Optimizing Bcl11b timing enhances natural killer cell differentiation and cytotoxicity from human pluripotent stem cells for effective immunotherapy

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Abstract Human hematopoietic stem cells (HSCs) derived from induced pluripotent stem cells (iPSCs) are revolutionizing regenerative medicine by providing hope for therapies, disease modeling, and drug discovery in hematological disorders and beyond. However, producing functional iPSC-derived HSCs with in vivo engraftment and multilineage potential is challenging. The transcription factor BCL11B is crucial for T-cell development and has roles in immune and aortic cell function. Recently, it's been identified in lymphoid-myeloid clusters and hemogenic endothelium (HE) from human iPSCs. However, its role in human hematopoiesis and lymphopoiesis is unknown. Although BCL11B expression in human NK cells has been studied, direct evidence of its role in developing lymphoid cells is lacking. Here, we genetically engineered hiPSCs with inducible overexpression of BCL11B via CRISPR/Cas9-mediated gene knock-in and investigated the temporal role of this transcription factor during hematopoietic differentiation. We demonstrated that transient induction of BCL11B during the mesoderm stage led to significant increases in the frequencies of multipotent HPCs that give rise to the erythroid, myeloid, and lymphoid lineages. We also observed that BCL11B induces the arterial hemogenic endothelium specification phenotype (CXCR4+DLL4+) and markedly upregulates transcription of genes encoding arterial markers (HES1, HEY1, and SOX17) and HSC markers (HOXA5, HOXA9, HOXA10, and RUNX1). Combined with conventional HSC markers, we also observed the upregulation of the HSC-specific marker CD49f (ITGA6) from dox-treated iBCL11B-iPSCs. CD49f+ cells are more efficient in generating long-term multilineage grafts than CD49f- cells. Moreover, our results demonstrated that BCL11B overexpression significantly promoted NK cell generation under chemically defined culture conditions. The resulting iBCL11B-hiPSC-derived NK cells exhibited mature NK-specific markers and displayed robust cellular cytotoxicity, with more rapid killing kinetics than NK cells derived from no-dox-treated iBCL11B-iPSCs. To assess the in vivo potential of iNK cells, NSG mice were inoculated intraperitoneally (IP) with 3x10^5 Luc2-eGFP+ SKOV3 cells, and engraftment was monitored using IVIS bioluminescent imaging. On day 4 post-SKOV3 injection, the mice were treated with either 10^7 wild-type or iBCL11B NK cells via IP injection. Results showed that BCL11B iNK cells mediated faster and more complete solid tumor cell killing in vitro and significantly better tumor control in vivo. The gene expression profile of Dox-treated versus non-Dox iNK cells showed a significant increase transcripts encoding of cytotoxic proteins and markers of activation (GZMA, GZMB, PRF1, GNLY, CD69, CTSW) and transcription factors essential for NK cell maturation. We also developed a synthetic BCL11B mRNA with enhanced stability that could be transfected into primary NK cells at high frequencies. Introduction of BCL11B mRNA slowed NK cell proliferation while simultaneously driving maturation and acquisition of cytotoxic granule components. Taken together, our findings suggest that activating BCL11B increases the population of HPs with lymphoid lineage potential, making it a better off-the-shelf cell source for targeted cancer immunotherapy.

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