Quantifying Tumor Immune Infiltrates by IHC

Hematoxylin and eosin (H&E) staining, and IHC were performed on 4 μm formalin-fixed paraffin-embedded (FFPE) pre- and post-treatment tissue samples. Stromal TILs were quantified manually by H&E-stained FFPE sections and scored as a percentage of stromal area according to the International Immuno-Oncology Working Group method for assessing TILs [23 (link)]. IHC staining on subsequent sections was performed using anti-CD4 (Abcam, cat#ab133616, 1:250), anti-Gr-B (11F1) (Leica Microsystems, cat#PA0291, ready-to-use), and anti-CD57 (BD Biosciences, cat# 347390, 1:40) antibodies. Sections were stained using the BondRX instrument and the Bond Polymer Refine Detection kit (Cat. #DS9800), and the IHC slides were scanned and digitized using the ScanscopeXT system (Aperio/Leica Technologies). Single stain IHC quantification analysis was performed by the pathologist using the HALO 2.3.2089.70 software (Indica Labs). The number of marker positive cells for each analysis area were calculated and expressed as density (number of positive cells/mm2) and densities were plotted using Prism V8.4.3 (GraphPad). Statistical analysis was done using a two-tailed, paired t-test, and P < 0.05 was considered statistically significant.