Facs lsrii sorp

PBMC were isolated by Pancoll gradient centrifugation (PAN-Biotech, Aidenbach, Germany). Monocytes were isolated by positive selection with anti-CD14 microbeads. The purity was analyzed by flow cytometry on a FACS LSRII SORP (BD Biosciences, Heidelberg, Germany) with anti-human CD14 and ranged from 90%-99% (S8B Fig). Remaining PBMC were frozen in RPMI 1640 supplemented with 20% FCS and 20% DMSO (both from Sigma-Aldrich, Munich, Germany) for subsequent isolation of autologous T cells.
Isolated monocytes were counted using trypan blue exclusion (Applichem Panreac, Darmstadt, Germany) and seeded at a density of 1.5*10⁶ cells/ml in RPMI 1640 (Gibco, Life science, Darmstadt, Germany), supplemented with 10% FCS (Sigma-Aldrich Chemie GmbH, Munich, Germany), 1% penicillin/streptomycin, 1% L-Glutamine and 1% HEPES buffer (all from Biochrom, Berlin, Germany), 50 μM 2-Mercaptoethanol (Sigma-Aldrich, Munich, Germany), 50 ng/ml human GM-CSF and 20 ng/ml human lL-4. Cells were incubated at 37°C and 5% CO₂, medium exchanged on after 3 days and cells harvested on day 6. Successful differentiation of monocytes into monocyte-derived dendritic cells (MoDC) (CD14^-, HLA-DR^high, CD11c⁺, CD83^dim) was confirmed by flow cytometric analysis (S8A Fig).

Phenotypic analysis of human B cell subsets was performed with the following antibodies: anti-CD19-PE-Cy7 (Beckman Coulter, Marseille, France), anti-CD27-BV421 (BD Biosciences, Heidelberg, Germany), anti-IgM-PerCP/Cy5.5 (BioLegend, CA, USA), anti-IgM-BV605 (BioLegend), anti-CD38-PE (BD Biosciences), anti-TNFR1(CD120a)-FITC (Miltenyi Biotech), anti-TNFR2(CD120b)-APC (R&D Systems, Inc., Minneapolis, MN, USA), and murine IgG_2A-APC (R&D Systems, Inc.) as isotype control where indicated. Cells were incubated in the dark for 30 min at 4°C in PBS with 0.5% FCS. Samples were acquired using a FACS LSRII SORP (BD Biosciences, Heidelberg, Germany), and cytometry data (LMD files) were analyzed with Kaluza software (Beckman Coulter). The aqua fluorescent reactive dye (LIVE/DEAD Fixable dead Cell stain Kit, Invitrogen, CA, USA) was used for definition of live and dead cells.
For staining of IL-10-producing B cells we used the IL-10 Secretion Assay (Miltenyi Biotech, Bergisch-Gladbach, Germany). B cells were stimulated for 40 h in culture medium with 1 µM CpG ODN 2006. Staining with anti-IL-10 was performed according to the protocol provided by the manufacturer with a prolonged incubation of cells labeled with IL-10 catch reagent (6 h) in presence of 0.25 µM CpG for restimulation. Cells were subsequently stained for expression of other surface markers before measurement on a flow cytometer.

In vitro differentiated and purified blood memory T_H17 cells were restimulated during 3 h with PMA and ionomycin using the Cell Stimulation Cocktail (eBioscience) and then stained with Alexa647-IL26, Alexa488-IL17A, Alexa568-IL22 and Alexa750-IFNγ probes using PrimeFlow RNA Assay (eBioscience) according to the manufacturer’s protocol. In some experiments, intracellular cytokine staining of re-stimulated T_H17 cells and blocked for secretion by Brefledin A after 1 h was performed using anti-human IL-9 PeCy7 (BioLegend, 1/20), IL-10 PE (BD Biosciences, 1/20), IL-13 BV421 (BioLegend, 1/20) and IL-21 PE (BioLegend, 1/20) antibodies before proceeding with the hybridization step of the Prime-Flow Assay. Cells were acquired on a FACS LSR II SORP (BD Biosciences) and analyzed with FlowJo_v10.7.1. A detailed gating strategy for this analysis is shown in Supplementary Fig. 8.