Female TgMISIIR-TAg transgenic mice were obtained by breeding female C57BL/6 mice with male TgMISIIR-TAg transgenic mice . The male MISIIR-TAg transgenic mice were obtained from Fox Chase Cancer Center (Philadelphia, PA, USA) . Female TgMISIIR-TAg transgenic mice 10 weeks of age were used in the experiments. These mice spontaneously developed ovarian tumors with complete tissue penetration. All animals were maintained under specific pathogen-free conditions, and all procedures were performed according to approved protocols and in accordance with recommendations for the proper use and care of laboratory animals by Johns Hopkins University Animal Care and Use Committee (protocol MO11M398).
The MOVCAR (mouse ovarian carcinoma) cell line was obtained from Fox Chase Cancer Center (Philadelphia, PA, USA). It was derived from ascites of a TgMISIIR-TAg transgenic mouse with ovarian tumors . All cell lines were maintained in RPMI 1640 complete medium supplemented with 10% heat-inactivated FBS (HyClone), 1% non-essential amino acid, 2 mM l-glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin, and 5 mM 2-ME (Invitrogen Life Technologies). To generate the MOVCAR-luc cell line, luciferase expressing MOVCAR tumor cells (MOVCAR-luc) were generated by transducing the MOVCAR cells with retrovirus containing luciferase, pLuci-thy1.1, and flow cytometry sorting following the previously described protocol . OVA-specific CD8+ T cells (OT-1) were produced via the stimulation of splenocytes obtained from OT-1 transgenic mice with irradiated EG.7 cells in the presence of interleukin-2 (20 IU/ml, Pepro-Tech, Rock Hill, NJ).
Plasmid DNA constructs and preparation
Pfuse-NKG2D-Fc was described previously . To generate pFuse-NKG2D-Fc-RO the oligos CTAGACGGGTGAAGCGGAGTATAATCAACTTTGAAAAACTGTAAC and TCGAGTTACAGTTTTTCAAAGTTGATTATACTCCGCTTCACCCGT were annealed and cloned into XbaI/Xho sites of Pfuse-NKG2D-Fc.
Transfection and protein purification
For purification of the NKG2D-FC and NKG2D-FC-RO, 50 μg of plasmid was transfected into 1 × 107 FD11 cells in T-150 flask using Lipofectamin 2000 (Invitrogen Corp., Carlsbad, CA, USA). After 3 days, cell culture media was accumulated, filtered using 0.22 μm syringe filter (Millipore, Billerica, MA, USA) and concentrated with Amicon cut-off 50 kDa Ultra -15 (Millipore, Billerica, MA, USA). Concentrated recombinant protein containing media was applied to a HiTrap Protein G HP column (GE Healthcare) following the vendor’s protocol. Protein concentrations were determined by the Coomassie Plus protein assay (Pierce, Rockford, IL, USA) and purity was estimated by SDS polyacrylamide gel electrophoresis.
Cell staining and flow cytometry analysis
For flow cytometry analysis, tumor cells were stained with Rae-1 antibody (BD Bioscience), or 0.5 μg of NKG2D-Fc and NKG2D-Fc-RO respectively and PE-conjugated anti-mouse antibody was used as a detection antibody (BD Bioscience). The percentage of OVA-specific IFN-γ secreting CD8+ T cells was determined using intracellular cytokine staining and FACScan analysis with CELLQuest software (Becton Dickinson Immunocytometry System, Mountain View, USA).
OVA-specific T cell activation and In vitro cytotoxicity assay
For T cell activation, MOVCAR cells were added to 48-well plates at a dose of 1 × 105 cells/well and incubated with 0.5 μg/ml of proteins. Eighteen hours later, treated tumor cells were incubated with 2 × 105 OVA-specific cytotoxic T cells (CTL). One day after activation, IFN-γ-secreting OVA-specific CTLs were identified by intracellular cytokine staining and analyzed by flow cytometry analysis. For the in vitro cytotoxicity experiment, 1 × 104 of luciferase-expressing MOVCAR (MOCAR-luc) cells were incubated with 0.5 μg/ml of one of the various proteins on 96-well plate for 6 hours and treated with 2 × 104 OVA-specific CTLs. The degree of CTL-mediated killing of the tumor cells was measured by the IVIS luminescence imaging system series 2000 as described previously .
In vivo tumor treatment experiments
Tumor growth was assessed in 10-week-old female TgMISIIR-TAg transgenic mice by visual inspection following open surgery. Mice (5 per group) with similar sized ovarian tumors were selected to receive treatment with 20 μg/mouse of NKG2D-Fc or NKG2D-Fc-RO protein every week for four weeks. Prior to treatment, mice to be treated with NKG2D-Fc had an average tumor size of 55.02 ± 21.18 mm3 and mice to be treated with NKG2D-Fc-RO had an average tumor size of 52.36 ± 27.04 mm3. Mice were intraperitoneally injected with 2.5x106 OT-1 CD8+ T cells twice, every other week. Female TgMISIIR-TAg transgenic mice without treatment were also included for comparison. Mice were sacrificed 1 week after the last treatment. Ovarian tumors were harvested for measurement. Tumor infiltrating lymphocytes (TILs) in the ovarian tumors were characterized for the presence of OVA-specific CD8+ T cells using OVA peptide-loaded H-2Kb tetramer staining and CD8 staining.
The data presented in this study are representative of at least two experiments performed, and are expressed as means ± standard deviation (S.D.). The number of samples in each group for any given experiment was >3. Results for intracellular cytokine staining with flow cytometry analysis and tumor treatment experiments were evaluated by analysis of variance (one-way ANOVA) and the Tukey-Kramer multiple comparison test. Comparisons between individual data points were performed using Student’s t-test. Statistical analysis was performed with GraphPad Prism 4.0 software and a p value < 0.05 was considered significant.