Cd25 apc cy7

Flow cytometry analysis was performed using a Guava 8HT (EMD Millipore Corporation, Billerica, MA USA). Splenic and blood cells from control and tolerant mice were collected and labeled with antimouse antibodies (BioLegend, San Diego, CA) for cell surface markers: CD4-PE and CD25- APC/Cy7. After cell membrane permeabilization with saponin, cells were intracellular labeled with antimouse antibodies: FOXP3-PE/Cy7, IL-17a- APC and IL-6-FITC (BioLegend).

Intestinal lamina propria cells and MLN cells were labeled with CD4-FITC, CD25-APC (eBioscience), CD8-APC-Cy7, CD3-PerCP (Biolegend), and B220-BV570 (BD Biosciences). Labeling of intracellular FoxP3 was performed after extracellular staining and fixation/permeabilization of cells. GM-CSF-derived dendritic cells were labeled with CD11b-PerCP Cy5.5 (BD Biosciences), CD11c-PE-Cy7, I-A/I-E-APC-Cy7, CD80-PE, CD86-APC, CD8-FITC, and B220-BV570 (Biolegend). Flt3L-derived dendritic cells were labeled with CD11c-PE-Cy7, CD11b- or Siglec-H-PerCP Cy5.5 (Biolegend), I-A/I-E-APC-Cy7, CD317-PE, CD40-Alexa Fluor 647, CD103-FITC, and B220-BV570. Coculture T cells were labeled with CD4-PerCP Cy5.5 (BD Biosciences), CD127-PE-Cy7, CD73-APC, CD195 (CCR5)-FITC, CD62L-BV570, and CD25-APC-Cy7 (Biolegend).

We isolated peripheral blood mononuclear cells (PBMC) from peripheral blood by Ficoll gradient and we stored them in liquid nitrogen for batched analysis. For multicolor flow cytometry analyses, we used the following monoclonal antibodies: from Becton Dickinson (BD, Franklin Lakes, NJ), CD3-FITC, CD3-PerCP-Cy5.5, CD4-APC-Cy7, CD8-BV510, CD45RO-FITC, CD45RA-APC, CD45RA-APC-Cy7, CD71-PE, IgD-PerCP-Cy5.5, CD25-APC-Cy7, CD138-BV421, CCR4-PE, CD27-PE, CD95-BV421, CD28-BV421, TIM3-BV421, CCR6-BV421, CCR7-AF700, CXCR3-PE, IL-2-PE, IL-17-BV786, and IFN-γ-PE-Cy7; from Biolegend (San Diego, CA), CD19-BV510, CD56-FITC, CD27-APC, CD127-FITC, CD57-PerCp-Cy5.5, PD-1-APC-Cy7, CXCR5-FITC, LAG3-APC, IL-10-PerCP-Cy5.5, and TNF-α-FITC. From eBioscience (San Diego, CA), CD4-PE-Cy7, CD21-PE, and CD24-APC-Cy7 were used. From Miltenyi Biotec (Auburn, CA), CD25-APC and anti-KLRG1-PE were used. From Beckman Coulter (Indianapolis, IN), CD38-PE-Cy7 was used.
We performed intracellular staining for IL-2, IL-4, IL-17, IFN-g, and TNF-a together with extracellular markers for CD4⁺, CD8⁺, and CD19⁺. Cells were fixed and permeabilized using Intracellular Fixation and Permeabilization Buffer Set (eBioscience) according to the manufacturer’s instructions.
Data were acquired (> 1 × 10⁶ events) on a 3-laser FACSLyric flow cytometer (BD Biosciences) and analyzed with FlowJo^® software.

Tofacitinib (Tofa, CP-690550, Selleck Chemical LLC, TX, USA) was dissolved in dimethylsulfoxide (DMSO) and then diluted in the culture medium at a final concentration of 10 (Tofa₁₀) or 100 μM (Tofa₁₀₀), so that the final concentration of DMSO would not exceed 1∶320 v/v. These concentrations were chosen on the basis of a previous work [7] (link) and did not induce unspecific cell toxicity, while permitting an effective reduction of proliferation.
Phytohaemagglutinin (PHA, Biochrom AG, Germany) was dissolved in water and then diluted 1∶200 in the culture medium at a final concentration of 1 μg/mL.
Oregon Green 488 carboxylic acid diacetate, succinimidyl ester (Carboxy-DFFDA, SE or CFSE, Invitrogen, Eugene, OR, USA) was dissolved at a concentration of 4.21 mM in DMSO and then used for proliferation assay at a final dye concentration of 5 μM.
7-aminoactinomycin D (7AAD) viability staining was from Immunostep (Salamanca, Spain).
All monoclonal antibodies for flow cytometric analysis were from Miltenyi Biotec (Bergisch Gladbach, Germany) except for CD25 APC-Cy7 (Biolegend, San Diego, CA, USA), HLA DR PE and CD19 FITC (Invitrogen, Carlsbad, CA, USA). 7AAD and monoclonal antibodies were used at concentrations indicated by the manufacturers.