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Aging and disease    2020, Vol. 11 Issue (1) : 118-128     DOI: 10.14336/AD.2019.1017
Orginal Article |
Tumor-Derived Soluble MICA Obstructs the NKG2D Pathway to Restrain NK Cytotoxicity
Qizhi Luo1, Weiguang Luo1,2, Quan Zhu1, Hongjun Huang3, Huiyun Peng1, Rongjiao Liu1, Min Xie1, Shili Li1, Ming Li1, Xiaocui Hu3, Yizhou Zou1,*
1Department of Immunology, Basic Medical School of Central South University, Changsha, Hunan, China.
2Department of Physiology, University of Texas Southwestern Medical Center at Dallas, TX, USA
3Cancer Hospital of Hunan, Xiangya Medical School, Central South University, Changsha, Hunan, China.
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The natural killer group 2D (NKG2D) receptor and its ligands play important roles in immune surveillance. In this study, we observed that the average serum soluble MICA (sMICA) concentration of 174 hepatocellular carcinoma (HCC) patients was significantly higher than that in 80 healthy subjects (602.17 ± 338.15 vs. 72.26 ± 87.88 pg/ml, t = 3.107, P=0.002). The levels of serum sMICA in 44 HCC patients with initial levels above 400 pg/ml declined significantly after surgical removal of the liver cancer tissue (P<0.001). Moreover, the mean survival time of HCC patients who had sMICA above 400 pg/ml was significantly shorter than that HCC patients with lower sMICA levels (P<0.001). Using the reporter cell line (NKG2D-2B4) in which activation of the NKG2D receptor pathway results in GFP expression based on the stimulation of immobilized rMICA, we showed that the number of GFP-expressing cells decreased sharply in presence of sMICA. Upon adding sMICA, the release of cytokines IFN-γ, TNF-α, and IL-8 by NK cell line (NKL) under stimulation of immobilized rMICA was blocked. Using MICA-expressing cells as the target cells, we observed that about 80% of target cells were killed by NKL at E:T of 10:1, but in presence of sMICAhigh serum of HCC patients, the dead target cells were reduced to 30.8%. Compared in presence of sMICAlow serum from HCC patients, there were 63.7% of target cells dead (p=0.043). Thus, our data suggested that sMICA obstructs the activation of NKG2D pathway to protect tumor cells from NK cell-mediated cytotoxicity.

Keywords hepatocellular carcinoma (HCC)      NKG2D      soluble MICA      NKG2D receptor pathway      tumor immune escape     
Corresponding Authors: Yizhou Zou   
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These authors contributed equally to this work.

Just Accepted Date: 28 June 2019   Issue Date: 15 January 2020
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Qizhi Luo
Weiguang Luo
Quan Zhu
Hongjun Huang
Huiyun Peng
Rongjiao Liu
Min Xie
Shili Li
Ming Li
Xiaocui Hu
Yizhou Zou
Cite this article:   
Qizhi Luo,Weiguang Luo,Quan Zhu, et al. Tumor-Derived Soluble MICA Obstructs the NKG2D Pathway to Restrain NK Cytotoxicity[J]. Aging and disease, 2020, 11(1): 118-128.
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Figure 1.  Serum sMICA concentration is associated with poor prognosis in HCC. (A) Standard curve for quantitative detection of sMICA using double-antibody sandwich ELISA made using purified rMICA*004 at eight concentrations ranging from 100 to 10000 pg/ml. Plotted is OD vs. lg[rMICA]. (B) sMICA concentrations (pg/ml) in serum of healthy controls (N=80), HCC patients (N=174), and HCC patients’ post-surgical resection of tumors (N=44). The horizontal lines indicate means. ** Indicates P<0.05. (C) Serum sMICA levels before and after surgery in HCC patients with serum sMICA ≥ 400 pg/ml before treatment (N=44). Serum samples were taken immediately prior to surgery and 15 days after surgery. Lines pair points from individual patients. ** Indicates P<0.05. (D) Survival curves of HCC patients not treated surgically divided based on serum sMICA concentration: sMICAlow < 400 pg/ml (N=70, red) and sMICAhigh ≥ 400 pg/ml (N=60, black). Log-rank statistical analysis was performed on the survival curves between the two groups; ** Indicates P<0.05.
Figure 2.  sMICA does not activate the NKG2D receptor pathway. (A) Diagram of NKG2D receptor reporter system. The NKG2D chimeric receptor is an engineered receptor composed of the human NKG2D extracellular domain and the mouse NKG2D membrane-spanning region. Upon binding of ligand to the chimeric NKG2D receptor, downstream signaling results in production of GFP. (B) Flow cytometry analysis of NKG2D-2B4 or control cells (2B4 cells) incubated with anti-NKG2D monoclonal antibody, anti-His tag monoclonal antibody, anti-HLA-I monoclonal antibody (w6/32), or mouse isotype IgG. (C) Percentage of GFP+ cells in NKG2D-2B4 and 2B4 cells incubated with BSA or soluble rMICA *004 or incubated in wells pre-coated with rMICA. (D) The cytokine responses of NK cells to anti-NKG2D monoclonal antibody coated (anti-NKG2D), soluble rMICA, and immobilized rMICA after 24 hours. The supernatants were collected and concentrations of IFN-γ, TNF-α, and IL-8 were determined (pg/ml). Plotted are results of triplicate experiments.
Figure 3.  Soluble MICA molecule blocks the activation of NKG2D pathway. (A) Binding of soluble rMICA*004-His to NKG2D-2B4 cells detected by staining of the PE-conjugated anti-His-tag antibody. The log of mean fluorescence intensity (lg[MFI]) as a function of rMICA*004-His is plotted on the right-hand axis. The blocking effect on NKG2D pathway signaling was determined by analysis of GFP+ cells; the percent GFP+ cells in the presence of each concentration of rMICA*004-His is plotted on the left-hand axis. (B) Percentage of GFP+ cells in the presence of sMICAhigh patient sera (N=10), the same sera pre-treated with anti-MICA mAb6B3 beads (Absorbed, N=10), and sera from healthy volunteers (NHS, N=10). * indicates statistically significant difference (P < 0.05). (C-E) Cytokines (C) IFN-γ, (D) TNF-α, and (E) IL-8 released from NKL incubated with immobilized rMICA. The concentrations of cytokines in supernatant of samples treated with NHS were defined as 100%. The ratio of sample treated with sMICAhigh and Absorbed are given. * Indicates statistically significant difference (P < 0.05).
Figure 4.  sMICA inhibits the cytotoxicity of NK cells toward MICA+ target cells. (A) Schematic of components involved in the MICA-NKG2D pathway. NK effector cells were incubated with MICA+ target cells stained with CSFE dye. sMICA interferes with cell killing mediated by NK cells. (B) Target cells (5000 cells per assay) with (MICA+ HFC) or without MICA expressed human fibroblasts (MICA- HFC) were co-cultured with NKL at different E: T ratios. The mean percentages of dead cells from three replicate experiments are plotted. (C) The percent dead target cells in the presence of soluble rMICA, BSA at finally concentrations diluted from 0.1 to 8.0 ng/ml at E: T of 10:1. Plotted are means of triplicate experiments. (D) The percent dead target cells in the presence of serum from sMICAhigh patients (N=10) and from sMICAlow patients (N=10) at E:T of 10:1. Normal health serum (NHS) was used as negative control and soluble NKG2D-Ig at concentration at 1.0 μg/ml were used as maximum blocking controls. Plotted are means of three experiments for each tested (C and D).
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