Anti hla dr clone l243

Antigen-pulsed mo-DCs were collected and washed with cold buffer A (PBS, 2 mM EDTA; pH 7.4–7.6). The cells were then lysed in cold buffer B (1% CHAPS, 1 mM EDTA, protease inhibitor cocktail) and incubated at 4° C on a rocking table for 15 minutes. This lysis step was repeated, insoluble material removed by centrifugation, and the supernatant was incubated with Protein G Dynabeads (Thermo Fisher) coated with 20 ug of anti-HLA-DR (clone L243, BioLegend) or isotype control (clone MOPC-173, BioLegend) antibodies at 4° C for one hour with inversion. The beads were then washed 4-times with lysis buffer B, and 3-times with buffer C (20 mM Tris, 150 mM NaCl, pH 7.4). The HLA-DR-bound peptides were eluted by incubating the beads with 0.1% TFA for 1 minute at room temperature and harvesting the eluate. This step was repeated once and the pooled eluate was centrifuged through a 10kD centrifugal filter (Microcon, Millipore). The filter was washed with 0.1 % TFA and pooled flow-through containing the eluted peptides was lyophilized by vacuum centrifugation. The peptides were then analyzed by mass spectrometry, as described in detail in the online supplementary material and methods.