Novel immunological tools to investigate CD4+ T cell activation in the canine species

Abstract

Abstract Opportunities to identify T cell epitopes in the dog have been thwarted by a lack of immunological tools specific for the species. As a result, antigen-specific triggers in diseases that affect dogs like autoimmune disorders and allergies remain unknown. Our objective was to develop immunological tools such as: IFN-γ enzyme-linked immunosorbent spot (ELISpot) and activation induced marker (AIM) assays, and an MHC class II tetramer to help overcome these hurdles. We collected splenocytes from dogs that underwent a routine splenectomy. We used these splenocytes to develop: an ELISpot assay detecting canine IFN-γ production by stimulated T cells, an AIM assay to assess early activation by CD40L expression on CD4+CD5+ canine splenocytes using a cross-reactive anti-human monoclonal antibody, and finally, we produced the first-ever canine MHC II tetramer specific for a common canine DR allele. We confirmed the efficacy of these tools in the presence of positive and negative controls, Staphylococcus aureus enterotoxin B (SEB) and dimethyl sulfoxide (DMSO), respectively. SEB induces a robust canine IFN-γ ELISpot readout quantifiable in canine splenocytes. Similarly, we discovered that canine CD40L expression peaks at 3 hours post-stimulation with SEB. Finally, we confirmed the development of the first canine pMHCII tetramer through size discriminatory SDS-PAGE analysis and peptide exchange assays. Our novel canine-specific reagents will help identify and characterize antigen-specific T cells and their cognate antigens driving canine MHC class II-restricted disease, thus guiding research towards the advancement of antigen-specific immunotherapies. Supported by a grant from the NIH (K01 OD027058)