Inflammatory Cytokines Regulate T-Cell Development from Blood Progenitor Cells in a Stage and Dose-Specifc 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 α (TNFα) as enhancers of proT-cell differentiation and expansion. Mechanistically, we found that TNFα induced T-lineage differentiation through the positive regulation of T-lineage genes GATA3, TCF7, and BCL11b. TNFα 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 TNFα. 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.

DLL4 and VCAM1 enhance the emergence of T cell-competent hematopoietic progenitors from human pluripotent stem cells

“…We compared these two newly optimised media formulations (PSC optima) against the previously developed cord blood-optimised media 53 (UCB opt, Figure 4a,b). PSC optima improved total cellularity compared to the CB control as early as day 14 and the magnitude of this effect was amplified over the course of the differentiation (Figure 4c).…”

“…(Figure 4a-g). We transferred PSC-derived hematopoietic progenitors into a serum-free media supplemented with a cytokine composition that we previously optimised for generating CD4+, CD8+ DP cells from umbilical cord blood (UCB) 53 (Figure 4b). Unfortunately, this media did not efficiently support T cell maturation from hPSCs.…”

“…We have previously shown that statistical dose response models are highly effective for optimizing stage-specific differentiation media formulations 53 . We conducted a 6 cytokine, 5 concentration factorial-type experiment over two stages of T cell development (Supplementary Figure 6).…”