Fabio Gallazzi

Identification of destabilizing residues in HLA class II-selected bacteriophage display libraries edited by HLA-DM

HLA‐DM (DM) functions as a peptide editor by catalyzing the release of class II‐associated invariant chain peptides (CLIP) and other unstable peptides, thus supporting the formation of stable class II‐peptide complexes for presentation. To investigate the general features that determine the DM susceptibility of HLA‐DR1/peptide complexes, we generated a large DM‐ensitive peptide repertoire from an M13 bacteriophage display library using a novel double selection protocol: we selected bacteriophage capable of binding to DR1 molecules and, subsequently, we enriched DR1‐bound bacteriophage susceptible to elution by purified DM molecules. Sequence and mutational analyses of the DR1/DM double‐selected peptides revealed that the amino acids Gly and Pro play a destabilizing role in the dissociation kinetics of DR1 ligands. This observation was confirmed also in natural peptide sequences such as CLIP 89 – 101, HA 307  –  319 and bovine collagen II (CII) 261–273. Our results demonstrate that DM susceptibility does not only depend on the number and nature of anchor residues, or the peptide length. Instead, less obvious sequence characteristics play a major role in the DM editing process and ultimately in the composition of peptide repertoires presented to T cells.