Anti-Tumor Immune Response Figure 7. Rescue of CY anti-tumor efficacy in nude mice by anti-DR5 antibodies. Tumor-bearing athymic nude mice (inoculated at day 0) were treated with CY or with PBS at day 9 (a) or day 7 (b) after tumor cell inoculation and were treated with anti-DR5 antibody (clone MD5-1) or PBS at day 8, 12 and 16 after tumor cell inoculation. Data shown are mean6SEM (n = 5/group for each experiment). * P,0.05 when CY is compared with CY + anti-DR5. The effect of anti-TRAIL Ab without CY is shown in (b). Two mice were disqualified from the graph as they were found dead early in the experiment. doi:10.1371/journal.pone.0006982.g007 production by T cells or NK cells could be an important readout to predict the efficacy of cancer vaccines or immunotherapies, possibly equally important as perforin-mediated cell apoptosis and IFN-c. Materials and Methods Ethical statement Animal experimentation was conducted according to University of Western Australia Animal Ethics Committee approvals and the NH&MRC code of conduct. Reagents and antibodies Cyclophosphamide, maphosphamide and gemcitabine were from Sigma-Aldrich, Baxter Oncology (Halle, Germany) and the Sir Charles Gairdner Hospital Pharmacy, respectively. FLIVO caspase staining kits were from Immunochemistry Technologies and used according to the instructions. Antibodies were from BD Biosciences, eBioscience and Caltag: TCRb-AF488 (H57-597), CD3e-FITC (145-2C11), CD4-PE/CD4-PECy7 (RM4-5), CD8- PECy5/CD8-APC-AF750 (53–6.7), CD19-PECy7 (eBio1D3), Ly6A/E-FITC (Sca1), Ki-67-FITC (B56), CD43-FITC (1B11) and DR5-PE (MD5-1). Flow cytometry was performed on FACSCalibur and FACSCanto II instruments and analyzed using Flowjo (TreeStar). Mice Female BALB/c (H-2d) wild-type and nude mice (6–8 wk old) were purchased from the Animal Resources Centre (Canning Vale, Australia). TCR transgenic CL4 mice, expressing a TCR specific for the H-2d-restricted peptide IYSTVASSL (residues 518–526) from the influenza virus A/PR8/8/34 HA protein, were generated and screened as described [15]. TRAIL-deficient mice were originally generated at Immunex Corporation [35,59] and were backcrossed to BALB/c at the Peter MacCallum Cancer Centre (Melbourne, Australia) (n = 12). Perforin-IFN-c double- deficient mice were bred at the Peter MacCallum Cancer Centre. BALB/c perforin-deficient mice were originally generated at the Peter MacCallum Cancer Centre [60] and bred at the Animal Care Unit at UWA (Perth, Australia). Tumor cell culture and inoculation Generation and maintenance of the BALB/c-derived mouse mesothelioma cell line AB1 and transfection with the HA gene PLoS ONE | www.plosone.org 9 September 2009 | Volume 4 | Issue 9 | e6982