Immunosuppressive effects of Transforming Growth Factor Beta (TGFβ) on cytotoxic function of human natural killer cells are reversible

Anjali Patel 1, Giuseppe Maiocco 1, Catherine Mirto 1, Donald S. Torry 1,2,3, and Andrew Wilber 1,3

1 Medical Microbiology, Immunology and Cell Biology, 2 Obstetrics and Gynecology, and 3 Simmons Cancer Institute, SIU School of Medicine, Springfield, IL 62702

Background: Natural killer (NK) cells are innate immune cells that are CD56pos/CD16pos and have anti-tumor function. We have shown that NK cells recovered from human kidney tumors are CD56pos/CD16dim/neg and poorly cytotoxic. These features are analogous to human decidual NK (dNK) cells, which are found at the maternal-fetal interface and support development of the placenta development during embryo implantation. The decidua and kidney tumor environment have high levels of transforming growth factor beta (TGFβ), which is known to convert peripheral blood NK (pNK) cells to dNK-like cells in vitro. We hypothesized that TGFβ-mediated inhibition of NK cell cytotoxic function is transient and therefore reversible.

Methods: pNK cells were isolated negative selection from blood of healthy donors (HD) or purchased from ATCC (NK-92; CRL-2407). RT-qPCR and flow cytometry were used to assess levels of TGFβ receptors (R1, R2, and R3) mRNA and protein, respectively. NK cells were cultured for 4-5 days with the proliferative cytokine IL2 without or with TGFβ (2-5 ng/mL). Cytotoxic function of NK cells (effectors) was quantified by lysis of human K562 cells engineered to express firefly luciferase (targets). For this, cells were mixed at 1:1, 1:2, and 1:5 target to effector ratio and cultured overnight. Statistics (ANOVA/t-test) were performed using GraphPad Prism with p≤0.05 considered significant.

Results: pNK cells from HD were CD56pos/CD16pos and NK-92 cells were CD56pos/CD16neg as reported. Both expressed equivalent levels of all three TGFβ receptors and were potently cytotoxic against K562 target cells. NK cells treated with TGFβ retained only 15% cytotoxic function compared with untreated cells (p≤0.05). Cytotoxic function was completely restored when TGFβ treatment was discontinued and NK cells cultured in standard conditions for 4-5 days.

Conclusions: Despite differences in CD16 expression, pNK cells and NK-92 cells are potently cytotoxic and represent an effective cancer therapy. TGFβ limits this potential in the tumor environment by converting pNK cells to non-cytotoxic dNK-like cells. Importantly, NK regained complete cytotoxic function when they were no longer exposed to TGFβ. Thus, inhibition of TGFβ signaling could preserve NK function in the tumor environment and have implications for anti-cancer therapies. Efforts to explore this possibility using selective inhibitors or genome editing technology are underway.