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.
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.
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).
Nolte-'t HoenEN, AlmeidaCR, CohenNR, NedvetzkiS, YarwoodH, et al. (2007). Increased surveillance of cells in mitosis by human NK cells suggests a novel strategy for limiting tumor growth and viral replication. Blood, 109: 670-673.
DaiKZ, RyanJC, NaperC, VaageJT (2018). Identification of MHC Class Ib Ligands for Stimulatory and Inhibitory Ly49 Receptors and Induction of Potent NK Cell Alloresponses in Rats. J Immunol, 200: 2847-2859.
JelencicV, SestanM, KavazovicI, LenarticM, MarinovicS, et al. (2018). NK cell receptor NKG2D sets activation threshold for the NCR1 receptor early in NK cell development. Nat Immunol, 19: 1083-1092.
ChampsaurM,LanierLL (2010). Effect of NKG2D ligand expression on host immune responses. Immunol Rev, 235: 267-285.
GrohV, RhinehartR, Randolph-HabeckerJ, ToppMS, RiddellSR, et al. (2001). Costimulation of CD8alphabeta T cells by NKG2D via engagement by MIC induced on virus-infected cells. Nat Immunol, 2: 255-260.
JamiesonAM, DiefenbachA, McMahonCW, XiongN, CarlyleJR, et al. (2002). The role of the NKG2D immunoreceptor in immune cell activation and natural killing. Immunity, 17: 19-29.
VernerisMR, KarimiM, BakerJ, JayaswalA, NegrinRS (2004). Role of NKG2D signaling in the cytotoxicity of activated and expanded CD8+ T cells. Blood, 103: 3065-3072.
BaconL, EagleRA, MeyerM, EasomN, YoungNT, et al. (2004). Two human ULBP/RAET1 molecules with transmembrane regions are ligands for NKG2D. J Immunol, 173: 1078-1084.
ZwirnerNW, Fernandez-VinaMA, StastnyP (1998). MICA, a new polymorphic HLA-related antigen, is expressed mainly by keratinocytes, endothelial cells, and monocytes. Immunogenetics, 47: 139-148.
GrohV, WuJ, YeeC, SpiesT (2002). Tumour-derived soluble MIC ligands impair expression of NKG2D and T-cell activation. Nature, 419: 734-738.
ZouY, BresnahanW, TaylorRT, StastnyP (2005). Effect of human cytomegalovirus on expression of MHC class I-related chains A. J Immunol, 174: 3098-3104.
LanierLL (2015). NKG2D Receptor and Its Ligands in Host Defense. Cancer Immunol Res, 3: 575-582.
GhadiallyH, BrownL, LloydC, LewisL, LewisA, et al. (2017). MHC class I chain-related protein A and B (MICA and MICB) are predominantly expressed intracellularly in tumour and normal tissue. Br J Cancer, 116: 1208-1217.
MorettaA, BottinoC, VitaleM, PendeD, CantoniC, et al. (2001). Activating receptors and coreceptors involved in human natural killer cell-mediated cytolysis. Annu Rev Immunol, 19: 197-223.
TakakiR, WatsonSR, LanierLL (2006). DAP12: an adapter protein with dual functionality. Immunol Rev, 214: 118-129.
WangT, SunF, WangY, JiangJ, PanM, et al. (2018). NKG2D Immunoligand rG7S-MICA Enhances NK Cell-mediated Immunosurveillance in Colorectal Carcinoma. J Immunother, 41: 109-117.
CaoW, XiX, HaoZ, LiW, KongY, et al. (2007). RAET1E2, a soluble isoform of the UL16-binding protein RAET1E produced by tumor cells, inhibits NKG2D-mediated NK cytotoxicity. J Biol Chem, 282: 18922-18928.
SalihHR, RammenseeHG, SteinleA (2002). Cutting edge: down-regulation of MICA on human tumors by proteolytic shedding. J Immunol, 169: 4098-4102.
WaldhauerI, GoehlsdorfD, GiesekeF, WeinschenkT, WittenbrinkM, et al. (2008), Tumor-associated MICA is shed by ADAM proteases. Cancer Res, 68: 6368-6376.
SunD, WangX, ZhangH, DengL, ZhangY (2011). MMP9 mediates MICA shedding in human osteosarcomas. Cell Biol Int, 35: 569-574.
ChitadzeG, LettauM, LueckeS, WangT, JanssenO, et al. (2016). NKG2D- and T-cell receptor-dependent lysis of malignant glioma cell lines by human gammadelta T cells: Modulation by temozolomide and A disintegrin and metalloproteases 10 and 17 inhibitors. Oncoimmunology, 5: e1093276.
JiangX, HuangJF, HuoZ, ZhangQ, JiangY, et al. (2012). Elevation of soluble major histocompatibility complex class I related chain A protein in malignant and infectious diseases in Chinese patients. BMC Immunol, 13: 62.
Huergo-ZapicoL, Gonzalez-RodriguezAP, ContestiJ, GonzalezE, Lopez-SotoA, et al. (2012). Expression of ERp5 and GRP78 on the membrane of chronic lymphocytic leukemia cells: association with soluble MICA shedding. Cancer Immunol Immunother, 61: 1201-1210.
KlossS, ChambronN, GardlowskiT, ArsenievL, KochJ, et al. (2015). Increased sMICA and TGFbeta1 levels in HNSCC patients impair NKG2D-dependent functionality of activated NK cells. Oncoimmunology, 4: e1055993.
ZouY, LuoW, GuoJ, LuoQ, DengM, et al. (2018). NK cell-mediated anti-leukemia cytotoxicity is enhanced using a NKG2D ligand MICA and anti-CD20 scfv chimeric protein. Eur J Immunol, 48: 1750-1763.
DuanS, GuoW, XuZ, HeY, LiangC, et al. (2019). Natural killer group 2D receptor and its ligands in cancer immune escape. Mol Cancer, 18: 29.
FrazaoA, RethackerL, MessaoudeneM, AvrilMF, ToubertA, et al. (2019). NKG2D/NKG2-Ligand Pathway Offers New Opportunities in Cancer Treatment. Front Immunol, 10: 661.
DengM, GuiX, KimJ, XieL, ChenW, et al. (2018). LILRB4 signalling in leukaemia cells mediates T cell suppression and tumour infiltration. Nature, 562: 605-609.
MingY, HuJ, LuoQ, DingX, LuoW, et al. (2015). Acute Antibody-Mediated Rejection in Presence of MICA-DSA and Successful Renal Re-Transplant with Negative-MICA Virtual Crossmatch. PLoS One, 10: e0127861.
LesokhinAM, HohlTM, KitanoS, CortezC, Hirschhorn-CymermanD, et al. (2012). Monocytic CCR2(+) myeloid-derived suppressor cells promote immune escape by limiting activated CD8 T-cell infiltration into the tumor microenvironment. Cancer Res, 72: 876-886.
LiT, YangY, HuaX, WangG, LiuW, et al. (2012). Hepatocellular carcinoma-associated fibroblasts trigger NK cell dysfunction via PGE2 and IDO. Cancer Lett, 318: 154-161.
SunC, XuJ, HuangQ, HuangM, WenH, et al. (2017). High NKG2A expression contributes to NK cell exhaustion and predicts a poor prognosis of patients with liver cancer. Oncoimmunology, 6: e1264562.
BugideS, GreenMR, WajapeyeeN (2018). Inhibition of Enhancer of zeste homolog 2 (EZH2) induces natural killer cell-mediated eradication of hepatocellular carcinoma cells. Proc Natl Acad Sci U S A, 115: E3509-E3518.
SheppardS, GuedesJ, MrozA, ZavitsanouAM, KudoH, et al. (2017). The immunoreceptor NKG2D promotes tumour growth in a model of hepatocellular carcinoma. Nat Commun, 8: 13930.
ZingoniA, VulpisE, CecereF, AmendolaMG, FuerstD, et al. (2018). MICA-129 Dimorphism and Soluble MICA Are Associated With the Progression of Multiple Myeloma. Front Immunol, 9: 926.
JiaHY, LiuJL, YuanMZ, ZhouCJ, SunWD, et al. (2015). Regulation Roles of MICA and NKG2D in Human Renal Cancer Cells. Asian Pac J Cancer Prev, 16: 3901-3905.
ZouY, YangX, JiangX, WangH, HaoQ, et al. (2009). High levels of soluble Major Histocompatibility Complex class I related chain A (MICA) are associated with biliary cast syndrome after liver transplantation. Transpl Immunol, 21: 210-214.
JiangX, ZouY, HuoZ, YuP (2011). Association of major histocompatibility complex class I chain-related gene A microsatellite polymorphism and hepatocellular carcinoma in South China Han population. Tissue Antigens, 78: 143-147.
AshiruO, BoutetP, Fernandez-MessinaL, Aguera-GonzalezS, SkepperJN, et al. (2010). Natural killer cell cytotoxicity is suppressed by exposure to the human NKG2D ligand MICA*008 that is shed by tumor cells in exosomes. Cancer Res, 70: 481-489.