Introduction

Induced pluripotent stem cells (iPSC) represent a promising platform for generating Natural Killer (NK) cells for standardized off-the-shelf immunotherapy. However, current feeder-free differentiation protocols typically yield immature iPSC-derived NK (iNK) cells. These iNK cells lack the expression of key receptors typically associated with final maturation stages like Killer Ig-Like Receptors (KIRs) and underperform when compared to peripheral blood derived NK (pNK) cells. This hinders further investment and clinical translation of iNK cells for immunotherapy. Although transcription factors such as T-bet are established regulators of NK cell maturation, their targeted application during iPSC to NK cell differentiation has not been completely elucidated. Here, we investigate whether temporally controlled T-bet expression can impact iNK cell differentiation to a more mature and cytotoxic phenotype.

Methods

A doxycycline inducible T-bet expression cassette was stably integrated into iPSCs via PiggyBac transposition. Cells were then differentiated to NK cell lineage using a feeder-free embryoid-based protocol. Doxycycline was administered from the hematopoietic progenitor (HPC) stage onwards to activate exogenous T-bet expression. Flow cytometry was performed longitudinally at weeks 3 to 5 of NK cell lineage commitment from HPC and at the very end of the differentiation protocol, to assess lineage progression, maturation (CD94, CD56 and KIRs) and activating receptors expression (CD16, NKG2D, NCRs). Functional assays included CD107a degranulation against K562, Raji and Daudi cells. Primary NK cells served as benchmarks.

Results

Unmodified iNK cells exhibited markedly lower expression of NKG2D (11.0% versus 99.8%), CD16 (4.6% versus 86.8%), and multiple KIRs (ranging from 0.5-2.1% versus 8.9-10.9%) compared to pNK cells. T-bet induction led to a 6- to 11-fold increase in the expression of KIRs when compared to unmodified iNKs, and a 1.6- to 3.3- fold increase when compared to pNK cells. The co-expression of CD56 and CD94 (an NK lineage commitment and maturation marker) was also significantly enhanced in modified cells compared to control iNK cells (78.9% versus 33.7%). Importantly, compared to unmodified controls, T-bet iNK cells demonstrated enhanced effector functions, with a 1.4-fold increase in cytotoxicity against K562 cells (measured by CD107a upregulation) and an average 3-fold increase in IFN-γ secretion across all target cell lines and upon PMA/Ionomycin stimulation.

Conclusion

Controlled T-bet induction during iPSC differentiation substantially enhances NK cell maturation, supported by increased expression of multiple KIRs and improved functional activity. These findings emphasize the advantage of transcription factor engineering to overcome maturation bottlenecks in iNK platforms. Ongoing single cell transcriptomic analysis will help to elucidate the downstream pathways modulated by T-bet and identify synergistic targets for further improving iNK functional fitness. Additionally, engineering iNK cells to express KIRs offers a tractable strategy to match patients' HLA profiles. This could enable iNK cell licensing, thereby improving cytotoxicity, cytokine production, and responsiveness to stress signals. These findings support transcriptional engineering as a potentially central pillar in advancing the clinical utility of iNK-cell based immunotherapies.

This content is only available as a PDF.
2025
Sign in via your Institution