MoDC Surface Stability and Turnover

Surface stability and half-life assessment were performed as described previously (Zarling et al., 2003 (link); Yarzabek et al., 2018 (link)). Briefly, monocytes were isolated and differentiated to moDCs for 7 d as described above. B*08:01⁺ or B*35:01⁺ donor moDCs were plated into a 96-well plate in duplicate for each condition. BFA treatment was added at negative time points: for a 4-hr time course, BFA was first added to cells for the 4-hr treatment time point, then 3 hr, etc. BFA was added at a concentration of 0.5 µg/mL to each well in media, and cells were incubated before centrifugation, washing with PBS, and staining with a monoclonal antibody cocktail of anti-CD11c-PE/Cy7, anti-HLA-DR-BV650, and anti-CD209-APC (all used at 1:200 and from Biolegend), as well as anti-Bw6-FITC (Biolegend, 1:40), for 30 min on ice. After staining, cells were washed twice with PBS, followed by staining with 7-AAD and analysis on a BD LSR Fortessa flow cytometer. Half-life values were extracted using a one-phase decay curve with a constrained plateau of zero.