Fig. 2: iNK cells generated via a feeder-free differentiation protocol can be engineered to stably express a synNotch-based genetic construct.

A Diagram of TIGIT.SynNotch.aCD73 genetic construct structure consisting of TIGIT-SynNotch-GAL4-VP64 recognition domain and UAS-aCD73 inducible scFv. B Schematic diagram of TIGIT.SynNotch.aCD73 activation and subsequent secretion of aCD73 scFv directly at the NK:cancer interface. C Gene expression percentage (left) and MFI (right) of TIGIT.SynNotch.aCD73 on iNK cells following differentiation measured via mCherry expression via flow cytometry (n = 4 independent experiments; unpaired two-tailed t-test). D TIGIT expression percentage (left) and MFI (right) measured on TIGIT.SynNotch.aCD73-engineered iNK cells via flow cytometry (n = 5 independent experiments; unpaired two-tailed t-test). E Purity of CD34 + , CD34 + CD43 + , and CD34 + CD45+ cells following 11 days of hematopoietic progenitor differentiation (n = 3 independent experiments; multiple unpaired two-tailed t-tests). F Representative dot plots of data depicted in Fig. 2E. G Engineered iNK cell purity following 39 days of NK cell differentiation (n = 5 independent experiments; unpaired two-tailed t-test). H Engineered iNK cell purity following 39 days of NK cell differentiation and an additional 2 weeks of iNK cell expansion (n = 4 independent experiments; unpaired two-tailed t-test). I–K Flow cytometry phenotype of engineered iNK cells following 39 days of NK cell differentiation (n = 5 independent experiments; multiple unpaired two-tailed t-tests) as well as following 2 additional weeks of iNK cell expansion (n = 4 independent experiments; multiple unpaired two-tailed t-tests). J–L Fold expansion and viability of engineered iNK cells for 10 days following NK cell differentiation (n = 3 independent experiments; two-way ANOVA, Sidak multiple comparison test). Data are presented as mean values +/- SEM. Source data are provided as a Source Data file.