T bet ebio4b10

The following antibodies and dyes were used for FACS analysis of human cells: CD4 (RPA-T4), Gata-3 (16E10A23), T-bet (4B10) (all BioLegend), and dead cell marker Viability Dye eFluor™ 780 (eBioscience). The following antibodies and dyes were used for FACS analysis of mouse cells: CD4 (RM4-5), Thy1.1 (Ox-7), Thy1.2 (30-H12), Gata-3 (16E10A23), IL-4 (11B11), IFNγ (XGM1.2), T-bet (eBio4B10) (all BioLegend), and dead cell marker Viability Dye eFluor™ 780 (eBioscience). For intracellular cytokine analysis, cells were restimulated with 5 ng/ml PMA and 500 ng/ml ionomycin for 4 h. 10 µg/ml BrefA were added after 2 h. Stainings were performed with up to 10⁶ cells from PBMC, lymph node, or erythrocyte depleted spleen cells, in 50 µl of FACS buffer (PBS/0.1% bovine serum albumin/0.02% NaN₃). After surface staining (30 min, 4°C), cells were fixed for 30 min at 4°C (fixation buffer, eBioscience), permeabilized (permeabilization buffer, eBioscience), and intracellularly stained for Gata-3 and T-bet or IL-4 and IFNγ expression for 45 min at room temperature. The cells were analyzed on a BD™ LSR II flow cytometer with the use of FACS Diva software (all Becton Dickinson). For further analyses of the data, FlowJo (TreeStar Inc.) software was used. Median fluorescence intensity (MFI) ratios of T-bet and Gata-3 were calculated by dividing the median fluorescence intensities of the two markers.

For T cell stimulations, cells were incubated with 5 μg/ml ESAT-6_1–20 peptide for 5 h at 37°C in the presence of brefeldin A and 1 mM aminoguanidine. Cells were stained with various combinations of the following fluorochrome-labeled antibodies: anti-CD4 (RM4-4), CD44 (IM7), CD45 (IM7), CD45.1 (A20), CD45.2 (104), CD69 (H1.2F3), CXCR3 (CXCR3-173), CX3CR1 (polyclonal), Foxp3 (FJK-16s), ICOS (15F9), IFN-γ (XMG1.2), KLRG1 (2F1/KLRG1), PD-1 (29F.1A12), T-bet (eBio4B10), Thy1.1 (OX-7), and Fixable Viability Dye eFluor 780 purchased from BioLegend, eBioscience (San Diego, CA), BD Biosciences (San Jose, CA), and R&D Systems (Minneapolis, MN). I-A^b ESAT-6_4–17 and I-A^b EsxG_46-61 MHC tetramers were produced by the NIAID Tetramer Core Facility (Emory University, Atlanta, GA). For staining with MHC class II tetramers, cells were incubated with tetramer at 1:50 dilution in complete medium containing 10% FCS, 1 mM aminoguanidine and monensin (eBioscience) at a 1:1000 dilution for 1 h at 37°C prior to staining with surface antibodies. All samples were acquired on an LSRFortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star, Ashland, OR).