TRAF6/mTOR双向调控巨噬细胞极化在放疗联合PD-1抗体诱导远隔效应中的作用及机制研究

Radiotherapy and immune checkpoint blockade αPD-1 cooperate to induce abscopal effect, with the mechanism remains to be fully understood. The “anti-tumor” phenotype of M1 and “pro-tumor” phenotype of M2 played pivotal parts in radiotherapy or immune checkpoint blockade therapy alone. However, the roles of M1 and M2 has not been reported in combined therapy inducing the abscopal effect. We have previously reported that M1 or M2 polarization are critically dependent on TRAF6 or mTOR, respectively, by using myeloid-specific deletion of TRAF6 or mTOR mice. Moreover, our preliminary results showed an increase of tumor infiltrated M1 and decrease of M2 in radiotherapy combined with αPD-1 induced abscopal model, suggesting various macrophage subpopulations may contribute differently to abscopal effect. Therefore, this project was designed to verify the roles of macrophage derived TRAF6-M1/mTOR-M2 axis in abscopal effect. By using the aforementioned mice, we aim to explore the bidirectional function of TRAF6 and mTOR in response to various polarization stimuli in vitro and complicated environmental milieus of combined therapy inducing abscopal model in vivo. Moreover, we will investigate the mechanisms of TRAF6-M1/mTOR-M2 axis in modulating abscopal effect, thus providing new strategies and insights into inducing successful abscopal effect in the clinic.

放疗联合检查点抑制剂αPD-1具有显著的协同作用，可诱导放疗远隔效应发生，其机制尚未完全明确。巨噬细胞不同亚群M1/M2在放疗或αPD-1治疗中表现出抗肿瘤效应及促肿瘤效应的双向性，但其在诱导远隔效应中的作用未见报道。申请人前期利用髓系特异性敲除TRAF6或mTOR小鼠证实，TRAF6及mTOR分别是M1/M2极化关键调控因子。同时申请人预实验发现，放疗联合抗αPD-1诱导远隔效应模型中M1显著增加，而M2减少。由此推测调控巨噬细胞中TRAF6-M1/mTOR-M2轴能影响远隔效应的发生。申请人拟利用这两种小鼠模型，在体外多种极化刺激物极化模型及在体内诱导远隔效应模型中进一步证实TRAF6调控M1极化而mTOR调控M2极化，并深入研究巨噬细胞中TRAF6-M1/mTOR-M2轴分别在诱导远隔效应中的作用及机制。本研究可能为临床诱导远隔效应提供新靶点和研究基础。

由放疗及检查点抑制剂诱导的远隔效应有良好的应用潜力，但其临床效果却差强人意。远隔效应的免疫学机制，尤其是由单核/髓系细胞介导的机制尚不明确。单核细胞由两群细胞以CCR2及CX3CR1表达区分为形态及功能各异的经典炎性单核细胞及非经典巡视单核细胞。单核细胞在微环境调控下进一步分化及转化为其他髓系细胞如树突状细胞及巨噬细胞。本项目研究不同单核细胞亚群参与调控放疗联合髓系检查点αCD40激动剂介导的远隔效应的作用及机制，并利用Ccr2RFP/+ Cx3cr1GFP/+ (R2X3), Ccr2RFP/RFPCx3cr1+/+ (R2-KO) 及Ccr2+/+Cx3cr1GFP/GFP (X3-KO) 小鼠观察髓系细胞迁移同时建立CCR2或CX3CR1缺失小鼠模型。放疗联合瘤内注射αCD40激动剂成功建立了远隔效应模型，上调肿瘤中趋化因子CCL2, CCL5, CCL7 and CCL12表达水平及外周血趋化因子受体CCR2及CX3CR1水平。分别在R2X3、R2-KO及X3-KO小鼠中构建远隔效应模型，观察到非治疗侧X3-KO小鼠肿瘤最小，R2X3小鼠肿瘤次之，R2-KO小鼠肿瘤最大。对三种小鼠体内肿瘤浸润免疫细胞的进一步分析提示，R2-KO小鼠中髓系细胞中树突状细胞及巨噬细胞显著减少。此外，体外功能学实验发现X3-KO小鼠巨噬细胞吞噬肿瘤细胞能力增强。本研究发现CX3CR1缺失小鼠增强巨噬细胞吞噬能力增强远隔效应，而CCR2缺失小鼠抗原递呈树突状细胞数量减少削弱远隔效应。本研究为为寻找肿瘤放疗联合治疗新靶点， 增强远隔效应提供新思路。