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Here, we report the first immunopeptidomics-derived binding motifs for DLA-DRB1*015:01 (DLA-DR15) and DLA-DRB1*012:01 (DLA-DR12), 2 alleles overrepresented in breeds predisposed to immune-mediated diseases. Because dogs co-express DLA-DR and DLA-DQ, the MHC class II Ab clone YKIX334.2 was validated to be DLA-DR-specific, enabling allele-selective immunoaffinity purification of DLA-DR molecules from homozygous DLA-DR15 and DLA-DR12 donor spleens. Mass spectrometry and GibbsCluster motif deconvolution of 838 DLA-DR15\u2013associated and 644 DLA-DR12\u2013associated peptides eluted from their respective peptide-binding grooves revealed distinct allele-specific binding motifs, with characterization of anchor residue preferences, peptide-length distributions, cross-species comparisons with human and murine MHC class II motifs, and source protein composition of the eluted self-peptidome. To evaluate the translational utility of these motifs, recombinant DLA-DR15 and DLA-DR12 molecules were used to screen rabies virus glycoprotein and nucleoprotein peptide libraries via fluorescence-based peptide competition assays, identifying high-affinity candidate binders for both alleles. Spearman rank correlation between immunopeptidomics-derived position-specific scoring matrix scores and peptide competition assay rankings demonstrated modest associations, consistent with these approaches capturing complementary dimensions of peptide\u2013MHC class II interaction. Ultimately, these findings establish what we believe is the first allele-specific peptide-binding motif framework for canine MHC class II, providing a foundation for DLA-allele\u2013informed CD4+ T-cell epitope discovery studies and Ag-specific immune response characterization in the dog.<\/jats:p>","DOI":"10.1093\/jimmun\/vkag243","type":"journal-article","created":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T17:29:19Z","timestamp":1789666159000},"source":"Crossref","is-referenced-by-count":0,"title":["Immunopeptidomics-driven MHC class II peptide-binding motif discovery for 2 common canine DR alleles"],"prefix":"10.1093","volume":"215","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3882-7276","authenticated-orcid":false,"given":"Haeree P","family":"Lang","sequence":"first","affiliation":[{"name":"Center for Immunology, University of Minnesota , Minneapolis, MN,","place":["United States"]},{"name":"Department of Veterinary Clinical Sciences, College of Veterinary Medicine, University of Minnesota , St. Paul, MN,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aysha","family":"Rana","sequence":"additional","affiliation":[{"name":"Center for Immunology, University of Minnesota , Minneapolis, MN,","place":["United States"]},{"name":"Department of Veterinary Clinical Sciences, College of Veterinary Medicine, University of Minnesota , St. Paul, MN,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Farah F","family":"Almeer","sequence":"additional","affiliation":[{"name":"Department of Veterinary Clinical Sciences, College of Veterinary Medicine, University of Minnesota , St. Paul, MN,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thamotharampillai","family":"Dileepan","sequence":"additional","affiliation":[{"name":"Center for Immunology, University of Minnesota , Minneapolis, MN,","place":["United States"]},{"name":"Department of 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