Illustration of MAPPs assay procedure and data output example.

<p>A: Illustration of MAPPs assay procedure. Monocytes are isolated from human buffy coats and differentiated to immature DCs in the presence of cytokines. Immature DCs are loaded with the model mAb and induced to maturation with lipopolysaccharide. After 24 hours, mature DCs are frozen. After lysis of mature DCs, HLA-DR:peptide complexes are isolated via immunoprecipitation using anti-HLA-DR coated beads. After several wash steps, peptides are eluted from HLA-DR complexes by pH shift and analysed by nano LC-MS with subsequent sequence identification via SEQUEST database search. B: MAPPs assay data output example. HLA-DR associated peptides can originate from different sequence regions of a protein and can occur in multiple length variants. Peptides in a sample with unique sequence are termed “different peptides”, highlighted with blue box. Nested sets of peptide length variants occurring within a sequence region sharing the same HLA-DR binding core are termed “clusters”, highlighted with green boxes. The number of different peptides per amino acid position can be summarized in a heatmap in which the cell colors are reflecting the number of different peptides, highlighted with red box. Different donors can differ in the pattern of presented peptides depending on binding propensities of their 2 HLA-DR alleles.