Foxp3 pe cy7

Peripheral blood mononuclear cells (PBMC) of patients and control subjects were isolated by Ficoll-Hypaque (GE Healthcare, Pittsburgh, PA) density-gradient centrifugation, and cellular viability was evaluated by trypan blue staining and it was always higher than 95%. Mononuclear cells were stained for 30 minutes in darkness at 4°C with the following monoclonal antibodies (mAbs): CD4-FITC (eBioscience, San Diego, CA) or CD4-APC/Cy7 (BioLegend, San Diego, CA), CD25-APC/Cy7 (Becton-Dickinson, Franklin Lakes, NJ), NKG2D-FITC (eBioscience), antilatency-associated peptide (LAP, a surrogate marker for TGF-β)-PerCp/Cy5.5 (BioLegend), and CD69-APC (eBioscience). Then, cells were washed and fixed and permeabilized with the Foxp3 Fix/Perm kit (eBioscience) for 30 minutes. Subsequently, mononuclear cells were stained with mAbs against IL-10 (PE) (BioLegend) and Foxp3 (PE/Cy7) (eBioscience). Doublet discrimination was performed by analyzing FSC-A versus FSC-W dot plots from the lymphocyte gate. In all cases, at least 1 × 10⁶ events were analyzed, and gates were set up by using fluorescence minus one controls and labeled mAb isotype controls. CD4⁺CD69⁺ and CD4⁺NKG2D⁺ cells were analyzed separately, and data were acquired in FACSCanto II flow cytometer (Becton Dickinson) and analyzed using the Flow Jo software v10 (Tree Star Inc., Ashland, OR).

MLN and spleen single-cell suspensions were prepared for flowcytometry analysis as previously described [61 (link)]. The cell suspensions were incubated with anti-mouse CD16/CD32 (Mouse BD Fc Block; BD Biosciences, Franklin Lake, NJ, USA) in PBS + 1% BSA for 15 min on ice to block unspecific binding sites. Afterwards, cells were stained with the following surface markers CD4-PerCp-Cy5.5, CD69-PE-Cy7, CXCR3-PE, CD25-AlexaFluor488, CD25-PE, (all purchased from eBioscience, San Diego, CA, USA) or T1ST2-FITC (MD Bioproducts, St. Paul, MN, USA) for 30 min on ice. Fixable Viability dye eFluor 780 (eBioscience) was used to exclude non-viable cells. Next, the cells were fixed and permeabilised with the Foxp3 Staining Buffer Set (eBioscience) according to the manufacturer’s protocol and then stained with the intracellular markers Foxp3-PE-Cy7, RORɣt-PE, IRF4-FITC, Tbet-eFluor660 or Gata3-eFluor660 (all purchased from eBioscience). Cells were measured on BD FACSCanto II flow cytometer, and results were analysed with FlowLogic software (Inivai Technologies, Mentone, Vic, Australia). The used gating strategy is shown in Figure S1.

Freshly isolated mesenteric lymph nodes (MLNs) were analyzed by flow cytometry (14 (link)). Identification of migratory DCs in the MLNs was performed by gating CD103+ cells out of CD11c+ MHC-II cells in the MLNs (the gating strategy is shown in Figure 2A). Cells obtained and resuspended in PBS with 1% bovine serum albumin were incubated with antimouse CD16/CD32 (Mouse BD Fc Block; BD Pharmingen) for 20 min on ice to block nonspecific binding sites. For surface staining, cells were incubated with CD4-PerCp-Cy5.5, CD69-PE, CD25-AlexaFluor488, CD11c-PerCp-Cy5.5, CD103-APC, CD40-FITC, CD86-PE-cy7, MHCII-PE, CD3-Percy5.5, CD27-PE, CD19-APC, B220-FITC (eBiosciences). Foxp3-PE-cy7, and Tbet-APC (eBioscience) were used for intracellular staining. Staining and flow cytometry were performed as described previously (14 (link)).

For assessment of intracellular cytokine expression in CD4⁺ T cells, the lymphocytes were incubated with 10 μg/mL brefeldin A (Sigma-Aldrich, St. Louis, MO, USA). After 6 hours, cells were harvested and stained with anti-CD4-FITC (eBioscience, Inc., San Diego, CA, USA) for 30 min at room temperature, followed by fixation and permeabilization using the Cytofix/Cytoperm Kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions. The intracellular cytokine staining was performed using the following monoclonal antibodies: IFN-γ-PE, IL-4-PEcy7, IL-17-PE, and Foxp3-PEcy7 (eBioscience, Inc., San Diego, CA, USA). Stained cells were analyzed by a cube-8 flow cytometer (Sysmex/Partec, Münster, Germany). Gates were applied to define the populations of interest, and the analysis was carried out using FCS Express 4 (De Novo Software, CA, USA). Isotype controls were used as negative controls.

Briefly, the small intestine was placed in Hank’s Balanced Salt Solution (HBSS) buffer supplemented with 10% fetal bovine serum. Peyers patches were excised and tissue was placed in digestion media containing 1 mM DTT, 1 mM EDTA in calcium/magnesium-free HBSS supplemented with 2% fetal calf serum and subsequently treated with Collagenase IV/Dnase digestion mix (0.5 mg/ml of collagenase IV and 200 μg/ml of Dnase). Lymphocytes were enriched using a 44/67% discontinuous Percoll (GE Lifesciences, Pittsburgh PA) gradient. Splenic lymphocytes were treated with ammonium chloride and washed. Cells were stimulated with phorbol 12-myristate 13-acetate and Ionomycin for 4 h at 37 °C in the presence of brefeldin A (GolgiPlug; BD Bioscience, San Jose, CA). Following stimulation, cells were stained with LIVE/DEAD Fixable Aqua (ThermoFisher Scientific, Waltham, MA), and the following antibody/fluorophore combinations APCC7-CD45, TCRβ-FITC, CD4-V500 (BD Bioscience), CD8-BV650, Foxp3-PECy7, IL17-PE, IFNγ-FITC (eBioscience, San Diego, CA), and fixed with fix/perm (eBioscience), were used according to the manufacturer’s instructions. Cells were acquired on an LSRII flow cytometer (BD Bioscience) and analyzed with FlowJo software (Tree Star, Ashland, OR); 100,000 events or greater were collected for each sample. Samples with yields less than 10,000 viable events were excluded from analysis.