Abstract: Background: Canine gliomas share many similarities with their human counterparts, including recruitment of immunosuppressive regulatory T-cells (Tregs) that inhibit host immune responses. We have shown previously the importance of the chemokine CCL2 and its high-affinity receptor CCR4 in recruitment of Tregs in a murine orthotopic glioma model and elevated CCL2 in canine high grade gliomas. However, mechanisms responsible for Treg recruitment in canine glioma are poorly defined. Hypotheses: 1) Canine Tregs posess canonical Treg markers and can be isolated for downstream assays; 2) Glioma-derived CCL2 is a potent chemoattractant for canine Tregs signalling via CCR4. Animals: Four healthy dogs. Methods: In vitro, mechanistic study. Canine Tregs were characterized by flow cytometry using canonical markers CD3, CD4, CD25, FOXP3 and CCR4 and sorted using fluorescent-activated cell sorting. CCL2 mRNA expression was assessed via RTqPCR in four canine glioma cell lines (1110, 0514, J3T-Bg, G06A). Treg migratory capacities were assessed using a Boyden chamber assay. Anti-CCL2 antibody and a CCR4 inhibitor (C021) were used in abrogation studies. Results: The canine CD4+CD25high T-cell population expressed canonical Treg markers FOXP3 and CCR4. Canine Treg migration was enhanced by CCL2 and glioma cell line-derived supernatant. Blockade of the CCL2/CCR4 axis abrogated migration of Tregs. CCL2 mRNA was expressed in all cell lines and expression increased when exposed to Tregs but not to conventional CD4+ T-cells. Conclusion: CD25high expression is critical for isolation of canine Tregs. Our study validated CCL2/CCR4 as a bi-directional Treg-glioma signalling axis and represents a rational target for immunotherapy in clinical patients.
