T-BET promotes inflammatory specialization without driving terminal maturation of iPSC-derived NK cells

Induced pluripotent stem cells (iPSCs) provide a scalable platform for off-the-shelf immune cell therapies; however, the mechanisms controlling maturation and functional specialization of iPSC-derived natural killer (iNK) cells are not fully understood. In this study, we combined single-cell transcriptomics, phenotypic profiling, and functional assays to characterize iNK cells and to determine whether temporally controlled expression of the transcription factor T-BET (TBX21) can promote iNK terminal specification and effector functions. Single-cell RNA sequencing and phenotypic profiling confirmed that baseline iNK cells form a transcriptionally distinct NK population and retain a developmental transcriptional signature. These iNK cells exhibited incomplete terminal maturation compared with peripheral blood NK cells, expressing much of the cytotoxic machinery but displaying limited acquisition of maturation-associated receptors. Despite induction of selected terminal specification markers, T-BET did not shift iNK cells toward a peripheral NK-like transcriptional identity. Instead, T-BET selectively triggered an inflammatory effector program characterized by elevated CCL3 , CCL5 and GZMA expression and increased IFNγ production across multiple tumor targets, without enhancement of terminal cytotoxic gene programs. These findings suggest a functional dissociation between inflammatory and cytotoxic programs in iPSC-derived NK cells. This work highlights transcriptional engineering as a strategy to modulate functional state in stem cell-derived NK cells.