TGF-β Decreases NK Cell Mobility and Cytotoxic Efficacy in Complex in vitro Models of the Leukemia Microenvironment

Abstract

Veronika Švubová,1,2 Lucie Janstová,1 Marek Jedlička,1,2 Eva Mašínová,1,2 Jana Szabová,1 Tereza Feglarová,1 Kateřina Kuglerová,1 Veronika Bosáková,3,4 Barbora Brodská,1 Kristýna Boráková,5 David Kundrát,1 Iva Trsová,1,6 Martina Böhmová,1 Kateřina Kuželová,1 Jiří Hrdý,7,8 Zdenka Gašová,1 Jan Vydra,1 Michaela Dostálová Merkerová,1 Marcela Hortová-Kohoutková,3,9 Jan Frič1,3,9 1Institute of Hematology and Blood Transfusion (IHBT), Prague, Czechia; 2Department. of Cell Biology, Faculty of Science – Charles University, Prague, Czechia; 3Department. of Cellular and Molecular Immunoregulation, International Clinical Research Center – St. Anne’s University Hospital in Brno (FNUSA-ICRC), Brno, Czechia; 4Department. of Biology, Faculty of Medicine – Masaryk University, Brno, Czechia; 5Pediatric-neonatal department, Institute for the Care of Mother and Child, Prague, Czechia; 6Department. of Genetics and Microbiology, Faculty of Science – Charles University, Prague, Czechia; 7Institute of Clinical Immunology and Allergology, First Faculty of Medicine, Charles University, Prague, Czechia; 8Institute of Clinical Immunology and Allergology, General University Hospital, Prague, Czechia; 9International Clinical Research Center, Faculty of Medicine, Masaryk University, Brno, CzechiaCorrespondence: Jan Frič, Department of Modern Immunotherapy, Institute of Hematology and Blood Transfusion, U Nemocnice 1, Prague, 128 00, Czechia, Email [email protected] Marcela Hortová-Kohoutková, International Clinical Research Center of St. Anne’s University Hospital in Brno (FNUSA-ICRC), Pekařská 53, Brno, 656 91, Czechia, Email [email protected]: 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.Keywords: 3D models, TGFbeta, acute myeloid leukemia, bone marrow niche, NK cells, immunotherapy

Keywords

• 3D models
• acute myeloid leukemia
• bone marrow niche
• TGFbeta
• NK cells
• immunotherapy.