<i><b>Supporting data</b></i><i><b> for</b></i> "Immunophenotyping of anti-PD-L1-treated hepatocellular carcinoma"

Hepatocellular carcinoma (HCC) is a deadly disease. The FDA recently approved the first immunotherapy, atezolizumab (anti-PD-L1) plus bevacizumab (anti-VEGF-A), for advanced HCC. Nonetheless, there are still patients not responding. Exploring how the tumour microenvironment is being altered upon anti-PD-L1 treatment helps unravel resistance mechanisms underlying anti-PD-L1 resistance. Using hydrodynamic tail vein injection (HDTVi), we established an anti-PD-L1 resistant HCC mouse model. We performed immunophenotyping of the tumour infiltrating immune cells utilising cytometry by Time-Of-Flight (CyTOF) and single-cell RNA sequencing (scRNA-seq). We discovered that anti-PD-L1 enhanced the infiltration of CD8⁺ T cells. Remarkably, several immune checkpoints were upregulated, accompanied by reduced effector functions. Anti-PD-L1 simultaneously induced T cell clonal expansion that shall aid in reinforcing immune response against tumour development. Together, these results suggest that anti-PD-L1 activates and expands T cells; however, these T cells are still in exhaustion due to other inhibitory receptors, rendering anti-PD-L1 ineffective in advanced HCC. Therefore, antibodies targeting the other immune checkpoints have considerable potential in restoring the therapeutic effects in combination with anti-PD-L1 to patients with advanced HCC. Submitted excel is the data for the figure included in the thesis, tab label corresponds to figure numbering肝细胞癌（Hepatocellular carcinoma, HCC）是一种致死性疾病。美国食品药品监督管理局（FDA）近日批准了首款免疫治疗联合方案：阿替利珠单抗（抗PD-L1）联合贝伐珠单抗（抗VEGF-A），用于晚期肝细胞癌的治疗。然而仍有部分患者对该疗法无应答。探究抗PD-L1治疗后肿瘤微环境的改变，有助于阐明抗PD-L1治疗耐药的潜在机制。本研究通过流体动力学尾静脉注射（HDTVi）构建了抗PD-L1耐药的肝细胞癌小鼠模型，利用飞行时间流式细胞术（CyTOF）与单细胞RNA测序（scRNA-seq）对肿瘤浸润免疫细胞开展免疫表型分析。研究发现，抗PD-L1治疗可增强CD8⁺ T细胞的肿瘤浸润能力。值得注意的是，多种免疫检查点分子表达上调，同时伴随T细胞效应功能减弱。此外，抗PD-L1治疗还可诱导T细胞克隆扩增，这一过程本应强化抗肿瘤免疫应答。综合上述结果表明，抗PD-L1可激活并扩增T细胞；但由于其他抑制性受体的作用，这些T细胞仍处于耗竭状态，使得抗PD-L1疗法在晚期肝细胞癌中疗效有限。因此，将靶向其他免疫检查点的抗体与抗PD-L1疗法联合使用，有望恢复晚期肝细胞癌患者的治疗效果。本次提交的Excel文件为本论文附图的配套数据，工作表标签与图表编号一一对应。