Quantitative Immunohistochemistry of Tumor Immune Markers

Tumor tissue was fixed in formalin and embedded in paraffin. For immunohistochemical staining, tissue was cut and mounted at a thickness of 4μm per slide. Slides were then stained with CD3 polyclonal (1:100, DAKO), CD4 clone 4B12 (1:80, Leica Biosystems), CD8 clone C8/144B (1:25, Thermo Scientific), PD-L1 clone E1L3N (1:100, Cell Signaling Technology), PD-1 clone EPR4877-2 (1:250, Abcam), CD45RO clone UCHL1 (ready-to-use, Leica Biosystems), FoxP3 clone 206D (1:50, BioLegend), and Granzyme B clone F1 (ready-to-use, Leica Biosystems)^{18 (link)}. Slides were then stained using diaminobenzidine as chromogen and the Leica Bond Polymer refine detection kit (Leica Biosystems). Slides were then counterstained with hematoxylin and scanned using an Aperio AT2 automated slide scanner (Leica Biosystems). Quantification was performed on 5 × 1 mm² regions per tumor sample within the tumor center and measuring the average density of positive cells per region as a count of positive cells/mm². For PD-L1, H-score was calculated by multiplying the proportion of positive cells in the sample (0–100%) by the intensity of staining (1⁺, 2⁺, or 3⁺) to obtain a score ranging between 1 and 300.