Tumor Immunotherapy Using NK-92Cells Carrying TGF-β Receptor Ⅱ And NKG2D

Objective：Tumor cells could inhibit NK and T cell function and promote tumor developmentthrough TGF-β1secretion. The capacity of NK cells to kill tumor cells depends on thecombined effect of suppressive and stimulatory signals delivered through surfacereceptors. NKG2D is one of the major activation receptors on NK cells. Therefore wefirst cloned the TGF-βRⅡ extracellular and transmembrane regions and intracellulardomain of NKG2D gene (TN), then we cloned the TN gene and infected the NK-92cells through lentiviral infection. We further evaluated the anti-tumor effects ofNK-92-TN cells through in vitro and in vivo experiments.Methods：（1） Overlap extension PCR was used to clone the extracellular andtransmembrane domains of human TGF-β receptorⅡand the intracellular domain ofNKG2D (TN) gene. The recombinant lentiviral shuttle plasmid was constructed andused to infect the293T cells through the superphosphate transfection to get thepseudotype virus to infect NK-92cells.（2） ELSIA method was used to detect the TGF-β1secertion by tumor cellsincluding SMMC7721and HepG2cells.（3） CCK-8assay, transwell assay analysis were used to analyze the viability andthe migration ability of the NK-92-TN cells.（4） Flow cytometry and cytotoxicity assay were used to analyze the activationand the killing capacity of the NK-92-TN cells in the presence or absence of TGF-β1.（5） CD4+T cells were cocultured with NK-92-TN cells in the presence ofTGF-β1to analyze their influence on regulatory T cell differentiation.（6） To evaluate the anti-tumor effects of the NK-92-TN cells with the nude micetumor model. Results:（1） The extracellular and transmembrane domains of human TGF-β receptorⅡwere combined with the intracellular domain of NKG2D (TN). NK-92stable cell linecarrying TN gene is generated through lentiviral infection.（2） TGF-β1was secreted by tumor cells, including SMMC7721and HepG2.（3） The results of CCK-8and transwell assay showed that NK-92-TN hasincreased viability and migration compared with the control cell line.（4） NK-92-TN cells expressed higher levels of NKG2D，which could not bedown-regulated by TGF-β1. TGF-β1could also stimulate IFN-γ production inNK-92-TN cells.（5） NK-92-TN cells could inhibit regulatory T cell differentiation in a dosedependment manner.（6） NK-92-TN cells alone or combined with radiotherapy could inhibit tumorgrowth in vivo.