Zombie nir live dead exclusion dye

This experiment was performed as previously described (Yang et al., 2020 (link)). Briefly, CD4⁺ T cells were isolated from PBMCs from five healthy donors (controls), the T-bet WT/M heterozygous father (TBX21 WT/M), and the T-bet M/M P (TBX21 M/M), with anti-CD4 Ab-coated beads (Miltenyi Biotec). This population of cells was then expanded with anti-CD3/CD28 Ab-coated Dynabeads (Thermo Fisher Scientific) at a 1:1 (cell:bead) ratio under Th0 cell–polarizing conditions, in the presence of 100 IU/ml proleukin IL-2 (PROMETHEUS) and anti-human IL-10 neutralizing Ab (Thermo Fisher Scientific). Cells were restimulated with anti-CD3/CD28 Ab-coated Dynabeads every 7–8 d. P’s CD4⁺ T cells were transduced with retroviruses obtained from pLZRS-ires-ΔCD271 plasmids with or without WT TBX21 or EV cDNA, as previously described (Martínez-Barricarte et al., 2016 (link); Yang et al., 2020 (link)). Cells were stained with Zombie NIR Live-Dead exclusion dye (BioLegend) and anti-CD271-FITC Ab (BioLegend) and were then fixed and permeabilized. They were stained with anti-TNF-α-BV510, anti-IL-13-APC, anti-IL-4-PE, anti-IL-17A-BV785, anti-IL-10-PE-Dazzle594, anti-IL-5-BV421, and anti-IFN-γ-PerCp-Cy5.5 Abs and subjected to flow cytometry in an Aurora cytometer (Cytek).

HVS-T cells from six healthy donors and P were cultured in the presence of IL-2 at 20 IU/ml (Roche). HVS-T cells were harvested, counted, and topped up to 10⁶ cells/ml without recombinant IL-2. 200 µl cells per well were plated to 96-well round bottom tissue-culture plates. After 24 h culture, cells were stimulated with 25 ng/ml PMA and 0.5 µM Ionomycin for 3 h. Protein transport inhibitor (eBioscience) was added to each well. After another 3 h, cells were harvested for surface staining with FcBlock, anti-CD271-FITC Abs, and Zombie-NIR live-dead exclusion dye (BioLegend). Cells were then fixed, permeabilized with FOXP3/perm kit (Thermo Fisher Scientific), and subjected to overnight ICS with anti-GATA3-BV421 (BD Biosciences), anti-IL-13-BV711 (BD Biosciences), anti-IFN-γ-eFluor450 (eBioscience), anti-IL-9-PERCP/Cy5.5 (BioLegend), anti-IL-5-PE (BioLegend), anti-T-bet-PE/Cy7 (BioLegend), anti-IL-10-PE/Dazzle594 (BioLegend), anti-IL-22-APC/Fire750 (BioLegend), anti-TNF-BV510 (BioLegend), anti-RORγ/RORγT-BV650 (BD Bioscience), anti-IL-17A-BV785 (BioLegend), and anti-IL-4-Alexa 647 (BioLegend), followed by flow cytometry analysis with an Aurora cytometer (Cytek).