Four-micrometer-thick sequential histologic tumor sections were obtained
from a representative formalin-fixed, paraffin-embedded tumor block and used for
IHC analysis. IHC was performed using an automated staining system (BOND-MAX;
Leica Microsystems) with antibodies against PD-L1 (clone E1L3N, dilution 1:100;
Cell Signaling Technology), CD3 (T-cell lymphocytes; dilution 1:100; Dako), CD4
(helper T cell; Novocastra; clone 4B12, dilution 1:80; Leica Biosystems), CD8
(cytotoxic T cell; clone CD8/144B, dilution 1:20; Thermo Fisher Scientific),
CD57 (natural killer T cell; clone HNK-1, dilution 1:40; BD Biosciences),
granzyme B (cytotoxic lymphocytes; clone F1, ready to use; Leica Biosystems),
CD45RO (memory T cell; clone UCHL1, ready to use; Leica Biosystems), PD-1 (clone
EPR4877-2, dilution 1:250; Abcam), FOXP3 (regulatory T cell; clone 206D,
dilution 1:50; BioLegend), and CD68 (macrophages; clone PG-M1, dilution 1:450;
Dako). Expression of all of the markers in cells was detected using a Novocastra
Bond Polymer Refine Detection kit (Leica Microsystems) with a diaminobenzidine
reaction to detect antibody labeling and hematoxylin counterstaining. Positive
and negative controls were used for PD-L1 IHC expression (human embryonic kidney
293 cell line transfected and nontransfected with PD-L1 gene and human placenta
and tonsil FFPE tissues) during each run IHC staining using autostainers. For
the TAIC IHC expression, human tonsil FFPE tissues with and without primary
antibody were used as positive and negative controls, respectively, with each
run IHC staining.