Flow cytometric cell analysis
Surface staining was performed on the MS column (first panel: Brilliant
violet 510™ anti-human CD4; RPA-T4, Brilliant Violet 421™ anti-human
CD197 (CCR7); G043H7, PE/Cy7 anti-human CD45RA; HI100, second panel:
Brilliant violet 510™ anti-human CD4; PE/Cy7 anti-human CD29/ß1; TS2/16,
PE anti-human ß7; FIB504, all from BioLegend (Koblenz, Germany), VioBlue
anti-human CD49d/α4; MZ18-24A9 from Miltenyi Biotec. (Bergisch Gladbach,
Germany) and human FC block (CSL Behring, Marburg, Germany)). The
enriched cell fraction was fixed using eBioscience™, FoxP3 staining
buffer, Thermo Fisher Scientific, Waltham, MA USA) and intracellular
staining was performed (APC anti-human IFNγ; 4S.B3, APC/Cy7 anti-human
IL-17A; BL168, PerCP/Cy5.5 anti-human TNFα; MAb11, all from BioLegend
and FITC anti-human CD154 (5C8) from Miltenyi Biotec. Flow cytometry
analysis was performed on the FACS Canto II device (BD Bioscience,
Heidelberg, Germany). Data were analyzed with FlowJo analysis software
(Ashland, OR, USA).