Jes5 16e3

The isolated PBMCs were resuspended in 2% FBS-PBS, and then stained with indicated antibodies against human CD3 (HIT3α, BioLegend), CD14 (63D3, BioLegend), IL-6 (UV4, BioLegend), IL-10 (JES5-16E3, eBioscience), TNF-α (Mab11, BioLegend), and detected by flow cytometry (BD FACS Calibur II, San Jose, CA, USA) and analyzed using FlowJo 7.6 software (TreeStar Inc., USA) as we previously described [24 (link)–26 (link)]. Mouse IgG1 (MOPC-21), IgG2b (27–35), IgG2a (G155–178), and rat IgG2b (κ) were used as isotype control.

Single cell suspensions from spleens and lymph nodes were obtained via passing through 70 μm cell strainers in cold PBS^−/−. Hearts were collected and digested with Liberase TM (Roche). Erythrocytes were removed with lysis buffer (BD Biosciences) from spleen and heart cell suspensions. Cells were stained at the following dilutions of stock reagents: Live/dead Aqua Fluorescent Reactive Dye 1:1,000 (Life Techonologies), anti-mouse CD16/32 1:100 (2.4G2, BD Pharmigen), anti-mouse 1:100 CD45 (30-F11, eBioscience), anti-mouse 1:100 CD3ɛ (145-2C11, BioLegend), anti-mouse 1:100 CD19 (eBio1D3, eBioscience), anti-mouse 1:100 CD11b (M1/70, Biolegend), 1:100 CD11c (Bu15, eBioscience), F4/80 1:100 (CI:A3-1, Serotec) in Supplementary Fig. 5d, F4/80 1:100 (BM8, eBioscience) in Fig. 5d,e and Supplementary Fig. 5c, IL-10 1:80 (JES5-16E3, eBioscience), FoxP3 1:100 (FJK-165, eBioscience), Ly6C 1:100 (HK1.4, eBioscience) or anti-CD25 1:100 (PC61.5, eBioscience). An eBioscience intracellular staining kit was used were applicable. Samples were acquired on a fluorescence-activated cell sorting Canto II (BD) and analysed with FlowJo10.

The following antibodies were used: Alexa Fluor 700-conjugated anti-CD4 antibody (RM4-5; BD Bioscience), fluorescein isothiocyanate-conjugated or allophycocyanin-conjugated anti-Foxp3 antibody (FJK-16S; eBioscience), phycoerythrin-conjugated anti-Helios antibody (22F6; Biolegend), phycoerythrin-conjugated anti-CD25 antibody (PC61 5.3; Invitrogen), phycoerythrin-conjugated anti-IL-4 antibody (11B11), Alexa Fluor 647-conjugated anti-IL-13 antibody (ebio13A; eBioscience), Pacific blue-conjugated anti-T cell receptor β (TCR-β) antibody (H57-597; Biolegend), allophycocyanin-conjugated anti-IL-10 antibody (JES5-16E3; eBioscience), and fluorescein isothiocyanate-conjugated anti-gamma interferon (IFN-γ) antibody (XMG1.2; BD Bioscience). Nonspecific binding was blocked with 4 μg of rat IgG per 1 × 10⁶ cells. Intracellular staining for Foxp3 and Helios was performed using a Foxp3-staining buffer kit (eBioscience). For intracellular cytokine staining, dead cells were excluded using the Live/Dead aqua dead cell stain kit (Molecular Probes), and cells were fixed and permeabilized using the BD Cytofix/Cytoperm kit. Flow cytometry was performed using a FACSCanto 2 or an LSR 2 (BD Biosciences), running FACSDiva software (BD Biosciences). Analysis was performed using FlowJo (Tree Star).