Flowlogic software version 8

Stimulated cells were transferred to FACS tubes and washed. The cells were stained extracellularly for 10 min at room temperature with Viobility 405/452 Fixable Dye, antibodies against CD14, CD20, both conjugated to VioBlue (Miltenyi Biotec, Bergisch Gladbach, Germany) and CD4 conjugated to PerCP (BD Biosciences). Subsequently, the stained cells were permeabilized using the BD Cytofix/Cytoperm kit (BD Biosciences) according to manufacturer’s instructions. Permeabilized cells were then stained intracellularly for 10 min at room temperature with antibodies against IL-2 conjugated to BV605 (BD Biosciences), TNF- α conjugated to PE-Vio 770, CD154 conjugated to PerCP-Vio 700, and IFN-γ conjugated to APC-Vio770 (Miltenyi Biotec) according to the methods of the supplier. Stained cells were quantified with a MACSQuant16^® analyzer flow cytometer (Miltenyi Biotec). FACS data were evaluated using FlowLogic software version 8.4 (Inivai Technologies, Victoria 3194 Australia). Reactive T cells were defined as CD4⁺ T cells expressing ≥2 of the following T_H1 activation markers: CD154, IFN-γ, IL-2 and TNF-α. DMSO background controls were subtracted from the data shown. The gating strategy used for all analyses can be found in the supplementary material (Figures S1 and S2).

After 24 h of IEC/PBMC co-culture, PBMCs were collected and stained for analysis with flow cytometry. Viability of the cells was determined using Fixable Viability Dye 780-APC Cyanine 7 (eBioscience, Saint Louis, MO, USA). Immunophenotyping and intracellular cytokine staining was performed using antibodies with appropriate isotypes (eBioscience, Saint Louis, MO, USA; Invitrogen, Saint Louis, MO, USA) (for the list of antibodies, clones and dilutions, see Table S1). Nonspecific binding was prevented by blocking for 15 min with PBS containing 2.5% FCS and Human FC Block (BD Biosciences, Franklin Lakes, NJ, USA) before extracellular and intracellular staining. Cells were fixated and permeabilized with the FoxP3/Transcription Factor Staining Buffer Set or Intracellular Staining Buffer Set (eBioscience, Saint Louis, MO, USA). PBMC measurements was performed using BD FACS Canto II (BD Biosciences, Franklin Lakes, NJ, USA), and data were analyzed by Flowlogic software, version 8.4 (Inivai Technologies, Melbourne, Australia).