BACKGROUND: Natural killer (NK) cell-based therapies represent a promising approach for acute myeloid leukemia (AML) relapse, yet their efficacy is hindered by immunosuppressive factors such as transforming growth factor beta (TGF-β) in the tumor microenvironment. This study investigated the effects of TGF-β on NK cell cytotoxicity and migration using 2D and 3D co-culture models that mimic the leukemic microenvironment. METHODS: TGF-β production was evaluated in AML-derived leukemic cell lines and mesenchymal stromal cells (hTERT-MSCs) using ELISA. Bulk RNA sequencing (RNA-seq) was performed to analyze global gene expression changes in TGF-β-treated primary human NK cells. NK cell cytotoxicity and migration were assessed in 2D monolayer and 3D spheroid co-cultures containing hTERT-MSCs and leukemic cells using flow cytometry and confocal microscopy. RESULTS: Both leukemic cells and MSCs produced TGF-β, with increased levels observed in MSCs after co-culture with primary AML blasts. RNA sequencing revealed that TGF-β altered key gene pathways associated with NK cell cytotoxicity, adhesion, and migration, supporting its immunosuppressive role. In functional assays, TGF-β exposure significantly reduced NK cell-mediated cytotoxicity in a time-dependent manner and impaired NK cell infiltration into 3D spheroids, particularly in models incorporating MSCs. Additionally, MSCs themselves provided a protective environment for leukemic cells, further reducing NK cell effectiveness in 2D co-cultures. CONCLUSION: TGF-β suppresses both NK cell cytotoxicity and migration, limiting their ability to eliminate leukemic cells and infiltrate the bone marrow niche (BMN). These findings provide novel insights into TGF-β-mediated immune evasion mechanisms and provide important insights for the future design of NK-based immunotherapies and clinical trials.

Department of Biology Faculty of Medicine Masaryk University Brno Czechia

Department of Cell Biology Faculty of Science Charles University Prague Czechia

Department of Cellular and Molecular Immunoregulation International Clinical Research Center St Anne's University Hospital in Brno Brno Czechia

Department of Genetics and Microbiology Faculty of Science Charles University Prague Czechia

Institute of Clinical Immunology and Allergology 1st Faculty of Medicine Charles University Prague Czechia

Institute of Clinical Immunology and Allergology General University Hospital Prague Czechia

Institute of Hematology and Blood Transfusion Prague Czechia

International Clinical Research Center Faculty of Medicine Masaryk University Brno Czechia

Pediatric neonatal department Institute for the Care of Mother and Child Prague Czechia

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