Achievement of immunocompetent and therapeutic T lymphopoiesis from pluripotent stem cells (PSCs) is a central aim in T cell regenerative medicine. To date, preferentially reconstituting T lymphopoiesis in vivo from PSCs remains a practical challenge. Here we documented that synergistic and transient expression of Runx1 and Hoxa9 restricted in the time window of endothelial-tohematopoietic transition and hematopoietic maturation stages in a PSC differentiation scheme (iR9-PSC) in vitro induced preferential generation of engraftable hematopoietic progenitors capable of homing to thymus and developing into mature T cells in primary and secondary immunodeficient recipients. Single-cell transcriptome and functional analyses illustrated the cellular trajectory of T lineage induction from PSCs, unveiling the T-lineage specification determined at as early as hemogenic endothelial cell stage and identifying the bona fide pre-thymic progenitors. The induced T cells distributed normally in central and peripheral lymphoid organs and exhibited abundant TCRαβ repertoire. The regenerative T lymphopoiesis restored immune surveillance in immunodeficient mice. Furthermore, gene-edited iR9-PSCs produced tumor-specific T cells in vivo that effectively eradicated tumor cells. This study provides insight into universal generation of functional and therapeutic T cells from the unlimited and editable PSC source.

Deletion of B cell master regulators reprogrammed B cells into T cells that were either functional defects or tumorigenic potential. Here we show that Hoxb5, which is expressed in uncommitted hematopoietic progenitors but absent in committed B and T lineages, could reprogram pro-pre-B cells into functional early T cell progenitors. The reprogramming started in bone marrow and completed in thymus, giving rise to T lymphocytes with transcriptomes, hierarchical differentiation, tissue distribution and immune functions closely resembling their natural counterparts. Hoxb5 repressed B cell master genes, activated T cell regulators and regulated crucial chromatin modifiers in pro-pre-B cells, ultimately driving B to T cell fate conversion. Our results provide a de novo paradigm for generating normal and functional T cells through reprogramming in vivo.