N-glycosylation by Mgat5 imposes a targetable constraint on immune-mediated tumor clearance.

The regulated glycosylation of the proteome has widespread effects on biological processes that cancer cells can exploit. Expression of N-acetylglucosaminyltransferase V (encoded by Mgat5 or GnT-V), which catalyzes the addition of ß1,6-linked N-acetylglucosamine to form complex N-glycans, has been linked to tumor growth and metastasis across tumor types. Using a panel of murine pancreatic ductal adenocarcinoma (PDAC) clonal cell lines that recapitulate the immune heterogeneity of PDAC, we found that Mgat5 is required for tumor growth in vivo but not in vitro. Loss of Mgat5 results in tumor clearance that is dependent on T cells and dendritic cells, with NK cells playing an early role. Analysis of extrinsic cell death pathways revealed Mgat5-deficient cells have increased sensitivity to cell death mediated by the TNF superfamily, a property that was shared with other non-PDAC Mgat5-deficient cell lines. Finally, Mgat5 knockout in an immunotherapy-resistant PDAC line significantly decreased tumor growth and increased survival upon immune checkpoint blockade. These findings demonstrate a role for N-glycosylation in regulating the sensitivity of cancer cells to T cell killing through classical cell death pathways.

Multiplexed Imaging Mass Cytometry Analysis Characterizes the Vascular Niche in Pancreatic Cancer.

Oncogenesis and progression of pancreatic ductal adenocarcinoma (PDAC) are driven by complex interactions between the neoplastic component and the tumor microenvironment, which includes immune, stromal, and parenchymal cells. In particular, most PDACs are characterized by a hypovascular and hypoxic environment that alters tumor cell behavior and limits the efficacy of chemotherapy and immunotherapy. Characterization of the spatial features of the vascular niche could advance our understanding of inter- and intratumoral heterogeneity in PDAC. In this study, we investigated the vascular microenvironment of PDAC by applying imaging mass cytometry using a 26-antibody panel on 35 regions of interest across 9 patients, capturing more than 140,000 single cells. The approach distinguished major cell types, including multiple populations of lymphoid and myeloid cells, endocrine cells, ductal cells, stromal cells, and endothelial cells. Evaluation of cellular neighborhoods identified 10 distinct spatial domains, including multiple immune and tumor-enriched environments as well as the vascular niche. Focused analysis revealed differential interactions between immune populations and the vasculature and identified distinct spatial domains wherein tumor cell proliferation occurs. Importantly, the vascular niche was closely associated with a population of CD44-expressing macrophages enriched for a proangiogenic gene signature. Taken together, this study provides insights into the spatial heterogeneity of PDAC and suggests a role for CD44-expressing macrophages in shaping the vascular niche. Significance: Imaging mass cytometry revealed that pancreatic ductal cancers are composed of 10 distinct cellular neighborhoods, including a vascular niche enriched for macrophages expressing high levels of CD44 and a proangiogenic gene signature.

Plasticity-induced repression of Irf6 underlies acquired resistance to cancer immunotherapy in pancreatic ductal adenocarcinoma.

Acquired resistance to immunotherapy remains a critical yet incompletely understood biological mechanism. Here, using a mouse model of pancreatic ductal adenocarcinoma (PDAC) to study tumor relapse following immunotherapy-induced responses, we find that resistance is reproducibly associated with an epithelial-to-mesenchymal transition (EMT), with EMT-transcription factors ZEB1 and SNAIL functioning as master genetic and epigenetic regulators of this effect. Acquired resistance in this model is not due to immunosuppression in the tumor immune microenvironment, disruptions in the antigen presentation machinery, or altered expression of immune checkpoints. Rather, resistance is due to a tumor cell-intrinsic defect in T-cell killing. Molecularly, EMT leads to the epigenetic and transcriptional silencing of interferon regulatory factor 6 (Irf6), rendering tumor cells less sensitive to the pro-apoptotic effects of TNF-α. These findings indicate that acquired resistance to immunotherapy may be mediated by programs distinct from those governing primary resistance, including plasticity programs that render tumor cells impervious to T-cell killing.

Plasticity-induced repression of Irf6 underlies acquired resistance to cancer immunotherapy.

Acquired resistance to immune checkpoint immunotherapy remains a critical yet incompletely understood biological mechanism. Here, using a mouse model of pancreatic ductal adenocarcinoma (PDAC) to study tumor relapse following immunotherapy-induced responses, we found that tumors underwent an epithelial-to-mesenchymal transition (EMT) that resulted in reduced sensitivity to T cell-mediated killing. EMT-transcription factors (EMT-TFs) ZEB1 and SNAIL function as master genetic and epigenetic regulators of this tumor-intrinsic effect. Acquired resistance was not due to immunosuppression in the tumor immune microenvironment, disruptions in the antigen presentation machinery, or altered expression of immune checkpoints. Rather, EMT was associated with epigenetic and transcriptional silencing of interferon regulatory factor 6 (Irf6), which renders tumor cells less sensitive to the pro-apoptotic effects of TNF-α. These findings show how resistance to immunotherapy in PDAC can be acquired through plasticity programs that render tumor cells impervious to T cell killing.

Efficacy of a Small-Molecule Inhibitor of KrasG12D in Immunocompetent Models of Pancreatic Cancer.

Mutations in the KRAS oncogene are found in more than 90% of patients with pancreatic ductal adenocarcinoma (PDAC), with Gly-to-Asp mutations (KRASG12D) being the most common. Here, we tested the efficacy of a small-molecule KRASG12D inhibitor, MRTX1133, in implantable and autochthonous PDAC models with an intact immune system. In vitro studies validated the specificity and potency of MRTX1133. In vivo, MRTX1133 prompted deep tumor regressions in all models tested, including complete or near-complete remissions after 14 days. Concomitant with tumor cell apoptosis and proliferative arrest, drug treatment led to marked shifts in the tumor microenvironment (TME), including changes in fibroblasts, matrix, and macrophages. T cells were necessary for MRTX1133's full antitumor effect, and T-cell depletion accelerated tumor regrowth after therapy. These results validate the specificity, potency, and efficacy of MRTX1133 in immunocompetent KRASG12D-mutant PDAC models, providing a rationale for clinical testing and a platform for further investigation of combination therapies. SIGNIFICANCE: Pharmacologic inhibition of KRASG12D in pancreatic cancer models with an intact immune system stimulates specific, potent, and durable tumor regressions. In the absence of overt toxicity, these results suggest that this and similar inhibitors should be tested as potential, high-impact novel therapies for patients with PDAC. See related commentary by Redding and Grabocka, p. 260. This article is highlighted in the In This Issue feature, p. 247.

Tetracycline degradation by nonthermal plasma: removal efficiency, degradation pathway, and toxicity evaluation.

Tetracyclines (TCs) are often discussed as one of the emerging contaminants detected in water matrices and studied for their persistence towards conventional water treatment technologies. In this work, the treatment of TC in aqueous solutions with nonthermal plasma gliding arc process was investigated. The degradation efficiency of TC was studied along with the effect of initial concentration, working gas, pH, and the presence of a radical scavenger. The generation of reactive oxidative species was characterized by the quantification of radical hydroxyl, hydrogen peroxide, ozone, nitrite, and nitrate. Mineralization efficiency was examined by assessment of Total organic carbon evolution. Experimental results have shown that the gliding arc plasma is effective for the treatment of TC. At an initial concentration of 5 mg/L: degradation rates of 94.95% and 60.45% were achieved, while mineralization rates were 81.3% and 57.34% under O2 and air plasma, respectively. O2 plasma exhibited an immense potential for the generation of reactive oxygen species. Meanwhile, air plasma showed better degradation performance in the presence of a radical scavenger. Moreover, degradation products were identified by mass spectroscopy analysis and degradation pathway was proposed. The gliding arc process proposed in this work is promising for the removal of TC antibiotics.

Oxidative Degradation of Hazardous Benzene Derivatives by Ferrate(VI): Effect of Initial pH, Molar Ratio and Temperature.

Two of the most hazardous benzene derivatives (HBD) that have polluted the aquatic environment are bromobenzene and chlorobenzene. Ferrate can degrade various pollutants quickly and efficiently without producing harmful byproducts. This study aims to determine the ability of ferrate to degrade harmful contaminants such as bromobenzene and chlorobenzene. A series of batch experiments were carried out, including for the molar ratio, initial pH solution, and temperature. The study was conducted at an initial pH of 3.6 to 9.6, a molar ratio of 2 to 8 and a temperature of 15 to 55 °C. The study will also examine the differences in functional groups in these pollutants. As a result of the experiments, the optimum conditions to oxidize HBD in a batch reactor was found to have an initial pH of 7.0, a molar ratio of 8, and a temperature of 45 °C, with a 10 min reaction time. Ferrate has a degradation ability against chlorobenzene greater than bromobenzene. The functional cluster in pollutants also significantly affects the degradation ability of ferrate. The results of the degradation experiment showed that ferrate(VI) could effectively oxidize hazardous benzene derivatives in a solution.

Calcium signaling induces a partial EMT.

Epithelial plasticity, or epithelial-to-mesenchymal transition (EMT), is a well-recognized form of cellular plasticity, which endows tumor cells with invasive properties and alters their sensitivity to various agents, thus representing a major challenge to cancer therapy. It is increasingly accepted that carcinoma cells exist along a continuum of hybrid epithelial-mesenchymal (E-M) states and that cells exhibiting such partial EMT (P-EMT) states have greater metastatic competence than those characterized by either extreme (E or M). We described recently a P-EMT program operating in vivo by which carcinoma cells lose their epithelial state through post-translational programs. Here, we investigate the underlying mechanisms and report that prolonged calcium signaling induces a P-EMT characterized by the internalization of membrane-associated E-cadherin (ECAD) and other epithelial proteins as well as an increase in cellular migration and invasion. Signaling through Gαq-associated G-protein-coupled receptors (GPCRs) recapitulates these effects, which operate through the downstream activation of calmodulin-Camk2b signaling. These results implicate calcium signaling as a trigger for the acquisition of hybrid/partial epithelial-mesenchymal states in carcinoma cells.

PTHrP Drives Pancreatic Cancer Growth and Metastasis and Reveals a New Therapeutic Vulnerability.

Pancreatic cancer metastasis is a leading cause of cancer-related deaths, yet very little is understood regarding the underlying biology. As a result, targeted therapies to inhibit metastasis are lacking. Here, we report that the parathyroid hormone-related protein (PTHrP encoded by PTHLH) is frequently amplified as part of the KRAS amplicon in patients with pancreatic cancer. PTHrP upregulation drives the growth of both primary and metastatic tumors in mice and is highly enriched in pancreatic ductal adenocarcinoma metastases. Loss of PTHrP-either genetically or pharmacologically-dramatically reduces tumor burden, eliminates metastasis, and enhances overall survival. These effects are mediated in part through a reduction in epithelial-to-mesenchymal transition, which reduces the ability of tumor cells to initiate metastatic cascade. Spp1, which encodes osteopontin, is revealed to be a downstream effector of PTHrP. Our results establish a new paradigm in pancreatic cancer whereby PTHrP is a driver of disease progression and emerges as a novel therapeutic vulnerability. SIGNIFICANCE: Pancreatic cancer often presents with metastases, yet no strategies exist to pharmacologically inhibit this process. Herein, we establish the oncogenic and prometastatic roles of PTHLH, a novel amplified gene in pancreatic ductal adenocarcinoma. We demonstrate that blocking PTHrP activity reduces primary tumor growth, prevents metastasis, and prolongs survival in mice.This article is highlighted in the In This Issue feature, p. 1601.

Epigenetic and Transcriptional Control of the Epidermal Growth Factor Receptor Regulates the Tumor Immune Microenvironment in Pancreatic Cancer.

Although immunotherapy has revolutionized cancer care, patients with pancreatic ductal adenocarcinoma (PDA) rarely respond to these treatments, a failure that is attributed to poor infiltration and activation of T cells in the tumor microenvironment (TME). We performed an in vivo CRISPR screen and identified lysine demethylase 3A (KDM3A) as a potent epigenetic regulator of immunotherapy response in PDA. Mechanistically, KDM3A acts through Krueppel-like factor 5 (KLF5) and SMAD family member 4 (SMAD4) to regulate the expression of the epidermal growth factor receptor (EGFR). Ablation of KDM3A, KLF5, SMAD4, or EGFR in tumor cells altered the immune TME and sensitized tumors to combination immunotherapy, whereas treatment of established tumors with an EGFR inhibitor, erlotinib, prompted a dose-dependent increase in intratumoral T cells. This study defines an epigenetic-transcriptional mechanism by which tumor cells modulate their immune microenvironment and highlights the potential of EGFR inhibitors as immunotherapy sensitizers in PDA. SIGNIFICANCE: PDA remains refractory to immunotherapies. Here, we performed an in vivo CRISPR screen and identified an epigenetic-transcriptional network that regulates antitumor immunity by converging on EGFR. Pharmacologic inhibition of EGFR is sufficient to rewire the immune microenvironment. These results offer a readily accessible immunotherapy-sensitizing strategy for PDA.This article is highlighted in the In This Issue feature, p. 521.

Endogenous TLR2 ligand embedded in the catalytic region of human cysteinyl-tRNA synthetase 1.

BACKGROUND: The generation of antigen-specific cytotoxic T lymphocyte (CTL) responses is required for successful cancer vaccine therapy. In this regard, ligands of Toll-like receptors (TLRs) have been suggested to activate adaptive immune responses by modulating the function of antigen-presenting cells (APCs). Despite their therapeutic potential, the development of TLR ligands for immunotherapy is often hampered due to rapid systemic toxicity. Regarding the safety concerns of currently available TLR ligands, finding a new TLR agonist with potent efficacy and safety is needed. METHODS: A unique structural domain (UNE-C1) was identified as a novel TLR2/6 in the catalytic region of human cysteinyl-tRNA synthetase 1 (CARS1) using comprehensive approaches, including RNA sequencing, the human embryonic kidney (HEK)-TLR Blue system, pull-down, and ELISA. The potency of its immunoadjuvant properties was analyzed by assessing antigen-specific antibody and CTL responses. In addition, the efficacy of tumor growth inhibition and the presence of the tumor-infiltrating leukocytes were evaluated using E.G7-OVA and TC-1 mouse models. The combined effect of UNE-C1 with an immune checkpoint inhibitor, anti-CTLA-4 antibody, was also evaluated in vivo. The safety of UNE-C1 immunization was determined by monitoring splenomegaly and cytokine production in the blood. RESULTS: Here, we report that CARS1 can be secreted from cancer cells to activate immune responses via specific interactions with TLR2/6 of APCs. A unique domain (UNE-C1) inserted into the catalytic region of CARS1 was determined to activate dendritic cells, leading to the stimulation of robust humoral and cellular immune responses in vivo. UNE-C1 also showed synergistic efficacy with cancer antigens and checkpoint inhibitors against different cancer models in vivo. Further, the safety assessment of UNE-C1 showed lower systemic cytokine levels than other known TLR agonists. CONCLUSIONS: We identified the endogenous TLR2/6 activating domain from human cysteinyl-tRNA synthetase CARS1. This novel TLR2/6 ligand showed potent immune-stimulating activity with little toxicity. Thus, the UNE-C1 domain can be developed as an effective immunoadjuvant with checkpoint inhibitors or cancer antigens to boost antitumor immunity.

GITR Agonism Triggers Antitumor Immune Responses through IL21-Expressing Follicular Helper T Cells.

Although treatment with the glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) agonistic antibody (DTA-1) has shown antitumor activity in various tumor models, the underlying mechanism is not fully understood. Here, we demonstrate that interleukin (IL)-21-producing follicular helper T (Tfh) cells play a crucial role in DTA-1-induced tumor inhibition. The administration of DTA-1 increased IL21 expression by Tfh cells in an antigen-specific manner, and this activation led to enhanced antitumor cytotoxic T lymphocyte (CTL) activity. Mice treated with an antibody that neutralizes the IL21 receptor exhibited decreased antitumor activity when treated with DTA-1. Tumor growth inhibition by DTA-1 was abrogated in Bcl6 fl/fl Cd4 Cre mice, which are genetically deficient in Tfh cells. IL4 was required for optimal induction of IL21-expressing Tfh cells by GITR costimulation, and c-Maf mediated this pathway. Thus, our findings identify GITR costimulation as an inducer of IL21-expressing Tfh cells and provide a mechanism for the antitumor activity of GITR agonism.

Differentiation of c-Kit+ CD24+ natural killer cells into myeloid cells in a GATA-2-dependent manner.

Myeloid progenitor cells have generally been considered the predominant source of myeloid cells under steady-state conditions. Here we show that NK cells contributed to a myeloid cell lineage pool in naïve and tumor-bearing mice. Using fate tracing of NKp46+ cells, we found that myeloid cells could be derived from NK cells. Notably, among mature CD11b+ CD27+ NK cells, c-Kit+ CD24+ NK cells were capable of differentiating into a range of myeloid lineages in vitro and produced neutrophils and monocytes in vivo. The differentiation was completely inhibited by NK-stimulating cytokines. In addition to the potential for differentiation into myeloid cells, c-Kit+ CD24+ NK cells retained NK cell phenotypes and effector functions. Mechanistically, GATA-2 was necessary for the differentiation of c-Kit+ CD24+ NK cells. Therefore, we discovered that GATA-2-dependent differentiation of c-Kit+ CD24+ NK cells contributes to myeloid cell development and identified a novel pathway for myeloid lineage commitment under physiological conditions.

Monocyte-Derived Dendritic Cells Dictate the Memory Differentiation of CD8+ T Cells During Acute Infection.

Monocyte-derived dendritic cells (moDCs) have been shown to robustly expand during infection; however, their roles in anti-infectious immunity remain unclear. Here, we found that moDCs were dramatically increased in the secondary lymphoid organs during acute LCMV infection in an interferon-γ (IFN-γ)-dependent manner. We also found that priming by moDCs enhanced the differentiation of memory CD8+ T cells compared to differentiation primed by conventional dendritic cells (cDCs) through upregulation of Eomesodermin (Eomes) and T cell factor-1 (TCF-1) expression in CD8+ T cells. Consequently, impaired memory formation of CD8+ T cells in mice that had reduced numbers of moDCs led to defective clearance of pathogens upon rechallenge. Mechanistically, attenuated interleukin-2 (IL-2) signaling in CD8+ T cells primed by moDCs was responsible for the enhanced memory programming of CD8+ T cells. Therefore, our findings unveil a specialization of the antigen-presenting cell subsets in the fate determination of CD8+ T cells during infection and pave the way for the development of a novel therapeutic intervention on infection.

Roles of NKT cells in cancer immunotherapy.

Cancer immunotherapy has emerged as an effective therapeutic strategy to treat cancer. Among diverse immune populations, invariant natural killer T (iNKT) cells have shown potent antitumor activity by linking innate and adaptive immune systems. Upon activation by lipid antigens on CD1d molecules, iNKT cells rapidly produce various cytokines and trigger antitumor immunity directly or indirectly by activating other antitumor immune cells. Administration of a representative iNKT cell ligand alpha-galactosylceramide (α-GalCer) or α-GalCer-pulsed APCs effectively stimulates iNKT cells and thereby induces antitumor effects. In this review, we will introduce the biology and importance of NKT cells in antitumor immunity. Previous studies have demonstrated that iNKT cells not only activate various immune cells but also reinvigorate exhausted immune cells in the tumor microenvironment. Furthermore, we will summarize the major clinical trials utilizing iNKT-based immunotherapies.

GM-CSF Promotes Antitumor Immunity by Inducing Th9 Cell Responses.

GM-CSF as an adjuvant has been shown to promote antitumor immunity in mice and humans; however, the underlying mechanism of GM-CSF-induced antitumor immunity remains incompletely understood. In this study, we demonstrate that GM-CSF potentiates the efficacy of cancer vaccines through IL9-producing Th (Th9) cells. GM-CSF selectively enhanced Th9 cell differentiation by regulating the COX2-PGE2 pathway while inhibiting the differentiation of induced regulatory T (iTreg) cells in vitro and in vivo GM-CSF-activated monocyte-derived dendritic cells converted tumor-specific naïve Th cells into Th9 cells, and delayed tumor growth by inducing antitumor CTLs in an IL9-dependent manner. Our findings reveal a mechanism for the adjuvanticity of GM-CSF and provide a rationale for the use of GM-CSF in cancer vaccines.

Activation of NKT Cells in an Anti-PD-1-Resistant Tumor Model Enhances Antitumor Immunity by Reinvigorating Exhausted CD8 T Cells.

PD-1-based cancer immunotherapy is a successful example of immune checkpoint blockade that provides long-term durable therapeutic effects in patients with cancer across a wide spectrum of cancer types. Accumulating evidence suggests that anti-PD-1 therapy enhances antitumor immunity by reversing the function of exhausted T cells in the tumor environment. However, the responsiveness rate of patients with cancer to anti-PD-1 therapy remains low, providing an urgent need for optimization and improvement. In this study, we designed an anti-PD-1-resistant mouse tumor model and showed that unresponsiveness to anti-PD-1 is associated with a gradual increase in CD8 T-cell exhaustion. We also found that invariant natural killer T cell stimulation by the synthetic ligand α-galactosylceramide (αGC) can enhance the antitumor effect in anti-PD-1-resistant tumors by restoring the effector function of tumor antigen-specific exhausted CD8 T cells. IL2 and IL12 were among the cytokines produced by αGC stimulation critical for reinvigorating exhausted CD8 T cells in tumor-bearing mice and patients with cancer. Furthermore, we observed a synergistic increase in the antitumor effect between αGC-loaded antigen-presenting cells and PD-1 blockade in a therapeutic murine tumor model. Our study suggests NKT cell stimulation as a promising therapeutic strategy for the treatment of patients with anti-PD-1-resistant cancer.Significance: These findings provide mechanistic insights into the application of NKT cell stimulation as a potent adjuvant for immunotherapy against advanced cancer. Cancer Res; 78(18); 5315-26. ©2018 AACR.

Effect of herbal extracts on bone regeneration in a rat calvaria defect model and screening system.

OBJECTIVES: The aim of this study was to evaluate the effects of herbal extracts on bone regeneration. Two known samples were screened. MATERIALS AND METHODS: We previously established a rat calvaria defect model using a combination of collagen scaffold and herbal extracts. An 8 mm diameter trephine bur with a low-speed dental hand piece was used to create a circular calvaria defect. The experimental group was divided into 4 classifications: control, collagen matrix, Danshen with collagen, and Ge Gan with collagen. Animals in each group were sacrificed at 4, 6, 8, and 10 weeks after surgery, and bone regeneration ability was evaluated by histological examination. RESULTS: Results revealed that both Danshen and Ge Gan extracts increased bone formation activity when used with collagen matrix. All groups showed almost the same histological findings until 6 weeks. However, after 6 weeks, bone formation activity proceeded differently in each group. In the experimental groups, new bone formation activity was found continuously up to 10 weeks. In the Danshen and Ge Gan groups, grafted materials were still present until 10 weeks after treatment, as evidenced by foreign body reactions showing multinucleated giant cells in chronic inflammatory vascular connective tissue. CONCLUSION: Histological analyses showed that Danshen and Ge Gan extractions increased bone formation activity when used in conjunction with collagen matrix.

Intracorporeal reduction of condylar fracture using both pedicled condylar and seperated ramal fragments after vertical ramal osteotomy.

The aim of this study is to introduce a surgical technique that can maintain blood supply to prevent condylar resorption in the extracorporeal reduction of condylar fracture. Neither the medial pterygoid muscle on the ramal bone nor the lateral pterygoid muscle on the condylar fragment was detached after vertical ramal osteotomy. Thus, reduction was performed in the intracorporeal state. Therefore, blood supply was expected to be maintained to the fragments of both the condylar and ramal bones. On postoperative radiographs, the anatomical outline of the fractured condyle was well restored, and the occlusion was stable. In the unilateral case, there were no signs of mandibular condylar resorption until postoperative 3 weeks. In the 2 bilateral cases, condylar displacements with plate fractures and screw loosening were observed at postoperative 1 month or 5 months, but radiodensity at the displaced fracture site increased during the follow-up period. Finally, complete remodeling of the condylar fragments with restored anatomic appearance was observed on 8-month or 2-year follow-up radiographs. All cases exhibited good healing aspects with no signs or symptoms of mandibular condylar dysfunction during the postoperative remodeling period after intracorporeal reduction of condylar fracture.

IL-21-mediated reversal of NK cell exhaustion facilitates anti-tumour immunity in MHC class I-deficient tumours.

During cancer immunoediting, loss of major histocompatibility complex class I (MHC-I) in neoplasm contributes to the evasion of tumours from host immune system. Recent studies have demonstrated that most natural killer (NK) cells that are found in advanced cancers are defective, releasing the malignant MHC-I-deficient tumours from NK-cell-dependent immune control. Here, we show that a natural killer T (NKT)-cell-ligand-loaded tumour-antigen expressing antigen-presenting cell (APC)-based vaccine effectively eradicates these advanced tumours. During this process, we find that the co-expression of Tim-3 and PD-1 marks functionally exhausted NK cells in advanced tumours and that MHC-I downregulation in tumours is closely associated with the induction of NK-cell exhaustion in both tumour-bearing mice and cancer patients. Furthermore, the recovery of NK-cell function by IL-21 is critical for the anti-tumour effects of the vaccine against advanced tumours. These results reveal the process involved in the induction of NK-cell dysfunction in advanced cancers and provide a guidance for the development of strategies for cancer immunotherapy.