Vol 44 # 4 December 2012 - Kma.org.kw

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309 Synergy between Dendritic Cell-Based Vaccine and Anti-CD137 Monoclonal ... December 2012 responses in 10 - 15% of patients, but is associated with severe toxicity [4,10] . Hence, new immunotherapeutic approaches with more effective anti-tumor activity and less toxicity are urgently required. The recent trend in cancer immunotherapy has been directed toward dendritic cell-based vaccine. It can induce anti-tumor immune response by activation of tumor-specific T lymphocytes [11] . In 14 clinical studies, 197 patients with metastatic renal cell carcinoma underwent therapy with dendritic cell-based vaccine [12] . According to the World Health Organization criteria, clinical responses to vaccine are defined as follows [13] . Complete response means complete disappearance of tumor; partial response means ≥ 50% decrease in tumor size without the appearance of new metastases; stable disease means < 50% decrease or < 25% increase in tumor size; and progressive disease means ≥ 25% increase in tumor size or the appearance of new metastases. Out of the 197 patients, 73 (37%) had clinical response with four complete responses, eight partial responses and 61 disease stabilizations [12] . Only mild side-effects, such as low-grade fever, fatigue, diarrhea, flulike symptoms, and local reactions around the injection site, were observed in these patients. Though dendritic cell-based vaccine had encouraging results in the treatment of metastatic renal cell carcinoma, clinical response rate remained limited. Dendritic cellbased vaccine was administered in combination with interleukin-2 to treat patients with metastatic renal cell carcinoma in order to improve vaccine efficacy [14] . However, clinical response rate was 40%, similar to single treatment with dendritic cell vaccine. Therefore, new approaches to significantly increase vaccine effect are needed. The CD137 (4-1BB), a member of tumor necrosis factor receptor (TNFR) superfamily, is an inducible costimulatory molecule expressed primarily on activated T cells [15] . Ligation of CD137 by anti-CD137 monoclonal antibody delivers a potent co-stimulatory signal to T cells, and enhances proliferation and activity of T cells, which have the ability to kill tumor cells [16] . Previous study has demonstrated that treatment of tumorbearing mice with anti-CD137 antibody can result in T cell-mediated tumor rejection [17] . We hypothesized that anti-CD137 antibody may potentiate anti-tumor effect of dendritic cell-based vaccine. The reason is as follows. Treatment with dendritic cell-based vaccine induces up-regulation of CD137 expression on T cells by activating T cells. Subsequent administration of anti- CD137 antibody further increases T cell proliferation and activity through ligation of CD137 on T cells, thereby strengthening anti-tumor effect. To confirm this hypothesis, we investigated the combined effect of dendritic cell-based vaccine with anti-CD137 antibody in renal cell carcinoma mouse model. MATERIALS AND METHODS Animals Balb/c mice, 8 - 10 weeks of age, were obtained from Shanghai Laboratory Animal Center (Shanghai City, China) and maintained under specific pathogenfree conditions. They were provided with sterilized food and water ad libitum. All animal experiments were conducted in accordance with the US National Institutes of Health guidelines for appropriate use of experimental animals and approved by Animal Research and Care Committee at our university. Preparation of dendritic cell-based vaccine Dendritic cell-based vaccine was prepared as described by Lim et al [18] . In brief, bone marrow cells of Balb/c mice were cultured in complete medium supplemented with 10 ng/ml recombinant mouse granulocyte macrophage colony-stimulating factor (R & D Systems, Minneapolis, MN, USA) and 10 ng/ml recombinant mouse interleukin-4 (R&D Systems, Minneapolis, MN, USA). Complete medium consisted of RPMI-1640 containing 10% heated-inactivated fetal bovine serum, 2 mmol/l L-glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin (all from Life Technologies, Grand Island, NY, USA). On day two, suspended cells were removed and adherent cells were cultured in fresh complete medium containing cytokines. Half of the medium was replaced with fresh medium containing cytokines every other day. On day seven, nonadherent and loosely adherent cells (i.e., immature dendritic cells) were harvested. The Balb/c-derived renal cell carcinoma cell line (Renca) was obtained from American Type Culture Collection (Manassas, VA, USA) and suspended in phosphatebuffered saline. Renca tumor cell lysate was prepared by four rapid freeze-thaw cycles (liquid nitrogen, 37 °C water bath). In order to induce dendritic cell maturation, immature dendritic cells were incubated with Renca tumor cell lysate at the ratio of three tumor cells lysate to one dendritic cell in complete medium. After 8 hours, 10 ng/ml recombinant mouse tumor necrosis factor-α (R & D Systems, Minneapolis, MN, USA) and 10 ng/ml recombinant mouse interferon-γ (R & D Systems, Minneapolis, MN, USA) were added to induce dendritic cell further maturation. After incubation for 16 hours, Renca tumor lysate-pulsed dendritic cells (i.e., mature dendritic cells) were harvested and used as dendritic cell-based vaccine in our study. Dendritic cells were evidenced by phenotype analysis. Phenotype analysis of dendritic cells Immature and mature dendritic cells were harvested, stained with fluorescein isothiocyanatelabeled anti-CD11c (dendritic cell marker) monoclonal
December 2012 KUWAIT MEDICAL JOURNAL 310 antibody (BD PharMingen, San Diego, CA, USA) and phycoerythrin-labeled anti-CD83 (maturation marker of dendritic cells) monoclonal antibody (BD PharMingen, San Diego, CA, USA), and then analyzed by flow cytometry for expressions of CD11c and CD83 on the cell surface. Analysis of CD137 expression on T cells Spleens from Balb/c mice were prepared as cell suspensions. T cells were isolated from splenocytes by the use of T cell separation reagents (Miltenyi Biotec,Auburn, CA, USA). Purified T cells (1 × 10 5 / well) were cultured alone or with Renca tumor lysatepulsed dendritic cells (1 × 10 4 /well) in round-bottom 96-well plates, harvested at 24-hour intervals for 7 days, and stained with fluorescein isothiocyanate-labeled anti-CD3 (T-cell marker) monoclonal antibody (BD PharMingen, San Diego, CA, USA) and phycoerythrinlabeled anti-CD137 monoclonal antibody (BD PharMingen, San Diego, CA, USA). CD137 expression on T cells was analyzed by flow cytometry. Tumor implantation and treatment The Balb/c mice were inoculated subcutaneously in the right flank with 5 × 10 5 Renca tumor cells on day 0. After three days, tumor-bearing mice were randomly divided into four groups. Control group received no treatment. In dendritic cell-treated group, mice were immunized subcutaneously in the left flank with Renca tumor lysate-pulsed dendritic cells (1 × 10 6 ) on days 3 and 10 [18] . Mice in antibody-treated group were injected intraperitoneally with anti-CD137 monoclonal antibody (100 μg; R & D Systems, Minneapolis, MN, USA) on days 3 and 10. Combination therapy group received both subcutaneous injection of Renca tumor lysate-pulsed dendritic cells (1 × 10 6 ) on days 3 and 10, and intraperitoneal administration of anti-CD137 monoclonal antibody (100 μg) on days 6 and 13. The maximal perpendicular diameters of tumor were measured using a caliper twice a week, and tumor size was recorded as tumor area (mm 2 ). Assessment of T cell proliferation Control and differently treated mice were killed 20 days after tumor implantation, and spleens were harvested. T cells were isolated from splenocytes by the use of T cell separation reagents (Miltenyi Biotec, Auburn, CA, USA). Purified T cells (1 × 10 5 /well) were co-cultured with Renca tumor lysate-pulsed dendritic cells (1 × 10 4 /well) in 96-well plates. On day five, cultures were pulsed with 1 μCi [3H]thymidine (PerkinElmer, Waltham, MA, USA) per well for 16 hours. The cells were collected on glass microfiber filters with a cell harvester. T cell proliferation was evaluated by measuring [3H]thymidine incorporation in a liquid scintillation counter. Results were expressed as counts per minute (cpm). Cytotoxicity assay A standard 51 Cr release assay was used to measure cell-mediated cytotoxicity [19] . In brief, Renca tumor cells were used as target cells. Target cells were labeled with 51 Cr (PerkinElmer, Waltham, MA, USA) for one hour at 37 °C and washed three times before use. Spleens were harvested from control and differently treated mice 20 days after tumor implantation. CD8 + T cells were isolated from splenocytes by the use of CD8 + T cell separation reagents (Miltenyi Biotec, Auburn, CA, USA), and stimulated in vitro with Renca tumor lysatepulsed dendritic cells at a 10:1 ratio. After five days, the primed CD8 + T cells (effector cells) were harvested, washed twice, and co-incubated with 51 Cr-labeled Renca tumor cells (target cells) at effector / target cell ratio of 100:1 in round-bottom 96-well plates. After four hours of incubation at 37 °C, culture supernatants were collected and 51 Cr release was measured using a gamma counter. The percentage of target cell specific lysis was calculated using the following formula: [(experimental 51 Cr release - spontaneous 51 Cr release) / (maximal 51 Cr release - spontaneous 51 Cr release)] × 100 [20] . Spontaneous release of 51 Cr was determined by incubating target cells alone. Maximal 51 Cr release was obtained by incubating target cells with 1% Triton X- 100 (Sigma-Aldrich, St. Louis, MO, USA). Measurement of interferon-γ production Twenty days after tumor implantation, spleens from control and differently treated mice were harvested. CD4 + T cells were isolated from splenocytes by the use of CD4 + T cell separation reagents (Miltenyi Biotec, Auburn, CA, USA), and stimulated with Renca tumor lysate-pulsed dendritic cells at a ratio of 10:1 in vitro. After 24 hours, culture supernatants were collected and determined for interferon-γ content by enzyme-linked immunosorbent assay (ELISA) kit (BD PharMingen, San Diego, CA, USA). Statistical analysis The data were presented as mean ± standard deviation (SD). Two-tailed Student’s t-test was used for data analysis. A p-value of less than 0.05 was considered statistically significant. GraphPad Prism software (GraphPad Software Inc, San Diego, CA, USA) was used for all statistical analysis. RESULTS Characteristics of dendritic cells As shown in Fig. 1, cultured cells highly expressed CD11c (dendritic cell marker), suggesting that these cells were dendritic cells. Renca tumor lysate-pulsed
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