Abstract: Objective　To investigate T cell subtypes and their functions in liver immune microenvironments among patients with alveolar echinococcosis (AE) using single⁃cell RNA sequencing (scRNA⁃seq).  Methods　Four AE patients that were admitted to Qinghai Provincial People's Hospital in 2023 for hepatic surgery for the first time were enrolled, and liver specimens were sampled 1 cm (peri⁃lesion, PL group) and > 5 cm from AE lesions (distal lesion, DL group) among each patient. Finally, a total of eight liver specimens were sampled from four AE patients for scRNA⁃seq analysis. Genome and transcriptome data of liver specimens were processed using the software Cell Ranger and R package. Differentially expressed genes (DEGs) and their biological functions were analyzed using gene ontology (GO) enrichment analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis, and the primary intercellular communication patterns and interaction mechanisms were identified among T cell subtypes in liver specimens using the CellChat package. In addition, the developmental stages of T cells were subjected to trajectory analysis with the monocle package to investigate the expression of genes associated with cell growth and tumor transformation, and to predict the developmental trajectories of T cells. Results　All four AE patients were female, with a mean age of (25.00 ± 9.06) years, and there were three cases from Jiuzhi County, Golog Tibetan Autonomous Prefecture and one case from Chengduo County, Yushu Tibetan Autonomous Prefecture, Qinghai Province. The viability of single⁃cell samples from eight liver specimens was 90.41% to 96.33%, and a total of 81 763 cells were analyzed, with 19 cell types annotated. Of these cell types, 13 were immune cells (87.60%), and T cells (33.13%), neutrophils (15.40%), and natural killer cells (11.92%) were the three most common cell types. Re⁃clustering of 27 752 T cells and proliferative T cells identified 10 distinct T cell subtypes, with CD8+ cytotoxic T cells (23.43%), CD8+ naive T cells (12.80%), and CD4+ effector memory T cells (17.73%) as dominant cell types. The proportions of T helper 2 (Th2) cells (5.19% vs. 3.63%; [χ2]  = 38.35, P < 0.01) and CD4+ effector memory T cells (21.59% vs. 13.67%; [χ2]  = 244.70, P < 0.01) were significantly higher in liver specimens in the PL group than in the DL group, and the proportion of CD4+ helper T cells was significantly lower in the PL group than in the DL group (7.50% vs. 14.75%; [χ2]  = 330.52, P < 0.01). KEGG pathway analysis revealed that Th2 cells were significantly enriched in cell apoptosis and multiple cancer⁃associated pathways, and CD4+ effector memory T cells were significantly enriched in the regulation of cytokines and chronic inflammation, while CD4+ helper T cells were significantly enriched in immune responses regulation. Trajectory analysis of T cells showed that CD4+ helper T cells were at an earlier developmental stage relative to Th2 cells and CD4+ effector memory T cells, and the expression of inhibitor of DNA binding 3 (ID3), thioredoxin interacting protein (TXNIP), Bcl2⁃associated athanogene 3 (BAG3) and heat shock protein family B (small) member 1 (HSPB1) genes appeared a tendency towards a decline over time. Conclusions　CD4+ effector memory T cells and CD8+ cytotoxic T cells are primary interacting cells in the liver specimens of AE patients. Reduced expression of Th2 cells and CD4+ helper T cells contributes to an inhibitory immune microenvironment, which promotes immune evasion by Echinococcus multilocularis, and Th2 cells are significantly enriched in multiple cancer⁃associated pathways, which may be linked to the invasive growth of E. multilocularis.

Key words: Alveolar echinococcosis, Single?cell RNA sequencing, Cell atlas, Liver, Immune microenvironment, T cell subtype 

摘要： 目的　应用单细胞转录组测序技术（single⁃cell RNA sequencing，scRNA⁃seq）探究泡型棘球蚴病（alveolar echinococcosis，AE）患者肝脏组织免疫微环境T细胞亚型及功能。方法　以青海省人民医院2023年收治的首次行肝脏手术的4例AE患者作为研究对象，取每例患者泡型棘球蚴寄生病灶外1 cm处肝组织作为病灶旁组（peri⁃lesion，PL）、距病灶> 5 cm的肝组织作为病灶远端组（distal lesion，DL），共收集4例AE患者的8份肝脏组织样本进行单细胞转录组测序。应用Cell Ranger软件和R软件读取、处理肝组织基因组及转录组数据，并利用基因本体论（gene ontology，GO）和京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes，KEGG）功能富集分析差异表达基因（differentially expressed genes，DEGs）及其生物学功能。使用Cellchat包分析肝组织T细胞亚型之间的主要通讯模式和相互作用机制，并通过Monocle包对T细胞的发育阶段进行拟时序分析，以明确细胞生长和肿瘤相关转化基因的表达情况，预测T细胞发育轨迹。结果　本研究4例AE患者均为女性，平均年龄为（25.00 ± 9.06）岁，分别来自青海省果洛藏族自治州久治县（3例）和玉树藏族自治州称多县（1例）。8份肝脏组织单细胞样本活性率为90.41%～96.33%，共获得81 763个单细胞；共注释出19种细胞类型，其中13种为免疫细胞（87.60%），占比居前3位的细胞类型分别为T细胞（33.13%）、中性粒细胞（15.40%）和自然杀伤细胞（11.92%）。对27 752个T细胞和增殖T细胞进行再聚类，注释出10种T细胞亚型，其中CD8+细胞毒性T细胞（23.43%）、CD8+幼稚T细胞（12.80%）和CD4+效应记忆T细胞（17.73%）占比较高。PL组肝组织中辅助型T细胞2（Th2细胞）和CD4+效应记忆T细胞占比分别为5.19%和21.59%，均显著高于DL组（3.63%和13.67%）（[χ2]  = 38.35、244.70，P均< 0.01）；PL组中CD4+辅助型T细胞（7.50%）占比低于DL组（14.75%）（[χ2]  = 330.52，P < 0.01）。富集分析结果显示，Th2细胞主要与细胞凋亡和多条癌症通路相关，CD4+效应记忆T细胞主要与细胞因子和慢性炎症调控相关，CD4+辅助型T细胞与免疫反应调控相关。T细胞发育轨迹分析结果显示，相较于Th2细胞和CD4+效应记忆T细胞，CD4+辅助型T细胞处于更早期发育阶段，且DNA结合抑制因子3（ID3）、硫氧还蛋白相互作用蛋白（TXNIP）、BAG家族伴侣蛋白3（BAG3）、热休克蛋白家族B（小分子）成员1（HSPB1）基因的表达量随时间延长呈下降趋势。结论　AE患者肝脏组织中，CD4+效应记忆T细胞和CD8+细胞毒性T细胞是主要的相互作用细胞。Th2细胞和CD4+辅助型T细胞的低表达形成了抑制性免疫微环境，促进了泡型棘球蚴的免疫逃逸。此外，Th2细胞富集了多条与癌症相关的通路，可能与泡型棘球蚴的浸润性生长相关。

关键词: 泡型棘球蚴病, 单细胞转录组测序, 细胞图谱, 肝脏, 免疫微环境, T细胞亚型