Pe cf594 cd25 m a251

DCs were plated at 50,000 cells per well in 96-well flat-bottom plates in CM and incubated with MAIT cells (20,000 cells per well, in triplicate) in the presence or absence of different concentrations of 5-A-RU/MG, bacterial supernatant, LPS, or R848. Depending on availability, autologous and/or allogeneic MAIT cells were used in different experiments because MR1 lacks polymorphism. For blocking experiments, DCs were incubated for 1 h with the ligands and then for 2 h with 30 μg/ml isotype controls, anti-MR1 (clone 26.5), anti-CD40L (clone 24.31), or anti–IL-12 (clone C8.6) Abs before the addition of MAIT cells. MAIT cell and DC activation was assessed by flow cytometry after 36 h with the following Abs: FITC CD83 (HB15e), allophycocyanin CD86 (FUN1), PE CD80 (L307.4; all from BD), PECy7 PDL1 (29E.2A3; BioLegend), and PE-CF594 CD25 (M-A251; BD). IFN-γ, IL-12p40, and IL-12p70 were also measured by ELISA (Abs from BD) on supernatants harvested after 36 h. We did not detect any IL-17 or IL-23 in our supernatants (ELISA Ab pairs from eBioscience, sensitivity 0.04 and 0.39 ng/ml, respectively).