Fig 2: Expression of immune-checkpoint molecule (ICM) ligands in head and neck squamous cell carcinoma (HNSCC) tissues. ICM ligands expression in the nest of case 11 is shown (A). Representative double immunostaining images with programmed cell death-1 (PD-1) in orange and cytokeratin in blue (upper right), galectin 9 in red and cytokeratin in blue (lower left) and CECAM-1 in green and cytokeratin in blue (lower right) are shown in the same area with HE images (upper left). ICM ligand expression in the stroma is shown (B). Representative double immunostaining images with PD-L1 in orange and CD204 in blue (upper right), CD68 in green and CD204 in blue (lower left) and galectin-9 in red and CD204 in blue (lower right) are shown in the same area with the HE images (upper left). Distribution of programmed cell death receptor ligand 1 (PD-L1) (orange) expressing macrophages along the periphery of the nest (cytokeratin in blue) was observed (C, upper panel) in an “immune-excluded” case (case 14). In an “immune-inflamed” case (case 24), high infiltration of PD-L1 (orange)-expressing macrophages into the nest (cytokeratin in blue) were observed (D, upper panel). These macrophages were positive for CD204 (C and D, lower panel). Upper right and lower right images of (C) and (D) indicate the expanded images of the white square left of the images. Magnifications are indicated in each image

Fig 3: Programmed cell death-1 (PD-1) expression on T-cells and interaction with programmed cell death receptor ligand 1 (PD-L1) expressing cells in head and neck squamous cell carcinoma (HNSCC) tissues. HNSCC tissues were double stained with PD-1 and CD3 and representative PD-1 expression on T-cells in HNSCC tissue is shown in (A) (upper; CD3 in blue, middle; PD-1 in green, lower; merge). PD-1 expressing T-cells were preferentially distributed in the nest of the “immune-excluded” case [case 15 (B, left)] and were equally distributed both in the nest and stroma [case 27 (B, right)]. Interactions of PD-L1 (orange) with cancer cells (cytokeratin+ in blue) (upper images) or macrophages (cytokeratin) with PD-1 (green) on lymphocytes are shown for case 24. Upper right and lower right images indicate the expanded images of the white square left of the images. White lines indicate the border between the nest and stroma. Magnifications are indicated in each image