Clone 10d6

Paraffin‐embedded tumor sections were dewaxed in xylene and ethanol and autoclaved for 15 min in an antigen retrieval solution to retrieve their antigen epitopes, and endogenous peroxidase activity was blocked with 3% H₂O₂. Tissue sections were incubated overnight at 4°C with primary antibodies, including rabbit polyclonal anti‐CD3 (1:200 dilution; ab4055, Abcam, Cambridge, UK) for CD3⁺ T lymphocytes, mouse monoclonal anti‐CD8 (1:300 dilution; clone G10F5, BD Pharmingen, San Diego, CA, USA) for CD8⁺ T lymphocytes, anti‐CD4 (1:300 dilution; clone 10D6, Novocastra, Newcastle, UK) for CD4⁺ T lymphocytes, and anti‐FOXP3 (1:300 dilution; clone 10D6, Novocastra) for FOXP3⁺ T lymphocytes. The secondary antibody was incubated in a ready‐for‐use EnVision–peroxidase system (Dako Japan, Tokyo, Japan). Sections were incubated with horseradish peroxidase‐labeled polymer (EnVision1kit, Dako, Carpinteria, CA, USA) for 30 min at 25°C and incubated with 3,30‐diaminobenzidine tetrahydrochloride (applied as a 0.02% solution containing 0.005% H₂O₂ in 0.05 M Tris–HCl; pH 7.6) at 25°C for 5–15 min and counterstained with hematoxylin. We counted each positive lymphocyte in the invasive tumor margin using a BZ‐X700 digital microscope at a magnification of 200× (Keyence) using hybrid cell count software (BZ‐H3C; Keyence; Figure S1).

An automated slide staining machine BOND RX (Leica Biosystems, Nußloch, BW, Germany) and an Opal 7-color Automation IHC kit (Perkin Elmer, Waltham, MA, USA) were used for mIHC staining. The following primary antibodies were used: CD68 (1:2000, clone KP-1, Agilent, Tokyo, Japan), CD163 (1:100, clone 10D6, Leica Biosystems), PD-L1 (1:100, clone SP142, abcam, Cambridge, MA, USA), CD20 (1:1, clone L26, Agilent, Tokyo, Japan), HLA class I (1:800, clone EMR8-5, abcam, Cambridge, MA, USA), and pan-cytokeratin (CK) (1:4, clone AE1/AE3, Agilent, Tokyo, Japan). Staining was performed using the Opal 7-color Automation IHC kit and BOND research detection kit (Leica Biosystems Nußloch, BW, Germany), according to the manufacturer’s instructions. Primary antibodies were incubated for 30 min at 25 °C. Slides were mounted using a ProLong Diamond Antifade Mountant (Thermo Fisher Scientific, Waltham, MA, USA).