To explore PD-1 expression in CD8+ T cells in the TME, multicolor-staining was utilized to assess the expression of PD-1 in immune cells, using PD-1 and CD8 by mIHC to analyze simultaneously, which helps to make predictions and accurately stratify patients compared with only CD8 staining or only PD-1 staining in ICC study [31]. This evidence concerns the gene CD8A and intrahepatic cholangiocarcinoma.