title: Uncovering the peptide-binding specificities of HLA-C: a general strategy to determine the specificity of any MHC class I molecule.
abstract: MHC class I molecules (HLA-I in humans) present peptides derived from endogenous proteins to CTLs. Whereas the peptide-binding specificities of HLA-A and -B molecules have been studied extensively, little is known about HLA-C specificities. Combining a positional scanning combinatorial peptide library approach with a peptide-HLA-I dissociation assay, in this study we present a general strategy to determine the peptide-binding specificity of any MHC class I molecule. We applied this novel strategy to 17 of the most common HLA-C molecules, and for 16 of these we successfully generated matrices representing their peptide-binding motifs. The motifs prominently shared a conserved C-terminal primary anchor with hydrophobic amino acid residues, as well as one or more diverse primary and auxiliary anchors at P1, P2, P3, and/or P7. Matrices were used to generate a large panel of HLA-C-specific peptide-binding data and update our pan-specific NetMHCpan predictor, whose predictive performance was considerably improved with respect to peptide binding to HLA-C. The updated predictor was used to assess the specificities of HLA-C molecules, which were found to cover a more limited sequence space than HLA-A and -B molecules. Assessing the functional significance of these new tools, HLA-C*07:01 transgenic mice were immunized with stable HLA-C*07:01 binders; six of six tested stable peptide binders were immunogenic. Finally, we generated HLA-C tetramers and labeled human CD8(+) T cells and NK cells. These new resources should support future research on the biology of HLA-C molecules. The data are deposited at the Immune Epitope Database, and the updated NetMHCpan predictor is available at the Center for Biological Sequence Analysis and the Immune Epitope Database.
authors.affiliation: Laboratory of Experimental Immunology, Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen 2200, Denmark;
authors.forename: Michael
authors.initials: M
authors.lastname: Rasmussen
authors.affiliation: Laboratory of Experimental Immunology, Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen 2200, Denmark;
authors.forename: Mikkel
authors.initials: M
authors.lastname: Harndahl
authors.affiliation: Laboratory of Experimental Immunology, Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen 2200, Denmark;
authors.forename: Anette
authors.initials: A
authors.lastname: Stryhn
authors.affiliation: INSERM, Unité 1016, Institut Cochin, Equipe Immunologie du Diabète, Groupe Hospitalier Cochin-Port-Royal, 75014 Paris, France;
authors.forename: Rachid
authors.initials: R
authors.lastname: Boucherma
authors.affiliation: Laboratory of Experimental Immunology, Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen 2200, Denmark;
authors.forename: Lise Lotte
authors.initials: LL
authors.lastname: Nielsen
authors.affiliation: INSERM, Unité 1016, Institut Cochin, Equipe Immunologie du Diabète, Groupe Hospitalier Cochin-Port-Royal, 75014 Paris, France;
authors.forename: François A
authors.initials: FA
authors.lastname: Lemonnier
authors.affiliation: Center for Biological Sequence Analysis, Technical University of Denmark, Lyngby 2800, Denmark; and Instituto de Investigaciones Biotecnológicas, Universidad Nacional de San Martín, 1650 San Martín, Buenos Aires, Argentina.
authors.forename: Morten
authors.initials: M
authors.lastname: Nielsen
authors.affiliation: Laboratory of Experimental Immunology, Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen 2200, Denmark; sbuus@sund.ku.dk.
authors.forename: Søren
authors.identifier: http://orcid.org/0000-0001-8363-1999
authors.initials: S
authors.lastname: Buus
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D019295:Computational Biology; D016208:Databases, Factual; D000939:Epitopes; D015870:Gene Expression; D015234:HLA-A Antigens / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D015235:HLA-B Antigens / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D015236:HLA-C Antigens / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D007694:Killer Cells, Natural / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D005810:Multigene Family; D019151:Peptide Library; D011485:Protein Binding; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism
chemical_list: D000939:Epitopes; D015234:HLA-A Antigens; D015235:HLA-B Antigens; D015236:HLA-C Antigens; D007457:Iodine Radioisotopes; D019151:Peptide Library; D011994:Recombinant Proteins