Lang, Haeree2025-03-212025-03-212024-12https://hdl.handle.net/11299/270580University of Minnesota Ph.D. dissertation. December 2024. Major: Comparative and Molecular Biosciences. Advisors: Steven Friedenberg, Marc Jenkins. 1 computer file (PDF); viii, 130 pages.Although dogs are diagnosed with many of the same immune-mediated diseases as their owners, canine immunology research — particularly the behavior and phenotypic characteristics of CD4+ T cells — remains limited. This dissertation seeks to bridge these gaps by investigating canine immune responses, specifically examining how healthy CD4+ T cells respond to certain antigens in vitro. Insights from this research have the potential to inform models of pathogenic, autoimmune, and allergic diseases in dogs. We reviewed the current state of canine T cell research, examining unique aspects that distinguish dogs from humans and mice and highlighting both the shortage of canine-specific immunological reagents and the reliance on human and mouse model paradigms. Our study of antigen-specific T cell responses to the rabies vaccine identified and characterized CD4+ and CD4+CD8+ double-positive T cells as the predominant subsets responding to rabies virus glycoprotein (RABV-G) restimulation in vitro. To support epitope discovery, we identified peptide binding motifs for two common DLA-DR alleles, DLA-DR15 and DLA-DR12. Derived from eluted and naturally processed peptides analyzed through mass spectrometry and GibbsCluster analysis, these motifs were used to predict immunodominant epitopes from RABV-G. Additionally, we developed canine-specific artificial antigen-presenting cells (aAPCs) engineered to expand antigen-specific CD4+ T cells in vitro. We showed that some of these aAPCs expressed high levels of DLA-DR15 MHC class II and that peptides could be loaded into their peptide binding groove. These aAPCs may serve as a valuable tool to expand T cells for adoptive cell transfer and CAR T cell therapy in canine patients. We also adapted an activation-induced marker assay using CD40L expression to help identify recently activated T cells in vitro and developed a cytokine-supplemented expansion protocol, reducing reliance on K562-mediated expansion alone. This research seeks to improve our understanding of canine CD4+ T cell responses to antigens and methods to identify them, introduces tools to study these responses in client-owned dogs, and may advance the development of personalized immunotherapies in veterinary medicine.enCanineCD4+ImmunologyMHC class IIVaccineVeterinaryThesis or Dissertation