W6 32

SPR was performed largely as described (Sim et al., 2020 (link)) with a BIAcore 3000 instrument and analyzed with BIAevalution software v4.1 (GE Healthcare). The HLA-A, -B, -C-specific mAb W6/32 (#311402, BioLegend) was immobilized to CM5 chips (Cytiva, USA) in 10 mM sodium acetate pH 5.5 at 5000–7000 response units (RU) by primary amine-coupling with a 2 μl/min flow rate. HLA-C was captured by W6/32 at 400–700 RU in PBS. Soluble TCR heterodimers were used as analytes in 10 mM HEPES pH 7.5 and 0.15 M NaCl with a flow rate of 50 μl/min. TCRs were injected for 2 min followed by a dissociation of 10 min. Binding was measured with serial dilutions of TCR from 80 μM to 1.25 μM for TCR10 and 1200 nM to 37.5 nM for TCR9a. Four independent TCR10-binding experiments were carried out with initial concentrations of 10 μM (two experiments), 40 μM (one experiment), and 80 μM (one experiment). Dissociation constants were obtained by modeling steady-state kinetics for TCR10 and kinetic curve fitting for TCR9a with BIAevaluation software.

Peripheral blood mononuclear cells were isolated from heparinized blood of HLA-A*2402-positive donors seropositive for HSV-1 using Ficoll density gradient centrifugation. CD8 + T cells were isolated from the mononuclear cells using a magnet beads based negative selection kit (IMag, BD Bioscience, San Jose, CA). To expand the CD8 + T cells, the HCEn cells infected with HSV-1 (KOS strain) at MOI 0.1 for 24 h were irradiated and cocultured with CD8 + T cells in RPMI supplemented with 10% fetal bovine serum and recombinant IL-2 (10 ng/ml) as described^{4 (link)}.
HLA-A*2402-restricted peptide, AYLPRPVEF, was used for the HSV-1 epitopes of UL37^{8 (link)}. The peptide-pulsed HCEn cells were co-cultured with the CD8 T cells at CD8 + T cells/HCEn cells ratio of 1/2. The supernatant collected after 6 h were measured for the released of granzyme B using a commercially available ELISA kit (Thermo Fisher, Waltham, MA). Anti-MHC class I antibody, W6/32 (Biolegend, San Diego, CA) or control antibody, were used for the blockade of MHC class I.
All of the procedures used in this study conformed to the tenets of the Declaration of Helsinki, and they were approved by the Institutional Review Board of Tottori University, Tottori, Japan. An informed consent was obtained from all of the participants.