2.4. Reverse transcription‐polymerase chain reactions assay

Total RNA was extracted from peripheral blood lymphocytes with Trizol reagents (Invitrogen, Carlsbad, CA), through centrifugation for 1 minute at 12 000g. After confirming RNA concentration and assessment of purity with ultraviolet absorbance at 260 nm on a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA), equal amounts of total RNA from each sample were reversely transcribed into cDNA using a reverse transcription kit (Takara, Kusatsu, Japan) in accordance with the manufacture's instructions. Equal amounts of cDNA for each sample were used as template for fluorescence quantitative reverse transcription‐polymerase chain reactions (RT‐PCR). The following sequence‐specific primers were designed by using Primer Premier 5.0 software (PREMIER Biosoft, Palo Alto, CA) for PCR amplification: (1) PD‐1 primers, 5'‐GCA CGA GGG ACA ATA GGA‐3' (forward) and 5'‐GAC AAT GGT GGC ATA CTC‐3' (reverse); (2) B7‐H1 primers, 5'‐CAG GGC ATT CCA GAA AGA‐3' (forward) and 5'‐CCT CCA TTT CCC AAT AGA C‐3' (reverse); (3) the housekeeping gene β‐actin primers, 5'‐CCT GGG CAT GGA GTC CTG TG‐3' (forward) and 5'‐TCT TCA TTG TGC TGG GTG CC‐3' (reverse). Thermal cycling was performed with an S1000 Thermal Cycler PCR detection system (Bio‐Rad, Hercules, CA). Each reaction contained 3 μL of cDNA, 0.96 μL of primer, 3 μL diethylpyrocarbonate and 6 µL Taq polymerase. The protocol included an initial denaturation step at 95°C for 3 minutes, followed by 40 cycles of 10 seconds at 95°C, 30 seconds at 57°C, and 30 seconds at 72°C, followed by 5 minutes at 72°C. Fluorescence signals were collected at 60°C. Each sample was assayed in triplicate and compared to arbitrary values assigned to standard melt curves generated for the target gene to obtain relative abundance of amplified products. These values were then normalized to those of β‐actin.