Anti cd3 hit3a

We performed the cytokine induction protocol as previously described (24 (link), 25 (link)). In brief, adult peripheral blood mononuclear cells (PBMCs) and cord blood mononuclear cells (CBMCs) (2 × 10⁶ cells/mL) were stimulated with or without purified phytohemagglutinin (PHA) (10 µg/mL), or 1 µg/mL anti-CD3 (HIT3a, Cat. #300314, BioLegend, San Diego, CA, USA) in combination with 1 µg/mL anti-CD28 (CD28.2, Cat. #302914, BioLegend), in 1-cm tissue culture plates with l-arginine-free medium (SILAC R1780 SIGMA, RPMI-1640 Medium) supplemented with 10% heat-inactivated fetal bovine serum, 1 mM glutamine, 1 mM sodium pyruvate, 50 mg/L l-leucine, 40 mg/L l-lysine, and 1× non-essential amino acids (Gibco cat. # 11140-035), 100 IE/mL penicillin, and indicated l-arginine (Sigma-Aldrich, St. Louis, MO, USA). After 72 h, cell-free culture supernatants were collected and assayed for cytokine production by enzyme-linked immunosorbent assay: TGF-β1 (R&D systems Inc., MN, USA) and IL-10 (R&D Systems).

MAIT cells were isolated from PBMC prepared from buffy coats of healthy donors, as previously described^{16 (link)}. Briefly, monocytes and CD4 T cells were sequentially depleted from PBMC by adhesion on polystyrene culture flasks for 3 h at 37 °C, and anti-CD4 microbeads (Miltenyi Biotech, France), respectively. Vα7.2⁺ cells were isolated from the non-monocyte and non-CD4 PBMC fraction, using an anti-Vα7.2⁺ antibody conjugated to FITC, followed by a positive selection with anti-FITC microbeads (Miltenyi Biotech, France) and used as enriched MAIT cells for co-culture experiments. The purity of isolated Vα7.2⁺ cells was >80% and >95% of the isolated Vα7.2⁺ cells were co-expressing CD161. More than 95% of the isolated CD161^hi Vα7.2⁺ cells were positive to APC-conjugated 5-OP-RU-loaded MR1 tetramers^{45 (link)} (NIH Tetramer Core Facility, Emory University Vaccine Center, Atlanta, USA) (Supplementary Fig. 3). MAIT cells added in co-culture experiments were either left nonactivated or were activated using anti-CD3 (HIT3a) at 2.5 μg/ml, soluble anti-CD28 (CD28.2) (1 μg/ml), and IL-7 at 10 ng/ml (BioLegend)^{21 (link)}.