Head and Neck Squamous Cell Carcinoma (HNSCC) is a deadly cancer with poor response to targeted therapy, largely driven by an immunosuppressive tumor microenvironment (TME). Here we examine the immune-modulatory role of the receptor tyrosine kinase EphA4 in HNSCC progression. Within the TME, EphA4 is primarily expressed on regulatory T cells (Tregs) and macrophages. In contrast ephrinB2, an activating ligand of EphA4, is expressed in tumor blood vessels. Using genetically engineered mouse models, we show that EphA4 expressed in Tregs promotes tumor growth, whereas EphA4 expressed in monocytes inhibits tumor growth. In contrast, ephrinB2 knockout in blood vessels reduces both intratumoral Tregs and macrophages. A novel specific EphA4 inhibitor, APY-d3-PEG4, reverses the accelerated tumor growth we had previously reported with EphB4 cancer cell knockout. EphA4 knockout in macrophages not only enhanced their differentiation into M2 macrophage but also increased Treg suppressive activity. APY-d3-PEG4 reversed the accelerated growth seen in the EphA4 knockout of monocytes but conferred no additional benefit when EphA4 was knocked out on Tregs. Underscoring an EphA4-mediated interplay between Tregs and macrophages, we found that knockout of EphA4 in Tregs not only decreases their activation but also reduces tumor infiltration of pro-tumoral M2 macrophages. These data identify Tregs as a primary target of APY-d3-PEG4 and suggest a role for Tregs in regulating macrophage conversion. These data also support the possible anti-cancer therapeutic value of bispecific peptides or antibodies capable of promoting EphA4 blockade in Tregs but not macrophages.
Significance: EphA4 in regulatory T cells has a pro-tumoral effect while EphA4 in macrophages plays an anti-tumoral role underscoring the necessity of developing biologically rational therapeutics.