Characterisation of an autochthonous mouse ccRCC model of immune checkpoint inhibitor therapy resistance

Many metastatic clear cell renal cell carcinomas (ccRCC) are resistant to immune checkpoint inhibitor therapies, however the mechanisms underlying sensitivity or resistance remain incompletely characterised. We demonstrate that ccRCCs in the Vhl/Trp53/Rb1 mutant mouse model are resistant to combined anti-PD-1/anti-CTLA-4 therapy alone and in combination with additional therapeutic agents that reflect current ccRCC clinical trials. However, in some animals in vivo checkpoint therapy allowed isolated splenic T cells to recognise cultured ccRCC cells from the same animal, implicating the tumour microenvironment in suppression of T cell activation. We identified putative immunosuppressive myeloid cell populations with features similar to myeloid cells in the microenvironment of human ccRCC. The expression patterns of immune checkpoint ligands in both the mouse model and in human ccRCC suggests that several checkpoint systems other than PD-1 and CTLA-4 are likely to represent the dominant T cell suppressive forces in ccRCC. Our findings characterise an autochthonous mouse ccRCC model of immune checkpoint inhibitor therapy resistance and pave the way for a systematic functional dissection of the identified potential molecular barriers to effective immune therapy of ccRCC. scRNA-seq analysis of a normal kidney and of 3 Vhl/Trp53/Rb1 mutant mouse ccRCC tumours, cells were sorted for CD45.1 and mixed in the ratio of 3:1 positive to negative prior to preparation for scRNA-seq to enrich for immune cells

多数转移性透明细胞肾细胞癌（clear cell renal cell carcinoma, ccRCC）对免疫检查点抑制剂疗法（immune checkpoint inhibitor therapies）存在耐药性，但目前对其敏感性或耐药性的潜在分子机制仍未完全阐明。本研究证实，Vhl/Trp53/Rb1突变小鼠模型中的ccRCC对单独使用抗PD-1/抗CTLA-4联合疗法，以及联合当前ccRCC临床试验中应用的其他治疗药物均表现出耐药性。但在部分小鼠体内，经检查点疗法处理后，其分离得到的脾脏T细胞可识别同一只小鼠体内培养的ccRCC细胞，这提示肿瘤微环境参与抑制T细胞活化。本研究鉴定出具有与人类ccRCC微环境中髓系细胞相似特征的推定免疫抑制性髓系细胞群。小鼠模型与人类ccRCC中的免疫检查点配体表达模式显示，除PD-1和CTLA-4外，其他多种检查点系统可能是ccRCC中主要的T细胞抑制因素。本研究明确了免疫检查点抑制剂疗法耐药的原位自发小鼠ccRCC模型，并为系统性功能解析ccRCC有效免疫治疗的潜在分子屏障奠定了基础。本研究针对正常肾脏及3株Vhl/Trp53/Rb1突变小鼠ccRCC肿瘤开展单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）分析：先对细胞按CD45.1进行分选，以3:1的阳性与阴性细胞比例混合，随后制备scRNA-seq文库以富集免疫细胞。