Live dead efluor780

Naive CD8⁺ T cells and CD4⁺ T cells were isolated from healthy donors’ PBMC using EasySep human Naïve CD8 or CD4 isolation kit according to manufacturer’s instructions (StemCell Technologies). APC (1×10⁴ cells per well) were cultured with naive CD8⁺ T cells (5×10⁴ cells per well) with or without naive CD4⁺ T cells from the same T cell donor (5×10⁴ cells per well) for 7 days in Yssel medium supplemented with 10% FCS. To analyze T cell proliferation, CD8⁺ T cells were stained with Cell Trace Violet (CTV, Thermo Fisher) prior to culture. To assess the expression of intracellular effector molecules, T cells were stimulated with PMA (50 ng mL⁻¹) and ionomycin (1 µg mL⁻¹) for 6 h in the presence of BFA (4 µg mL⁻¹) for 6 h (all from Sigma). After washing, cells were stained for surface CD4 for 30 min at 4 °C, washed and stained with Live/dead eFluor780 (Thermo Fisher Scientific) for 20 min at 4 °C. Then the cells were fixed and permeabilized (Intracellular Fixation & Permeabilization Buffer Set, eBioscience) and stained for intracellular proteins (Granzyme A, Perforin, and IFN-γ) at room temperature for 45 min in a buffer containing 2% of normal mouse serum. The samples were acquired on a FACSVerse instrument (BD Biosciences).

For intracellular cytokine staining, CD4⁺ T cells or CD4⁺ T cell/monocyte cocultures were stimulated for 3 h in the presence of PMA (50 ng/ml; Sigma-Aldrich), ionomycin (750 ng/ml; Sigma-Aldrich), and GolgiStop (as per the manufacturer’s instructions; BD Biosciences). Cells were washed and stained with CD3-PE Cy7 (UCHT1; BioLegend) and LIVE/DEAD efluor 780 (Thermo Fisher Scientific). Cells were then fixed in 2% PFA and permeabilized with 0.5% saponin (Thermo Fisher Scientific). Cells were subsequently stained for the following cytokines: IL-10–Alexa Fluor 488 (JES3-9D7; BioLegend), IL-17A–PE (BL168; BioLegend), IFN-γ–Pacific blue (4S.B3; BioLegend), and TNF–allophycocyanin (MAb11; BioLegend).
For intranuclear staining of IKZF3, cells were fixed and permeabilized with FOXP3 staining buffer (BioLegend) for 15 min at room temperature before being stained for CD3-PE Cy7, IL-10–Alexa Fluor 488, IL-17A–PE, IFN-γ–Pacific blue, TNF-BV605 (MAb11; BioLegend), and either IKZF3-Alexa Fluor 647 (EPR9342[B]; Abcam) or isotype control (EPR25A; Abcam) for 30 min. Standard gating strategy for intracellular cytokine staining is shown in Supplemental Fig. 1A–C.

T cells were re-stimulated on day 6 with phorbol 12-myristate 13-acetate (PMA, 50 ng/mL, Sigma) and ionomycin (Iono; 1 μg/mL; Merck Calbiochem, Burlington, MA, USA) for 6 h in the presence of brefeldin A (BFA; 1×; eBioscience ThermoFisher, Carlsbad, CA, USA) at 37 °C, 5% CO₂. Non-specific binding was blocked using TruStain (Biolegend, San Diego, CA, USA) for 10 min. Cell viability was assessed using live/dead eFluor780 (ThermoFisher, Carlsbad, CA, USA) for 20 min at 4 °C (Table 1). Then the cells were fixed and permeabilized (Intracellular Fixation & Permeabilization Buffer Set; ThermoFisher, Carlsbad, CA, USA) and stained for rat anti-CD3, a cytoplasmic epitope of CD3 (Bio-Rad, Hercules, CA, USA) and selected intracellular proteins (anti-mouse TNF-α; anti-rat IFN-γ) (Table 1), for 45min at RT in permeabilization wash buffer (eBioscience ThermoFisher, Carlsbad, CA, USA). The samples were analyzed using the BD FACS Symphony instrument (BD Biosciences, San Jose, CA, USA) and FlowJo v10.