High-throughput and Sensitive Immunopeptidomics Platform Reveals Profound Interferon-Mediated Remodeling of the Human Leukocyte Antigen (HLA) Ligandome* □ S Chloe Chong‡§ §§, Fabio Marino‡§ §§, HuiSong Pak‡§, Julien Racle‡§**, Roy T. Daniel¶, Markus Mu ¨ ller, David Gfeller‡§**, George Coukos‡§, and Michal Bassani-Sternberg‡§‡‡ Comprehensive knowledge of the human leukocyte anti- gen (HLA) class-I and class-II peptides presented to T- cells is crucial for designing innovative therapeutics against cancer and other diseases. However methodolo- gies for their purification for mass-spectrometry analysis have been a major limitation. We designed a novel high- throughput, reproducible and sensitive method for se- quential immuno-affinity purification of HLA-I and -II pep- tides from up to 96 samples in a plate format, suitable for both cell lines and tissues. Our methodology drastically reduces sample-handling and can be completed within five hours. We challenged our methodology by extracting HLA peptides from multiple replicates of tissues (n  7) and cell lines (n  21, 10 8 cells per replicate), which re- sulted in unprecedented depth, sensitivity and high repro- ducibility (Pearson correlations up to 0.98 and 0.97 for HLA-I and HLA-II). Because of the method’s achieved sensitivity, even single measurements of peptides purified from 10 7 B-cells resulted in the identification of more than 1700 HLA-I and 2200 HLA-II peptides. We demonstrate the feasibility of performing drug-screening by using ovarian cancer cells treated with interferon gamma (IFN). Our analysis revealed an augmented presentation of chymot- ryptic-like and longer ligands associated with IFN in- duced changes of the antigen processing and presenta- tion machinery. This straightforward method is applicable for basic and clinical applications. Molecular & Cellular Proteomics 17: 10.1074/mcp.TIR117.000383, 533–548, 2018. The rich repertoire of peptides presented by HLA class I (HLA-I) 1 and HLA class II (HLA-II) complexes, referred to as the immunopeptidome, reflects the health state of a cell. HLA-bound peptides (HLAp) derived from cancer-specific and mutated proteins, pathogens and self-peptides in case of autoimmunity, serve as leading targets for T-cell recognition. In recent years the remarkable clinical efficacy of immune checkpoint blockade therapies has motivated researchers to discover immunogenic T-cell epitopes that mediate disease control (1) or improved survival for development of personal- ized vaccines (2–5). Presently, mass spectrometry (MS) is the only unbiased methodology to comprehensively interrogate the in vivo nat- urally presented HLAp repertoire (6), in human cell lines (7–9), tumor tissues (10 –12) and body fluids such as plasma (13). Importantly, pioneering proof-of-concept studies have shown that this technology has matured to the extent that identifica- From the ‡Ludwig Institute for Cancer Research, University of Lausanne, 1066 Epalinges, Switzerland; §Department of Oncology, University Hospital of Lausanne, 1011 Lausanne, Switzerland; ¶Ser- vice of Neurosurgery, University Hospital of Lausanne, 1011 Laus- anne, Switzerland; Vital IT, Swiss Institute of Bioinformatics, 1015 Lausanne, Switzerland; **Swiss Institute of Bioinformatics, 1015 Lau- sanne, Switzerland Author’s Choice—Final version free via Creative Commons CC-BY license. Received October 5, 2017, and in revised form, December 12, 2017 Published, MCP Papers in Press, December 14, 2017, DOI 10.1074/mcp.TIR117.000383 Author contributions: C.C., F.M., H.-S.P., and M.B.-S. designed research; C.C., F.M., H.-S.P., G.C., and M.B.-S. performed research; C.C., F.M., H.-S.P., J.R., M.M., D.G., and M.B.-S. analyzed data; C.C., F.M., and M.B.-S. wrote the paper; R.T.D. contributed new reagents/analytic tools. 1 The abbreviations used are: HLA-I, human leukocyte antigen class I; HLAp, HLA-bound peptides; a.a., amino acid; APPM, antigen processing and presentation machinery; 2m, beta-2-microglobulin; CV, coefficient of variation; FA, formic acid; FDR, false discovery rate; HLA, human leukocyte antigen; HLA-II, human leukocyte antigen class II; HCD, higher-energy collision dissociation; HCl, hydrochloric acid; IEDB, immune epitope database; IFN, Interferon gamma; IP, immunoaffinity purification; LFQ, label-free quantification; NaCl, so- dium chloride; NH 4 AcO, ammonium acetate; Pro-A, protein-A sep- harose 4B; SCX, strong-cation-exchange; MS, mass spectrometry; FBS, fetal bovine serum; TIL, tumor infiltrating lymphocyte; IAA, io- doacetamide; ACN, acetonitrile; TFA, trifluoroacetic acid; AMBIC, ammonium bicarbonate. Technological Innovation and Resources Author’s Choice © 2018 by The American Society for Biochemistry and Molecular Biology, Inc. This paper is available on line at http://www.mcponline.org los Molecular & Cellular Proteomics 17.3 533