2.7 Generation of HMy2.CIR Cell Lines Expressing the Indicated HLA-A Molecule

HMy2.CIR cell line was purchased (Zhongqiao xinzhou Biotech, Shanghai) and maintained in complete IMDM medium with 10% FCS and 1% penicillin/streptomycin. Total mRNA was extracted from PBMCs of healthy donor with the indicated HLA-A alleles by RNAprep Pure Hi-Blood Kit (TIANGEN, Beijing, China), and the cDNA was generated by using HiScriptII1^st Strand cDNA Synthesis Kit (Vazyme, China) at RT. Each HLA-A allele was then amplified in PCR using the primer combination AF/AR, and the 5’ and 3’ ends of PCR product contained the same sequences as that of the linearized vector. Linearized pcDNATM3.1/myc-His (–)AMCS was amplified in PCR using the primer combination P-1F/P-1R. Finally, the routine construction of pcDNATM3.1 recombinant plasmids were obtained with ClonExpressII One Step Cloning Kit (Vazyme, China).

Primers were synthesized by Sangon Biotech (Shanghai) and displayed in Table S1 . After electrotransfection, the cell lines stably expressing HLA-A molecule were screened by G418, and then stained with PE-anti-HLA-ABC (clone W6/32, eBioscience), FITC-anti-HLA-A24 (clone 17A10, MBL) or PE-anti-HLA-A2 (clone BB7.2, BD Bioscience). The resulting cells highly expressing the indicated HLA-A allotypes were positively sorted using a fluorescence activated cell sorter (FACS, BD FACSAriaIISORP), and followed by cell pure culture and gene sequencing analyses.