Tumor biopsies from advanced stage (III/IV) patients with human papillomavirus (HPV)-negative HNSCC were obtained at the time of standard-of-care surgical resection under institutional review board-approved protocol NCT03429036 with informed consent. Sections were stained using Opal multiplex kits, according to the manufacturer’s protocol (PerkinElmer), for a panel of 4′,6-diamidino-2-phenylindole (DAPI), pan-cytokeratin (clone AE1/AE3), HLA class I (EMR8-5) and CD3 (SP7). Positive and negative controls included normal human tonsil and unstained sections (no primary antibody), respectively. After optimization, staining of all analyzed sections was done in a single automated assay. Images were captured by a PerkinElmer Vectra Polaris. Multispectral fluorescent images were analyzed using QuPath software.20 (link) Following annotation of entire tumor sections into tumor and stromal tissue based on cytokeratin staining, HLA positivity and CD3+ cells were quantified using automated detection based on universally applied fluorescence detection thresholds.