Bifunctional Role of Monocyte Subsets in Modulating Radiotherapy Combined Intra-Tumor αCD40 Agonist Induced Abscopal Effect

Abstract

Abscopal effect induced by radiotherapy and immune checkpoint blockade is a promising yet far from satisfactory strategy in clinical. The underlying immune mechanism, especially driven by monocytes remains poorly undefined. Monocytes consist of two phenotypically and functionally distinct subsets distinguished by expression of chemokine receptors CCR2 and CX3CR1: classical inflammatory Ly6ChiCCR2hi monocytes and nonclassical patrolling Ly6CloCCR2loCX3CR1hi monocytes. Monocytes differentiate and transit to other myeloid cells such as dendritic cells and macrophages according to various environmental cues. Herein we investigated the roles of monocyte subsets in modulating tumor control consisting of combination RT and myeloid checkpoint agonist αCD40 to specifically ignite myeloid cell activation. To establish abscopal model, contralateral tumors were implanted in each mouse, while only one side were treated with RT (8 Gy × 3) + αCD40 agonist (50 μg, intra-tumor). Tumor volume and mice survival were compared in each group (control, RT, αCD40 and RT + αCD40). Ccr2RFP/+ Cx3cr1GFP/+ (R2 × 3), Ccr2RFP/RFPCx3cr1+/+ (R2-KO) and Ccr2+/+Cx3cr1GFP/GFP (X3-KO) mice were used for cell tracking and to dissect chemokine receptor CCR2 and CX3CR1 on monocyte. Tumor infiltrating immune cells were analyzed by flowcytometry and RNA-seq. RT combined with αCD40 significantly dampened tumor growth on both ipsilateral and contralateral sides in abscopal model (p< 0.01), accompanied by upregulation of chemokine receptors CCR2 and CX3CR1 on myeloid cells were both increased in tumor and peripheral blood. Chemokine ligands CCL2, CCL3, CCL5, CCL7, CCL12 and CX3CL1 were upregulated in tumor after RT and αCD40 treatment, recruiting CCR2 and CX3CR1 expressing monocytes in situ. To elucidate the roles of CCR2 and CX3CR1 in mediating local and systemic anti-tumor immunity, R2 × 3, R2-KO and X3-KO mice with combined treatment were used. Tumor size on ipsilateral leg were similar among groups. However, tumor growth was significantly delayed on contralateral side in X3-KO mice while accelerated in R2-KO mice compared with that in R2 × 3 mice. Mechanistically, remarkable decrease of antigen presenting dendritic cells (MHCII+Ly6ChiCD11c+) were observed in R2-KO mice. Moreover, phagocytosis was strengthened in macrophages (F4/80+CD11b+) of X3-KO mice. CX3CR1 deletion ignite anti-tumor immunity elicited by RT and αCD40 through enhanced phagocytosis in macrophages, while CCR2 deletion renders inferior tumor control through reduction of dendritic cells. Preferential targeting nonclassical patrolling monocyte may lead to enhanced local and systemic tumor control.