library_construction_protocol: Tumors were subjected to tumor-infiltrating lymphocytes (TIL) isolation. CD4+ T cells were isolated by using Mouse CD4 (L3T4) MicroBeads (Miltenyi Biotec) according to the manufacturer's protocol. Intra-tumoral Treg cells (CD4+ YFP+) were sorted from the isolated CD4+ T cells by using BD FACSAria Cell Sorter. Conventional CD4+ T cells (CD4+YFP-) and Treg cells (CD4+YFP+) sorted from Hepa1-6 tumors and tumor-free LN of Foxp3YFP-cre mice respectively were included for comparison. Cells were pooled from multiple mice per sample. BD Rhapsody 8-lanes cartridges (664887 BD Biosciences) were loaded with 80,000 cells to capture 50,000-60,000 cells per sample. Single cells were isolated with the BD Rhapsody HT Xpress Single-Cell Analysis System using the BD RhapsodyTM Enhanced Cartridge Reagent Kit (633773 BD Biosciences) and BD Rhapsody cDNA Kit (633733 BD Biosciences) according to the manufacturer's recommendations (BD Biosciences). cDNA libraries were prepared using the Whole Transcriptome Analysis Amplification Kit (633801 BD Biosciences) following the BD Rhapsody System mRNA Whole Transcriptome Analysis (WTA) Preparation Protocol (BD Biosciences). All the libraries were sequenced in a paired-end mode (PE150) on DNBseq-T7 (GenePlus).