CD4+ T cells were isolated from splenocytes using anti-mouse CD4 magnetic particles (BD Biosciences). For RNA-seq to analyze the effects of IL-10 on CD4+ T cells (Fig. 5), cells were treated by immobilized anti-CD3 antibodies with or without recombinant IL-10 (Peprotech) for 48 h. RNA was extracted with TRIzol reagent (Invitrogen) and purified with the Direct-zol RNA MiniPrep Kit (Zymo Research). Library preparation and high-throughput sequencing were performed at Macrogen using TruSeq RNA Sample Prep Kit v2 and HiSeq 2000 (Illumina) or GeneWiz using the NEBNext Ultra II Directed RNA library kit (New England BioLabs) and HiSeq X (Illumina). RNA-seq data were mapped to the GRCm38/mm10 using HISAT2 (60 (link)). Differently expressed genes were analyzed using HTSEq (61 (link)) and edgeR (62 (link)) or DESeq2 (63 (link)). Volcano plots and heat maps were drawn using ggplot2 (64 ) and gplots (65 ). Expression tiling of RNA-seq was visualized using the Integrative Genomics Viewer (66 (link)). Functional annotation of gene lists with Gene Ontology terms and KEGG pathways was performed with the DAVID tools (29 (link), 30 (link)) and GSEA (44 (link), 45 (link)).