Impact of dipeptidyl peptidase 9 on the MHC class I immunopeptidome

Introduction: Dipeptidyl peptidase 9 (DPP9) is an intracellular amino-dipeptidase with a wide-ranging physiological role including DNA repair, mitochondria homeostasis, and immune system regulation. The dysregulation of DPP9 has been implicated in immune-associated defects and cancer, including acute myeloid leukemia, non-small-cell lung cancer, colorectal cancer, clear cell renal cell carcinoma, and breast cancer. Notably, DPP9's first identified natural substrate was an antigen peptide, linking it to the presentation of antigens on MHC class I molecules for cytotoxic T lymphocytes.

This study aims to deepen our understanding of DPP9's impact on peptide presentation by MHC I molecules through a comprehensive immunopeptidome analysis using mass spectrometry. By conducting high-coverage immunopeptidomic analysis, we can examine in which way modulation of DPP9 activity affects the diversity of peptides bound to MHC class I molecules.

Methods: We established and optimized a workflow for enriching MHC I bound antigens, utilizing automated affinity purification with crosslinked monoclonal antibodies on AssayMAP Bravo platform. This optimization included successful downscaling of antibody use and protein input while maintaining detection efficiency. The workflow, adaptable to a variety of sample types, was applied to different HEK cell line conditions, including DPP9 knock-out, overexpression, and wild-type. This comparison gives the possibility to investigate DPP9's role in MHC I-bound peptide presentation.

Results and Outlook: The MHC I enrichment method successfully identified over 1000 peptides in each cell line sample, without additional enhancement of the immune response. This high coverage facilitates robust analysis of the immunopeptidome, revealing significant differences across the three cell line conditions. Preliminary results indicate substantial variations in the peptide repertoire linked to DPP9 modulation. Further studies integrating additional proteomic data will provide deeper insights into DPP9's role in antigen processing and presentation dynamics on MHC I molecules.