Inflammatory Cytokines Regulate T-cell Development from Blood Progenitor Cells in a Stage and Dose-Specific Manner

Abstract

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 (TNF) as enhancers of proT-cell differentiation and expansion. Mechanistically, we found that TNF induced T-lineage differentiation through the positive regulation of T-lineage genes GATA3, TCF7, and BCL11b. TNF 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. 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.