Anti cd3 percp cy5.5 ucht1

MAIT cell cloning was performed as described by Cansler et al. (22 ). Briefly, MR1 tetramer-positive T cells were sorted from cryopreserved bronchoalveolar lavage cells and rested overnight. Limiting dilution assay was performed to seed single cells in 96-well plates in RPMI media containing 10% human serum (HuS), 2% L-glutamine, 0.1% gentamycin and irradiated feeder cells (lymphoblastoid cell lines and peripheral blood mononuclear cells). Media was supplemented with recombinant human (rh)IL-2 (5 ng/mL), rhIL-7 (0.5 ng/mL), rhIL-12 (0.5 ng/mL), rhIL-15 (0.5 ng/mL) (BioLegend) and anti-CD3 (0.03 µg/mL) (eBiosciences). Plates were assessed weekly for growth with clones taking 2 – 3 weeks to grow. Clones were taken forward for subsequent analyses only from plates, which had growth in approximately less than 30% of the wells. Resulting clones were subjected to surface MR1 5-OP-RU tetramer and monoclonal antibody staining with 1:200 of Live/dead viability dye (Aqua, Life Technologies), 1:25 of anti-CD3 PerCP/Cy5.5 (UCHT1, BioLegend), 1:25 of anti-CD4 BV785 (OKT4, BioLegend), 1:50 of anti-CD8 (FITC, RPA-T8) and 1:25 of anti-CD26 PE-Cy7 (BA5b, BioLegend), as well as IFN-γ ELISPOT assay to confirm MAIT cell identify and clonal purity.

For isolation of PBMC, heparinized blood was taken from healthy donors. This procedure and the respective consent documents were approved by the “Ethik Kommission” of the University Hospital Goethe-University Frankfurt. PBMC were isolated from peripheral blood using Histopaque-1077 (Sigma-Aldrich) according to the manufacturer’s instructions. Untouched CD3⁺ T-cell were isolated from PBMC using the Pan-T-cell isolation kit according to the manufacturer’s instructions (Miltenyi, Bergisch Gladbach, Germany). Mean purity was 96.0 ± 0.7% (n = 37) determined by FACS analysis using anti-CD3-PerCP/Cy5.5-#UCHT1 (BioLegend). Monocyte-depleted PBMC were generated using anti-CD14 beads (Miltenyi) with a mean depletion efficiency of 98.1 ± 0.7% (n = 7) as assessed by FACS analysis using an anti-CD14eFluor450-#61D3 antibody (eBioscience, Frankfurt, Germany). Cells were resuspended in RPMI 1640 supplemented with 10 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, and 1% human serum (Life Technologies, Darmstadt, Germany) and seeded at 3 × 10⁶ cells/ml in round-bottom polypropylene tubes (Greiner, Frickenhausen, Germany).