Single-cell RNA sequencing (scRNA-Seq) analysis of tumor-infiltrating lymphocytes (TILs) from B16-F0 melanoma in mice

To comprehensively analyze the influences on the tumor infiltrated immune cells after deficiency of APPL2 specifically in Tregs, we employed single-cell RNA sequencing (scRNAseq) to profile CD45+ TILs from both Foxp3YFP-Cre Appl2f/f and Foxp3YFP-Cre control mice. To our surprise, tumor infiltrated B cells were dramatically decreased in Foxp3YFP-Cre Appl2f/f mice. Hereby, there was a trend of increase for most of the other immune subsets. These data indicated that the infiltrated B cells, as a whole, were tumor supportive and restricting protumoral B cell infiltration was critical for Appl2-KO Tregs in suppressing tumor growth. Overall design: For scRNA-Seq analysis, TILs from mice bearing B16-F0 melanoma were suspended in RPMI-1640 medium containing 2% FBS and DAPI solution ( BD, Cat: 564907, 1:3000). CD45+ DAPI- cells were sorted and prepared as approximately 1 × 106 cells/ml, cell viability was above 97%. Cell suspensions were loaded onto a 10×Genomics Chromium Next GEM Single Cell 3' Reagent Kits v3.1 according to the manufacturer's instructions.