T bet pe

The following antibodies and reagents from eBioscience or BD were used: CD3e-FITC (145-2C11), CD4-PE (RM4-5), CD4-PErCP (RM4-5), CD8-APC (53-6.7), CXCR3-PE (CXCR3-173), T-bet-PE (eBio4B10), Foxp3-PE (MF23), CD25-APC (PC61), and CD16/32 (93). Before staining, all cell preparations were incubated with anti-mouse CD16/32 (Fc receptor block) for 15 min on ice to reduce nonspecific antibody binding. For surface staining, cells were incubated with cocktails of mAbs in flow cytometry buffer. For intracellular staining, live cells were incubated with PMA (200 ng/ml; Solarbio, China) and ionomycin (1 μg/ml; Cayman Chemical, USA) in the presence of brefeldin A (1:1000; eBioscience, USA) for 5 h at 37°C in 5% CO₂. Cell suspensions were first stained with surface antibodies, then treated with Foxp3/Transcription Factor Staining Buffer Set (eBioscience, USA) according to the manufacturer’s instructions before staining intracellularly with anti-mouse T-bet or Foxp3. The single-color controls and isotype-control Abs were used to validate the flow cytometry results. Samples were acquired using a Canto II flow cytometer (BD) and the data were analyzed using FlowJo software version 7.6.1.

The following monoclonal antibodies were purchased from eBioscience: CD278 (ICOS)-biotin, CD27-PeCy7, Foxp3-FITC, RORγt-PE, T-bet-PE; or from BD Biosciences: PD1-PECF594, CXCR3-APC, CD24-PECF594, CD25-BB515, CD44-PECy7, CD4-A700, CD8-A700, CD4-PB, CD62L-A700, GATA3-PE, RORγt-PECF594, STAT1 (pY701)-A488, IFNγ-PE, IL-10-APC, IL-17-PerCP-Cy5.5, streptavidin-PECy7. Live/dead fixable near-IR stain (Thermo Fisher) was used to exclude dead cells. For transcription factor staining, cells were stained for surface markers, followed by fixation and permeabilization before nuclear factor staining according to the manufacturer’s protocol (Foxp3 staining buffer set from eBioscience). For cytokine staining, cells were stimulated in media containing phorbol 12-myristate 13-acetate (50 ng/ml, Sigma-Aldrich), ionomycin (250 ng/ml, Sigma-Aldrich), and brefeldin-A (1/100, eBioscience) for 3 hr. After stimulation, cells were stained for surface markers, followed by fixation and permeabilization before intracellular staining according to the manufacturer’s protocol (cytokine staining buffer set from BD Biosciences). For phosphorylation staining, cells were stimulated with IFN-γ (50 ng/ml, PeproTech) for 30 min, fixed with formaldehyde, and permeabilized with methanol before staining. Flow cytometric analysis was performed on a Canto II (BD Biosciences) and analyzed using FlowJo software (Tree Star).

For surface staining, frozen PBMCs were thawed at 37°C and washed 2 times with phosphate-buffered saline containing 1% fetal bovine serum. Cells were incubated with Human BD Fc Block™ (10 minutes at room temperature) followed by staining with directly conjugated mAbs for 30 minutes at 4°C. Cells were then washed and resuspended in staining buffer before flow cytometry analysis. The monoclonal antibodies used were anti-human CD3-BV605, CD4-BV711, CD8-APC-H7, CD45RA-AF700, CD26-PE-CF594 or CD26-BV421, Ki67-AF488, Granzyme B-AF700, T-bet-PE, TCF-7/TCF-1-AF647, CD95-BV421, Annexin V-PE, hCD45-BV605 (BD Biosciences, San Diego, CA, USA), CCR7-BV421, PD-1-BV785, CD226-FITC, TIM-3-PE-Cy7, Perforin-APC (Biologend, San Diego, CA, USA), TIGIT-APC, Eomes-PE-eF610, TOX-PE, AITR/GITR-PE (invitrogen, Carlsbad, CA, USA) antibodies and corresponding isotype controls. Data were acquired using an LSR Fortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star, Ashland, OR, USA).