E1j2j

IHC staining was performed on representative surgical tissue sections from FFPE tissue blocks of LUSC patients using anti–human PD-L1 antibodies: E1J2J, Cell Signaling Technology; 28-8, Abcam; 22C3, Dako; SP142, Spring Bioscience; anti–human CD8 antibodies (C8/144B; Nichirei Bioscience); anti–human PD-1 antibodies (NAT105; Abcam); anti–human β2M antibodies (A0072; Dako); and anti–human HLA class I-A/B/C antibodies (EMR8-5; Hokudo). The detection of immunostaining was performed using Histofine Simple Stain MAX-PO (Nichirei Bioscience) or BOND polymer Refine Detection kit (Leica Biosystems). Some of the sections were stained with the PD-L1 22C3 pharmDx kit (Dako). PD-L1 positivity was defined as membranous or cytoplasmic staining in at least 1% of tumor cells. The staining results of β2M and HLA class I-A/B/C of tumor cells were categorized as negative (0%), focally positive (<50%), or positive (≥50%).

IHC for PD-L1 was performed on formalin-fixed paraffin-embedded tissue sections using antibodies directed against the N-terminal (E1J2J, Cell Signaling Technology, Beverly, MA, USA) and C-terminal (SP142, Spring Bioscience, Fremont, CA, USA) domains of PD-L1. IHC for Ki-67 was performed using a mouse monoclonal antibody (MIB-1, Dako, Tokyo, Japan). The antigen–antibody complexes were visualized with Histofine Simple Stain MAX PO (Nichirei Bioscience, Tokyo, Japan). Some sections were double-stained with PD-L1 (E1J2J) and CD3, using a mouse monoclonal antibody (F7.2.38, Dako) with PolyView IHC reagent (mouse-AP, Enzo Life Sciences, Farmingdale, NY, USA) (Supplemental Figure S2). A tumor sample was considered positive for PD-L1 expression, when >5% of tumor cells were stained, where PD-L1 expression in tumor cells was discriminated from that in immune cells on the basis of cell morphology and/or cytoplasmic CD3 expression. LMP1 expression was detected using a mouse monoclonal antibody (CS1-4, Dako) with the EnVision system (Dako). EBER-ISH was performed on the Leica Bond-III Automatic Stainer (Leica Microsystems, Wetzlar, Germany) using Bond Ready-to-Use ISH EBER Probe and Bond Ready-to-Use Anti-Fluorescein Antibody (Leica Biosystems, Wetzlar, Germany).

The cells were fixed with 10% formaldehyde for 30 min and permeabilized with buffer containing 0.3% Triton X-100 and 1× Blocking One solution (Nacalai Tesque) for 1 h at RT. The cells were labeled with aPD-L1 antibodies (E1J2J, Cell Signaling Technology; 22C3, Dako; and SP142, Spring Bioscience) in buffer containing 0.3% Triton X-100 and 1× Blocking One solution (Nacalai Tesque) overnight at 4°C. They were then labeled with goat anti-Mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 488 (A11001; Thermo Fisher Scientific) or goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 488 (A11034; Thermo Fisher Scientific) in buffer containing 0.3% Triton X-100 and 1× Blocking One solution (Nacalai Tesque) for 1 h at RT. The nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific). The images were captured by a FLUOVIEW FV1000 laser scanning microscope (Olympus).