PBMCs were resuspended in RPMI 1640 culture medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 5% heat-inactivated human AB serum (MP Biomedicals, Eschwege, Germany), 2 mM L-glutamine (Sigma-Aldrich), and 1% penicillin/streptomycin solution (Sigma-Aldrich). CD3/CD28 Dynabeads (Life technologies, Oslo, Norway) were added to obtain a bead-to-cell ratio of 1:1. The cultures were incubated for 7 days at 37°C in 5% CO2.

Activated PBMCs were washed twice in phosphate-buffered saline (PBS), resuspended in 500 μL of Pierce® IP lysis LDE225 in vitro buffer (Pierce Biotechnology, Rockford, IL, USA), and homogenized using FastPrep®-24 (MP Biomedicals). Lysates were cleared by a 10-min centrifugation at 10 000 × g at 4°C. Protein concentration was 2.8 μg/μL by using the Protein Assay Dye Reagent Concentrate (Bio-Rad, Hercules, CA, USA). Briefly, 200 μL of human sera diluted 1:10 in PBS with 0.02% Tween 20 were incubated with Dynabeads Protein G (Invitrogen Dynal AS, Oslo, Norway) for 1 h at room temperature. The beads were washed with PBS and incubated with 200 μL PBS containing 20 μg activated PBMC lysate for 1 h at room temperature. Beads were then washed with PBS, and retained proteins were eluted with 2 × SDS sample buffer (20% glycerol, 4% sodium dodecyl sulfate (SDS),

125 mM tris-HCl pH 6.8, 12% 2-mercaptoethanol, 0.004% bromophenol blue). Proteins were Selleck NVP-BKM120 diglyceride separated by SDS-polyacrylamide gel electrophoresis. The separated components were electroblotted onto an Immobilon-P polyvinylidene difluoride membrane (Millipore Corporation, Billerica, MA, USA). Blot membranes were blocked in polyvinylidene fluoride (PVDF) blocking reagent for Can Get Signal (Toyobo, Ltd., Tokyo, Japan). The blots were washed with tris-buffered saline with Tween

20 and reacted with a 1:1000 dilution of antihuman PD-1 antibody (R&D Systems, Minneapolis, MN, USA) for 1 h at room temperature, washed and then reacted with HRP (Horseradish peroxidase)-conjugated antimouse immunoglobulin G (IgG) for 1 h. The blots were then developed by electrochemi-luminescence immunoassay (ECL) (GE Healthcare) according to the manufacturer’s instructions. Titers of serum anti-PD-1 antibodies were measured by indirect enzyme-linked immunosorbent assay (ELISA) using Protein Detector ELISA Kit (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA). All serum samples were tested in duplicate. Briefly, 96-well U-bottom microtiter plates (Greiner Bio-One GmbH, Frickenhausen, Germany) were coated with 100 μL of 1 μg/mL recombinant PD-1 (Abnova, Taipei, Taiwan) in PBS at room temperature for 1 h. Unbound antigen was removed, nonspecific binding sites were blocked by incubation with 1% bovine-serum albumin (BSA) in PBS, and the wells were incubated with 100 μL of human sera diluted 1:20 in PBS with 1% of BSA for 1 h.