Fig. 1

Summary of workflow. A) A cohort of 35 HNSCC patients was selected, with serial sections per patient. In step 1, 15 serial sections were processed using a 53-plex panel of antibodies with PCF to visualize PD-1 and PD-L1 expression as well as the distribution of various cell types across tissues (B, left panel). In step 2, serial sections from 16 patients were stained using isPLA kit and scanned by PCF to detect PD-1/PD-L1 interactions (B, middle panel). the resulting images from steps 1 and 2 were registered to ensure isPLA signals corresponded to regions with PD-1 and PD-L1 expression. In step 3, an exploratory cohort of 28 serial sections were stained with a 6-plex panel and imaged using the PhenoImager system to identify interacting cells through PD-1/PD-L1 across whole tissues and per different response groups to immunotherapy (B, right panel)