Gene expression profiles of peripheral blood mononuclear cells (PBMC) from children with active idiopathic nephrotic syndrome (INS) and healthy controls (HC) [HC_vs_INS]

An autoimmune B cell origin for childhood idiopathic nephrotic syndrome (INS) is predicted based on the efficacy of rituximab (RTX) at maintaining long-term remission from proteinuria. Knowledge regarding the nature of the culprit B cell response is very limited. In particular, no transcriptomics work has been performed to evaluate the B cell response in INS. Using single-cell RNA-sequencing (scRNAseq) on peripheral blood mononuclear cells (PBMC) isolated from four affected children with active INS and four age/sex-matched health controls (HC), we demonstrate that a B cell transcriptional program poised for effector functions represents the major immune perturbation in the blood of children with active INS. We show that this nephrotic B cell signature is conferred by the engagement of memory B cells through an extrafollicular developmental route defined by expression of the interfollicular-homing gene GPR183 and the expansion of atypical B cells and marginal zone-like B cells. Moreover, we show that genes involved in APRIL signaling, which promotes extrafollicular antibody-secreting cell development, were substantially upregulated in B cells, monocytes, and dendritic cells from INS children. Collectively, our study provides evidence for an extrafollicular origin for humoral immunity in active INS. PBMC were isolated from the blood of four children at either first onset or relapse of INS and four healthy age-matched controls. Live lymphocytes and monocytes were sorted by fluorescence-activated cell sorting (FACS) and analyzed by single-cell RNA-sequencing (scRNAseq).

鉴于利妥昔单抗（rituximab, RTX）可维持蛋白尿的长期缓解，本研究推测儿童原发性肾病综合征（childhood idiopathic nephrotic syndrome, INS）的发病机制存在自身免疫性B细胞源性异常。目前关于致病B细胞应答的本质相关认知仍十分有限，尤其尚无针对INS患者B细胞应答开展的转录组学研究。
本研究对4例活动性INS患儿及4例年龄、性别匹配的健康对照（healthy controls, HC）的外周血单个核细胞（peripheral blood mononuclear cells, PBMC）开展单细胞RNA测序（single-cell RNA-sequencing, scRNAseq），结果显示，具备效应功能潜能的B细胞转录程序是活动性INS患儿外周血中最主要的免疫扰动特征。本研究证实，该肾病相关性B细胞特征源于记忆B细胞经由滤泡外发育通路的激活——该通路以滤泡归巢基因GPR183的表达为特征，同时伴随非典型B细胞及边缘区样B细胞的扩增。此外，本研究发现，在INS患儿的B细胞、单核细胞及树突状细胞中，参与增殖诱导配体（APRIL）信号通路（可促进滤泡外抗体分泌细胞发育）的基因均显著上调。综上，本研究为活动性INS患者体液免疫的滤泡外起源提供了实验依据。
本研究采集了4例初发或复发的INS患儿及4例年龄匹配的健康对照的外周血以分离PBMC，通过荧光激活细胞分选术（fluorescence-activated cell sorting, FACS）分选出活淋巴细胞与单核细胞，并开展单细胞RNA测序（scRNAseq）分析。