IDO-orchestrated crosstalk between pDCs and Tregs inhibits autoimmunity

Lippens, Carla; Duraes, Fernanda V.; Dubrot, Juan; Brighouse, Dale; Lacroix, Mathilde; Irla, Magali; Aubry-Lachainaye, Jean-Pierre; Reith, Walter; Mandl, Judith N.; Hugues, Stéphanie

For flow cytometry analysis, single cell suspensions were incubated with FcBlock (anti-CD16/32 FcγRII-RIII) for 10 min, at 4 °C and stained with antibodies. Intracellular cytokine stainings were done using the Intracellular Fixation & Permeabilisation buffer set (eBioscience) or with the Fix & Perm kit (BD Biosciences) for IL-10. Cell proliferation was assessed by flow cytometry using anti–mouse Ki67 and respective isotype control (Rat IgG2a, kappa). For IFN-γ, IL-17 and IL-10 staining, cells were re-stimulated in complete RPMI containing PMA/ionomycin, and incubated 4 h at 37 °C, 5% CO2. Golgi stop solution (BD Biosciences) was added to the last 2.5 h of culture. Data were acquired with a Cyan™ ADP or a Gallios (Beckman Coulter) and analysed using FlowJo software (FlowJo company). cDCs were defined as CD11chiPDCA-1- and pDCs as CD11cintPDCA-1+ or CD11cintSiglec-H+. B cells and macrophages were defined respectively as CD19+ and CD11b+Ly6C+.