Large granular lymphocyte leukemia (LGLL) is a rare lymphoproliferative disorder characterized by clonal expansion of cytotoxic CD8⁺ T cells or NK cells. Somatic activating mutations in STAT3 occur in over 50% of patients, yet their functional consequences remain poorly understood. A major limitation has been the lack of disease-relevant models for mechanistic studies of STAT3-driven leukemogenesis. To investigate STAT3 mutations in cell lines, patient samples, and T cells, we generated an expression vector with an EF1α promoter to drive expression of STAT3 fused to a C-terminal Strep-tagII followed by a ribosomal skip, P2A-linked truncated CD19 (tCD19) marker to enable antibody-based enrichment of modified cells. We validated this system in HEK293 cells with twelve STAT3 mutants spanning the coiled-coil, DNA-binding, and SH2 domains identified from genomic sequencing of LGLL patient samples. Western blot analysis confirmed equal Strep-tagII expression and elevated phosphorylation of STAT3 at Y705, a marker of activation, in mutants relative to wild-type. Preliminary data demonstrate that primary T cells can be efficiently transduced with our new STAT3 vector, with transgene expression confirmed by CD19 detected via flow cytometry. Ongoing work focuses on expanding these T cells to investigate how STAT3 mutations alter activation, protein-protein interactions, and DNA binding using mass spectrometry and CUT&Tag sequencing. In parallel, the HEK293 system is being used to characterize underexplored STAT3 variants through a transcriptional reporter assay. This dual approach provides a comprehensive foundation to dissect the contributions of STAT3 mutations in LGLL.
