Fingerprints of Hepatocellular Carcinoma-Infiltrated ?dT Cells Revealed by Single-Cell Sequencing

We report the application of single cell RNA sequencing for profiling the fingerprints of ?dT cells of hepatocellular carcinoma compared with healthy donors. By comparing the transcriptome and TCR clonality of ?dT cells from 3 HCC patients and expanded ?dT cells from 3 healthy donors, we found that expanded ?d T cells derived from different donors shared great transcriptomic similarity and we found higher enrichments of major ?d TCRs in expanded ?d T cells compared to infiltrated but not peripheral ones in HCC. Analysis of differentiation trajectory demonstrated a branched structure in expanded ?d T cells, implying functional and differentiation stage diversity, and ?d T cells from HCC patients are localized to the far end of the trajectory. Furthermore, GVSA analyses made on metabolic pathways demonstrated strong enrichments in major metabolic pathways such as OXPHOS, glycolysis, and fatty acid metabolism in expanded ?d T cells versus peripheral ones of HCC. The comparison indicated higher cytotoxicity score of expanded ?d T cells compared to infiltrated or peripheral ones of HCC. The successful application of allogeneic ?d T cells immunotherapy in late-stage liver cancer patients was supported by our current scRNA transcriptomic analyses, which suggested strong anti-tumor functional complementation by the ex-vivo expanded ?d T cells. Overall design: We report the application of single cell RNA sequencing for profiling the fingerprints of ?dT cells of hepatocellular carcinoma compared with healthy donors. Transcriptomic and TCR clonality analysis were both applied. The results showed that expanded ?d T cells derived from different donors shared great transcriptomic similarity and higher enrichments of major ?d TCRs in expanded ?d T cells. Analysis of differentiation trajectory demonstrated a branched structure in expanded ?d T cells, implying functional and differentiation stage diversity, and ?d T cells from HCC patients are localized to the far end of the trajectory. Furthermore, GVSA analyses made on metabolic pathways demonstrated strong enrichments in major metabolic pathways such as OXPHOS, glycolysis, and fatty acid metabolism in expanded ?d T cells versus peripheral ones of HCC. The comparison indicated higher cytotoxicity score of expanded ?d T cells compared to infiltrated or peripheral ones of HCC.

本研究报道了单细胞RNA测序（single cell RNA sequencing, scRNA-seq）在肝细胞癌（hepatocellular carcinoma, HCC）患者γδT细胞（γδ T cells）指纹图谱分析中的应用，并与健康供体进行对照。通过对3例肝细胞癌患者体内γδT细胞与3例健康供体体外扩增γδT细胞的转录组及T细胞受体（T cell receptor, TCR）克隆性开展比较分析，本研究发现：不同供体来源的体外扩增γδT细胞具有高度相似的转录组特征；与肝细胞癌患者肿瘤浸润及外周血γδT细胞相比，体外扩增γδT细胞中主要γδ TCR的富集程度更高。分化轨迹分析显示，体外扩增γδT细胞的分化轨迹呈现分支结构，提示其功能与分化阶段存在异质性；而肝细胞癌患者体内的γδT细胞则位于该分化轨迹的远端。此外，针对代谢通路的GVSA分析显示，与肝细胞癌患者外周血γδT细胞相比，体外扩增γδT细胞在氧化磷酸化（OXPHOS）、糖酵解及脂肪酸代谢等主要代谢通路上均呈现显著富集。对比分析还表明，体外扩增γδT细胞的细胞毒性评分高于肝细胞癌患者体内的肿瘤浸润及外周血γδT细胞。本研究的单细胞RNA转录组分析结果为异基因γδT细胞免疫疗法在晚期肝癌患者中的成功应用提供了理论支撑，提示体外扩增γδT细胞具备强大的抗肿瘤功能互补能力。

整体实验设计：本研究采用单细胞RNA测序技术，对肝细胞癌患者与健康供体的γδT细胞指纹图谱进行分析，并同步开展转录组与TCR克隆性检测。结果显示：不同供体来源的体外扩增γδT细胞具有高度相似的转录组特征，且其主要γδ TCR的富集程度更高；分化轨迹分析表明，体外扩增γδT细胞的分化轨迹呈分支结构，提示功能与分化阶段存在异质性，肝细胞癌患者体内的γδT细胞则位于该轨迹远端；代谢通路GVSA分析显示，与肝细胞癌患者外周血γδT细胞相比，体外扩增γδT细胞在氧化磷酸化、糖酵解及脂肪酸代谢等核心代谢通路中富集显著；对比分析进一步证实，体外扩增γδT细胞的细胞毒性评分高于肝细胞癌患者体内的肿瘤浸润及外周血γδT细胞。