New findings in The Journal of Immunology establish the first allele-specific peptide binding motif framework for canine MHC class II. Dr. Haeree Lang, a post-doctoral fellow in the College of Veterinary Medicine at North Carolina State University, defined two peptide-binding motifs of common canine MHC class II alleles, DLA-DR15 and DLA-DR12. These alleles were highly prevalent in Standard Poodles and Golden Retrievers, respectively, as two breeds predisposed to immune-mediated and autoimmune diseases and cancer.
“Much of the canine immunology field still relies on tools and assumptions from the 1980s, even as human and mouse immunology have moved into the era of single-cell RNA sequencing, high-throughput TCR sequencing, and antigen-specific tetramer technology. Closing that gap is a central motivation behind this work,” shared Dr. Lang.
In dogs, MHC class II genes are located within the Dog Leukocyte Antigen (DLA) region, which includes two functional molecules: DLA-DR and DLA-DQ. As dogs co-express DLA-DR and DLA-DQ on every antigen-presenting cell, the research team first had to confirm that their purification antibody recognized only DLA-DR. They validated this using engineered cell lines expressing each molecule separately, which allowed them to isolate DLA-DR-bound peptides with confidence. Then, using immunopeptidomics, they isolated naturally processed and presented peptides from canine spleen tissue and used mass spectrometry to generate unique binding motifs for each allele.
Given rabies vaccination is legally mandated for dogs in the US and drives strong, long-lasting T cell responses, the research team applied the identified motifs to rabies vaccine proteins. They confirmed several predicted epitopes through competition binding assays. Building on this, they also found that canine DLA-DR motifs resemble human HLA-DR far more closely than they resemble murine I-A.
“All together, I hope this gives canine immunologists a predictive framework: whether the protein of interest is a vaccine antigen, tumor neoantigen, allergen, or a self-protein implicated in autoimmune disease, we can predict which epitopes are likely to be displayed on DLA-DR15 and/or DLA-DR12 molecules and could drive a robust immune response,” said Dr. Lang.
To build on this work, Dr. Lang plans to expand this approach to additional DLA alleles and move from predicting which peptides bind these alleles to directly confirming and characterizing the T cell responses themselves. This will include testing predicted epitopes against canine CD4+ T cells and tetramerizing recombinant DLA-DR15 and DLA-DR12 monomers into peptide:MHC class II tetramers, to can identify, track, and characterize antigen-specific T cells the same way it’s routinely done in human and mouse immunology. From here, the researchers want to apply this framework to specific diseases in the breeds predisposed to them, to start characterizing what these antigen-specific responses look like in a clinical setting.
“The long-term goal is to use this as a foundation for identifying and characterizing antigen-specific CD4+ T cells, regardless of their source, which will allow us to work toward better immunotherapies and vaccines. I hope dogs can be embraced more as a comparative model for human immunology. They’re already man’s best friend in our daily lives, and given how closely their immune systems, environmental factors, and lifestyles resemble ours, I’d love to see them recognized as one of our best scientific allies too.”
