Abstract
T cells are pivotal effectors of the immune system and can be harnessed as therapeutics for regenerative medicine and cancer immunotherapy. An unmet challenge in the field is the development of a clinically relevant system that is readily scalable to generate large numbers of T-lineage cells from hematopoietic stem/progenitor cells (HSPCs). Here, we report a stromal cell-free, microbead-based approach that supports the efficient in vitro development of both human progenitor T (proT) cells and T-lineage cells from CD34+cells sourced from cord blood, GCSF-mobilized peripheral blood, and pluripotent stem cells (PSCs). DL4-μbeads, along with lymphopoietic cytokines, induce an ordered sequence of differentiation from CD34+ cells to CD34+CD7+CD5+ proT cells to CD3+αβ T cells. Single-cell RNA sequencing of human PSC-derived proT cells reveals a transcriptional profile similar to the earliest thymocytes found in the embryonic and fetal thymus. Furthermore, the adoptive transfer of CD34+CD7+ proT cells into immunodeficient mice demonstrates efficient thymic engraftment and functional maturation of peripheral T cells. DL4-μbeads provide a simple and robust platform to both study human T cell development and facilitate the development of engineered T cell therapies from renewable sources.
Highlights
T cells are pivotal effectors of the immune system and can be harnessed as therapeutics for regenerative medicine and cancer immunotherapy
The development of stromal-cell free systems recapitulates the requirement for Notch signaling, such as the use of plate-bound Notch ligands[7,14,15,16,17,18] and adhesion molecules, like VCAM119, for the generation of T-lineage cells and progenitor T cells, with proT cells being capable of thymus engraftment in immunodeficient mice
Previous stromal cell-free strategies have relied on two-dimensional (2D) tissue culture platforms using Delta-like 4 (DLL4) Fc-fusion proteins (DL4-Fc) immobilized on tissue-culture plates[7,9,18,19]
Summary
T cells are pivotal effectors of the immune system and can be harnessed as therapeutics for regenerative medicine and cancer immunotherapy. Several stromal-cell based approaches mimic the thymic environment by providing Notch ligands, including the OP9-DL4 coculture system[7,12] and the recently developed artificial thymic organoid system[8,13] These approaches enable Notch signaling to initiate the T cell program in hematopoietic stem/progenitor cells (HSPCs) in the absence of the thymus. The development of stromal-cell free systems recapitulates the requirement for Notch signaling, such as the use of plate-bound Notch ligands[7,14,15,16,17,18] and adhesion molecules, like VCAM119, for the generation of T-lineage cells and progenitor T cells (proT), with proT cells being capable of thymus engraftment in immunodeficient mice. The DL4-μbead culture system provides an avenue for the clinically relevant generation of T cells from engineered PSC lines
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