37°C, 5% CO2 atmosphere for 24 h, 48 h, or 72 h, and supernatants were harvested for the detection of cytokines by ELISA

Enhanced antitumor effects after treatment with Ad-Lptn

Three days after s.c. inoculation with 5 × 105 EG7 or EG7/IL-10 cells, tumor-bearing mice were intratumorally injected with PBS, Ad-LacZ, or Ad-Lptn. The results (Fig. 4a) demonstrated that tumor growth in mice treated with Ad-Lptn was markedly less than that in mice treated with PBS or Ad-LacZ (P < 0.05, days 22). The inhibition of tumor growth was observed most significantly in EG7/IL-10 tumor-bearing mice after treatment with Ad-Lptn when compared with that of EG7/IL-10 tumor-bearing mice or that of EG7 tumor-bearing mice treated with Ad-Lptn alone (P < 0.05, days 22). Three tumor-bearing mice in each group were killed 5 days after the last injection of Ad-Lptn, and the tumors in the lateral rear leg were extracted and photographed (Fig. 4b). The results (Fig. 4c) illustrated that the tumor weight was also significantly less in mice that received the combined treatment with IL-10 and Lptn than treated with IL-10 or Lptn alone (0.20 vs. 0.61 vs. 0.33 g P < 0.01, days 22).

Fig. 4.

Fig. 4

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Enhanced antitumor effects after IL-10 gene transfer and treated with Ad-Lptn. C57BL/6 mice were inoculated s.c. with 5 × 105 EG7 or EG7/IL-10 cells on day 0. The tumor-bearing mice were divided into six groups (each group containing ten mice) and injected intratumorally with PBS, Ad-LacZ, or Ad-Lptn. Injection of 2 × 109 PFU/0.1 ml Ad-Lptn or Ad-Lacz was performed on days 3, 10, and 17. The length and width of the tumor mass were measured with caliper every other day. Tumor-bearing mice in each group were observed for their survival time. a, b Significantly inhibition of tumor growth in EG7/IL-10-bearing mice after treatment with Ad-Lptn. c Tumor weight of EG7/IL-10-bearing mice with intratumoral injection of Ad-Lptn. d Survival period of tumor-bearing mice after treatment with IL-10 and Ad-Lptn

Seven tumor-bearing mice left in each group were observed for their survival period. The results (Fig. 4d) showed that tumor-bearing mice treated with Ad-Lptn survived much longer than mice treated with PBS or Ad-LacZ (P < 0.05). The survival time was still longer in EG7/IL-10 tumor-bearing mice that received the therapy of Ad-Lptn than that of EG7/IL-10 tumor-bearing mice or that of EG7 mice treated with Ad-Lptn alone (P < 0.05). Six of seven mice after combined treatment with IL-10 and Lptn were tumor-free and survived more than 90 days. These data suggested that combined treatment with IL-10 and Lptn elicited more potent and specific antitumor effect in vivo than IL-10 or Lptn used alone.

Increased NK-cell activity after treatment with Ad-Lptn

Five days after the last therapy, the splenocytes isolated from killed tumor-bearing mice of different groups were used in cytotoxic assay against YAC-1 cells at E:T ratios at 25:1, 50:1, and 100:1 by LDH release assay. As is shown (Fig. 5a), NK-cell activity in EG7 tumor-bearing mice treated with Ad-Lptn increased when compared with those in EG7 tumor-bearing mice injected with PBS or Ad-Lacz (47.64 vs. 16.42 vs. 17.63% P < 0.01), while the splenic NK-cell activity increased most significantly in EG7/IL-10 tumor-bearing mice after treatment with Ad-Lptn when compared with those in EG7/IL-10 tumor-bearing mice or EG7 tumor-bearing mice treated with Ad-Lptn alone (56.67 vs. 39.47 vs. 47.64% P < 0.05). These data suggested that the enhancement of non-specific immunity might also be involved in the antitumor response of Lptn.

Fig. 5.

Fig. 5

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Induction of NK and CTL activity after treatment with IL-10 and Ad-Lptn. Tumor-bearing mice were intratumoral injected with a dose of 2 × 109 PFU/0.1 ml Ad-Lptn three times. Three mice in different groups were killed 5 days after the last injection. a Their splenocytes were isolated and used in cytolytic assays against YAC-1 cells. b Their splenocyte were restimulated in vitro for 7 days with inactivated EG7 cells or EG7/IL-10 cells in the presence of recombinant murine IL-2 (20 U/ml) and assayed for their cytotoxicity against EG7 cells or EG7/IL-10 cells, respectively. Data are representative of three independent experiments

More potent specific CTL cytotoxicity after treatment with Ad-Lptn

The splenocytes derived from mice of various groups were restimulated in vitro with inactivated EG7 or EG7/IL-10 tumor cells for 7 days in the presence of recombinant murine IL-2 (20 U/ml) and then collected as CTL effector cells. EG7 or EG7/IL-10 cells were used as target cells, respectively. The CTL activity was determined at E:T ratios of 25:1, 50:1, and 100:1 by LDH release assay. Consistent with the protection against the challenge of T lymphoma cells, the EG7 tumor-bearing mice treated with Ad-Lptn exhibited a T lymphoma-specific CTL response which was higher than that of EG7-bearing mice treated with PBS or Ad-Lacz (60.18 vs. 39.32 vs. 42.22% P < 0.01), but it was less potent than that induced by combined treatment with IL-10 and Lptn (81.23% P < 0.05). The highest CTL activity against EG7/IL-10 cells was induced in EG7/IL-10 tumor-bearing mice treated with Ad-Lptn when compared with that in mice of other groups (Fig. 5b).

More Th1 type cytokine production after treatment with Ad-Lptn

Five days after the last injection of Ad-Lptn, we determined the production of IL-2 (for 24 h), IL-4, IL-10 (for 48 h), and IFN-γ (for 72 h) by splenocytes stimulated in vitro with inactivated EG7 or EG7/IL-10 cells at a 10:1 ratio in a total volume of 1 ml at 37°C, 5% CO2. The data (Fig. 6) showed that splenocytes from mice treated with Ad-Lptn produced significantly higher levels of IL-2 and IFN-γ and lower levels of IL-10 than those from mice treated with Ad-Lacz or PBS (420 vs. 282 vs. 257 pg/ml, 1,658 vs. 1,157 vs. 853 pg/ml, 240 vs. 321 vs. 317 pg/ml P < 0.05). The splenocytes from EG7/IL-10 tumor-bearing mice secreted higher levels of IL-4 and IL-10 and lower levels of IFN-γ compared with those from EG7 tumor-bearing mice (342 vs. 219 pg/ml, 478 vs. 321 pg/ml, 330 vs. 1,157 pg/ml P < 0.05).

Fig. 6.

Fig. 6

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Production of IL-2, IL-4, IL-10, and IFN-γ by lymphocytes derived from tumor-bearing mice treated with IL-10 and Ad-Lptn. Mice in different groups were killed 5 days after the last injection. 5 × 106 splenocytes were incubated with inactivated EG7 or EG7/IL-10 cells at a 10:1 ratio in a total volume of 1 ml at 37°C, 5% CO2 atmosphere for 24 h, 48 h, or 72 h, and supernatants were harvested for the detection of cytokines by ELISA

Efficient expression of IP-10 and Mig after treatment with Ad-Lptn in vivo

Five days after the last intratumoral injection of Ad-Lptn, the IP-10, Mig, VEGF, and MMP-2, mRNA expression was analyzed by semiquantitative RT–PCR. As is shown (Fig. 7), IP-10 and Mig were markedly observed in the tumor tissues of EG7/IL-10 tumor-bearing mice or mice treated with Ad-Lptn, accompanying with weak expression of VEGF and MMP-2. The intratumoral expression of VEGF and MMP-2 mRNA in EG7/IL-10 tumor-bearing mice after injection of Ad-Lptn was significantly less than any other group. This indicated that combined treatment with IL-10 and Lptn could efficiently inhibit angiogenesis in the tumor tissues.

Fig. 7.

Fig. 7

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The intratumoral IP-10, Mig, VEGF, and MMP-2 mRNA expression in different group. Five days after the last intratumoral injection of Ad-Lptn, total RNA was extracted from subcutaneous tumor tissues by Trizol Reagent. The intratumoral IP-10, Mig, VEGF, and MMP-2 mRNA expression was analyzed by semiquantitative RT–PCR. A PCR product of IP-10, Mig, VEGF, MMP-2, and β-actin was visualized by electrophoresis in a 2.5% agarose gel containing 0.5 μg/ml ethidium bromide. Lane1 EG7/PBS; Lane2 EG7/Lacz; Lane3 EG7/Lptn; Lane4 EG7-IL10/PBS; Lane5 EG7-IL10/Lacz; Lane6 EG7-IL10/Lptn. One of three independent experiments with similar results is shown

Effects of Lptn on angiogenesis in the tumor tissues

In EG7/PBS or EG7/Lacz controls, many brown particles, for which localization was confirmed by VEGF staining, were found on vascular endothelium and in the mesenchymas of the tumor (Fig. 8Aa, Ab). However, in the IL-10 gene transfer or treated with Ad-Lptn group, few brown particles were found in the tumor tissues (Fig. 8Ac, Ad, Ae); in the combined therapy group, brown particles were hardly found in the tumor tissues (Fig. 8Af). The ratio of VEGF expression in each group was shown (Fig. 8B) according to the above calculations. The expression ratio of VEGF in EG7 tumor-bearing mice treated with PBS or Ad-Lacz was markedly higher than any other group (37.4 vs. 36.2% P < 0.05). The expression ratio of VEGF in EG7/IL-10 tumor-bearing mice after injection of Ad-Lptn was significantly lower than any other group (7.2% P < 0.05).

Fig. 8.

Fig. 8

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Immunohistochemical staining for VEGF (A) and calculations for the ratio of VEGF expression (B) in the tumor issues 5 days after the last intratumoral injection of Ad-Lptn. Magnification: ×400. (a) EG7/PBS, (b) EG7/Lacz, (c) EG7/Lptn, (d) EG7-IL10/PBS, (e) EG7-IL10/Lacz, and (f) EG7-IL10/Lptn

Manifest necrosis in the tumor tissue of treatment with Ad-Lptn

Subcutaneous tumor nodules were taken from tumor-bearing mice that had been killed 22 days after tumor challenge. Histological examination of tumor mass showed that the most obvious tumor necrosis was present inside the tumors of the EG7/IL-10 tumor-bearing mice treated with Ad-Lptn. Medium necrosis was present in EG7/IL-10 tumor-bearing mice or EG7 tumor-bearing mice treated with Ad-Lptn alone. However, little necrosis was found inside tumors of EG7 tumor-bearing mice treated with PBS or Ad-Lacz (Table 1).

Table 1.

Pathological analysis of tumor mass in T lymphoma-bearing mice treated with IL-10 and Ad-Lptn

Groups EG7/PBS EG7/Lacz EG7/Lptn EG7-IL10/PBS EG7-IL10/Lacz EG7-IL10/Lptn
Necrosis − − ++ + + +++
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Tumor necrosis: −, no necrosis; +, less than 1/3 of the tumor size

++, between 1/3 and 2/3 of tumor size; +++, more than 2/3 of the tumor size

Lptn gene therapy downregulated the expression of CD4+Foxp3+ Treg cells and Gr1+CD11b+ MDSCs in mice

To define the underlying mechanism involved in interleukin-10 and lymphotactin-induced tumor growth suppression, we analyzed the expression of immune suppressive cells (CD4+Foxp3+ Treg cells and Gr1+CD11b+ MDSCs) in spleen of tumor-bearing mice via flow cytometry. After 5 days of last treatment, spleens from tumor-bearing mice were stained with anti-CD4 FITC and anti-Foxp3 PE-cy5 or with anti-Gr1 PE and anti-CD11b PE-cy5, and splenocytes were analyzed by flow cytometry. We found that CD4+Foxp3+ Treg cells decreased significantly in the IL-10 gene transfer group (21.52%) and Lptn gene therapy group (18.41%), compared with control (25.27% vs. 24.79%) (ratio in the CD4+ T cells) (Fig. 9a). The number of CD4+Foxp3+ Treg cells decreased most significantly in the combined treatment with IL-10 and Lptn group (15.14%) (ratio in the CD4+ T cells). Similarly, Gr1+CD11b+ MDSCs decreased significantly in the IL-10 gene transfer group (2.79% vs. 3.83%) and Lptn gene therapy group (0.75%), compared with control (4.42% vs. 4.16%) (Fig. 9b). Decreased expression of immune suppressive cells partially contributed to the enhanced host antitumor immune response.

Fig. 9.

Fig. 9

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Interleukin-10 and lymphotactin gene transfer downregulated CD4+Foxp3+ Tregs and Gr1+CD11b+ MDSCs in mice. a Splenocytes from killed mice were stained with mouse anti-CD4 fluorescein isothiocyanate (FITC) and anti-Foxp3 phycoerythrin-cy5 (PE-cy5) antibodies, and splenocytes were analyzed by flow cytometry. Similarly, splenocytes from killed mice were stained with mouse anti-Gr1 phycoerythrin (PE) and anti-CD11b phycoerythrin-cy5 (PE-cy5) antibodies, and splenocytes were analyzed by flow cytometry in (b). These profiles are representative results from three independent experiments

The roles of T cells and NK cells in the induction of antitumor immunity by Lptn

To investigate the responsibility of T-cell subpopulations and NK cells in the protective immunity induced by IL-10 and Lptn, the mice were depleted of CD4+ T cells, CD8+ T cells, or NK cells during tumor challenge. As is shown (Fig. 10), EG7 tumor-bearing mice depleted of CD8+ T cells during tumor challenge abrogated the protective immunity induced by Lptn (24.4 ± 1.0 mm). Mice depleted of CD4+ T cells during tumor challenge showed slightly larger tumors than mice in the control group (17.9 ± 1.2 mm P < 0.05), but slightly smaller than mice depleted of NK cells (18.1 ± 0.8 mm). These data confirmed that CD8+ T cells were the predominant effector cells in antitumor immunity induced by Lptn and that CD4+ T cells and NK cells were less important than CD8+ T cells in the effector phase. EG7/IL-10 tumor-bearing mice depleted of NK cells during tumor challenge showed much larger tumors than mice depleted of CD8+ T cells (18.4 ± 1.0 vs. 11.9 ± 1.5 mm). Mice depleted of CD4+ T cells during tumor challenge showed slightly larger tumors than mice in the control group (P < 0.05) and slightly smaller tumors than mice depleted of CD8+ T cells, indicating that NK cells were the predominant effector cells in antitumor immunity induced by IL-10 and Lptn and that CD4+ T cells and CD8+ T cells were necessary in the effector phase.

Fig. 10.

Fig. 10

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Determination of immune subsets responsible for the protective immunity induced by IL-10 and Ad-Lptn. Normal C57BL/6 mice were inoculated s.c. with 5 × 105 EG7 or EG7/IL-10 cells, followed by intratumoral injected with 2 × 109 PFU/0.1 ml Ad-Lptn 3 days later. Four days before tumor challenge, the mice (n = 10 per group) started to receive a total of five intraperitoneal injections (0.1 ml per mouse per injection) of ascites from hybridoma-bearing mice at intervals of 3 days. The antibodies used were GK1.5 (anti-CD4), 2.43 (anti-CD8), and PK136 (anti-NK) McAbs. Normal rat IgG was given as control Ab. Depletion of T-cell subsets and NK cells was monitored by flow cytometry, which showed >90% specific depletion in splenocytes. Tumor measurements were made 5 days after the last injection of Ad-Lptn. Columns represent mean tumor diameters

Discussion

Although IL-10 is commonly regarded as an immunosuppressive cytokine that favored tumor escape from immune surveillance, a wealth of evidence is accumulating that IL-10 also possesses some immunostimulating properties. The production of IL-10 within the tumor microenvironment can be sustained by malignant cells and tumor-infiltrating macrophages and lymphocytes, including NK cells and T cells. It has been associated with tumor regression in some preclinical models, although the molecular mechanisms underlying this effect have not been well understood yet. Transfection of mouse carcinoma [3, 7] and melanoma cell lines [11] with IL-10 elicits loss of tumorigenicity and increases immunogenicity accompanied by a strong lymphocyte and antibody-dependent immune memory. Other investigators not only have reported that IL-10 secreting murine tumor cells can be highly immunogenic when compared with unmodified parental cells but also have shown that IL-10 does not inhibit IFN-γ production by CD8+ cytotoxic T cells, opposite to what is observed with CD4+ Th1 cells [12]. Yet, exogenous IL-10 administration can mediate regression of established melanoma and breast cancer metastases in various preclinical in vivo models [4–6, 10, 13]. It is noticeable that tumor rejection is inhibited by viral IL-10, and cellular IL-10 (cIL-10) favors the eradication of cancer cells, confirming that the double function of this cytokine can be split and linked to different domains of the molecule [14]. In particular, investigators have shown that a single amino acid substitution can abrogate the ability of cIL-10 to mediate tumor regression [15].

In fact, the antitumor effects of tumor cell line genetically modified with cytokine gene alone are still far from satisfactory. The reasons may be manifold and partly due to the failure to recruit lymphocytes of the appropriate specificity. Many studies have demonstrated that gene transfer with a variety of chemokines elicits potent antitumor activity. Lymphotactin is a unique member of the C class of chemokines. Ex vivo experiments have suggested that Lptn is particularly important for the recruitment of T cells and NK cells to the site of an immune response [16–19]. It has been demonstrated that transfectin of Lptn gene into tumor tissue induces potent antitumor immunity. Adenoviral vectors expressing Lptn and IL-2 or Lptn and IL-12 synergize to facilitate tumor regression in murine breast cancer models [20]. In our previous studies, DC genetically modified with Lptn and gp100 gene exhibited enhanced preferential chemotaxis to T cells and NK cells and were capable of delivering tumor antigen peptide more efficiently to induce tumor rejection in B16 melanoma model [21, 22]. We have also shown that the therapeutic efficacy of cytosine deaminase suicide gene therapy is potentiated by Ad-Lptn gene transfer in murine CT-26 colon carcinoma or B16 melanoma models [23]. Therefore, the intratumoral injection of Ad-Lptn may have the potential to overcome the above limitation and may be a promising approach to improve the therapeutic efficacy of cytokines.

In this study, we selected EG7 cell line (EL-4 transfected with ovabumin gene), because IL-10 served as adjuvant to peptide-pulsed APC vaccine and induced much more effective immune response in the ovabumin-expressing subcutaneous implants model [24]. The intratumoral Lptn gene transfer caused local accumulation of more NK cells and T cells that can subsequently be activated by EG7 cells, which will contribute to induce more potent antitumor effect of IL-10 gene therapy. Our results demonstrated that IL-10 gene transfer into EG7 cells could inhibit its tumorigenesis and prolonged survival of tumor-bearing mice (2 of 7 mice treated with IL-10 were tumor-free and survived more than 90 days) (Fig. 1). Moreover, testing showed that inhibition of tumor growth after treatment with Ad-Lptn in EG7 tumor-bearing mice was observed most significantly in a therapeutic setting when compared with that in mice treated with PBS or Ad-Lacz control (Fig. 4a). The survival time was further prolonged in mice; 6 of 7 mice treated with IL-10 and Lptn were tumor-free and survived more than 90 days (Fig. 4d).

The mechanisms involved in the improved antitumor efficacy of the IL-10 and Lptn might be related to the following aspects. First, effective induction of cell-mediated immune response by IL-10 or Lptn might play an important role. Some authors have suggested that the IL-10 antitumor activity observed in animal models might be mainly mediated by NK cells [4, 6, 25]. The involvement of either NK cells or T-cell subset in an antitumor response was proposed to be related to MHC class I expression by the tumor cells. Downregulation of MHC class I levels was associated with increased NK activity [25, 26]. NK cell in vitro cytotoxicity has been reported to be enhanced by IL-10 [27–32]. IL-10 is a potent antimetastatic agent that is effective in immunocompromised hosts. This effect thus appears to be relatively independent of T-cell function but is dependent on NK-cell activity. In contrast, the inhibitory effect of IL-10 on tumorigenesis relies on T-cell function [4, 10]. However, in our study, the splenic NK cells and CTL cytotoxicities were enhanced significantly after IL-10 gene transfer or treated with Ad-Lptn (39.47 vs. 50.42%, 47.64 vs. 60.18% Figs. 2, 5). And the highest NK cells and CTL activity was induced in the EG7/IL-10 tumor-bearing mice treated with Ad-Lptn (56.67 vs. 81.23%). It suggested that the non-specific and specific antitumor immunity were activated potently and might be involved in the antitumor response of IL-10 and Lptn. In vivo depletion experiments with anti-NK1.1, anti-CD4, and anti-CD8 monoclonal antibodies illustrated that CD8+ T cells were the predominant effector cells in EG7 tumor-bearing mice treated with Ad-Lptn, and NK cells were the predominant effector cells in EG7/IL-10 tumor-bearing mice treated with Ad-Lptn (Fig. 10). CD4+ T cells were necessary in the effector phase but were less important. These results suggested that priming of NK cells and CTL after IL-10 gene transfer or treated with Ad-Lptn induced effective antitumor immunity.

Second, IL-10 inhibited angiogenesis in vivo. Malignant tumors do not grow beyond 2–3 mm3 and cannot metastasize unless they stimulate the formation of new blood vessels and thus provide a route for the increased inflow of nutrients and oxygen and outflow of waste products [33]. Some authors thought that IL-10 can also inhibit tumor-infiltrating macrophages (TIM) and decrease tumor-induced angiogenesis and enhance the production of tumor-toxic molecules which leads to tumor regression in some preclinical models [5, 8, 34]. In vivo, the decrease in neovascularization found in IL-10 secreting tumors is most likely due to the ability of IL-10 to downregulate the synthesis of VEGF, IL-1α, TNF-α, IL-6, and MMP-9 in tumor-associated macrophages, which are known to be promoters of angiogenesis [35]. In our study, IP-10 and Mig were strongly expressed, while VEGF and MMP-2 were weakly expressed in EG7 tumor tissues after IL-10 gene transfer or treated with Ad-Lptn (Fig. 7). And the most significant expression was observed in the EG7/IL-10 tumor-bearing mice treated with Ad-Lptn. Moreover, immunohistochemical staining of VEGF in the tumor issues showed that the expression of VEGF in EG7 tumor tissues after IL-10 gene transfer or treated with Ad-Lptn was weaker when compared with control (Fig. 8). In the EG7/IL-10 tumor-bearing mice treated with Ad-Lptn, the expression of VEGF in the tumor was hardly found. Furthermore, the results of histological examination also showed that manifest necrosis was observed most significantly to be present in the tumor mass after IL-10 gene transfer and treated with Ad-Lptn (Table 1). These results illustrated that IL-10 or Lptn inhibited angiogenesis through increasing IP-10 and Mig expression and reducing VEGF and MMP-2 expression and finally caused manifest necrosis of tumor tissues for lack of enough nutriments. As is well known, IP-10 and Mig are potent chemoattractants for Th1 and induce T lymphocytes to the tumor site and result in tumor regression, which is associated with IFN-gamma-dependent antiangiogenesis involved in tumor rejection by CD4+ and CD8+ T-cell effectors [36]. This increase in IP-10 and Mig correlated with the increased CTL cytotoxicity (Figs. 2, 5).

Thirdly, it is accepted that the balance between Th1 and Th2 cells plays an important role in antitumor immune response [37]. IL-2 and IFN-γ, produced by Th1 cells and NK cells, are critically important for the induction of antitumor cellular immunity in vivo [38]. Therefore, induction of Th1 type cytokines can be beneficial to tumor eradication. Here, we showed that the production of IL-2 and IFN-γ from splenocytes increased in mice treatment with Ad-Lptn (420 vs. 1,658 pg/ml Fig. 6a, b). The reason may be related to the fact that more T cells and NK cells were attracted by Lptn and activated by the DC presenting tumor antigens. Then, more Th1 cytokines were secreted. However, assessment of the immune status of the animals demonstrated that mice inoculated with EG7/IL-10 cancer cells showed prevalence of a systemic and tumor-specific Th2 response. Spleen cells obtained from these mice showed an increased production of IL-4 and IL-10 (342 vs. 479 pg/ml Fig. 3c, d), characteristic of a Th2 response. This T-cell response occurred in the context of a shift toward a Th2 response, which agreed with previous study [7]. But it seems a little confused that splenocytes from the combined treatment group produced the highest levels of IL-10 (752 pg/ml Fig. 6d), while accompanied with higher levels of IL-2 and IFN-γ (342 vs. 1,112 pg/ml Fig. 6a, b). This might be partially attributed to the chemotactic effect of Lptn, which leads to large amount of IL-10 production in the combined treatment group. Further investigation is still needed to confirm it.

Lastly, downregulation of immune suppressive cells (CD4+Foxp3+ Treg cells and Gr1+CD11b+ MDSCs) contributes to enhanced host antitumor immune response. The recognition that immune suppression is crucial to promote tumor progression, which might explain the failure of some cancer vaccines, has resulted in a paradigm shift regarding approaches to cancer immunotherapy. Two major immunosuppressive cell types are mainly involved in tumor-induced immunosuppression: Tregs and MDSCs. The CD4+Foxp3+ Treg cells have a crucial role in tumor immune pathogenesis, and they also modulate immune therapeutic efficacy [39, 40]. Myeloid-derived suppressor cells (MDSC) have been identified as a population of immature myeloid cells with the ability to suppress T-cell activation in humans and mice [41, 42]. MDSC are well-known inhibitors of CD8+ T-cell antigen-specific reactivity by different mechanisms, mainly through their capacities to produce nitric oxide and radical oxygen species [43, 44]. We explored the mechanism underlying IL-10 and Lptn gene therapy for cancer and found that CD4+Foxp3+ Treg cells from spleens of mice were significantly decreased (85.2%) in IL-10 gene transfer group and Lptn gene therapy group (74.3%) (Fig. 9a). Similarly, significant decrease in the expression of Gr1+CD11b+ MDSCs was also observed in the IL-10 gene transfer group (22.8%) and Lptn gene therapy group (82.5%) (Fig. 9b). This decrease in Treg cell development correlated with the increased NK cells and CTL cytotoxicity (Figs. 2, 5).

Overall, IL-10 gene transfer into EG7 cells could induce therapeutic antitumor immunity more potently after injection of Ad-Lptn. Efficient induction of specific and non-specific antitumor immunity might be responsible for the enhanced efficacy of the IL-10. These observations outline a potent strategy to improve the efficacy of IL-10 and Ad-Lptn.

Acknowledgments

We thank Dr. Qingqing Wang, Dr. Jianping Pan, and Dr. Weilin Chen for helpful discussion, Dr. Huarong Chen and Mr. Ying Zhong for technical assistance. This work was supported by grants from the National Natural Science Foundation of China (30471588), Zhejiang Provincial Natural Science Foundation of China (R207443), and National Key Basic Research Program of China (2007CB512807 and 2007CB512400).

Footnotes

Jianbin Zhang is the first author and Zhidong Zhou is co-first author.

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