The CCL2-CCR4 Axis Promotes Regulatory T Cell Trafficking to Canine Glioma Tissues

Abstract

Purpose Spontaneously occurring glioma in pet dogs is increasingly recognized as a valuable translational model for human glioblastoma. Canine high grade glioma and human glioblastomas share many molecular similarities, including accumulation of immunosuppressive regulatory T cells (Tregs) that inhibit anti-tumor immune responses. Identifying in dog mechanisms responsible for Treg recruitment may afford targeting the cellular population driving immunosuppression, the results providing a rationale for translational clinical studies in human patients. Our group has previously identified C-C motif chemokine 2 (CCL2) as a glioma-derived T-reg chemoattractant acting on chemokine receptor 4 (CCR4) in a murine orthotopic model of glioma. Recently, we demonstrated a robust increase of CCL2 in the brain tissue of canine patients bearing high-grade glioma. Methods We performed a series of in vitro experiments using canine Tregs and patient-derived canine glioma cell lines (GSC 1110, GSC 0514, J3T-Bg, G06A) to interrogate the CCL2-CCR4 signaling axis in the canine. Results We established a flow cytometry gating strategy for identification and isolation of FOXP3⁺ Tregs in dogs. The canine CD4 + CD25^high T-cell population was highly enriched in FOXP3 and CCR4 expression, indicating they are bona fide Tregs. Canine Treg migration was enhanced by CCL2 or by glioma cell line-derived supernatant. Blockade of the CCL2-CCR4 axis significantly reduced migration of canine Tregs. CCL2 mRNA was expressed in all glioma cell lines and expression increased when exposed to Tregs but not to CD4 + helper T-cells. Conclusion Our study validates CCL2-CCR4 as a bi-directional Treg-glioma immunosuppressive and tumor-promoting axis in canine high-grade glioma.