Il 12

Healthy adult blood was obtained from the blood bank of the University Leipzig, approved by the local ethics committee (Ref. #079-15-09032015). Peripheral blood mononuclear cells were isolated from male human donors at age 20-50 by Ficoll-Paque™ density-gradient (GE Healthcare, UK) centrifugation. CD161+ TCR Vα7.2+ MAIT cells and CD14+ monocytes were obtained by positive magnetic separation using respective microbeads (Miltenyi Biotec, Germany). For RNA-Seq experiments, monocytes were loaded with fixed E. coli at a MOI of 10 for 3h, followed by intense washing in PBS to remove E. coli. MAIT cells and E. coli-loaded monocytes were incubated at a ratio of 1:1 for 16h. In some experiments, MAIT cells were stimulated with 50ng/ml IL-12 and 50ng/ml IL-18 (both from MBL International, MA, USA), 10µg/ml plate-bound anti-CD3 and 1µg/ml soluble anti-CD28 (both from Biolegend, CA, USA) or a combination of both for 16h. For TWIST1 inhibition experiments, the small molecule inhibitor harmine (Sigma-Aldrich, Germany) was used at a concentration of 20 or 40µM, DMSO served as solvent control.

PBMCs were thawed and one million cells were plated per stimulation condition. Stimulation conditions included media alone, 25 ng/ml IL-2 (Roche), 50 IU/ml of IL-12 (MBL international, Woburn, Massachusetts), 50 IU/ml of IL-18 (MBL international) and 25 ng/ml IL-15 (R&D Systems, Minneapolis, Minnesota). Cells were stimulated for six days in an incubator at 37°C with 5% CO₂. After stimulation, the cells were washed and stained for viability with AARD cultured with surface phenotype panel against CD3, CD4, CD8, TIGIT or isotype control antibody and acquired on the flow cytometer as above.