Anti hla dr bv650

Surface HLA-B thermal stability was assessed based on previously established methods. In brief, moDCs were incubated for either 1 or 2 hr at RT, 37 or 42°C. Following incubation, moDCs were washed and stained 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 Red live/dead fixable dye and analysis on a BD LSR Fortessa flow cytometer. Expression values for each allotype were normalized to 37°C, and each allotype was compared using unpaired t tests.

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.

Cells were stained with LIVE/DEAD reagent (Invitrogen, MA, USA), anti-CD3 APC-eFluor780 (ThermoFisher, MA, USA), anti-CD4-BUV395, anti-CD8-BV786, anti-CD27-BUV737, anti-CD45RA-APC-H7 and anti-HLADR-BV650 (BioLegend, CA, USA), anti-CD161-PE, and anti-ϒδ TCR-PE-CY7 (BD Life Sciences, NJ, USA) and anti-Vδ1-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany) (see Supplemental Table 1). Data were acquired on an LSR-Fortessa II, and BD FACSDiva Software (BD Life Sciences, NJ, USA).