Identification of synergistic HLA class II–restricted myeloma-associated antigens. (A) Overlap analysis of HLA class II ligand source proteins of primary MM samples (n = 7), MCLs (n = 5), and HV samples (total, n = 23: PBMC [n = 13], BMNC [n = 5], granulocytes [n = 5]). (B) Statistical analysis of false-positive myeloma-antigen identifications at different threshold values, as described in Figure 2. Randomized virtual ligandome sizes were set to 226 proteins and TAAs were defined based on simulated cohorts of 12 MM vs 23 HV samples. (C) Comparative profiling of HLA class II ligand source proteins based on the frequency of HLA-restricted presentation in MM and HV ligandomes. Frequencies of MMs/HVs positive for HLA-restricted presentation of the respective source protein (x-axis) are indicated on the y-axis. (D) Overlap analysis of HLA class I TAAs (n = 58) and HLA class II MM-exclusive antigens (n = 1135). (E) HLA class I TAAs, which also yield potentially synergistic HLA class II ligands. (F) Overlap analysis comprising the entire HLA class I and II ligand source proteomes of MM samples. sum, summary; rep., representation.