Cd69 fn50

All antibodies used in this study were purchased from BD Biosciences unless stated otherwise. The clone numbers are indicated in parentheses. PBMC were harvested for flow cytometry after 24 and 48 h post-establishment of the co-culture. Cells were first washed with PBS and incubated at room temperature for 15 min with Fixable Live/Dead Blue Viable Dye (Thermo Scientific). After incubation, the cells were washed in PBS buffer containing 0.5% BSA and 2 mM EDTA. Subsequently, the cells were incubated with the following antibodies: Vδ1 TCR (TS-1, Thermo Scientific) FITC, Vδ2 TCR (B6, Biolegend) PerCP, CD3 (UCHT1) V500, CD8 (SK1) APC-Cy7, CD14 (MϕP9) PE-CF594, CD56 (B159) PE-Cy7, CD69 (FN50, Biolegend) BV421, CD83 (HB15e) APC, CD161 (HP-3g10, Biolegend) BV605, Vα7.2 (3C10, Biolegend) PE, and CD38 (HB7) BUV395 for 15 min at 4°C. Assays on NK cells were performed additionally with the following antibodies: CD3 (UCHT1) V500, CD16 (3G8, Biolegend) FITC, CD56 (B159) PE-Cy7, CD38 (HB7) BUV395, CD69 (FN50 Biolegend) PE. Flow cytometry analyses were performed using a BD LSRII flow cytometer (BD). Subsequent gating analyses were performed using FlowJo software version 9.9.6 or 10.5.2 (Tree Star Inc.).

Single cell suspensions were stained with fixable viability dye eFluor^® 780 (eBioscience) for 30 min at 4°C, and Fc receptors were blocked with anti-CD16/32 (BioLegend) blocking antibody prior to surface staining with antibodies. Antibodies for surface staining used are listed, mouse: CD45 (30-F11, BioLegend), TCR β (H57-597, BioLegend), CD69 (H1.2F3, eBioscience), CD1d tetramer (NIH); human: CD45 (HI30, eBioscience), TCRα/β (IP26, BioLegend), CD69 (FN50, BioLegend), Vα24 (6B11, BD Bioscience). For intracellular staining, single cell suspensions were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich), 1 μg/mL ionomycin (Sigma-Aldrich) and 1 μg/mL Golgi stop A (BD Biosciences) for 4 h. After surface staining, cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD biosciences) and further stained intracellularly with human IFNγ (B27, BioLegend) and human IL-4 (MP4-25D2, BioLegend). For Ki67 staining, cells were fixed and permeabilized with Cytofix/Cytoperm solution (eBioscience), and further stained with Ki67 (SolA15, eBioscience). Data were acquired using LSR II (BD Biosciences) and analyzed using the FlowJo software (BD Biosciences).

The following fluorochrome-conjugated antibodies were used: CD4 (RPA-T4, Biolegend), CD8 (SK1, Biolegend), CD19 (HIB19, Biolegend), CD69 (FN50, Biolegend), CD25 (BC96, Biolegend), CXCR5 (J252D4, Biolegend), PD-1 (EH12.2H7, Biolegend), IFNγ (4S.B3, Biolegend), IL-4 (MP4-25D2, Biolegend), IL-17A (BL168, Biolegend), FOXP3 (PCH101, Invitrogen), and EZH2 (11/EZH2, BD Biosciences). For surface staining, cells were stained with antibodies at 4 °C in the dark for 30 min. For intracellular cytokine staining, cells were stimulated with a leukocyte activation cocktail containing GolgiPlug (BD Bioscience) for 4 h, then fixed and permeabilized using Cytofix/Cytoperm (BD Bioscience), followed by incubation with antibodies. For FOXP3 and EZH2 staining, cells were fixed and permeabilized using the FOXP3 staining kit (Thermo Fisher) according to the instructions of the manufacturer. Cells were analyzed with a BD Aria II Flow Cytometer (BD Bioscience) and data were processed using FlowJo X (Tree Star).