Total RNA from the liver and spleen of WT and PD-1-deficient mice was isolated by Trizol (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. RNA samples were quantitated by measurement of optical density at 260 nm. Total mRNA (2 mg) was reverse-transcribed to cDNA using the RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas China, Shenzhen City, China), in accordance with the manufacturer's instructions. qPCR was performed to quantitatively analyze the gene transcription levels of fgl2, IFN-c and TNF-a genes. The primers for fgl2 were: sense 59-TGGACAACAAAGTGG-CAAATCT-39 and anti-sense 59-TGGAACACTTGCCATC-CAAA-39. The primers for IFN-c were: sense 59-TCAAGTGG-CATAGATGTGGAAG-39, and anti-sense 59-CGCTTATG-TTGTTGCTGATGG-39. The primers for TNF-a were: sense 59-CACGCTCTTCTGTCTACTGAAC-39 and anti-sense 59-ATCTGAGTGTGAGGGTCTGG-39. The primers for b-actin (internal control) were: sense 59-CACTATCGGCAATGAG-CGGTTCC-39 and anti-sense 59-CAGCACTGTGTTGGCA TAGAGGTC-39. The qPCR was performed at 95uC for 10 s followed by 40 cycles of 95uC for 5 s, 60uC for 15 s, and 72uC for 15 s. The specificity of PCR product was examined by a dissociation curve, and results were analyzed by the 2 2DDCT method [35] .