单细胞测序揭示自然杀伤细胞在青年和老年重症肺结核患者中的差异

Single-cell sequencing reveals differences in natural killer cells between young and elderly patients with severe pulmonary tuberculosis

目的:探讨青年和老年重症肺结核患者中的免疫细胞亚群差异,重点关注自然杀伤细胞(NK细胞)亚群相关功能和通路。方法:通过与公开研究文献的通信作者联系,获取4例重症肺结核患者[其中,2例青年患者(年龄≤40岁),2例老年患者(年龄≥65岁)]的单细胞测序数据和其他临床相关指标。对研究对象单细胞RNA测序数据进行质控、统计,去除不合格的细胞后,进一步进行非线性降维和聚类。利用公认的细胞标志物对相关免疫细胞进行注释,统计各样本和分组中的细胞类型百分比,比较各细胞亚型在不同分组中的变化趋势。同时,比较不同细胞类型之间的差异基因,通过京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genome,KEGG)富集分析探索这些基因在生物过程和通路中的作用。结果:对来源于重症肺结核青年患者中的15359个细胞和老年患者的9571个细胞进行注释,共鉴定到11个细胞亚群,占比最多的细胞类型分别是单核细胞(61%)、CD4^(+)T细胞(17%)、CD8^(+)T细胞(8%)、巨核细胞(5%)、B细胞(4%)及NK细胞(3%)。分析发现,青年患者NK细胞百分比[中位数(四分位数):4.339%(3.955%,4.722%)]高于老年患者[中位数(四分位数):0.822%(0.813%,0.831%)],但差异未见统计学意义(Z=-0.431,P=0.667);利用观察到的细胞数与期望细胞数的比值(Ro/e)统计学方法分析同样显示NK细胞在青年组中呈现增多趋势。进一步对NK细胞聚类分析揭示了3种不同NK细胞亚群,青年患者中高表达CD16的NK细胞百分比[中位数(四分位数)]为44.409%(41.672%,47.147%),略高于老年患者[中位数(四分位数):35.172%(32.169%,38.174%)],但差异未见统计学意义(Z=-0.431,P=0.667)。对比CD16高表达的NK细胞和中表达的NK细胞发现存在435个明显变化的基因,其中高表达的基因388个,低表达的基因47个。KEGG结果显示差异表达基因主要集中作用在细胞感知、细胞因子、B细胞受体、抗原加工提呈等信号通路。结论:本研究通过分析青年和老年重症肺结核患者外周血免疫细胞之间的差异,揭示了青年重症肺结核患者中NK细胞百分比,以及CD16^(+)NK细胞百分比增多,为肺结核患者的分级临床治疗提供了新的理论依据和潜在治疗靶点。

Objective:To investigate the differences in immune cell subsets between young and elderly patients with severe pulmonary tuberculosis,with particular emphasis on the functions and pathways of natural killer(NK)cell subsets.Methods:Single-cell sequencing data and other clinically relevant indicators were obtained by contacting the corresponding authors of publicly available research literature.Data were collected from four patients with severe pulmonary tuberculosis,comprising two young patients(age≤40 years)and two elderly patients(age≥65 years).The single-cell RNA sequencing data underwent quality control and statistical analysis.After excluding unqualified cells,the data were subjected to nonlinear dimensionality reduction and clustering.Recognized cellular markers were used to annotate immune cells and determine the percentage of each cell type in each sample and group.This enabled the comparison of cell subtype trends across different groups.Differential gene expression between cell types was analyzed,and the roles of these genes in biological processes and pathways were investigated using enrichment analysis in the Kyoto Encyclopedia of Genes and Genomes(KEGG).Results:A total of 15359 cells from young patients and 9571 cells from elderly patients with severe pulmonary tuberculosis were analyzed,leading to the identification of 11 distinct cell subpopulations.The most prevalent cell types included monocytes(61%),CD4^(+)T cells(17%),CD8^(+)T cells(8%),megakaryocytes(5%),B cells(4%),and NK cells(3%).The analysis revealed that the percentage of NK cells was higher in young patients(median(IQR):4.339%(3.955%,4.722%))compared to elderly patients(median(IQR):0.822%(0.813%,0.831%)),although this difference was not statistically significant(Z=-0.431,P=0.667).Statistical analysis using the ratio of observed to expected cell numbers indicated an increasing trend of NK cells in the young group.Further clustering analysis identified three distinct NK cell subgroups.The median percentage of NK cells with high CD16 expression was 44.409%(IQR:41.672%,47.147%)in young patients,slightly higher than the 35.172%(IQR:32.169%,38.174%)observed in elderly patients;however,this difference was not statistically significant(Z=-0.431,P=0.667).A comparison between NK cells with high CD16 expression and those with moderate expression revealed 435 significantly altered genes,comprising 388 upregulated genes and 47 downregulated genes.KEGG pathway analysis indicated that these differentially expressed genes were predominantly involved in signaling pathways related to cell perception,cytokine signaling,B cell receptor activity,and antigen processing and presentation.Conclusion:This study examined the differences in peripheral blood immune cells between young and elderly patients with severe pulmonary tuberculosis,highlighting an increased percentage of NK cells and CD16^(+)NK cells in young patients.These findings may offer a new theoretical basis and identify potential therapeutic targets for the stratified clinical management of tuberculosis patients.