MHC-II complex immunoprecipitation

A20 cells were cultured in T₇₅ cell culture flasks (Corning, NY, USA). The expanded cells were pulsed with H. pylori lysates in combination with adjuvants MPLA, MDP, or CpG for 12 hr, collected, washed twice with sterile PBS, and divided into two fractions. One fraction, approximately 10⁷ cells, was used for whole-proteome analysis. The remaining 10⁸ cells were lysed in cold lysis buffer (1.0% w/v CHAPS, Protease Inhibitor tablet, and PMSF) for MHC-II complex immunoprecipitation. The cell lysates were centrifuged at 18,000×g for 20 min. The supernatant (containing the MHC–peptide complexes) was transferred into a new 1.5 mL microcentrifuge tube (Corning) containing a mixture of Sepharose CNBr-activated beads (Cytivia, Utah, USA) and 2 mg Anti-Mouse H2-IA^d/IE^d (M5/114) antibody (BioXcell, NH, USA). The immune complexes were captured on the beads by incubating on a rotor at 4℃ for 18 hr. Sequentially, the immune complexes were transferred to a polypropylene column (Bio-Rad Laboratories, Hercules, CA, USA) and washed with 10 mL buffer A (150 mM NaCl, 20 mM Tris, pH 8.0), 10 mL buffer B (400 mM NaCl, 20 mM Tris, pH 8.0), 10 mL buffer A, and 10 mL buffer C (Tris 20 mM, pH 8.0). The MHC-II–peptide complexes were eluted with 300 µL 10% glacial acetic acid (Macklin, Shanghai, China) three times. The eluate was stored at −80℃ until mass spectrometry analysis was performed.