MR1-Dependent Immune Activation Assay

PBMCs were seeded in U-bottom 96-well plates (10⁶ per 200 μl per well) and cultured for 16–18 h (37°C, 5% CO₂) in RPMI 1640 complete medium with 5% human AB serum. Cells were either left unstimulated or stimulated with whole Escherichia coli bacteria (ATCC strain 25922, Manassas, VA, USA) fixed with 1% paraformaldehyde for 5 min [39 (link)], or with a combination of IL-12 and IL-18 (both at 50 ng/ml, Peprotech, Cranbury, NJ, USA). Some samples were preincubated either with anti-MR1 blocking antibody (20 μg/ml, clone 26.5, BioLegend, San Diego, CA, USA) or with IgG2a isotype control (20 μg/ml, clone MPOC-173, BioLegend) prior to E.coli stimulation.