Reprogramming the Intrahepatic Cholangiocarcinoma Immune Microenvironment by Chemotherapy and CTLA-4 Blockade Enhances Anti-PD1 Therapy (bulk RNA-seq)

Intrahepatic cholangiocarcinoma (ICC) has limited therapeutic options and a dismal prognosis. Adding blockade of the PD1 pathway to gemcitabine/cisplatin chemotherapy has recently shown efficacy in biliary tract cancers but with low response rates. Here, we studied the effects of anti-CTLA-4 when combined with anti-PD1 and gemcitabine/cisplatin in orthotopic murine models of ICC. This combination therapy led to substantial survival benefits and reduction of morbidity in two aggressive ICC models that were resistant to immunotherapy alone. Gemcitabine/cisplatin treatment increased tumor-infiltrating lymphocytes and normalized the ICC vessels, and when combined with dual CTLA-4/PD1 blockade, increased the number of activated CD8+Cxcr3+IFN?+ T cells. CD8+T cells were necessary for the therapeutic benefit because the efficacy was compromised when CD8+ T cells were depleted. Expression of Cxcr3 on CD8+ T cells is necessary and sufficient since CD8+ T cells from Cxcr3+/+ but not Cxcr3–/– mice rescued efficacy in T cell?deficient mice. Finally, rational scheduling of anti-CTLA-4 “priming” with chemotherapy followed by anti-PD1 therapy achieved equivalent efficacy with reduced overall drug exposure. These data suggest that this combination approach should be clinically tested to overcome resistance to current therapies in ICC patients. Overall design: Using tumor tissues collected in a time-matched manner, we employed bulk RNA-Seq to evaluate the transcriptional changes in murine 425-ICC tissues after 8 days of treatment with GC alone, dual ICB alone, and GC/dual ICB versus IgG control.肝内胆管癌（Intrahepatic cholangiocarcinoma, ICC）的治疗选择有限且预后极差。在吉西他滨（gemcitabine）/顺铂（cisplatin）化疗基础上联用PD-1通路阻断治疗，近期在胆道系统肿瘤中展现出疗效，但应答率较低。本研究在ICC原位小鼠模型（orthotopic murine models）中，探究了抗CTLA-4治疗联合抗PD-1治疗与吉西他滨/顺铂化疗的协同效应。该联合疗法可在两株对单一免疫治疗耐药的侵袭性ICC模型中，带来显著的生存获益并降低并发症负担。吉西他滨/顺铂治疗可增加肿瘤浸润淋巴细胞（tumor-infiltrating lymphocytes, TILs）的浸润，并使ICC血管正常化；与CTLA-4/PD-1双重阻断疗法联用时，可进一步增加活化CD8+Cxcr3+IFNγ+ T细胞的数量。CD8+T细胞是该治疗获益的必要条件：耗竭CD8+T细胞会显著削弱治疗效果。CD8+T细胞表面的Cxcr3表达既是治疗起效的必要条件，也是充分条件：来自CXCR3野生型（Cxcr3+/+）而非CXCR3基因敲除（Cxcr3-/-）小鼠的CD8+T细胞，可在T细胞缺陷小鼠中恢复治疗疗效。最后，采用“抗CTLA-4预致敏联合化疗，随后予以抗PD-1治疗”的合理给药方案，可在降低总药物暴露量的同时达到等效的治疗效果。本研究结果提示，该联合疗法应开展临床研究，以克服ICC患者对当前治疗的耐药性。 总体实验设计：本研究采用时间匹配方式收集肿瘤组织，通过批量RNA测序（bulk RNA-Seq）评估小鼠425-ICC组织在接受以下8天治疗后的转录组变化：吉西他滨/顺铂单一治疗组、双重免疫检查点阻断（ICB）单一治疗组、吉西他滨/顺铂联合双重ICB治疗组，并以IgG对照组（IgG control）作为参照。