Isolation of the MHCII peptidome.

We followed our original procedure⁸, with modifications⁵¹. Cells isolated from islets, pLNs or spleens were suspended in lysis buffer (40mM MEGA 8, 40mM MEGA 9, 1mM PMSF, 0.2mM Iodoacetamide, 20ug/ml Leupeptin, Roche Complete Mini Protease cocktail in PBS) and rocked for 1hr at 4°C. The cell lysate was spun in a centrifuge at 20,000 xg for 30min at 4°C. In order to eliminate non-specific binding of peptides, the supernatant was first incubated with polyclonal mouse IgG (BioXcell; 1.5 mg antibody/sample) bound to Sepharose 4B at 4°C for 30min. The unbound material containing MHCII-peptide complexes was collected and was then added to a tube containing PBS-washed sepharose conjugated to the anti-I-A^g7 antibody (AG2.42.7, 1.5 mg/sample) and incubated at 4°C overnight. The I-A^g7-sepharose was applied to a column and washed four times as follows: 10ml 150mM NaCl, 20mM Tris pH7.4; 10ml 400mM NaCl, 20mM Tris pH7.4; 10ml 150mM NaCl, 20mM Tris pH7.4; 10ml 20mM Tris pH8.0. Peptides were eluted with 10% acetic acid and dried with a SpeedVac. Eluted peptides were passed over detergent removal spin columns (Pierce) to remove traces of remaining detergent and were cleaned by using the C18 Spin Columns from ThermoScientific (Pierce).