基于单细胞测序筛选川崎病静脉注射丙种球蛋白无反应的关键生物指标

Screening the key biomarkers of intravenous immunoglobulin-unresponsive Kawasaki disease based on single cell sequencing

目的 应用单细胞测序(SCS)技术探索川崎病静脉注射丙种球蛋白(IVIG)治疗无反应患儿免疫细胞及表达变化,筛选相关生物指标。方法 选择2022年12月—2023年8月上海市儿童医院收治的川崎病患儿4例,其中3例为IVIG敏感患儿,1例为IVIG无反应患儿。抽取川崎病患儿急性期及治疗后体温平稳(恢复期)时的外周血2 mL,提取外周血单个核细胞,进行SCS数据分析。比较川崎病患儿的基本临床信息,SCS数据分析结果(IVIG无反应与IVIG敏感患儿急性期和恢复期各细胞类型占比和细胞数量)。进行基因本体论(GO)通路富集分析IVIG敏感和IVIG无反应患儿急性期和恢复期B淋巴细胞富集结果,以及B淋巴细胞差异表达基因(DEG)表达情况。结果 4例川崎病患儿中,男3例、女1例,年龄2.1~5.5岁,IVIG使用时发热天数5 d 2例、6 d 1例、4 d 1例,IVIG治疗无反应1例、治疗敏感3例,无冠状动脉(CAL)3例、CAL 1例(IVIG治疗无反应患儿同时并发CAL)。IVIG无反应患儿和IVIG敏感患儿急性期单核细胞数量占比均高于恢复期。IVIG无反应患儿急性期B淋巴细胞占比高于恢复期,而IVIG敏感患儿急性期B淋巴细胞占比低于恢复期;IVIG无反应患儿和IVIG敏感患儿急性期自然杀伤(NK)细胞、CD4~+T淋巴细胞、CD8~+T淋巴细胞占比均低于恢复期。IVIG无反应患儿恢复期CD8~+T淋巴细胞数量(4 603)多于急性期(3 555个),B淋巴细胞数量(329个)少于急性期(2 165个)。IVIG无反应患儿急性期和恢复期B淋巴细胞数量分别少于IVIG敏感患儿急性期(6 646)和恢复期(7 986)。IVIG无反应患儿恢复期与急性期CD8~+T淋巴细胞增幅大于IVIG敏感患儿。基因富集结果显示,IVIG敏感患儿治疗后的B淋巴细胞相关的免疫活化通路及免疫球蛋白受体DEG表达下调,包括免疫应答调节细胞表面受体信号通路、免疫应答激活信号通路、抗原受体介导的信号通路等;而IVIG无反应患儿恢复期的DEG富集与B细胞活化通路及免疫球蛋白受体表达通路无关。IVIG敏感患儿在不同疾病阶段的B恢复期细胞DEG P值差异显著的HSPH1、MS4A1、FCRLA、VPREB3、CD72、LAT2、HSP90AA1、RCSD1、CD79A、FKBP4、HLA-DRB1、MTND3、HSP90AB1、PTPRCAP、CD37(共15个基因)与B淋巴细胞的免疫、增殖、分化或功能有关,其中CD79A、MS4A1、FCRLA与B细胞受体的信号传导有关。结论 B淋巴细胞数量变化及DEG表达与IVIG治疗效果相关,CD8~+T淋巴细胞数量增多在川崎病患儿CAL发生机制中起重要作用。

Objective To screen the biological indicators related to intravenous immunoglobulin(IVIG)-unresponsive Kawasaki disease by single cell sequencing(SCS)and explore the expression of immune cells.Methods Four children with Kawasaki disease who were admitted to Shanghai Children's Hospital from December 2022 to August 2023 were enrolled in this study.There were 3 children sensitive to IVIG therapy and one childunresponsive to IVIG therapy.Peripheral venous blood(2 mL)was collected at acute stage and after body temperature was steady.Peripheral blood mononuclear cell(PBMC)was extracted,and the changes of immune cell were analyzed by SCS.The basic clinical characteristics of the patients and the results of SCS data analysis(ratio and cell counts of each cell type during acute phase and convalescent phase in IVIG unresponsive group and IVIG sensitive group)were compared.The Gene Ontology(GO)database was used to analyze the enrichment of B cells and the expression of differentially expressed genes(DEG)in the acute phase and convalescent phase.Results There were three boys and one girl,aged 2.1 to 5.5 years.When IVIG was used,the fever continued for 5 days in two patients,6 days in one,and 4 days in one.The patient who did not respond to IVIG therapy suffered from coronary artery lesion(CAL).The ratio of monocytes in the acute phase was higher than in the convalescent phase in the four patients.The ratio of B cells in the acute phase was higher than that in the convalescent phase inthe IVIG-unresponsive patient,while in the IVIG-sensitive patients,the ratio of B cells in the acute phase was lower than that in the convalescent phase.The ratio of natural killer(NK)cells,CD4+T cells,and CD8+T cells in the acute phase were all lower than those in the convalescent phase in all the patients.In the IVIG-unresponsive patient,the number of CD8+T cells(4603)increased and the number of B cells decreased(329)after treatment,which were significantly different from those in the acute phase(3555 and 2165 cells,respectively).The number of CD8+T cells and B cells in the IVIG-unresponsive patient were significantly lower than those in IVIG-sensitive patients in both acute phase and convalescent phase(6646 and 7986 cells,respectively).The increased number of CD8+T lymphocytes in convalescent and acute phase of IVIG-unresponsive patients was greater than that in IVIG-sensitive patients.Gene enrichment results showed that DEG related to immune activation pathways and immunoglobulin receptor expression in B cells were downregulated in IVIG-sensitive patients after IVIG treatment,including immune response regulatory cell surface receptor signaling pathways,immune response activation signaling pathways,and antigen receptor-mediated signaling pathways.In contrast,DEG in the IVIG-unresponsive patient showed no association with B cell activation pathways or immunoglobulin receptor expression pathways after treatment.Fifteen DEG with significant differences in P value at different stages of Kawasaki disease,including HSPH1,MS4A1,FCRLA,VPREB3,CD72,LAT2,HSP90AA1,RCSD1,CD79A,FKBP4,HLA-DRB1,MTND3,HSP90AB1,PTPRCAP,and CD37,were associated with immunity,proliferation,differentiation,and function of B cells.CD79A,MS4A1,and FCRLA were directly related to B cell receptor signaling.Conclusion Quantity change of B cells and DEG expressionare related to IVIG therapeutic effect,and increased CD8+T cells play an important role in the mechanism of CAL in children with Kawasaki disease.