a The relationship between PD-L1 and PD-L2 expression status. There was no significant correlation between PD-L1 and PD-L2 expression (P = 0.18) (n = 437). b Double immunohistochemical staining of PD-L1 and PD-L2. Green areas show PD-L1 expression and brown areas show PD-L2 expression. (Left) Cancer cells expressing only PD-L2, (Middle) cancer cells expressing both PD-L1 and PD-L2 and (Right) cancer cells expressing only PD-L1. c Multiplex immunohistochemical staining of PD-L1 and PD-L2. FITC was used to visualise PD-L2 (green colour), Cy5 was used to visualise PD-L1 (red colour) and DAPI was used to visualise nuclei (blue colour). (Top) Cancer cells expressing PD-L2 and PD-L1 were mixed. (Bottom) PD-L2- and PD-L1-expressing cells have different location. d Kaplan–Meier curves for overall survival in oesophageal cancer patients according to PD-L1 and PD-L2 expression status. Both PD-L1- and PD-L2-negative group showed significantly favourable overall survival compared with the other groups (log-rank P = 0.001).