Cd4 4b12

Specimens from 20 patients obtained between January 2000 and December 2013 were fixed with 20% nonbuffered formalin, and those obtained from 19 patients between January 2014 and October 2017 were fixed with 10% buffered formalin (Table S1). Formalin-fixed paraffin-embedded tissues were sectioned at 5 μm, deparaffinized, rehydrated, and stained in an automated system (Ventana Benchmark ULTRA System; Roche, Tucson, AZ, USA) using commercially available detection kits and antibodies against PD-L1 (28–8, ab205921; Abcam, Tokyo, Japan), CD4 (4B12; Nichirei, Tokyo, Japan), CD8 (D1M8I; Cell Signaling Technology, Danvers, MA, USA), and CD3 (LN10; Leica Biosystems, Richmond, IL, USA). PD-L1 is primarily located in the cell membrane of tumor cells, and its expression was evaluated semi-quantitatively by two pathologists based on the proportion of PD-L1-positive tumor cells. A PD-L1 expression rate of 1% or greater was defined as PD-L1-positive. CD3 and CD8 were utilized as a marker for pan T lymphocytes and cytotoxic T lymphocytes, respectively. Thus, CD3+ and CD8+ lymphocytes were counted in the tumors, and the percentage of CD8+ lymphocytes in CD3+ lymphocytes was calculated as the TIL fraction, as previously reported [28 (link),29 (link),30 (link)].

The mIF analysis was performed using 4‐μm‐thick formalin‐fixed, paraffin‐embedded (FFPE) sections (Figure S2a). mIF was performed using an Opal seven‐color immunohistochemistry (IHC) kit (Akoya Biosciences). FFPE sections were fixed in 10% neutral‐buffered formalin (NBF) before paraffin block, then deparaffinized, rehydrated, and fixed one more time in 10% NBF buffer for 20 min before peroxide blocking. Antigen retrieval was performed using either citrate buffer or 0.1% sodium azide (pH 9) buffer (Nichirei Biosciences) and microwave treatment for 15 min. The slides were washed with TBST/0.5% Tween (three times, 2 min each) and incubated with 3% H₂O₂ for 10 min. Then, slides were incubated with the following primary antibodies: CD4 (4B12, Nichirei Biosciences, Ready to use/Opal 520), CD8 (C8/144B, Nichirei Biosciences, Ready to use/Opal 570), PD‐L1 (E1J2J, Cell Signaling Technology; 1:200/Opal 540), FoxP3 (236A/E7, Abcam; 1:100/Opal 620), pan‐CK (C11, Cell Signaling Technology; 1:500/Opal 690). CD8/CD4/PD‐L1/pan‐CK was used for membrane staining, FoxP3 for nuclear staining. Tyramide signal amplification solution was applied after the corresponding secondary HRP‐conjugated polymer for each mAb from the Opal seven‐color IHC kit. Nuclei were stained with spectral 4′, 6‐diamino‐2‐phenylindole (DAPI) and mounted using ProLong Gold Antifade Reagent (Cell Signaling Technology).