Antigen-specific TCR-T cells from Rag2 gene-deleted pluripotent stem cells impede solid tumour growth in a mouse model.

The technology of adoptive transfer of T-cell receptor (TCR) engineered T cells is wildly investigated as it has the potential to treat solid cancers. However, the therapeutic application of TCR-T cells is hampered by the poor quality derived mainly from patients' peripheral blood, as well as heterogeneous TCRs caused by the mismatch between transgenic and endogenous TCRs. To improve the homogeneity, antigen-specificity and reduce possible autoreactivity, here we developed a technique to generate antigen-specific T cells from Rag2 gene-deleted pluripotent stem cells (PSCs) and further measured their anti-tumour efficacy. PSCs were first targeted with OT1 TCR into the Rag2 locus to prevent TCR rearrangement during T-cell development. The engineered PSCs were then differentiated through a two-step strategy, in vitro generation of haematopoietic progenitor cells, and in vivo development and maturation of TCR-T cells. Finally, the response to tumour cells was assessed in vitro and in vivo. The regenerated OT1-iT displayed monoclonal antigen-specific TCR expression, and phonotypic normalities in the spleen and lymph node tissues. Importantly, the OT1-iT cells eliminated tumour cells while releasing specific cytokines in vitro. Furthermore, adoptive transfer of OT1-iT cells suppresses solid tumour growth in tumour-bearing animals. Our study presents a novel and straightforward strategy for producing antigen-specific TCR-T cells in vivo from PSCs, allowing for allogeneic transplantation and therapy of solid tumours.

Co-Expression of Runx1, Hoxa9, Hlf, and Hoxa7 Confers Multi-Lineage Potential on Hematopoietic Progenitors Derived From Pluripotent Stem Cells.

The intrinsic factors that determine the fundamental traits of engraftment ability and multi-lineage potential of hematopoietic stem cells (HSCs) remain elusive. The induction of bona fade HSCs from pluripotent stem cells (PSCs) in dishes is urgently demanded but remains a great challenge in translational medicine. Runx1, Hoxa9, Hlf, and Hoxa7 are developmentally co-expressed during endothelial-to-hematopoietic transition and adult haematopoiesis. However, the expression of these factors fails to be turned on during in vitro hematopoietic induction from PSCs. Here, we established an inducible gene over-expression embryonic stem cell (ESC) line in which exogenous Runx1, Hoxa9, Hlf, and Hoxa7 genes were tandemly knocked in. A population of induced hematopoietic progenitor cells (iHPCs) expressing Kit and Sca1 surface markers were successfully obtained in vitro from the gene edited-ESC line. Upon transplantation of the Runx1-Hoxa9-Hlf-Hoxa7 ESC-derived iHPCs into irradiated immunodeficient mice, they can dominantly contribute to B cells, low proportions of T cells and myeloid cells. However, Runx1-Hoxa9-Hlf ESC-derived iHPCs only produced B lineage cells with extremely low contributions. Our study unveils that the coordination of Runx1, Hoxa9, Hlf, and Hoxa7 led to generation of the hematopoietic progenitors with the capacity of multi-lineage hematopoietic reconstitution in the immunodeficient recipient mice.