Švubová Veronika, Janstová Lucie, Jedlička Marek, Mašínová Eva, Szabová Jana, Feglarová Tereza, Kuglerová Kateřina, Bosáková Veronika, Brodská Barbora, Boráková Kristýna, Kundrát David, Trsová Iva, Böhmová Martina, Kuželová Kateřina, Hrdý Jiří, Gašová Zdenka, Vydra Jan, Dostálová Merkerová Michaela, Hortová-Kohoutková Marcela, Frič Jan
Institute of Hematology and Blood Transfusion (IHBT), Prague, Czechia.
Department. of Cell Biology, Faculty of Science - Charles University, Prague, Czechia.
Immunotargets Ther. 2025 Jun 18;14:589-604. doi: 10.2147/ITT.S512700. eCollection 2025.
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.
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.
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.
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.
基于自然杀伤（NK）细胞的疗法是急性髓系白血病（AML）复发的一种有前景的治疗方法，然而肿瘤微环境中的免疫抑制因子如转化生长因子β（TGF-β）阻碍了其疗效。本研究使用模拟白血病微环境的二维和三维共培养模型，研究了TGF-β对NK细胞细胞毒性和迁移的影响。
使用酶联免疫吸附测定（ELISA）评估AML来源的白血病细胞系和间充质基质细胞（hTERT-MSCs）中TGF-β的产生。进行批量RNA测序（RNA-seq）以分析TGF-β处理的原代人NK细胞中的全局基因表达变化。使用流式细胞术和共聚焦显微镜在包含hTERT-MSCs和白血病细胞的二维单层和三维球体共培养中评估NK细胞的细胞毒性和迁移。
白血病细胞和间充质基质细胞均产生TGF-β，与原代AML母细胞共培养后，间充质基质细胞中TGF-β水平升高。RNA测序显示，TGF-β改变了与NK细胞细胞毒性、粘附和迁移相关的关键基因途径，支持其免疫抑制作用。在功能试验中,TGF-β暴露以时间依赖性方式显著降低NK细胞介导的细胞毒性，并损害NK细胞向三维球体的浸润，特别是在包含间充质基质细胞的模型中。此外，间充质基质细胞本身为白血病细胞提供了一个保护环境，进一步降低了二维共培养中NK细胞的有效性。
TGF-β抑制NK细胞的细胞毒性和迁移，限制了它们消除白血病细胞和浸润骨髓龛（BMN）的能力。这些发现为TGF-β介导的免疫逃逸机制提供了新的见解，并为未来基于NK细胞的免疫疗法设计和临床试验提供了重要的见解。
