Cd3 clone 145 2c11

To perform surface staining, 1 × 10⁶ cells were placed in individual wells of a 96-well round bottom plate and incubated with the appropriate antibody cocktails for 15 min at 4°C on a slow rocker. After the staining, cells were fixed in a solution of 2% ultrapure formaldehyde (Polysciences, Inc., Warrington, PA, USA) in FACS buffer for 20 min on ice, washed twice, and analyzed the following day on the Canto II (BD Biosciences) or FACSCalibur (BD Biosciences). Intracellular staining was performed using Cytofix/Cytoperm Fixation/Permeabilization Solution Kit with BD GolgiStop (BD biosciences) according to the manufacturer’s instruction. Flow cytometry acquisition was performed on an LSRIISorp. Data were analyzed using FACS Express or FlowJo software (Tree Star, Inc., Ashland, OR, USA). Antibodies against CD45 (clone 30-F11, BD Pharmingen), CD3 (clone 145-2C11, BD Pharmingen), CD4 (clone GK1.5, BD Pharmingen), CD8 (clone 5H10, Biolegend), T-bet (clone eBio4B10, eBioscience), IL-17A (clone ebio17B7, eBioscience), IL-4 (clone B11B, Biolegend), IFNγ (clone XMG 1.2, eBioscience), IL-22 (clone A3.6M, eBioscience and clone poly5164, Biolegend), TGF-β (clone 11A5, Biolegend), IL-17F (clone ebio18F10, eBioscience), NKP46 (clone 29A 1.4, Biolegend), c-Kit (clone 2B8, Biolegend), Sca-1 (clone D7, BD Pharmingen), and CD127 (clone A7R34, eBioscience) were used.

Spleen and heart fragments were collected to evaluate IFN-γ-producing CD4⁺ T cells, IL-10-producing Tregs, and IL-10-producing Tr1 cells. To isolate mononuclear cells from cardiac tissues, the hearts were removed at 15 d.p.i. washed (to remove blood clots), minced with scissors into small fragments, extensively washed, and subjected to enzymatic digestion with 500 mg/mL of Liberase solution (Roche Applied Science, Indianapolis, IN, USA) for 1 h at 37°C. The tissues were washed with RPMI 10% FCS and total cells that passed through the cell strainer were subsequently counted. Two million leukocytes from heart tissue or from spleen were stimulated in vitro with PMA/Ionomycin/Golgi Stop for 6 h. The leukocytes were stained with specific antibodies for Cd11b (clone MI/70 BD), I-A/E (clone 269 BD), CD3 (clone 145-2C11), CD4 (clone RM4-5 BD), Foxp3 (clone MF 23 BD), IFN-γ (clone XMG 1.2 BD), IL-10 (clone JESS-16E3 BD), and IL-27p28 (clone MM27-7b1 Biolegend), and evaluated by flow cytometry.