Transmembrane TNF-TNFR2 axis promotes generation of human T cell precursors.

The goal of this study is to dissect the impact of soluble (sTNF) and transmembrane (tmTNF) forms of tumour necrosis factor on the differentiation of human hematopoietic stem and progenitor cells (HSPCs) into T cell precursors. To achieve this, we utilized scRNA-sequencing technologies to compare the transcriptome profiles of HSPCs that had been differentiated in the artificial thymic organoid (ATO) system in the absence (control) or presence of TNF signal (soluble or transmembrane). CD3-CD14-CD19-CD56-CD34+ umbilical cord blood HSPCs from 4 donors were pooled and induced to undergo T cell development using the ATO system. In the absence of TNF signal (control), ATOs were supplemented with interleukin-7 and FMS-like tyrosine kinase 3 ligand. For sTNF-activated ATOs, organoids were supplemented additionally with 0.25 ng/mL of sTNF. For tmTNF-activated ATOs, 1 % of the stromal cells that were used to assemble organoids were engineered to express tmTNF. At day 10 post culture, CD45+ differentiating HSPCs were sorted from all three conditions and analysed using scRNA-seq.

本研究旨在解析可溶性肿瘤坏死因子（soluble TNF, sTNF）与跨膜型肿瘤坏死因子（transmembrane TNF, tmTNF）两种亚型对人类造血干祖细胞（human hematopoietic stem and progenitor cells, HSPCs）向T细胞前体分化的影响。为达成该研究目标，本研究采用单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）技术，对比人工胸腺类器官（artificial thymic organoid, ATO）系统中，分别在无（对照组）、存在TNF信号（可溶性或跨膜型）的培养条件下分化的HSPCs的转录组谱。本研究将来自4名供体的CD3⁻、CD14⁻、CD19⁻、CD56⁻、CD34⁺脐带血造血干祖细胞混合后，通过ATO系统诱导其向T细胞发育。其中，无TNF信号的对照组ATO体系仅添加白细胞介素-7（interleukin-7, IL-7）与FMS样酪氨酸激酶3配体（FMS-like tyrosine kinase 3 ligand）；sTNF激活组的ATO体系额外添加0.25 ng/mL的sTNF；tmTNF激活组的ATO体系中，1%的组装类器官所用的基质细胞（stromal cells）经工程化改造以表达tmTNF。培养至第10天时，从三种培养条件下均分选得到CD45⁺的分化HSPCs，采用scRNA-seq进行分析。