2015 ACVIM Forum Research Abstract Program

Variants in the DLA-79 gene may be associated with multiple autoimmune diseases in dogs. Further studies are warranted to confirm these findings more broadly, and to determine the specific mechanism by which the identified variants may alter canine immune system function. Type 1 regulatory T (Tr1) cells play a pivotal role in the maintenance of peripheral tolerance. In contrast to their extensive characterization in humans and mice, little is known about Tr1 cells in the dog, an important veterinary species and an animal model for various human diseases. Secretion of high-level interleukin-10 (IL-10) is considered to be a defining hallmark of Tr1 cells. To date, a well-validated assay for intracellular IL-10 in the dog has not been described. Our goal was to develop a robust flow cytometric assay for canine intracellular IL-10 as a precursor to the study of Tr1 cells in this species. We screened three commercially available antibodies (Abs): a monoclonal antihuman IL-10 Ab (clone JES3-9D7) marketed for use in flow cytometry and Western blots, and both polyclonal (catalog number AF735) and monoclonal (clone 138128) anti-canine IL-10 Abs marketed only for use in Western blots. Cross-reactivity of the anti-human monoclonal Ab was predicted on the basis of 81% amino acid sequence identity of the canine and human proteins. Canine IL-10 was cloned into two vectors, pcDNA3 and pMIG. Both constructs were used to transfect human embryonic kidney (HEK) 293T cells. Surprisingly, canine IL-10 expressed by transfected cells failed to be detected by the anti-human monoclonal Ab by either Western blots or flow cytometry. Both anticanine IL-10 Abs detected canine IL-10 expressed by transfected HEK 293T cells, the polyclonal Ab performing better in the setting of Western blots and the monoclonal Ab performing better in the setting of flow cytometry. In summary, two commercially available Abs have been validated for the detection of canine intracellular IL-10. Studies examining the expression of this cytokine by both in vitro-generated IL-10-producing cells and the peripheral blood monoclonal cells of dogs with immune-mediated and neoplastic diseases are ongoing. The use of mesenchymal stem cell (MSC) therapy to treat clinical disease may be optimized through selection and pre-conditioning methods that would enhance their immunomodulatory functions. We investigated the effects of several pre-conditioning methods and gender of the tissue donor on parameters of feline adipose-derived MSC in vitro that may be applicable to and opti-mize the clinical effect of MSC application in this species. Passage 3 feline MSC were generated from cryopreserved subcutaneous adipose tissue from 6 healthy cats, three males and three females. MSC were subjected to six different pre-conditioning agents or conditions for 24 hours: control, poly I:C, IFNc, TGFb, serum-free, and hypoxia. Supernatants from the MSC were harvested after 24 hours, aliquoted, and frozen for batch analysis. Levels of IL-6, IL-8, IL-10, and MCP-1 in the supernatants were determined by ELISA. Both pretreatment and gender of donor significantly affected measured cytokine levels in the 24 hour supernatants from feline adipose-derived MSC: IL-6 and IL-10 were affected by donor gender whereas all of the measured cytokines were affected by at least one of the pretreatments. Although further study is necessary, this initial study shows that specific pre-conditioning methods hold promise for tailoring and augme