Quantifying Immune Checkpoint Receptor Expression

Total RNA was extracted from fresh PBMCs by the RNeasy Mini Kit (Qiagen, Germany), which was used to synthesize cDNA by a reverse transcription reagent kit (Takara, China). Real-time quantitative PCR was used to detect the expression levels of target genes in triplicate by Takara SYBR Supermix (Takara, China) by ABI QuantStudio 5 (Applied Biosystems, United States) as previously described (Shen et al., 2020 (link)). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control in this study. The sequences of the specific primers are listed as follows: PD-1 forward 5′-AAGGCGCAGATCAAAGAGAGCC-3′ and reverse 5′-CAACCACCAGGGTTTGGAACTG-3′; TIM-3 forward 5′-GACTCTAGCAGACAGTGGGATC-3′ and reverse 5′-GGTGGTAAGCATCCTTGGAAAGG-3′; LAG-3 forward 5′-GCAGTGTACTTCACAGAGCTGTC-3′ and reverse 5′-AAGCCAAAGGCTCCAGTCACCA-3′; CTLA-4 forward 5′-ACGGGACTCTACATCTGCAAGG-3′ and reverse 5′-GGAGGAAGTCAGAATCTGGGCA-3′; and GAPDH forward 5′-GTCTCCTCTGACTTCAAC AGCG-3′ and reverse 5′-ACCACCCTGTTGCTGTAG CCAA-3′.