Enhanced interaction between natural killer cells and lung cancer cells: involvement in gefitinib-mediated immunoregulation.
BACKGROUND
Natural killer (NK) cells can kill tumor cells in a non-MHC-restricted manner. However, cancer cells frequently escape from the attack of NK cells by multiple ways. In this study, we investigated the effect of gefitinib on the interaction between NK cells and lung cancer cells.


METHODS
⁵¹Cr release assay, CD107a assay, and IFN-γ secretion assay were performed to detect the sensitivity of lung cancer cell lines A549 and H1975 to NK cells cytotoxicity in the presence of gefitinib. Human NK cells were co-cultured with A549 and H1975 cell lines in the presence of gefitinib. NKG2D ligands, ULBP1, ULBP2, MICA, and MHC-I on tumor cells, and NKG2D, NKp44 and NKp46 on NK cells were evaluated with flow cytometry. 51Cr release assay was performed when NKG2D antibody were added into the co-culture system. Expressions of stat3 and LC3 I/II on tumor cells were determined with western blot after co-cultured with NK cells. After treated with gefitinib, mannose-6-phosphate receptor (MPR) on H1975 cells was evaluated by flow cytometry. ⁵¹Cr release assay were performed when MPR antagonist were used.


RESULTS
Gefitinib increased cytotoxicity of NK cells to human lung cancer H1975 cells with EGFR L858R + T790M mutations, while not in A549 cells with wild type EGFR. Gefitinib could block the immune escape by up-regulating the expression of NKG2D ligands ULBP1, ULBP2 or MICA on tumor cells and NKG2D on NK cells in the co-culture system. Gefitinib and NK cells up-regulated MHC-I expression in A549 while not in H1975 cells. NKG2D antibody blocked the enhanced NK cytotoxicity by gefitinib. The combination of NK cells and gefitinib could significantly down-regulate stat3 expression. Furthermore, NK cells-mediated tumor cell autophagy was observed in A549 cells while not in H1975 cells. Notably, gefitinib increased autophagy and MPR expression in H1975 cells, which improved the sensitivity to NK cell-based immunotherapy.


CONCLUSIONS
Gefitinib greatly enhanced NK cell cytotoxicity to lung cancer cells with EGFR L858R + T790M resistance mutation. Combination of EGFR tyrokinase inhibitors and NK cells adoptive immunotherapy may represent a potentially effective strategy for patients with non-small cell lung cancer.
Department of Thoracic Oncology, Cancer Center, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, PR China. wangys75@gmail.com.
Sisi
S
He
Tao
T
Yin
Dan
D
Li
Xiang
X
Gao
Yang
Y
Wan
Xuelei
X
Ma
Tinghong
T
Ye
Fuchun
F
Guo
Jianhong
J
Sun
Ziqiang
Z
Lin
Yongsheng
Y
Wang
D001343:Autophagy / Q000187:drug effects / Q000276:immunology; D002450:Cell Communication / Q000187:drug effects / Q000276:immunology*; D015550:Cell Degranulation / Q000187:drug effects; D045744:Cell Line, Tumor; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D066246:ErbB Receptors / Q000235:genetics; D000077156:Gefitinib; D015395:Histocompatibility Antigens Class I / Q000378:metabolism; D006801:Humans; D056747:Immunomodulation / Q000187:drug effects; D007371:Interferon-gamma / Q000096:biosynthesis; D007694:Killer Cells, Natural / Q000187:drug effects / Q000276:immunology* / Q000502:physiology; D008024:Ligands; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000276:immunology* / Q000473:pathology; D009154:Mutation / Q000235:genetics; D055655:NK Cell Lectin-Like Receptor Subfamily K / Q000378:metabolism; D011799:Quinazolines / Q000494:pharmacology / Q000627:therapeutic use*; D017527:Receptor, IGF Type 2 / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000378:metabolism
D015395:Histocompatibility Antigens Class I; C527085:KLRK1 protein, human; D008024:Ligands; D055655:NK Cell Lectin-Like Receptor Subfamily K; D011799:Quinazolines; D017527:Receptor, IGF Type 2; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D007371:Interferon-gamma; C512478:EGFR protein, human; D066246:ErbB Receptors; D000077156:Gefitinib