Anti cd3

Cell staining and the flow cytometry‐based phenotypic analyses of PBMCs and cells were performed according to standard flow cytometry methods (Martin et al, 2014; Izawa et al, 2017). The following monoclonal antibodies were conjugated to phycoerythrin‐cyanin7 (PE‐Cy7) Brilliant Violet 785 (BV785), Brilliant Violet 510 (BV510), Brilliant Violet (BV650), phycoerythrin (PE), phycoerythrin‐cyanin5 (PE‐Cy5), Brilliant Violet 451 (BV421), Peridinin‐chlorophyll‐cyanin5.5 (PerCP‐Cy5.5): anti‐CD25 (BC96; dilution 1:40), anti‐CD3 (OKT3; dilution 1:40), anti‐CD4 (OKT4; dilution 1:40), anti CD8 (RPA‐T8; dilution 1:40), anti‐CD27 (O323; dilution 1:40), anti‐CD45RA (HI100; dilution 1:40), anti‐CD161 (HP‐3G10; dilution 1:20), anti‐TCR Vα7.2 (3C10; dilution 1:20) all purchased from Sony Biotechnology Inc.; anti‐TCR Vα24 (C15; dilution 1:20), anti‐TCR Vβ11 (C21; dilution 1:20), anti‐TCR γδ (IMMU510; dilution 1:40) from Beckman Coulter; and anti‐CD19 (HIB19; dilution 1:40) and anti‐CD57 (NK‐1; dilution 1:200) from BD Biosciences. For intracellular staining of CTPS1, cells were fixed and permeabilized using the Intraprep kit (Beckman Coulter) according to the manufacturer's instructions. Cells were stained with an anti‐CTPS1 antibody (EPR8086B, Abcam; dilution 1:200) or an isotype‐matched antibody and then labeled with a FITC‐goat anti‐rabbit secondary antibody.

Cells were stained with fixable viability dye (eBioscience-Thermo Fisher Scientific, Bleiswijk, The Netherlands) and then with the following antibodies for surface staining: anti-TCRVα7.2, anti-CD161, anti-CD3, anti-ST2, anti-CD4 and anti-CD8 (SONY biotechnology, Weybridge, United Kingdom) (eBiosciences-Thermo Fisher Scientific) (Biolegend, Amsterdam, The Netherlands). MAIT cells were identified as TCRVα7.2⁺CD161⁺ or MR1 5-OP-RU tetramer⁺ cells. MR1 6-FP tetramer⁺ was used as negative control (NIH Tetramer Facility, Atlanta, GA, USA) (Figure S1). For intracellular cytokine staining, cells were fixed in 4% paraformaldehyde, washed, and permeabilized with 0.5% saponin (Sigma-Aldrich), and then incubated with anti-IFNγ, anti-TNFα, anti-Granzyme B or isotype controls (eBiosciences-Thermo Fisher Scientific, SONY biotechnology or BioLegend). In some experiments, MAIT cells were stained intracellularly with anti-Tbet (BD Biosciences, Grenoble, France) and anti-PLZF (R&D Systems) antibodies using a transcription factor buffer set (BD Biosciences). Events were acquired on a FACS LSR Fortessa (BD Biosciences) and analyzed using FlowJo software v10.7.1 (Becton, Dickinson and Company, Ashland, OR, USA).