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Abstract：
Background: Hypofractionated radiotherapy (hRT) can induce a T cell-mediated abscopal effect on non-irradiated tumor lesions, especially in combination with immune checkpoint blockade (ICB). However, clinically, this effect is still rare, and ICB-mediated adverse events are common. Lenalidomide (lena) is an anti-angiogenic and immunomodulatory drug used in the treatment of hematologic malignancies. We here investigated in solid tumor models whether lena can enhance the abscopal effect in double combination with hRT. Methods: In two syngeneic bilateral tumor models (B16-CD133 melanoma and MC38 colon carcinoma), the primary tumor was treated with hRT. Lena was given daily for 3 weeks. Besides tumor size and survival, the dependence of the antitumor effects on CD8+ cells, type-I IFN signaling, and T cell costimulation was determined with depleting or blocking antibodies. Tumor-specific CD8+ T cells were quantified, and their differentiation and effector status were characterized by multicolor flow cytometry using MHC-I tetramers and various antibodies. In addition, dendritic cell (DC)-mediated tumor antigen cross-presentation in vitro and directly ex vivo and the composition of tumor-associated vascular endothelial cells were investigated. Results: In both tumor models, the hRT/lena double combination induced a significant abscopal effect. Control of the non-irradiated secondary tumor and survival were considerably better than with the respective monotherapies. The abscopal effect was strongly dependent on CD8+ cells and associated with an increase in tumor-specific CD8+ T cells in the non-irradiated tumor and its draining lymph nodes. Additionally, we found more tumor-specific T cells with a stem-like (TCF1+ TIM3- PD1+) and a transitory (TCF1- TIM3+ CD101- PD1+) exhausted phenotype and more expressing effector molecules such as GzmB, IFNγ, and TNFα. Moreover, in the non-irradiated tumor, hRT/lena treatment also increased DCs cross-presenting a tumor model antigen. Blocking type-I IFN signaling, which is essential for cross-presentation, completely abrogated the abscopal effect. A gene expression analysis of bone marrow-derived DCs revealed that lena augmented the expression of IFN response genes and genes associated with differentiation, maturation (including CD70, CD83, and CD86), migration to lymph nodes, and T cell activation. Flow cytometry confirmed an increase in CD70+ CD83+ CD86+ DCs in both irradiated and abscopal tumors. Moreover, the hRT/lena-induced abscopal effect was diminished when these costimulatory molecules were blocked simultaneously using antibodies. In line with the enhanced infiltration by DCs and tumor-specific CD8+ T cells, including more stem-like cells, hRT/lena also increased tumor-associated high endothelial cells (TA-HECs) in the non-irradiated tumor. Conclusions: We demonstrate that lena can augment the hRT-induced abscopal effect in mouse solid tumor models in a CD8 T cell- and IFN-I-dependent manner, correlating with enhanced anti-tumor CD8 T cell immunity, DC cross-presentation, and TA-HEC numbers. Our findings may be helpful for the planning of clinical trials in (oligo)metastatic patients.
摘要：
背景：大分割放疗（hRT）可以诱导T细胞介导的非照射肿瘤病灶的外视效应，特别是与免疫检查点阻断（ICB）联合使用。然而,临床上,这种效果仍然很少见，ICB介导的不良事件很常见。来那度胺（lena）是一种抗血管生成和免疫调节药物，用于治疗血液恶性肿瘤。我们在实体肿瘤模型中研究了lena与hRT双重组合是否可以增强腹腔镜效果。方法：在两种同基因的双侧肿瘤模型（B16-CD133黑色素瘤和MC38结肠癌）中，原发性肿瘤用hRT治疗。Lena每天服用3周。除了肿瘤大小和生存率,抗肿瘤作用对CD8+细胞的依赖性，I型IFN信号传导，和T细胞共刺激与耗竭或阻断抗体测定。肿瘤特异性CD8+T细胞定量，通过使用MHC-I四聚体和各种抗体的多色流式细胞术对其分化和效应物状态进行表征。此外,研究了树突状细胞（DC）介导的肿瘤抗原在体外和直接离体的交叉呈递以及肿瘤相关血管内皮细胞的组成。结果：在两种肿瘤模型中，hRT/lena双重组合诱导了显著的消隐效应。未照射的继发性肿瘤的控制和存活率明显优于各自的单一疗法。远视效应强烈依赖于CD8细胞，并且与未照射肿瘤及其引流淋巴结中肿瘤特异性CD8T细胞的增加有关。此外,我们发现更多的肿瘤特异性T细胞具有干细胞样（TCF1+TIM3-PD1+）和暂时性（TCF1-TIM3+CD101-PD1+）耗竭表型，并且表达更多的效应分子，如GzmB，IFNγ,和TNFα。此外，在未照射的肿瘤中，hRT/lena治疗也增加了交叉呈递肿瘤模型抗原的DC。阻断I型IFN信号传导，这对于交叉展示至关重要，完全废除了abscopal效应。骨髓来源的DC的基因表达分析显示，lena增加了IFN应答基因和与分化相关的基因的表达，成熟（包括CD70，CD83和CD86），迁移到淋巴结，和T细胞激活。流式细胞术证实在照射的肿瘤和横隔肿瘤中CD70+CD83+CD86+DC的增加。此外，当使用抗体同时阻断这些共刺激分子时,hRT/lena诱导的远视效应减弱.与DC和肿瘤特异性CD8+T细胞的浸润增强一致，包括更多的干细胞样细胞，hRT/lena还增加了未照射肿瘤中的肿瘤相关高内皮细胞（TA-HECs）。结论：我们证明lena可以以CD8T细胞和IFN-I依赖性方式增强小鼠实体肿瘤模型中hRT诱导的远视效应，与增强的抗肿瘤CD8T细胞免疫相关，DC交叉演示，和TA-HEC号码。我们的发现可能有助于（寡）转移性患者的临床试验计划。
