Allogeneic T cell/B cell coculture assay

B cells were isolated from PBMCs using B Cell Isolation Kit II, human (Miltenyi Biotec) and T cells from an allogeneic donor were isolated using CD2 MicroBeads, human (Miltenyi Biotec), and cell isolation was performed using the Miltenyi Biotec MidiMACS separator following the manufacturer’s instructions. B cells were cultured with 5-OP-RU at indicated concentrations for 4 hours. The B cells were then washed twice before addition of BFA and allogeneic (MHC-mismatched, as MR1 is monomorphic) T cells at a 1:1 effector:target ratio and cultured for 18 hours. Cells were collected and stained with viability dye before fixation and permeabilization using the eBioscience Intracellular Fixation and Permeabilization Buffer Set (Invitrogen). Antibodies diluted in permeabilization buffer were then incubated for 45 min at room temperature.