Abstract
The generation of T-cells from stem cells in vitro could provide an alternative source of cells for immunotherapies. T-cell development from hematopoietic stem and progenitor cells (HSPCs) is tightly regulated through Notch pathway activation by Delta-like (DL) ligands 1 and 4. Other molecules, such as stem cell factor (SCF) and interleukin (IL)-7, play a supportive role in regulating the survival, differentiation, and proliferation of developing T-cells. Numerous other signaling molecules influence T-lineage development in vivo, but little work has been done to understand and optimize their use for T-cell production. Using a defined engineered thymic niche system, we undertook a multi-stage statistical learning-based optimization campaign and identified IL-3 and tumor necrosis factor α (TNFα) as a stage- and dose-specific enhancers of cell proliferation and T-lineage differentiation. We used this information to construct an efficient three-stage process for generating conventional TCRαβ+CD8+ T-cells expressing a diverse TCR repertoire from blood stem cells. Our work provides new insight into T-cell development and a robust system for generating T-cells to enable clinical therapies for treating cancer and immune disorders.
Highlights
T-cell immunotherapies using cancer antigen-specific T-cell receptors (TCRs) or chimeric antigen receptors (CARs) have emerged as a potent treatment option for diseases such as B-cell acute lymphoblastic leukemia (B-ALL) and diffuse large B-cell lymphoma[1]
1234567890():,; We have previously described a defined engineered thymic Interactions between tumor necrosis factor α (TNFα) and the Notch pathway enhances niche (ETN) system for generating proT-cells from human umbilical cord blood (UCB)-derived CD34+ HSPCs22
To elucidate a mechanism for the synergistic effect of IL-3 and TNFα, we examined the expression of the IL-3 receptor (CD123) after stimulation of CD34+ hematopoietic stem and progenitor cells (HSPCs) with TNFα (Fig. 3e)
Summary
T-cell immunotherapies using cancer antigen-specific T-cell receptors (TCRs) or chimeric antigen receptors (CARs) have emerged as a potent treatment option for diseases such as B-cell acute lymphoblastic leukemia (B-ALL) and diffuse large B-cell lymphoma[1]. Cell development requires reduced Notch pathway activation around the β-selection checkpoint[30], we titrated DL4 and reduced the concentration 7.5-fold while maintaining similar proportions of proT-cells (Supplementary Fig. 5). On day 7, large populations of proT and CD4ISP cells were present in cultures These cells responded strongly to increasing concentrations of SCF, IL-3, and TNFα (Fig. 4b). An optimized three-stage process for T-cell generation from blood stem cells In order to define a set of preferred conditions for each step in the differentiation, we optimized the RSMs to find the cytokine concentrations that maximized the number of cells in different populations for each 7-day interval. The three-stage optimum cytokines enhance survival and/or proliferation to provide a substantial increase in CD3+TCRαβ+ cells expressing a diverse TCR repertoire These results validate the optimization methodology used for predicting dynamic cytokine signaling requirements during development. Cells generated using this technology can mature into phenotypically elaborated CD8SP T-cells that are capable of secreting cytokines upon nonspecific TCR stimulation
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