STAT3 Activation Promotes NK Cell Proliferation, NKG2D Expression, and NK Cell Antitumor Activity
Authors: Phatarpekar Prasad , Zhu Shiguo , Denman Cecele J. , Nguyen-Jackson Nam , Watowich Stephanie S. , Cooper Laurence J.N. , Lee Dean A. 
Published: October 2019

Abstract
Abstract 105

Background:
Signal Transducer and Activator of Transcription 3 (STAT3) activation plays an important role in carcinogenesis, and is recognized as a therapeutic target in many human malignancies. STAT3 was also found to be an important negative regulator in cellular immunity, and in some animal models inhibition of STAT3 has been effective in improving NK antitumor efficacy. However, there has been little direct assessment for the role of STAT3 signaling in human NK cells. We previously described robust ex vivo expansion and activation of NK cells by cocultivation with K562-based artificial antigen presenting cells (aAPC) genetically modified to express membrane bound IL-21 (mIL21). We found that mIL21 results in greater proliferation, longer telomere length, and less senescence than NK cells expanded with mIL15. Since STAT3 is predominantly activated by IL-21 whereas IL-15 predominantly activates STAT5, we hypothesized that activation of STAT3 plays a critical role in NK cell expansion and anti-tumor activity. We also found that the histone deacetylase (HDAC) inhibitor MS275 upregulates NKG2D expression, whereas the HDAC inhibitor valproic acid downregulates NKG2D expression. We found that STAT3 is constitutively phosphorylated in NK cells, and valproic acid, but not MS275, efficiently inhibits STAT3 tyrosine phosphorylation. This led us to hypothesize that constitutive STAT3 phosphorylation is required for NKG2D expression and NK cell antitumor activity.

Objective:
To assess the role of STAT3 phosphorylation in NK cell proliferation, NKG2D expression, and antitumor activity.

Methods:
In human NK cells, small molecule STAT3 inhibitors JSI-124 and S3I-201 were applied to block STAT3 phosphorylation. In mice, floxed STAT3 was deleted in hematopoietic cells by Cre expression under the Tie2 promoter. NK cell proliferation was induced by K562 aAPCs expressing mIL21. IL10 and IL21 were applied to induce STAT3 phosphorylation. STAT3 phosphorylation was detected by western blot and phospho-flow. NKG2D expression and NK cell cytoxicity were evaluated by flow cytometry and Calcein release assay. STAT3 binding to DNA upstream of NKG2D was assessed by ChIP assay. Statistical comparison was performed by paired Student's t test using GraphPad Prism.

Results:
Analysis of phosphorylated STAT3 in NK cells by phospho-flow showed increased levels of activated STAT3 in NK cells stimulated with mIL-21 compared to mIL-15. We found that inhibition of STAT3 strongly suppressed the expansion of NK cells and significantly reduced cytotoxicity of expanded NK cells. We found that NKG2D surface expression on NK cells was significantly downregulated by STAT3 inhibitors, and accordingly, NK cell mediated killing activity was decreased. Conversely, STAT3 activation in primary NK cells by IL10 and IL21 upregulated NKG2D expression, resulting in increased NK cell mediated tumor lysis that could be reversed by small molecule STAT3 inhibition. We then analyzed NKG2D expression on NK cells in mice, and found that NKG2D expression in STAT3 knockouts was significantly lower than on wild type mice. By ChIP assay, we found that phosphorylated STAT3 directly binds upstream of NKG2D. IL10 and IL21 increased this binding, and STAT3 inhibition decreased this binding.

Conclusions:
Our finding of an immune-activating role for STAT3 in NK cells differs from previous reports, and is for the first to show that NK cell proliferation, NKG2D expression, and cytotoxicity is regulated by STAT3 phosphorylation. This novel role for STAT3 signaling may be important in the application of targeted STAT3 therapies and in improving NK cell therapeutic efficacy for patients with cancer. Work is in progress to delineate downstream signaling of STAT3 in NK cells.

Disclosures:
No relevant conflicts of interest to declare.