Immune checkpoint inhibitors targeting PD-1/PD-L1 axis have shown promising results in patients with non-small-cell lung cancer (NSCLC); however, the objective response rate is less than 30%. One major PD-L1 inducer, interferon γ (IFNg), has anti-tumour properties and is secreted by T cells and NK cells. IFNg-induced PD-L1 is considered to be a major mechanism that facilitates cancer cell escape from host immunity. Therefore, a better understanding of how IFNg stimulus influences the regulation of PD-L1 expression in cancer cells is warranted.
NSCLC cell lines: A549, PC-9, and LC2/ad were used for in vitro assays. Comparative analysis between IFNg and EGF was conducted in LC2/ad cells because EGF stimuli also enhanced PD-L1 expression in this cell line. PD-L1 expression was evaluated using flow cytometry while cell signalling pathway analysis was assessed using phospho-receptor tyrosine kinase arrays. To assess the NK cell-mediated cytotoxicity, NK cells were collected from healthy donors. NK killing was evaluated by lactate dehydrogenase release assay. Apoptotic cells were assessed via flow cytometry using Annexin V.
IFNg stimulus significantly upregulated PD-L1 expression in all cell lines. In LC2/ad cells, IFNg activated STAT1 signalling while EGF activated AKT, MAPK, and ribosomal protein S6 (RPS6). Although IFNg-induced PD-L1 was clearly blocked by the JAK-STAT inhibitor, tofacitinib, EGF-induced PD-L1 was not. Both IFNg- and EGF-induced PD-L1 were blocked by PI3K and MAPK inhibitors; however, they were not blocked by the RPS6 inhibitor. Interestingly, IFNg downregulated NK cell-activating ligand expression while upregulating MHC class I molecules, the phenotype of which can theoretically escape NK cells easily. Thus, we confirmed that IFNg stimulus attenuated NK cell-mediated cytotoxicity in LC2/ad cells; however, tofacitinib blocked IFNg-reduced NK killing. We also confirmed that tofacitinib, at concentrations less than 3 μM, did not cause NK cell apoptosis.
Tofacitinib blocks IFNg-induced immunoescape from NK cells in NSCLC cells, thereby suggesting that NK cell therapy combined with tofacitinib might be a promising strategy in overcoming tumour immune escape.
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Legal entity responsible for the study
Japan Society for the Promotion of Science (JSPS) Kakenhi Grant (25462189 and 16K10696),.
M. Nakata: Research grant / Funding (institution), The sponsor had no control over the interpretation or presentation of this work.: Kyowa Kirin. All other authors have declared no conflicts of interest.