The concentration of peptide required to generate 50% of the maximal response was used as a measure of avidity. Mice were sacrificed 14 days after a single priming vaccination. Single-cell suspensions from individual spleens were cultured in complete medium in 25 cm2 upright flasks (3×106 cells/mL) supplemented with 10−8 M of the corresponding PSMA HLA-A*0201-binding peptide and 20 IU/mL IL-2 (R&D Systems, Abingdon, UK). Following 6 days stimulation in vitro, the cytolytic activity of the CTL cultures was assessed in Kinase Inhibitor Library price a standard

5-h 51Cr-release assay. Target cells were labeled with 51Cr with or without peptide for 1 h. Target (T) cells (5×103) were then cultured with effector (E) T cells at different E:T ratios.

Specific % lysis was calculated by the formula: (release by CTL−release by targets alone)/(release by 4% NP40−release by targets alone)×100. Splenocytes harvested from naïve HHD mice were pulsed with 1 mM PSMA27, PSMA663, or control HLA-A*0201-binding anti-PD-1 antibody inhibitor peptide (VLHDDLLEA) at a concentration of 2×107/mL in PBS. The cells were then labeled with either 0.5 or 5 μM CFSE (Molecular Probes, Invitrogen) for 8 min at 37°C before adding FCS to a final concentration of 20% to quench the reaction. After washing, the cells were mixed at a 1:1 ratio such that each prevaccinated mouse received 1×107 cells pulsed with PSMA peptide and the same number pulsed with control peptide in 0.1 mL PBS by intravenous injection. Splenocytes were harvested from individual mice 20 h later, lymphocytes were isolated using density gradient centrifugation and CFSE staining was analyzed by FACS Canto (BD Pharmingen). Lymphocytes from the same mice were also used in an ELISpot assay as described. HHD mice were vaccinated with p.DOM-PSMA27, p.DOM-PSMA663, or p.DOM control vaccine 13 days prior to the assay. TRAMP-PSMA+ HHD+, and TRAMP-HHD+ cells were labeled with 10 and 1 μM CFSE, respectively, as described above and then mixed in a 1:1 ratio. The cell suspension was then mixed

with Matrigel® (BD Biosciences) at a ratio of 1:1 so that each mouse received 1×106 of each population in a total volume of 150 μL by subcutaneous injection. Matrigel® cell suspensions were kept on ice 3-mercaptopyruvate sulfurtransferase until the time of injection, according to the manufacturer’s protocol. After 5 days, the Matrigel® plugs were harvested and digested with 1 mg/mL collagenase/dispase and 0.5 mg/mL DNAseI. CFSE staining of the cells released from the plug was analyzed using a FACS Calibur (BD). The spleens of the same mice were used in an ELISpot assay, as described, to identify those responding to vaccination; animals with an IFN-γ response of less than twice the background or <50 SFCs/106 cells were excluded from the analysis. Experimental groups were compared using a Mann–Whitney U-test. In vivo tumor lysis was analyzed using Fisher’s exact test.