K562/HLA-E cells binding assay
The stable transformed K562/HLA-E*0103 or K562/HLA-E*0101 cells were respectively incubated with 10 μM each indicated peptide and 1 μM human β2-microglobulin (β2m, Sigma) in serum-free RPMI 1640 medium for 16 h at 26°C with 5% CO2, while K562/HLA-E cells with no peptide incubation under the same culture conditions were used as blank control. These cells binding with peptides were further incubated at 37°C for 2 h for thermal stability. We detected the expression of HLA-E molecules on surface of K562/HLA-E cells by staining with PE-labeled anti-HLA-E monoclonal antibody (mAb) (3D12; BioLegend, USA), using ACEA NovoExpress system (Agilent Technologies, USA). The results are presented as the fluorescence index (FI). FI ≥ 1 represents high-affinity peptide, indicating that the stable combination of the peptide with HLA-E molecules on surface of K562/HLA-E cells could increase the mean fluorescence of the HLA-E molecules by at least one-fold.