Flowlogic software version 7

After IEC/moDC co-culture, ccDC were collected and stained for flow cytometry analysis using CD11c-PerCP eFluor 710 (clone 3.9), CD14-APC (clone 61D3), HLA-DR-PE (clone LN3), CD80-FITC (clone 2D10.4) and CD86-PE Cyanine 7 (clone IT2.2) (all from eBioscience, San Diego, CA, USA). Viability was determined using Fixable Viability Dye 780-APC Cyanine 7 (eBioscience). Non-specific binding sites were blocked using PBS supplemented with 5% FCS before extracellular antibody staining. Flow cytometry measurements were done using BD FACS Canto II (Becton Dickinson, Franklin lakes, NJ, USA) and data were analyzed using Flowlogic software version 7 (Inivai Technologies, Mentone, VIC, Australia).

After DC/T-cell assay immune cells were stained for flow cytometry analysis. Cells from DC/T-cell cultures were incubated with Fc receptor blocking solution (Biolegend, San Diego, CA, USA) for 10 min on ice. Then, cells were washed in PBS supplemented with 1% BSA and incubated for 30 min on ice with the following antibodies: CD4-PerCp Cy5.5, CD69-eFluor 450 and CXCR3-Alexa Fluor 488 (all from Thermofisher except CXCR3 from BD). Dead cells were excluded using Fixable Viability Dye eFluor^® 780 (Thermofisher). Stained cells were measured by FACS Canto II (BD Biosciences) and analyzed using Flowlogic software version 7 (Inivai Technologies, Mentone, VIC, Australia).

Spleen and MLN single cell suspensions (0.5–1 × 10⁶ cells/well) were incubated with anti-mouse CD16/CD32 (Mouse BD Fc Block, BD Biosciences, San Jose, CA, USA) in PBS supplemented with 1% BSA and 5% FCS for 15 min on ice to block non-specific binding sites. Subsequently, cells were incubated for 30 min with the following antibodies: CD4-PerCP Cy5.5, CCR6-PE (both from Biolegend, San Diego, CA, USA) CD8a-PECy7, CD69-PE, CD25-Alexa Fluor 488, CD3-PerCP Cy5.5, CD27-PE, CD19-APC and B220-FITC (all from Thermo Fisher). For intracellular staining, cells were first fixated and permeabilized with Foxp3 Staining buffer set (Thermo Fisher) according to manufacturer’s protocol, followed by incubation with Foxp3-PECy7 (Thermo Fisher), RORγT-Alexa Fluor 647 (BD) or Tbet-eFluor 660 (Biolegend). Dead cells were excluded using Fixable Viability Dye eFluor^® 780 (Thermo Fisher). Stained cells were measured by FACS Canto II (BD Biosciences) and analyzed using Flowlogic software version 7 (Inivai Technologies, Mentone, VIC, Australia).