αcd25 pe cy5 pc61

Differentiation of murine T cells was performed as previously described (40 (link)). Splenic T cells were isolated by magnetic activated cell sorting using the “pan T cell isolation kit II” according to the manufacturer’s instructions (Miltenyi Biotech). Cells were fluorescently stained with an antibody cocktail containing αCD4-FITC (RM4-5, eBioscience), αCD44-PE (IM7, Biolegend), αCD62L-APC (MEL-14, eBioscience), and αCD25-PE-Cy5 (PC61.5, eBioscience) and were subsequently isolated by fluorescence activated cell sorting on MoFlo (Beckman-Coulter, Brea, CA, USA) in the FACS-core unit of the University Hospital Erlangen. Sorted naive T cells (CD4⁺CD62L⁺CD44^lowCD25⁻) were stimulated by plate-bound anti-CD3 (2 µg/ml, 145-2C11, BD Pharmingen, San Diego, CA, USA) and soluble anti-CD28 (2 µg/ml, 37.51, BD Pharmingen) and cultured for 4 days in the presence of IL-6 (40 ng/ml, R&D Systems, Minneapolis, MN, USA) and TGFβ1 (2 ng/ml, Biolegend) for Th17, IL-12p70 (20 ng/ml,R&D Systems) and anti-IL-4 (10 µg/ml, Biolegend) for Th1 or TGFβ1 (10 ng/ml, Biolegend) alone for Treg differentiation. T cell frequencies were analyzed by flow cytometry (FACSCantoII, BD Biosciences) using the antibodies listed in the section “Murine FACS analysis.”

Splenic T cells were isolated by magnetic-activated cell sorting using the “pan T cell isolation kit II” according to the manufacturer’s instructions (Miltenyi Biotech, Bergisch Gladbach, Germany). Cells were fluorescently stained with an antibody cocktail containing αCD4-FITC (RM4-5, eBioscience, San Diego, CA), αCD44-PE (IM7, BioLegend, San Diego, CA), αCD62L-APC (MEL-14, eBioscience), and αCD25-PECy5 (PC61.5, eBioscience) and were subsequently isolated by fluorescence-activated cell sorting on MoFlo (Beckman-Coulter, Brea, CA) in the FACS core unit in Erlangen, Germany.