T-cell development from hematopoietic stem and progenitor cells (HSPCs) is tightly regulated through Notch pathway activation by the Notch ligands Delta-like (DL) 1 and 4 and Jagged-2. Other molecules, such as stem cell factor (SCF), FMS-like tyrosine kinase 3 ligand (Flt3L) and interleukin (IL)-7, play a supportive role in regulating the survival, differentiation, and proliferation of developing progenitor (pro)T-cells. Numerous other signaling molecules are known to instruct T-lineage development in vivo, but little work has been done to optimize their use for T-cell production in vitro. Using a defined T-lineage differentiation assay consisting of plates coated with the Notch ligand DL4 and adhesion molecule VCAM-1, we performed a cytokine screen that identified IL-3 and tumor necrosis factor @[alpha] (TNF@[alpha]) as enhancers of proT-cell differentiation and expansion. Mechanistically, we found that TNF@[alpha] induced T-lineage differentiation through the positive regulation of T-lineage genes GATA3, TCF7, and BCL11b. TNF@[alpha] also synergized with IL-3 to induce proliferation by upregulating the expression of the IL-3 receptor on CD34+ HSPCs, yielding 753.2 (532.4-1026.9; 5-95 percentile)-fold expansion of total cells after 14 days compared to 8.9 (4.3-21.5)-fold expansion in conditions without IL-3 and TNF@[alpha]. We then optimized cytokine concentrations for T-cell maturation. Focusing on T-cell maturation, we used quantitative models to optimize dynamically changing cytokine requirements and used these to construct a three-stage assay for generating CD3+CD4+CD8+ and CD3+CD4-CD8+ T-cells. Our work provides new insight into T-cell development and a robust in vitro assay for generating T-cells to enable clinical therapies for treating cancer and immune disorders.

中文翻译：

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炎症细胞因子以阶段和剂量特定的方式调节血液祖细胞的T细胞发育。

来自造血干细胞和祖细胞（HSPC）的T细胞发育通过Notch配体Delta-like（DL）1和4和Jagged-2的Notch途径激活而受到严格调节。其他分子，例如干细胞因子（SCF），FMS样酪氨酸激酶3配体（Flt3L）和白介素（IL）-7，在调节发育祖细胞（pro）T的存活，分化和增殖中起辅助作用-细胞。已知许多其他信号分子可指导体内T谱系发育，但很少有工作来优化它们在体外T细胞生产中的用途。使用定义的T谱系分化分析，该分析由涂有Notch配体DL4和粘附分子VCAM-1的板组成，我们进行了细胞因子筛选，鉴定了IL-3和肿瘤坏死因子α（TNFα）作为proT细胞分化和扩增的增强剂。从机理上讲，我们发现TNFα通过对T谱系基因GATA3，TCF7和BCL11b的正调控来诱导T谱系分化。TNFα也与IL-3协同作用，通过上调CD34 + HSPC上的IL-3受体的表达来诱导增殖，与14天后相比，总细胞扩增了753.2倍（532.4-1026.9； 5-95％）在没有IL-3和TNFα的条件下扩增至8.9（4.3-21.5）倍。然后，我们优化了T细胞成熟所需的细胞因子浓度。专注于T细胞的成熟，我们使用了定量模型来优化动态变化的细胞因子需求量，并使用它们构建了一个用于生成CD3 + CD4 + CD8 +和CD3 + CD4-CD8 + T细胞的三阶段测定法。我们的工作为T细胞的发展提供了新的见识，并为生成T细胞提供了强大的体外测定方法，从而使临床疗法能够治疗癌症和免疫疾病。