T2 Cell Binding Assay

The lymphoblast cell line T2 (174 × CEM. T2) was purchased from the American Type Culture Collection (cat. no. CRL-1992™; Manassas, VA, USA). T2 cells are transporter-associated cells with antigen processing (TAP)-deficient and defective for endogenous MHC class I presentation. Although T2 cells express a very low level of the HLA-A*0201 molecule under normal culture conditions, they express HLA-A*0201 molecules at much higher levels after binding to appropriate peptides that stabilize the expression of HLA-A*0201 on the cell surface (40). To further determine the binding capability of predicted HTNV GP nonapeptides to HLA-A*0201 molecules, the T2 cell binding assay was performed as described elsewhere (41). Briefly, T2 cells were incubated with 50 µmol/L peptides and 1 µmol/L human β2-microglobulin (β2 m, Sigma) in serum-free RPMI 1640 medium for 18 h at 37°C with 5% CO₂. The expression of HLA-A*0201 molecules on the surface of T2 cells was then determined by staining with PE-labeled anti-HLA-A*02 mAb (Clone BB7.2; BioLegend) and detected by flow cytometry (FACScan; BD Biosciences). The results are presented as the fluorescence index (FI), which was determined as follows: FI = (mean PE fluorescence with the given peptide − mean PE fluorescence without peptide)/(mean PE fluorescence without peptide) (6). FI ≥ 1 represents high-affinity peptides, indicating that the stable combination of these peptides with HLA-A*0201 molecules on the surface of T2 cells could increase the mean fluorescence of the HLA-A*0201 molecules by at least onefold.