Foxp3 transcription factor buffer set

For cell-surface staining, cells were incubated with antibodies at 4 °C for 30 min in the dark. For intracellular staining, cells were fixed and permeabilized by the FoxP3 Transcription Factor buffer set (ThermoFisher Scientific, 00-5523-00). Antibodies were incubated for 1 h at room temperature. In most cases, Zombie Violet Fixable viability Kit was used to exclude dead cells. Samples were acquired on LSRFortessa and analyzed with FlowJo v.10 software. For cell sorting, cells were stained as described above and sorted using the flow cytometry FACSAria III system. Post-sort purity was over 95%.

Peripheral blood samples were first stained for surface epitopes [αCD8 AF700 (53-6.7), αCD28 BV510 (CD28.2), αCD69 FITC (FN50), αCD160 PE-Cy7 (By55), αPD-1 PerCP-Cy5.5 (EH12.2H7) from BioLegend] including a live/dead staining reagent (LIVE/DEAD Fixable Blue Dead Cell Stain Kit for UV excitation from ThermoFisherScientific) for 30 min at 4°C. Afterwards, lymphocytes were fixed and red blood cells (RBCs) were lysed (1-step Fix/Lyse solution from eBioscience). Fixation and permeabilisation of cells was performed using the Foxp3/Transcription Factor Buffer Set (ThermoFisherScientific) according to the manual. To block unspecific binding of antibodies, cells were incubated for 10 min at 4°C with CohnII and subsequently, antibodies directed against intracellular epitopes (GzmB AF647 (GB11), CTLA-4 PE (L3D10), Ki67 AF488 (Ki-67), CD3 APC-Cy7 (HIT3a) from BioLegend, Perforin BV421 (δG9) from BD) were added and further incubated for 20 min at 4°C. Samples were recorded using the LSRII (BD) and analyzed using the FlowJo X 10.0.7r2 Treestar software. Gates were set according to fluorescence minus one (FMO) controls. Gating strategy is depicted in Supplementary Figure 5.

Fresh liver non-parenchymal cells or splenocytes from individual mice were stained with antibodies against B220 (RA3-6B2), CD95 (Jo2), CD3 (SP34-2), CD4 (L200), CD45 (30-F11) were purchased from BD Biosciences (San Jose, CA) and intracellular staining made use of the FoxP3/Transcription factor buffer set (Thermofisher). Data were obtained with a BD Aria II Flow cytometer and were analyzed with the FlowJo Software, version 10.7 (www.flowjo.com). (BD Biosciences).

Isolated cells were stained with Live/Dead Fixable Dead Cell Stain (ThermoFisher Scientific) and marker staining performed with panels of surface or intracellular marker antibodies (Table 1). Intracellular FACS staining was performed using the FoxP3 transcription factor buffer set (ThermoFisher Scientific). Whole blood samples were stained with surface antibodies, antibodies, afterwards erythrolysis was performed using FACS Lysing solution (BD Biosciences) according, to the manufacturer’s instructions. Samples were analysed with a Cytoflex LX or Cytoflex SI Flow Cytometer (Beckman Coulter) and evaluated using FlowJo (Biosciences, Ashland, OR, USA). For tSNE plots and histograms, CD45 positive cells were downsampled to equal numbers of events and samples concatenated (min 3 mice per group) to cluster the cells according to their marker and physical similarity.

TCDD was kindly provided by Dr. Steve Safe (Institute of Biosciences & Technology, Texas A&M Health Sciences Center, College Station, Texas). FICZ was purchased from Enzo Life Sciences (Farmingdale, NY). Both TCDD and FICZ were dissolved in DMSO and diluted in corn oil for use. Sodium butyrate, corn oil, and mBSA were purchased from Sigma-Aldrich (St. Louis, MO). RPMI 1640, l-glutamine, penicillin-streptomycin, HEPES, PBS, and FBS were purchased from Invitrogen Life Technologies (Carlsbad, CA). Fluorophore labeled monoclonal antibodies (mAbs), such as BV785-conjugated anti–CD45, FITC–conjugated anti-CD3, APC/cy7-conjugated anti-CD4, and Alexa Fluor 700-conjugated anti-CD8 used for the flow cytometry, were purchased from Bio Legend (San Diego, CA) and Thermo Fisher (Grand Island, NY). For FoxP3 staining, we used FoxP3 Transcription Factor Buffer Set, and for IL-17 we used intracellular (IC) Fixation and Permeabilization Buffer from Thermo Fisher (Grand Island, NY).

Splenocytes at 1 × 10⁶ cells/well were exposed to an RBD peptide pool (2 μg/mL for each peptide) or 5 μg/mL of each peptide in the presence of a protein transport inhibitor containing brefeldin A (BD Biosciences) for 12 h at 37 °C. Intracellular cytokine staining was performed according to a protocol modified from a previous paper [8 (link)]. The treated cells were incubated with LIVE/DEAD Fixable Red Dead Cell stain (Invitrogen) for 20 min at room temperature. The cells were washed, incubated with an anti-CD16/32 antibody (2.4G2) solution to block the Fc receptor, and stained for surface markers (CD3ε, CD4, and CD8 molecules), followed by intracellular IFN-γ cytokine staining using the Foxp3/transcription factor buffer set (eBioscience). The antibodies used were anti-CD3ε BUV737 (17A2), anti-CD4 APC (RM4–5), anti-CD8α PE-Cy7 (53–6.7), and anti-IFN-γ BV650 (XMG1.2). All the antibodies were obtained from BD Biosciences. The stained cells were acquired on a FACS Aria Fusion (BD Biosciences) and were analyzed via FlowJo^TM V10 (TreeStar, Ashland, OR, USA).