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

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