Purpose: The use of costimulatory molecules targeting distinct T-cell signaling pathways has provided a means for triggering and enhancing antitumor immunity; however, it is still not fully understood what types of costimulatory molecules are suitable for the combination in tumor therapy. Our purpose in this study is to establish an effective antitumor immune approach by using costimulatory molecule 4-1BBL in combination with soluble PD-1.

Experimental Design: The murine H22 hepatocarcinoma served as an ectopic tumor model. Local gene transfer was done by injection with naked plasmid p4-1BBL and/or psPD-1. The synergistic mechanism of dual-gene therapy was elucidated by detecting the change of gene expression of immunoregulatory factors in tumor microenvironment. The effects of immunotherapy were evaluated by testing the function of tumor-specific T cells, measuring tumor weight or volume, survival of mice, and H&E staining of tissues.

Results: 4-1BBL expressed by normal nonimmune cells effectively enhanced antitumor immune response but up-regulated PD-L1 and did not reduce IL-10 and transforming growth factor-β (TGF-β). sPD-1 synergized with 4-1BBL to establish efficient antitumor immune environment, including down-regulation of IL-10 and TGF-β, further up-regulation of interleukin (IL)-2 and IFN-γ, and higher CD8+ T-cell infiltration. The combined treatment by 4-1BBL/sPD-1 eradicated tumors from mice with small amounts of preexistent tumor cells or tumors from ∼60% of individuals with larger amounts of preexistent tumor cells.

Conclusions: Our findings in this report imply a great potential of 4-1BBL in combination with sPD-1 in tumor therapeutics with the in vivo existent tumor cells as antigens.

The application of defined tumor antigens as therapeutic cancer vaccines has not yet resulted in a significant efficacy in cancer patients (1–4), which is partly explained by the existence of other undefined epitopes of tumor antigens in many cases (5) so that the therapeutic tumor vaccines may not cover all of residual tumor cells after different treatments. Therefore, it might be more valuable to use the in vivo existent tumor cells as the repertoire of tumor antigens for the generation of the pool of tumor-specific T cells to eradicate the small amount of residual tumor cells in vivo. The obstacle for this strategy is that the immunogenicities of self/tumor antigens on tumor cells in vivo are too weak to effectively induce the activation of tumor-specific T cells (5). However, the utilization of costimulatory molecules targeting distinct T-cell signaling pathways provides a means to trigger an effective antitumor immune response with tumor cells as antigens.

4-1BB, an attractive costimulatory molecule for tumor immunotherapy, is expressed on activated T cells and antigen-presenting cells such as dendritic cells (6–9). Therapies by utilizing the 4-1BBL/4-1BB signaling pathway have been shown to generate antitumor effects in various model systems (10–13). Recently, 4-1BBL and anti–4-1BB antibody have been used in tumor therapy in combination with other therapeutic elements such as cytokine interleukin (IL)-12, tumor vaccines, radiation therapy, and anti–CTLA-4 antibody (6, 13–16). The activation of 4-1BB signaling usually results in the production of a large amount of IFN-γ, which is capable of inducing or up-regulating the expression of PD-L1 (also B7-H1) on both immune cells and tumor cells (17–19). As coinhibitory molecules, PD-L1 and its receptor PD-1 mediate the inhibition of T-cell response (20, 21) and thus play an important role in down-regulating the antitumor immunity at effector phase. These findings imply a possibility of further improvement of antitumor effect of 4-1BBL by reducing the related immunosuppressive elements.

Blockade of the PD-L1/PD-1 pathway with antibody or soluble PD-1 (sPD-1) has been proved to enhance antitumor immunity (22–24) and inhibit tumor growth (18, 25). Our previous study has also confirmed that sPD-1 can reduce IFN-γ–mediated immunosuppressive effect (26). In this study, we tried to use sPD-1 to counteract the negative immunoregulatory elements existent in the process of tumor immunotherapy with 4-1BBL. 4-1BBL alone effectively enhanced antitumor immune response but up-regulated PD-L1 and did not reduce IL-10 and TGF-β in tumor microenvironment. Such disadvantages were compensated by sPD-1. In addition, a more ideal curative effect on tumor-bearing mice was realized by using 4-1BBL in combination with sPD-1. These findings verify that the modulation of 4-1BBL treatment with sPD-1 can trigger robust antitumor immunity with in vivo existent tumor cells as antigens.

Animals and cell lines. Six- to 8-week-old female BALB/c mice were purchased from Center of Medical Experimental Animals of Hubei Province (Wuhan, China). The animals were maintained in Tongji Medical Animal Facilities under pathogen-free conditions. All studies involving mice (including tumor cell inoculation and tumor weight in experiments) were approved by the institute's Animal Care and Use Committee. Baby hamster kidney (BHK) cell line, mouse H22 hepatocarcinoma cell line (BALB/c background), and mouse melanoma B16F1 cell line (C57BL/6 background) were purchased from China Center for Type Culture Collection (Wuhan, China) and cultured in DMEM with 10% FCS.

Plasmids. Eukaryotic expression vector p4-1BBL carrying full-length cDNA of murine 4-1BBL, psPD-1 carrying the cDNA encoding extracellular domain of murine PD-1 (sPD-1; ref. 25), and pGRA carrying the cDNA encoding extracellular domain of mouse IFN-γ receptor α-chain (26) were constructed by insertion of cDNA into plasmid pcDNA3.1 (Invitrogen, Carlsbad, CA) and were preserved in our laboratory.

Gene transfection in vitro and in vivo. Plasmids were prepared and analyzed as previously described (23, 25). The in vitro transfection of BHK cells was done with FuGENE 6 reagent (Roche Diagnostics, Indianapolis, IN) following the manufacturer's instructions. G418 (500 μg/mL) was used to select the stable transfection clones.

The in vivo transfection was done by direct local injection. Briefly, naked plasmid (100 μg in 100-μL saline) was injected directly into muscle (i.m. injection) of inoculation site. The plasmids were injected every 3 days because the mRNA expression by the vector decreased 3 days after i.m. injection of plasmids as observed in the preliminary experiments and our previous studies.

Conventional reverse transcription-PCR and real-time PCR. Total RNAs were isolated, using TRIzol reagent (Invitrogen) according to the manufacturer's instructions, from cells or muscle tissues of normal mice or tumor marginal tissues of tumor-bearing mice. A reverse transcription-PCR (RT-PCR) procedure was used to determine relative quantities of mRNA (OneStep RT-PCR kit, Qiagen, Valencia, CA). Twenty-eight PCR cycles were used for all of the analyses and the mRNA of β-actin was used as the internal control. The sequences of primers for detection of 4-1BBL mRNA were as follows: sense, 5′-CTGTGTTCCTATCTTCACCC-3′; antisense, 5′-TGTCTTCTTCGTACCTCAG-3′. The primers for other genes were previously described (25).

Quantitative real-time PCR for PD-L1, IL-2, IFN-γ, IL-10, and TGF-β1 genes was done using primers and procedures previously described (23, 25). The results were expressed as the expression level of each gene relative to that of housekeeping gene β-actin.

Western blot analysis. Seventy-two hours after transfection, cells and tissue samples were lysed or homogenized for Western blot as described (27). Rat anti-mouse 4-1BBL monoclonal antibody (TKS-1) was kindly provided by Prof. Hideo Yagita (Juntendo University, Tokyo, Japan). Antimouse PD-1 polyclonal antibody was purchased from R&D Systems (Minneapolis, MN).

Proliferation assay. BHK cells transfected with p4-1BBL, pcDNA3.1, or without transfection were cultured and fixed in 96-well culture plates with glutaric dialdehyde. Mouse splenocytes were cultured in wells and stimulated with HSP70-H22 peptide complexes (23, 25). To determine the proliferation of T cells, 1.0-μCi [3H]thymidine was added during the last 10 h of 72-h culture and then the incorporation of [3H]thymidine was measured in a MicroBeta TriLux liquid scintillation counter (Wallac, Turku, Finland).

ELISA. Mouse spleen cells were stimulated in the same way as mentioned earlier. The cells were passaged 72 h later and cultured for another 48 h. The levels of IFN-γ and IL-2 in the supernatants were assessed by ELISA using murine IFN-γ or IL-2 ELISA Kit (eBioscience, San Diego, CA) according to the manufacturer's protocol.

Cytotoxicity assay. Splenocytes were stimulated with HSP70-H22 peptide complexes in the presence or absence of BHK-expressed 4-1BBL, as described above, and cultured for 7 days in the presence of 20 units/mL IL-2. Then, the cells were used as effector cells for cytotoxicity assay. In other experiments, splenocytes from tumor-bearing mice were individually stimulated with HSP70-H22 peptide complexes in vitro for 3 days and used as effector cells. The flow cytometry–based method (27) was used for cytotoxicity assay. Briefly, carboxyfluorescein diacetate succinamidyl ester (CFSE)–labeled H22 and B16F1 target cells were incubated with effectors at different effector-to-target ratios at 37°C for 4 h. After staining with dye 7-amino-actinomycin D (7-AAD) to mark dead or dying cells, cytotoxicity was evaluated by flow cytometry. Cytolysis was determined by 7-AAD+CFSE+ cells / total CFSE+ cells.

Animal experiments and treatment protocol. BALB/c mice were inoculated with H22 cells by injection of 1 × 105 cells into right hind thigh muscle. On day 2 after inoculation, mice from treatment groups received 100 μg of plasmid DNA by i.m. injection (local naked DNA transfection). Fifty micrograms of p4-1BBL and 50 μg of psPD-1 were used in combination treatment. Gene delivery was done every 3 days for the indicated times in different experiments. The mice of control groups received an equal volume of saline or equal amount of pcDNA3.1 plasmid. In some experiments, HSP70-H22 peptide complexes (25) were used as tumor vaccine and injected together with plasmids as indicated. Mice were sacrificed and tumors were dissected and weighed on the indicated day after inoculation. In some experiments, the incidence of tumor in mice and the survival of mice were recorded.

Histology and immunohistochemistry. Mouse muscle tissues of inoculation sites of the treated mice were surgically excised, fixed for 12 to 24 h in 4% formalin, embedded in paraffin, and sectioned for H&E staining. For indirect immunostaining, fresh tissues were embedded in optimum cutting temperature solution and cut into 10-μm sections. Sections were fixed by acetone and incubated overnight at 4°C with rat anti-mouse CD8 monoclonal antibody diluted at 1/100. Biotinylated rat anti-mouse immunoglobulin G was used as secondary antibody, followed by streptavidin-conjugated horseradish peroxidase in the third step. The antibodies were purchased from Abcam, Inc. (Cambridge, MA).

Statistics. Results were expressed as mean ± SD and interpreted by ANOVA repeated measures test and Kaplan-Meier analysis. Differences were considered to be statistically significant when P < 0.05.

Normal nonimmune cell–expressed 4-1BBL possesses antitumor immune function. Agonistic 4-1BB antibody is generally used to trigger 4-1BB signal in tumor immunity. We constructed a 4-1BBL–expressing vector (p4-1BBL) as an alternative strategy to activate 4-1BB signal. BHK cells were transfected with p4-1BBL and expression of p4-1BBL was identified by RT-PCR and Western blot (Fig. 1A). Thereby, mouse splenocytes were stimulated with HSP70-H22 peptide complexes in the presence or absence of BHK-expressed 4-1BBL. The results showed that both the cell proliferation (Fig. 1B) and the production of IL-2 and IFN-γ (Fig. 1C) were significantly increased in the presence of BHK-expressed 4-1BBL. When the splenocytes were stimulated with HSP70-H22 peptide complexes for 7 days, BHK-expressed 4-1BBL also significantly enhanced the specific cytotoxicity of the activated lymphocytes to H22 tumor cells but not to melanoma B16 cells (Fig. 1D). To validate the in vitro results in vivo, we then, on the basis of identifying the expression of p4-1BBL in muscle (Fig. 1A), treated H22 tumor-bearing mice by i.m. injection with p4-1BBL. The significant inhibition of tumor growth was observed at different time points in p4-1BBL group (Fig. 1E). Taken together, these results indicate that 4-1BBL expressed by normal nonimmune cells can trigger antitumor immune response and inhibit tumor growth; however, the effect of 4-1BBL/4-1BB signal alone is limited and not enough to eradicate tumor in vivo.

Fig. 1.
Fig. 1. The immunoregulatory effect of 4-1BBL expressed in nontumor nonimmune cells. A, expression of 4-1BBL in BHK cells or muscle after transfection with p4-1BBL. The expression of 4-1BBL mRNA and protein was determined by RT-PCR and Western blot 72 h after the transfection. B and C, activation of spleen cells was augmented by BHK-expressed 4-1BBL. Spleen cells from three naive mice were separately stimulated in triplicate with HSP70 peptide complexes in the presence or absence of BHK-expressed 4-1BBL. Control lymphocytes were cultured in the absence of HSP70 peptides and BHK-expressed 4-1BBL. Cell proliferation was determined by [3H]thymidine incorporation assay (B) and IL-2 and IFN-γ in the supernatants were detected by ELISA (C). D, cytotoxicity of spleen cells to H22 cells. Spleen lymphocytes from three naive mice were activated individually in vitro with HSP70-H22 peptide complexes in the presence or absence of BHK-expressed 4-1BBL and then incubated in triplicate with either H22 cells or melanoma B16F1 cells. Control lymphocytes were cultured in the absence of HSP70 peptides and BHK-expressed 4-1BBL before the incubation with target cells. The cytotoxicity of spleen cells to tumor cells was measured. E, inhibitory effect of in vivo transfection of p4-1BBL on tumor growth. Mice (n = 8 per group at each time point) were inoculated with H22 hepatocarcinoma cells and treated with p4-1BBL (four times for 14-d observation and eight times for 28-d observation) as described in Materials and Methods. Data are representative of two independent experiments. *, P < 0.05, compared with control group. **, P < 0.05, compared with pcDNA3.1 group.
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The immunoregulatory effect of 4-1BBL expressed in nontumor nonimmune cells. A, expression of 4-1BBL in BHK cells or muscle after transfection with p4-1BBL. The expression of 4-1BBL mRNA and protein was determined by RT-PCR and Western blot 72 h after the transfection. B and C, activation of spleen cells was augmented by BHK-expressed 4-1BBL. Spleen cells from three naive mice were separately stimulated in triplicate with HSP70 peptide complexes in the presence or absence of BHK-expressed 4-1BBL. Control lymphocytes were cultured in the absence of HSP70 peptides and BHK-expressed 4-1BBL. Cell proliferation was determined by [3H]thymidine incorporation assay (B) and IL-2 and IFN-γ in the supernatants were detected by ELISA (C). D, cytotoxicity of spleen cells to H22 cells. Spleen lymphocytes from three naive mice were activated individually in vitro with HSP70-H22 peptide complexes in the presence or absence of BHK-expressed 4-1BBL and then incubated in triplicate with either H22 cells or melanoma B16F1 cells. Control lymphocytes were cultured in the absence of HSP70 peptides and BHK-expressed 4-1BBL before the incubation with target cells. The cytotoxicity of spleen cells to tumor cells was measured. E, inhibitory effect of in vivo transfection of p4-1BBL on tumor growth. Mice (n = 8 per group at each time point) were inoculated with H22 hepatocarcinoma cells and treated with p4-1BBL (four times for 14-d observation and eight times for 28-d observation) as described in Materials and Methods. Data are representative of two independent experiments. *, P < 0.05, compared with control group. **, P < 0.05, compared with pcDNA3.1 group.

Fig. 1.
Fig. 1. The immunoregulatory effect of 4-1BBL expressed in nontumor nonimmune cells. A, expression of 4-1BBL in BHK cells or muscle after transfection with p4-1BBL. The expression of 4-1BBL mRNA and protein was determined by RT-PCR and Western blot 72 h after the transfection. B and C, activation of spleen cells was augmented by BHK-expressed 4-1BBL. Spleen cells from three naive mice were separately stimulated in triplicate with HSP70 peptide complexes in the presence or absence of BHK-expressed 4-1BBL. Control lymphocytes were cultured in the absence of HSP70 peptides and BHK-expressed 4-1BBL. Cell proliferation was determined by [3H]thymidine incorporation assay (B) and IL-2 and IFN-γ in the supernatants were detected by ELISA (C). D, cytotoxicity of spleen cells to H22 cells. Spleen lymphocytes from three naive mice were activated individually in vitro with HSP70-H22 peptide complexes in the presence or absence of BHK-expressed 4-1BBL and then incubated in triplicate with either H22 cells or melanoma B16F1 cells. Control lymphocytes were cultured in the absence of HSP70 peptides and BHK-expressed 4-1BBL before the incubation with target cells. The cytotoxicity of spleen cells to tumor cells was measured. E, inhibitory effect of in vivo transfection of p4-1BBL on tumor growth. Mice (n = 8 per group at each time point) were inoculated with H22 hepatocarcinoma cells and treated with p4-1BBL (four times for 14-d observation and eight times for 28-d observation) as described in Materials and Methods. Data are representative of two independent experiments. *, P < 0.05, compared with control group. **, P < 0.05, compared with pcDNA3.1 group.
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The immunoregulatory effect of 4-1BBL expressed in nontumor nonimmune cells. A, expression of 4-1BBL in BHK cells or muscle after transfection with p4-1BBL. The expression of 4-1BBL mRNA and protein was determined by RT-PCR and Western blot 72 h after the transfection. B and C, activation of spleen cells was augmented by BHK-expressed 4-1BBL. Spleen cells from three naive mice were separately stimulated in triplicate with HSP70 peptide complexes in the presence or absence of BHK-expressed 4-1BBL. Control lymphocytes were cultured in the absence of HSP70 peptides and BHK-expressed 4-1BBL. Cell proliferation was determined by [3H]thymidine incorporation assay (B) and IL-2 and IFN-γ in the supernatants were detected by ELISA (C). D, cytotoxicity of spleen cells to H22 cells. Spleen lymphocytes from three naive mice were activated individually in vitro with HSP70-H22 peptide complexes in the presence or absence of BHK-expressed 4-1BBL and then incubated in triplicate with either H22 cells or melanoma B16F1 cells. Control lymphocytes were cultured in the absence of HSP70 peptides and BHK-expressed 4-1BBL before the incubation with target cells. The cytotoxicity of spleen cells to tumor cells was measured. E, inhibitory effect of in vivo transfection of p4-1BBL on tumor growth. Mice (n = 8 per group at each time point) were inoculated with H22 hepatocarcinoma cells and treated with p4-1BBL (four times for 14-d observation and eight times for 28-d observation) as described in Materials and Methods. Data are representative of two independent experiments. *, P < 0.05, compared with control group. **, P < 0.05, compared with pcDNA3.1 group.

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The immune response profile is improved by using 4-1BBL in combination with sPD-1. One of the possibilities for the limitation of 4-1BBL–mediated antitumor effect is the up-regulation of PD-L1 by 4-1BBL–promoted production of IFN-γ. To verify this, we first checked the effect of 4-1BBL on PD-L1 by analyzing the transcription activity of PD-L1 gene in tumor tissues after treatment by i.m. transfection with p4-1BBL or in combination with plasmid pGRA, the expression vector of a soluble IFN-γ receptor. The treatment with p4-1BBL significantly increased the transcription activities of both IFN-γ and PD-L1 genes, whereas the transcription of PD-L1 gene was significantly reduced by soluble IFN-γ receptor (Fig. 2A). The results confirmed that the up-regulation of PD-L1 by 4-1BBL is mediated by IFN-γ.

Fig. 2.
Fig. 2. Effect of 4-1BBL and sPD-1 on the expression profile of PD-L1 and four cytokine genes. A, indirect influence of 4-1BBL on the expression of PD-L1 gene in tumor microenvironment. Mice (n = 3 per group) were inoculated with H22 cells and treated with pcDNA 3.1, p4-1BBL, pGRA, and p4-1BBL/pGRA. On day 14 after tumor inoculation, total RNAs were isolated from tumor marginal tissues and the expression of IFN-γ and PD-L1 genes was analyzed with real-time PCR. B, expression of p4-1BBL and psPD-1 in muscle tissue after local gene transfection. Naked plasmid DNA was injected into the muscles of mice (n = 3 per group) and mRNA and protein were determined by RT-PCR and Western blot, respectively, 72 h after transfection. C and D, effect of 4-1BBL and sPD-1 on the expression of PD-L1 and four cytokine genes in tumor microenvironment. Mice were inoculated with H22 cells and treated with pcDNA3.1, psPD-1, p4-1BBL, and p4-1BBL/psPD-1. On days 14 and 28 after tumor inoculation, total RNAs were isolated from tumor marginal tissues of three mice in each group at each time point, and the transcription activities of PD-L1 gene (C) and IL-2, IFN-γ, IL-10, and TGF-β genes (D) were analyzed with RT-PCR and real-time PCR. Data are representative of two independent experiments. *, P < 0.05, compared with pcDNA3.1 group.
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Effect of 4-1BBL and sPD-1 on the expression profile of PD-L1 and four cytokine genes. A, indirect influence of 4-1BBL on the expression of PD-L1 gene in tumor microenvironment. Mice (n = 3 per group) were inoculated with H22 cells and treated with pcDNA 3.1, p4-1BBL, pGRA, and p4-1BBL/pGRA. On day 14 after tumor inoculation, total RNAs were isolated from tumor marginal tissues and the expression of IFN-γ and PD-L1 genes was analyzed with real-time PCR. B, expression of p4-1BBL and psPD-1 in muscle tissue after local gene transfection. Naked plasmid DNA was injected into the muscles of mice (n = 3 per group) and mRNA and protein were determined by RT-PCR and Western blot, respectively, 72 h after transfection. C and D, effect of 4-1BBL and sPD-1 on the expression of PD-L1 and four cytokine genes in tumor microenvironment. Mice were inoculated with H22 cells and treated with pcDNA3.1, psPD-1, p4-1BBL, and p4-1BBL/psPD-1. On days 14 and 28 after tumor inoculation, total RNAs were isolated from tumor marginal tissues of three mice in each group at each time point, and the transcription activities of PD-L1 gene (C) and IL-2, IFN-γ, IL-10, and TGF-β genes (D) were analyzed with RT-PCR and real-time PCR. Data are representative of two independent experiments. *, P < 0.05, compared with pcDNA3.1 group.

Fig. 2.
Fig. 2. Effect of 4-1BBL and sPD-1 on the expression profile of PD-L1 and four cytokine genes. A, indirect influence of 4-1BBL on the expression of PD-L1 gene in tumor microenvironment. Mice (n = 3 per group) were inoculated with H22 cells and treated with pcDNA 3.1, p4-1BBL, pGRA, and p4-1BBL/pGRA. On day 14 after tumor inoculation, total RNAs were isolated from tumor marginal tissues and the expression of IFN-γ and PD-L1 genes was analyzed with real-time PCR. B, expression of p4-1BBL and psPD-1 in muscle tissue after local gene transfection. Naked plasmid DNA was injected into the muscles of mice (n = 3 per group) and mRNA and protein were determined by RT-PCR and Western blot, respectively, 72 h after transfection. C and D, effect of 4-1BBL and sPD-1 on the expression of PD-L1 and four cytokine genes in tumor microenvironment. Mice were inoculated with H22 cells and treated with pcDNA3.1, psPD-1, p4-1BBL, and p4-1BBL/psPD-1. On days 14 and 28 after tumor inoculation, total RNAs were isolated from tumor marginal tissues of three mice in each group at each time point, and the transcription activities of PD-L1 gene (C) and IL-2, IFN-γ, IL-10, and TGF-β genes (D) were analyzed with RT-PCR and real-time PCR. Data are representative of two independent experiments. *, P < 0.05, compared with pcDNA3.1 group.
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Effect of 4-1BBL and sPD-1 on the expression profile of PD-L1 and four cytokine genes. A, indirect influence of 4-1BBL on the expression of PD-L1 gene in tumor microenvironment. Mice (n = 3 per group) were inoculated with H22 cells and treated with pcDNA 3.1, p4-1BBL, pGRA, and p4-1BBL/pGRA. On day 14 after tumor inoculation, total RNAs were isolated from tumor marginal tissues and the expression of IFN-γ and PD-L1 genes was analyzed with real-time PCR. B, expression of p4-1BBL and psPD-1 in muscle tissue after local gene transfection. Naked plasmid DNA was injected into the muscles of mice (n = 3 per group) and mRNA and protein were determined by RT-PCR and Western blot, respectively, 72 h after transfection. C and D, effect of 4-1BBL and sPD-1 on the expression of PD-L1 and four cytokine genes in tumor microenvironment. Mice were inoculated with H22 cells and treated with pcDNA3.1, psPD-1, p4-1BBL, and p4-1BBL/psPD-1. On days 14 and 28 after tumor inoculation, total RNAs were isolated from tumor marginal tissues of three mice in each group at each time point, and the transcription activities of PD-L1 gene (C) and IL-2, IFN-γ, IL-10, and TGF-β genes (D) were analyzed with RT-PCR and real-time PCR. Data are representative of two independent experiments. *, P < 0.05, compared with pcDNA3.1 group.

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Because the blockade of IFN-γ activity in the process of tumor immunotherapy is unreasonable, and we have found that the effective blockade of PD-L1/PD-1 pathway can be realized by the utilization of sPD-1 (23–26), here we hypothesized that sPD-1 could be used to improve 4-1BBL–mediated antitumor immune response. To test this, we first identified the expression of 4-1BBL and sPD-1 after dual-gene delivery (Fig. 2B) and then analyzed the transcription activity of PD-L1 gene in tumor tissue after different treatments. The results showed that the transcription activity of PD-L1 gene in tumor tissue was increased (Fig. 2C) even in the presence of sPD-1. The transcription activities of IFN-γ and IL-2 genes were further increased, whereas the transcription activities of IL-10 and TGF-β genes were significantly decreased, which were not decreased by 4-1BBL alone (Fig. 2D). The modulation of IL-10 and TGF-β in the tumor microenvironment is obviously due to the presence of sPD-1, not 4-1BBL. These data indicate that the combination of 4-1BBL and sPD-1 can remodulate tumor microenvironment and make it bias to antitumor immunity.

sPD-1 enhances 4-1BBL–mediated antitumor immunity. The above data revealed that the immune response profile can be improved if 4-1BBL is used in combination with sPD-1. We then analyzed whether this biased antitumor immunity could generate stronger cytotoxicity to tumor cells and result in the accumulation of more lymphocytes in tumor microenvironment. The cytotoxicity of splenocytes to tumor cells was analyzed on day 14 after tumor inoculation. The results showed that the cytotoxicity of splenocytes to H22 tumor cells (not B16 melanoma cells) was further increased in 4-1BBL/sPD-1 combination group, compared with that of single-gene (4-1BBL or sPD-1) treated group (Fig. 3A). Immunohistochemical studies on day 28 after tumor inoculation revealed a significant difference in the amount of tumor-infiltrating T cells between control and treated mice. The amount of CD8+ cells in the tumor marginal tissue of 4-1BBL/sPD-1 combination group was significantly increased as compared with that of single-gene treated groups (Fig. 3B). The curative effect was also observed at different time points. Tumor growth in each treatment group was significantly suppressed compared with that in pcDNA3.1 control group (Fig. 3C), and the suppression of tumor growth by 4-1BBL/sPD-1 treatment was much stronger than that by single-gene treatment, indicating a synergistic antitumor effect of 4-1BBL and sPD-1.

Fig. 3.
Fig. 3. Antitumor effect of 4-1BBL and sPD-1. Mice were inoculated with H22 hepatocarcinoma cells and treated with pcDNA3.1, psPD-1, p4-1BBL, and p4-1BBL/psPD-1. A, cytotoxicity of spleen cells to H22 cells. On day 14 after tumor inoculation, spleen cells were isolated from tumor-bearing mice (n = 6 per group) for cytotoxicity assay with either H22 or B16F1 cells as target cells. pcDNA3.1-treated group was used as control. The cytotoxicity of spleen cells to tumor cells was measured as described in Materials and Methods. B, on day 28, tumor marginal tissues from different groups (n = 6 per group) were analyzed by immunohistochemistry as described in Materials and Methods. The relative amount of CD8+ T cells was expressed by the average number of CD8+ T cells per high-power lens focus (hpf). C, inhibitory effect of different treatments on tumor growth. Tumors were removed and weighed on day 14 (eight mice in each group) and day 28 (another eight mice in each group) after inoculation. Data are representative of two independent experiments. *, P < 0.05, compared with pcDNA3.1 group. **, P < 0.05, compared with either psPD-1 group or p4-1BBL group.
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Antitumor effect of 4-1BBL and sPD-1. Mice were inoculated with H22 hepatocarcinoma cells and treated with pcDNA3.1, psPD-1, p4-1BBL, and p4-1BBL/psPD-1. A, cytotoxicity of spleen cells to H22 cells. On day 14 after tumor inoculation, spleen cells were isolated from tumor-bearing mice (n = 6 per group) for cytotoxicity assay with either H22 or B16F1 cells as target cells. pcDNA3.1-treated group was used as control. The cytotoxicity of spleen cells to tumor cells was measured as described in Materials and Methods. B, on day 28, tumor marginal tissues from different groups (n = 6 per group) were analyzed by immunohistochemistry as described in Materials and Methods. The relative amount of CD8+ T cells was expressed by the average number of CD8+ T cells per high-power lens focus (hpf). C, inhibitory effect of different treatments on tumor growth. Tumors were removed and weighed on day 14 (eight mice in each group) and day 28 (another eight mice in each group) after inoculation. Data are representative of two independent experiments. *, P < 0.05, compared with pcDNA3.1 group. **, P < 0.05, compared with either psPD-1 group or p4-1BBL group.

Fig. 3.
Fig. 3. Antitumor effect of 4-1BBL and sPD-1. Mice were inoculated with H22 hepatocarcinoma cells and treated with pcDNA3.1, psPD-1, p4-1BBL, and p4-1BBL/psPD-1. A, cytotoxicity of spleen cells to H22 cells. On day 14 after tumor inoculation, spleen cells were isolated from tumor-bearing mice (n = 6 per group) for cytotoxicity assay with either H22 or B16F1 cells as target cells. pcDNA3.1-treated group was used as control. The cytotoxicity of spleen cells to tumor cells was measured as described in Materials and Methods. B, on day 28, tumor marginal tissues from different groups (n = 6 per group) were analyzed by immunohistochemistry as described in Materials and Methods. The relative amount of CD8+ T cells was expressed by the average number of CD8+ T cells per high-power lens focus (hpf). C, inhibitory effect of different treatments on tumor growth. Tumors were removed and weighed on day 14 (eight mice in each group) and day 28 (another eight mice in each group) after inoculation. Data are representative of two independent experiments. *, P < 0.05, compared with pcDNA3.1 group. **, P < 0.05, compared with either psPD-1 group or p4-1BBL group.
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Antitumor effect of 4-1BBL and sPD-1. Mice were inoculated with H22 hepatocarcinoma cells and treated with pcDNA3.1, psPD-1, p4-1BBL, and p4-1BBL/psPD-1. A, cytotoxicity of spleen cells to H22 cells. On day 14 after tumor inoculation, spleen cells were isolated from tumor-bearing mice (n = 6 per group) for cytotoxicity assay with either H22 or B16F1 cells as target cells. pcDNA3.1-treated group was used as control. The cytotoxicity of spleen cells to tumor cells was measured as described in Materials and Methods. B, on day 28, tumor marginal tissues from different groups (n = 6 per group) were analyzed by immunohistochemistry as described in Materials and Methods. The relative amount of CD8+ T cells was expressed by the average number of CD8+ T cells per high-power lens focus (hpf). C, inhibitory effect of different treatments on tumor growth. Tumors were removed and weighed on day 14 (eight mice in each group) and day 28 (another eight mice in each group) after inoculation. Data are representative of two independent experiments. *, P < 0.05, compared with pcDNA3.1 group. **, P < 0.05, compared with either psPD-1 group or p4-1BBL group.

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Tumor vaccine cannot fortify the antitumor effect of 4-1BBL/sPD-1. Either 4-1BBL or sPD-1 has been reported to enhance the antitumor efficacy of tumor vaccines (tumor-specific antigen or antigen-pulsed dendritic cells) by augmenting the response of immune cells to antigen (17, 23, 28, 29). We then asked whether tumor vaccine was still needed to trigger more efficient antitumor immune response in vivo if 4-1BBL is used in combination with sPD-1. In our animal tumor model system, both 4-1BBL and sPD-1 did have a synergistic effect with tumor vaccine (HSP70-H22 peptide complexes; Fig. 4A), indicating that the presentation of more tumor antigens is necessary if 4-1BBL or sPD-1 is used alone. However, the cocktail by joining tumor vaccine, 4-1BBL, and sPD-1 did not further enhance the antitumor effect, which was evaluated by tumor suppression after the treatment (Fig. 4B), the cytotoxicity of splenocytes to H22 cells (Fig. 4C), and the transcription activities of different cytokine genes (Fig. 4D). Moreover, after 4-week treatment, the incidence of tumor was significantly decreased in both 4-1BBL/sPD-1 and 4-1BBL/sPD-1/vaccine groups, and tumor formation was also delayed; however, there was no significant difference between these two groups (Fig. 4E). These data indicate that the presentation of more tumor antigens is not necessary any more if 4-1BBL is used in combination with sPD-1 in tumor-bearing host.

Fig. 4.
Fig. 4. Tumor vaccine could not fortify the antitumor effect of 4-1BBL/sPD-1. A, synergistic effect of tumor vaccine with either 4-1BBL or sPD-1. Mice (n = 6 per group) were inoculated with H22 hepatocarcinoma cells and treated with saline, vaccine (HSP70-H22 peptide complexes), plasmid, and vaccine/plasmid. Tumors were removed and weighed on day 28 after inoculation. B to E, mice were inoculated with H22 hepatocarcinoma cells and treated with saline, vaccine, p4-1BBL/psPD-1, and vaccine/p4-1BBL/psPD-1, respectively. B, inhibitory effects of different treatments on tumor growth. After a 4-wk treatment, tumors were removed and weighed on days 28 and 56 after inoculation in different groups (n = 6 per group at each time point). C, assay of cytotoxicity of spleen cells to H22 cells. On day 14 after tumor inoculation, spleen lymphocytes were isolated from tumor-bearing mice (n = 6 per group). The cytotoxicity of spleen cells to H22 tumor cells was measured as described in Materials and Methods. D, expression of IL-2, IFN-γ, IL-10, and TGF-β in tumor microenvironment on days 14 and 28 after tumor inoculation (n = 3 per group at each time point). The transcription activity of genes was analyzed with real-time PCR. E, effect of different treatments on the incidence of tumor in mice (n = 8 per group). The time of the formation of palpable tumor in each mouse was recorded and the incidence was calculated at different time points after tumor inoculation. Data are representative of two independent experiments. *, P < 0.05, compared with control group. **, P < 0.05, compared with vaccine group.
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Tumor vaccine could not fortify the antitumor effect of 4-1BBL/sPD-1. A, synergistic effect of tumor vaccine with either 4-1BBL or sPD-1. Mice (n = 6 per group) were inoculated with H22 hepatocarcinoma cells and treated with saline, vaccine (HSP70-H22 peptide complexes), plasmid, and vaccine/plasmid. Tumors were removed and weighed on day 28 after inoculation. B to E, mice were inoculated with H22 hepatocarcinoma cells and treated with saline, vaccine, p4-1BBL/psPD-1, and vaccine/p4-1BBL/psPD-1, respectively. B, inhibitory effects of different treatments on tumor growth. After a 4-wk treatment, tumors were removed and weighed on days 28 and 56 after inoculation in different groups (n = 6 per group at each time point). C, assay of cytotoxicity of spleen cells to H22 cells. On day 14 after tumor inoculation, spleen lymphocytes were isolated from tumor-bearing mice (n = 6 per group). The cytotoxicity of spleen cells to H22 tumor cells was measured as described in Materials and Methods. D, expression of IL-2, IFN-γ, IL-10, and TGF-β in tumor microenvironment on days 14 and 28 after tumor inoculation (n = 3 per group at each time point). The transcription activity of genes was analyzed with real-time PCR. E, effect of different treatments on the incidence of tumor in mice (n = 8 per group). The time of the formation of palpable tumor in each mouse was recorded and the incidence was calculated at different time points after tumor inoculation. Data are representative of two independent experiments. *, P < 0.05, compared with control group. **, P < 0.05, compared with vaccine group.

Fig. 4.
Fig. 4. Tumor vaccine could not fortify the antitumor effect of 4-1BBL/sPD-1. A, synergistic effect of tumor vaccine with either 4-1BBL or sPD-1. Mice (n = 6 per group) were inoculated with H22 hepatocarcinoma cells and treated with saline, vaccine (HSP70-H22 peptide complexes), plasmid, and vaccine/plasmid. Tumors were removed and weighed on day 28 after inoculation. B to E, mice were inoculated with H22 hepatocarcinoma cells and treated with saline, vaccine, p4-1BBL/psPD-1, and vaccine/p4-1BBL/psPD-1, respectively. B, inhibitory effects of different treatments on tumor growth. After a 4-wk treatment, tumors were removed and weighed on days 28 and 56 after inoculation in different groups (n = 6 per group at each time point). C, assay of cytotoxicity of spleen cells to H22 cells. On day 14 after tumor inoculation, spleen lymphocytes were isolated from tumor-bearing mice (n = 6 per group). The cytotoxicity of spleen cells to H22 tumor cells was measured as described in Materials and Methods. D, expression of IL-2, IFN-γ, IL-10, and TGF-β in tumor microenvironment on days 14 and 28 after tumor inoculation (n = 3 per group at each time point). The transcription activity of genes was analyzed with real-time PCR. E, effect of different treatments on the incidence of tumor in mice (n = 8 per group). The time of the formation of palpable tumor in each mouse was recorded and the incidence was calculated at different time points after tumor inoculation. Data are representative of two independent experiments. *, P < 0.05, compared with control group. **, P < 0.05, compared with vaccine group.
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Tumor vaccine could not fortify the antitumor effect of 4-1BBL/sPD-1. A, synergistic effect of tumor vaccine with either 4-1BBL or sPD-1. Mice (n = 6 per group) were inoculated with H22 hepatocarcinoma cells and treated with saline, vaccine (HSP70-H22 peptide complexes), plasmid, and vaccine/plasmid. Tumors were removed and weighed on day 28 after inoculation. B to E, mice were inoculated with H22 hepatocarcinoma cells and treated with saline, vaccine, p4-1BBL/psPD-1, and vaccine/p4-1BBL/psPD-1, respectively. B, inhibitory effects of different treatments on tumor growth. After a 4-wk treatment, tumors were removed and weighed on days 28 and 56 after inoculation in different groups (n = 6 per group at each time point). C, assay of cytotoxicity of spleen cells to H22 cells. On day 14 after tumor inoculation, spleen lymphocytes were isolated from tumor-bearing mice (n = 6 per group). The cytotoxicity of spleen cells to H22 tumor cells was measured as described in Materials and Methods. D, expression of IL-2, IFN-γ, IL-10, and TGF-β in tumor microenvironment on days 14 and 28 after tumor inoculation (n = 3 per group at each time point). The transcription activity of genes was analyzed with real-time PCR. E, effect of different treatments on the incidence of tumor in mice (n = 8 per group). The time of the formation of palpable tumor in each mouse was recorded and the incidence was calculated at different time points after tumor inoculation. Data are representative of two independent experiments. *, P < 0.05, compared with control group. **, P < 0.05, compared with vaccine group.

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Tumor cell amount and treatment time influence immunotherapeutic efficacy. The above results showed that 4-1BBL/sPD-1 effectively established antitumor environment in tumor-bearing mice, but the tumors were not completely suppressed in some mice. Therefore, we further analyzed the influence of tumor cell amount and the time of treatment on the therapeutic effect of 4-1BBL/sPD-1. When the mice were inoculated with 1 × 104 H22 tumor cells, the tumors were completely inhibited by a 4-week treatment, evaluated by the incidence of tumor and the survival of mice (Fig. 5A). When the mice were inoculated with a 10-fold dosage of tumor cells (1 × 105), all of the mice in control group died within 40 days, but tumors, after 4-week treatment, were completely inhibited in ∼40% of mice, which survived for more than 100 days. Compared with 4-week treatment, the prolonged treatment (8 weeks) significantly reduced the incidence of tumor and increased the survival rate to ∼60% (P < 0.05, Kaplan-Meier analysis; Fig. 5A).

Fig. 5.
Fig. 5. Therapeutic effects of local transfection with p4-1BBL/psPD-1 in different situations. A, incidence and survival rate follow-up after p4-1BBL/psPD-1 treatment. Mice were inoculated with 1 × 104 or 1 × 105 H22 cells and treated with p4-1BBL/psPD-1 (50 μg each) every 3 d for 4 wk (T-4w) or 8 wk (T-8w). Animals were monitored every week (for incidence of tumor in mice) or every 3 d (for the survival of mice). Data were the combination of two independent experiments (n = 8 in each experiment) in which similar results were obtained. B and C, effect of treatment on tumor growth and histologic analysis of tissues from inoculation site. Mice (n = 16 in 8-wk treatment group, n = 8 in other groups) were inoculated with 1 × 105 H22 cells and treated as described above. The sizes of individual tumors (only for tumor-bearing mice) were monitored, starting from the day of the formation of palpable tumor, and calculated according to the formula V = (a × b2) / 2, with a as the larger diameter and b as the smaller diameter (B). C, 4 wk (w4; four mice), 8 wk (w8; four mice), or 12 wk (w12; five mice) after the inoculation of tumor cells, the tissues from inoculation site of the mice without palpable tumor were analyzed by H&E staining (×200). Details were given in Results.
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Therapeutic effects of local transfection with p4-1BBL/psPD-1 in different situations. A, incidence and survival rate follow-up after p4-1BBL/psPD-1 treatment. Mice were inoculated with 1 × 104 or 1 × 105 H22 cells and treated with p4-1BBL/psPD-1 (50 μg each) every 3 d for 4 wk (T-4w) or 8 wk (T-8w). Animals were monitored every week (for incidence of tumor in mice) or every 3 d (for the survival of mice). Data were the combination of two independent experiments (n = 8 in each experiment) in which similar results were obtained. B and C, effect of treatment on tumor growth and histologic analysis of tissues from inoculation site. Mice (n = 16 in 8-wk treatment group, n = 8 in other groups) were inoculated with 1 × 105 H22 cells and treated as described above. The sizes of individual tumors (only for tumor-bearing mice) were monitored, starting from the day of the formation of palpable tumor, and calculated according to the formula V = (a × b2) / 2, with a as the larger diameter and b as the smaller diameter (B). C, 4 wk (w4; four mice), 8 wk (w8; four mice), or 12 wk (w12; five mice) after the inoculation of tumor cells, the tissues from inoculation site of the mice without palpable tumor were analyzed by H&E staining (×200). Details were given in Results.

Fig. 5.
Fig. 5. Therapeutic effects of local transfection with p4-1BBL/psPD-1 in different situations. A, incidence and survival rate follow-up after p4-1BBL/psPD-1 treatment. Mice were inoculated with 1 × 104 or 1 × 105 H22 cells and treated with p4-1BBL/psPD-1 (50 μg each) every 3 d for 4 wk (T-4w) or 8 wk (T-8w). Animals were monitored every week (for incidence of tumor in mice) or every 3 d (for the survival of mice). Data were the combination of two independent experiments (n = 8 in each experiment) in which similar results were obtained. B and C, effect of treatment on tumor growth and histologic analysis of tissues from inoculation site. Mice (n = 16 in 8-wk treatment group, n = 8 in other groups) were inoculated with 1 × 105 H22 cells and treated as described above. The sizes of individual tumors (only for tumor-bearing mice) were monitored, starting from the day of the formation of palpable tumor, and calculated according to the formula V = (a × b2) / 2, with a as the larger diameter and b as the smaller diameter (B). C, 4 wk (w4; four mice), 8 wk (w8; four mice), or 12 wk (w12; five mice) after the inoculation of tumor cells, the tissues from inoculation site of the mice without palpable tumor were analyzed by H&E staining (×200). Details were given in Results.
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Therapeutic effects of local transfection with p4-1BBL/psPD-1 in different situations. A, incidence and survival rate follow-up after p4-1BBL/psPD-1 treatment. Mice were inoculated with 1 × 104 or 1 × 105 H22 cells and treated with p4-1BBL/psPD-1 (50 μg each) every 3 d for 4 wk (T-4w) or 8 wk (T-8w). Animals were monitored every week (for incidence of tumor in mice) or every 3 d (for the survival of mice). Data were the combination of two independent experiments (n = 8 in each experiment) in which similar results were obtained. B and C, effect of treatment on tumor growth and histologic analysis of tissues from inoculation site. Mice (n = 16 in 8-wk treatment group, n = 8 in other groups) were inoculated with 1 × 105 H22 cells and treated as described above. The sizes of individual tumors (only for tumor-bearing mice) were monitored, starting from the day of the formation of palpable tumor, and calculated according to the formula V = (a × b2) / 2, with a as the larger diameter and b as the smaller diameter (B). C, 4 wk (w4; four mice), 8 wk (w8; four mice), or 12 wk (w12; five mice) after the inoculation of tumor cells, the tissues from inoculation site of the mice without palpable tumor were analyzed by H&E staining (×200). Details were given in Results.

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We then repeated the above experiment by inoculating mice with 1 × 105 H22 cells and further analyzed the efficacy of 4-1BBL/sPD-1. In the treatment groups, some mice formed tumor, and further treatment could delay the development of tumor, but the tumors eventually reached the size similar to that of control group (Fig. 5B). After a 4-week treatment, there was no palpable tumor in some mice, and H&E staining of the tissues from the inoculation sites of these mice showed the existence of a few tumor cells in some individuals and abundant immune cells, but no tumor cells, in other mice without palpable tumor (Fig. 5C). The existence of residual tumor cells revealed the reason why some mice developed tumor after 4-week treatment. After 8-week treatment, some “cured” mice were sacrificed for H&E staining. Tumor cells were not observed; however, the immune cells still existed (Fig. 5C). After 8-week treatment, some cured mice were fed for 4 more weeks. H&E staining of the tissue from the inoculation site showed that the immune cells were gone and the tissues recovered (Fig. 5C), indicating that tumor was really eradicated by the treatment of p4-1BBL/psPD-1 if tumor was not formed after 8-week treatment.

Many tumor immunotherapy efforts are focused on the generation of strong T-cell response against tumor by combination approaches including the utilization of costimulatory molecules. The proper combination of different therapeutic molecules can only be realized on the basis of the choice of suitable molecules and the elucidated mechanism through which they synergize each other. In this study, we chose 4-1BBL and sPD-1 to enhance the activation and function of T cells at both early and late phases of antitumor immune response. Our data show that a proper combination of immunoregulatory factors can establish an antitumor environment with in vivo existent tumor as the source of antigens. In the presence of 4-1BBL and sPD-1, the endogenous tumor antigens can be very efficiently utilized to induce antitumor immune responses that the responses cannot be further amplified by the addition of exogenous antigens such as tumor vaccine. Therefore, in our animal tumor model, 4-1BBL and sPD-1 synergistically established efficient antitumor immune environment to eradicate tumors from mice with small amounts of preexistent tumor cells or to eradicate tumors from more than half of individuals with larger amounts of preexistent tumor cells.

In this study, 4-1BBL is chosen for the purpose of augmentation of T-cell activation through costimulation. We show that the 4-1BBL–expressing vector does not have to enter into specific cells such as tumor cells or immune cells, and the expression of 4-1BBL in nontumor/nonimmune cells can efficiently exert its function in the experiments both in vitro and in vivo. 4-1BBL is capable of enhancing the expression of B7 molecules on dendritic cells to augment the dendritic cell–mediated second signal (B7/CD28 signaling) for T-cell activation (7, 8). In particular, 4-1BBL/4-1BB signaling, unlike B7/CD28, is very important for the effector phase of T-cell response (30–32). The stimulation of 4-1BBL can prevent the apoptosis of CD8+ cells after several cell division cycles (28). More importantly, many CD8+ T cells lose CD28 on their surface (32–34) in the later phase of immune response, and, without further activation, these CD8+CD28− T cells can be converted into suppressor T cells (35) to inhibit immune response at the effector phase. The CD8+CD28− T cells produced in immune response can only be further activated by the 4-1BBL/4-1BB pathway but not by the B7/CD28 pathway (32).

Although 4-1BBL has been shown to produce antitumor effects in various model systems (10–13), our present study revealed that 4-1BBL–induced IFN-γ production resulted in the up-regulation of PD-L1 in our tumor model, and 4-1BBL did not effectively decrease the expression of immunosuppressive cytokines IL-10 and TGF-β in tumor microenvironment. These data indicate that there is still plenty of room for further improvement of immune response when 4-1BBL is used alone. Thus, we further chose sPD-1 to cooperate with 4-1BBL for a better therapeutic efficacy.

Our data show that the blockade of PD-L1 by sPD-1 not only increased the transcription activities of IL-2 and IFN-γ genes in priming phase (first 2 weeks after tumor inoculation) but also improved antitumor immune response at later phase (second 2 weeks). The combination of 4-1BBL with sPD-1 significantly decreased the expression of IL-10 and TGF-β in the treated mice, resulting in the further increased expression of IL-2 and IFN-γ and the accumulation of CD8+ T cells in tumor microenvironment 4 weeks after tumor inoculation. The reduced expression of both IL-10 and TGF-β by sPD-1 favors the prevention of the formation of suppressor T cells, as we previously reported that blockade of the PD-L1/PD-1 pathway with sPD-1 resulted in the reduced amount of regulatory T cells in tumor microenvironment, which was evaluated on the basis of the down-regulation of foxp3 gene expression in tumor environment (23). In addition, it has been reported that blockade of the B7/CTLA-4 pathway enhances the antitumor immune response concomitant with the formation of autoimmune response (36), but that is not the case when the PD-L1/PD-1 pathway is blocked (25). In this study, we further showed that maintaining a relative long treatment period with 4-1BBL/sPD-1 was more beneficial without the induction of autoimmunity because we did not observe any abnormal changes in treated mice, including the functional test of liver and kidney (data not shown).

In summary, the therapeutic strategy with 4-1BBL in combination with sPD-1 boosts not only the chance of the development of a successful means for tumor immunotherapy but also the understanding of the undisclosed roles played by immune system in the face of malignancies. It implicates a potent benefit for the clearance of the residual tumor cells after surgical removal of tumor.

Grant support: National Development Program (973) for Key Basic Research of China, no. 2002CB513100, and the National Natural Science Foundation of China, no. 30471587.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: H. Xiao and B. Huang contributed equally to this work.

1
Wrzesinski C, Restifo NP. Less is more: lymphodepletion followed by hematopoietic stem cell transplant augments adoptive T-cell-based anti-tumor immunotherapy.
Curr Opin Immunol
 
2005
;
17
:
195
–201.
2
Rosenberg SA, Yang JC, Restifo NP. Cancer immunotherapy: moving beyond current vaccines.
Nat Med
 
2004
;
10
:
909
–15.
3
Nestle FO, Farkas A, Conrad C. Dendritic-cell-based therapeutic vaccination against cancer.
Curr Opin Immunol
 
2005
;
17
:
163
–9.
4
Overwijk WW. Breaking tolerance in cancer immunotherapy: time to ACT.
Curr Opin Immunol
 
2005
;
17
:
187
–94.
5
Yu Z, Theoret MR, Touloukian CE, et al. Poor immunogenicity of a self/tumor antigen derives from peptide-MHC-I instability and is independent of tolerance.
J Clin Invest
 
2004
;
114
:
551
–9.
6
Kudo-Saito C, Hodge JW, Kwak H, Kim-Schulze S, Schlom J, Kaufman HL. 4-1BB ligand enhances tumor-specific immunity of poxvirus vaccines.
Vaccine
 
2006
;
24
:
4975
–86.
7
Futagawa T, Akiba H, Kodama T, et al. Expression and function of 4-1BB and 4-1BB ligand on murine dendritic cells.
Int Immunol
 
2002
;
14
:
275
–86.
8
Kim YJ, Li G, Broxmeyer HE. 4-1BB ligand stimulation enhances myeloid dendritic cell maturation from human umbilical cord blood CD34+ progenitor cells.
J Hematother Stem Cell Res
 
2002
;
11
:
895
–903.
9
Wilcox RA, Chapoval AI, Gorski KS, et al. Cutting edge: Expression of functional CD137 receptor by dendritic cells.
J Immunol
 
2002
;
168
:
4262
–7.
10
Yurkovetsky ZR, Shurin GV, Barry DA, Schuh AC, Shurin MR, Robbins PD. Comparative analysis of antitumor activity of CD40L, RANKL, and 4-1BBL in vivo following intratumoral administration of viral vectors or transduced dendritic cells.
J Gene Med
 
2006
;
8
:
129
–37.
11
Cheuk AT, Mufti GJ, Guinn BA. Role of 4-1BB:4-1BB ligand in cancer immunotherapy.
Cancer Gene Ther
 
2004
;
11
:
215
–26.
12
Zhang H, Merchant MS, Chua KS, et al. Tumor expression of 4-1BB ligand sustains tumor lytic T cells.
Cancer Biol Ther
 
2003
;
2
:
579
–86.
13
Xu DP, Sauter BV, Huang TG, Meseck M, Woo SL, Chen SH. The systemic administration of Ig-4-1BB ligand in combination with IL-12 gene transfer eradicates hepatic colon carcinoma.
Gene Ther
 
2005
;
12
:
1526
–33.
14
Zhang H, Knutson KL, Hellstrom KE, Disis ML, Hellstrom I. Antitumor efficacy of CD137 ligation is maximized by the use of a CD137 single-chain Fv-expressing whole-cell tumor vaccine compared with CD137-specific monoclonal antibody infusion.
Mol Cancer Ther
 
2006
;
5
:
149
–55.
15
Shi W, Siemann DW. Augmented antitumor effects of radiation therapy by 4-1BB antibody (BMS-469492) treatment.
Anticancer Res
 
2006
;
26
:
3445
–53.
16
Kocak E, Lute K, Chang X, et al. Combination therapy with anti-CTL antigen-4 and anti-4-1BB antibodies enhances cancer immunity and reduces autoimmunity.
Cancer Res
 
2006
;
66
:
7276
–84.
17
Blank C, Kuball J, Voelkl S, et al. Blockade of PD-L1 (B7-H1) augments human tumor-specific T cell responses in vitro.
Int J Cancer
 
2006
;
119
:
317
–27.
18
Tsushima F, Tanaka K, Otsuki N, et al. Predominant expression of B7-H1 and its immunoregulatory roles in oral squamous cell carcinoma.
Oral Oncol
 
2006
;
42
:
268
–74.
19
Blank C, Brown I, Peterson AC, et al. PD-L1/B7H-1 inhibits the effector phase of tumor rejection by T cell receptor (TCR) transgenic CD8+ T cells.
Cancer Res
 
2004
;
64
:
1140
–5.
20
Dong H, Chen L. B7-H1 pathway and its role in the evasion of tumor immunity.
J Mol Med
 
2003
;
81
:
281
–7.
21
Nurieva R, Thomas S, Nguyen T, et al. T-cell tolerance or function is determined by combinatorial costimulatory signals.
EMBO J
 
2006
;
25
:
2623
–33.
22
Xu L, Liu Y, He X. Expression and purification of soluble human programmed death-1 in Escherichia coli.
Cell Mol Immunol
 
2006
;
3
:
139
–43.
23
Geng H, Zhang GM, Xiao H, et al. HSP70 vaccine in combination with gene therapy with plasmid DNA encoding sPD-1 overcomes immune resistance and suppresses the progression of pulmonary metastatic melanoma.
Int J Cancer
 
2006
;
118
:
2657
–64.
24
He L, Zhang G, He Y, Zhu H, Zhang H, Feng Z. Blockade of B7-H1 with sPD-1 improves immunity against murine hepatocarcinoma.
Anticancer Res
 
2005
;
25
:
3309
–13.
25
He YF, Zhang GM, Wang XH, et al. Blocking programmed death-1 ligand-PD-1 interactions by local gene therapy results in enhancement of antitumor effect of secondary lymphoid tissue chemokine.
J Immunol
 
2004
;
173
:
4919
–28.
26
He YF, Wang XH, Zhang GM, Chen HT, Zhang H, Feng ZH. Sustained low-level expression of interferon-γ promotes tumor development: Potential insights in tumor prevention and tumor immunotherapy.
Cancer Immunol Immunother
 
2005
;
54
:
891
–7.
27
Huang B, Zhao J, Li HX, et al. Toll-like receptors on tumor cells facilitate evasion of immune surveillance.
Cancer Res
 
2005
;
65
:
5009
–14.
28
Grunebach F, Kayser K, Weck MM, Muller MR, Appel S, Brossart P. Cotransfection of dendritic cells with RNA coding for HER-2/neu and 4-1BBL increases the induction of tumor antigen specific cytotoxic T lymphocytes.
Cancer Gene Ther
 
2005
;
12
:
749
–56.
29
Wiethe C, Dittmar K, Doan T, Lindenmaier W, Tindle R. Provision of 4-1BB ligand enhances effector and memory CTL responses generated by immunization with dendritic cells expressing a human tumor-associated antigen.
J Immunol
 
2003
;
170
:
2912
–22.
30
Bertram EM, Dawicki W, Sedgmen B, Bramson JL, Lynch DH, Watts TH. A switch in costimulation from CD28 to 4-1BB during primary versus secondary CD8 T cell response to influenza in vivo.
J Immunol
 
2004
;
172
:
981
–8.
31
Dawicki W, Watts TH. Expression and function of 4-1BB during CD4 versus CD8 T cell responses in vivo.
Eur J Immunol
 
2004
;
34
:
743
–51.
32
Bukczynski J, Wen T, Watts TH. Costimulation of human CD28− T cells by 4-1BB ligand.
Eur J Immunol
 
2003
;
33
:
446
–54.
33
Vallejo AN. CD28 extinction in human T cells: altered functions and the program of T-cell senescence.
Immunol Rev
 
2005
;
205
:
158
–69.
34
Chamberlain WD, Falta MT, Kotzin BL. Functional subsets within clonally expanded CD8+ memory T cells in elderly humans.
Clin Immunol
 
2000
;
94
:
160
–72.
35
Filaci G, Fravega M, Negrini S, et al. Nonantigen specific CD8+ T suppressor lymphocytes originate from CD8+CD28− T cells and inhibit both T-cell proliferation and CTL function.
Hum Immunol
 
2004
;
65
:
142
–56.
36
Blansfield JA, Beck KE, Tran K, et al. Cytotoxic T-lymphocyte-associated antigen-4 blockage can induce autoimmune hypophysitis in patients with metastatic melanoma and renal cancer.
J Immunother
 
2005
;
28
:
593
–8.
American Association for Cancer Research
2007