Yongyan Chen

NK cell-based immunotherapy for malignant diseases.

Natural killer (NK) cells play critical roles in host immunity against cancer. In response, cancers develop mechanisms to escape NK cell attack or induce defective NK cells. Current NK cell-based cancer immunotherapy aims to overcome NK cell paralysis using several approaches. One approach uses expanded allogeneic NK cells, which are not inhibited by self histocompatibility antigens like autologous NK cells, for adoptive cellular immunotherapy. Another adoptive transfer approach uses stable allogeneic NK cell lines, which is more practical for quality control and large-scale production. A third approach is genetic modification of fresh NK cells or NK cell lines to highly express cytokines, Fc receptors and/or chimeric tumor-antigen receptors. Therapeutic NK cells can be derived from various sources, including peripheral or cord blood cells, stem cells or even induced pluripotent stem cells (iPSCs), and a variety of stimulators can be used for large-scale production in laboratories or good manufacturing practice (GMP) facilities, including soluble growth factors, immobilized molecules or antibodies, and other cellular activators. A list of NK cell therapies to treat several types of cancer in clinical trials is reviewed here. Several different approaches to NK-based immunotherapy, such as tissue-specific NK cells, killer receptor-oriented NK cells and chemically treated NK cells, are discussed. A few new techniques or strategies to monitor NK cell therapy by non-invasive imaging, predetermine the efficiency of NK cell therapy by in vivo experiments and evaluate NK cell therapy approaches in clinical trials are also introduced.

Natural killer cells in liver disease

Natural killer (NK) cells are enriched in lymphocytes within the liver and have unique phenotypic features and functional properties, including tumor necrosis factor–related apoptosis‐inducing ligand–‐dependent cytotoxicity and specific cytokine profiles. As a key component of innate immunity in the liver, NK cells perform critical roles in host defense against pathogens and tumors through their natural cytotoxicity and cytokine production, and they also act as regulatory cells by engaging in reciprocal interactions with other types of liver cells through cell‐to‐cell contact and the production of cytokines. Accumulating evidence from the last decade suggests that NK cells play an important role in controlling viral hepatitis, liver fibrosis, and liver tumorigenesis, but also contribute to the pathogenesis of liver injury and inflammation. The characterization of intrahepatic NK cell functions has not only helped us to better understand the pathogenesis of liver disease, but has also revealed new therapeutic targets for managing this disease. (HEPATOLOGY 2013;57:1654–1662)

Increased susceptibility to liver injury in hepatitis B virus transgenic mice involves NKG2D‐ligand interaction and natural killer cells

The innate immunopathogenesis responsible for the susceptibility to hepatocyte injury in chronic hepatitis B surface antigen carriers is not well defined. In this study, hepatitis B virus (HBV) transgenic mice (named HBs‐Tg) were oversensitive to liver injury after immunologic [polyinosinic:polycytidylic acid or concanavalin A (ConA)] or chemical (CCl4) triggering. It was then found that the nonhepatotoxic low dose of ConA for wild‐type mice induced severe liver injury in HBs‐Tg mice, which was dependent on the accumulated intraheptic natural killer (NK) cells. Expressions of NKG2D ligands (Rae‐1 and Mult‐1) in hepatocytes were markedly enhanced upon ConA stimulation in HBs‐Tg mice, which greatly activated hepatic NK cells via NKG2D/Rae‐1 or Mult‐1 recognition. Interestingly, the presence of NK T cells was necessary for NK cell activation and worked as positive helper cell possibly by producing interferon‐γ and interleukin‐4 in this process. Conclusion: Our findings for the first time suggested the critical role of NKG2D recognition of hepatocytes by NK cells in oversensitive liver injury during chronic HBV infection. (HEPATOLOGY 2007.)

Antiapoptotic Activity of Autocrine Interleukin-22 and Therapeutic Effects of Interleukin-22-Small Interfering RNA on Human Lung Cancer Xenografts

Purpose: Non–small cell lung carcinoma (NSCLC) is one of most common malignant diseases and usually is resistant against apoptosis-inducing chemotherapy. This study is to explore the antiapoptotic mechanisms of interleukin (IL)-22 in human lung cancer. Experimental Design: Nineteen cases with stage I to III NSCLC were collected to determine the expression of IL-22. Stable transfection of human IL-22 cDNA into A549 and PG cells and transfection of IL-22-RNA interference (RNAi) into these cancer cell lines were done to reveal the molecular mechanisms of IL-22. Results: It was found that IL-22 was highly expressed in primary tumor tissue, malignant pleural effusion, and serum of patients with NSCLC. IL-22R1 mRNA was also detected in lung cancer tissues as well as lung cancer cell lines. Overexpression of IL-22 protected lung cancer cell lines from serum starvation-induced and chemotherapeutic drug-induced apoptosis via activation of STAT3 and its downstream antiapoptotic proteins such as Bcl-2 and Bcl-xL and inactivation of extracellular signal-regulated kinase 1/2. Exposure to blocking antibodies against IL-22R1 or transfection with the IL-22-RNAi plasmid in vitro resulted in apoptosis of these lung cancer cells via STAT3 and extracellular signal-regulated kinase 1/2 pathways. Furthermore, an in vivo xenograft study showed that administration of IL-22-RNAi plasmids significantly inhibited the human tumor cell growth in BALB/c nude mice. Conclusions: Our study indicates that autocrine production of IL-22 contributes to human lung cancer cell survival and resistance to chemotherapy through the up-regulation of antiapoptotic proteins.

Activation and function of hepatic NK cells in hepatitis B infection: an underinvestigated innate immune response

Summary.  Natural killer (NK) cells are abundant in the normal liver, accounting for around one‐third of intrahepatic lymphocytes and are important in the defence against hepatitis B virus (HBV) infection as innate immune responses. In this review, we discuss the mechanisms of hepatic NK cell activity against HBV. Whether directly activated by HBV infection or indirectly activated by other lymphocytes such as NKT cells or antigen‐presenting cells (APCs), hepatic NK cells exert their anti‐viral functions by natural cytotoxicity and production of high levels of cytokines. However, activated NK cells play an important role in regulating adaptive immune responses by interaction with other lymphocytes such as T, B and APCs. In addition, NK cells may contribute to the lymphocyte‐mediated liver injury during HBV infection that was previously considered to be mediated only by CD8+ T cells or/and NKT cells.

Accelerated liver fibrosis in hepatitis B virus transgenic mice: involvement of natural killer T cells.

The immunopathogenic process from hepatitis B virus (HBV) infection to liver fibrosis is incompletely understood because it lacks an animal model. In this study we observed the development of liver fibrosis in HBV transgenic (HBV‐tg) mice and found the roles of natural killer T (NKT) cells in HBV‐related liver fibrosis. We found liver fibrosis spontaneously developed in HBV‐tg mice with the elevated transcription of col1a1, matrix metalloproteinase (MMP)2, and tissue inhibitor of metalloproteinase (TIMP)1. Mice were then injected with repetitive hepatotoxin carbon tetrachloride (CCl4) to induce prominent liver fibrosis. After chronic CCl4 treatment, the serum alanine aminotransferase (ALT) was higher, the liver regenerative nodules became more and bigger, and the fibrosis area was remarkably increased in HBV‐tg mice than in C57BL/6 mice. Moreover, the increase in col1a1 and MMP2 transcription was greater, with a sustaining high level of TIMP1 and a greater activation of hepatic stellate cells (HSCs) in the livers of CCl4‐treated HBV‐tg mice. Our data also showed that there were more liver mononuclear cells (MNCs) in HBV‐tg mice after CCl4 injection, and Rag1−/− mice adoptive transferred lymphocytes from HBV‐tg mice displayed increased collagen deposition. Further study demonstrated the number of liver NKT cells increased after CCl4 treatment and NKT cells were overactivated in HBV‐tg mice in the long term. It was further confirmed that NKT cells were critical for HSCs activation by depletion of NKT cells of HBV‐tg mice and adoptive transfer of purified NKT cells from HBV‐tg mice into recipient Rag1−/− mice. The inflammatory cytokines IL‐4 and IL‐13 produced by NKT cells played a pivotal role in HSCs activation in an in vitro coculture experiment. Conclusion: These data suggest that NKT cells from HBV‐tg mice induce the HSC activation in liver fibrogenesis. (HEPATOLOGY 2011;.)

γδT Cells Drive Myeloid-Derived Suppressor Cell–Mediated CD8+ T Cell Exhaustion in Hepatitis B Virus–Induced Immunotolerance

The mechanisms of liver hepatitis B virus (HBV)–induced systemic immune tolerance are still elusive, and the role of γδT cells has not yet been described. We examined the function of γδT cells in HBV-carrier mice––immunocompetent mice with plasmid-mediated persistent HBV expression in the liver. In this study, we found that γδT cell deficiency led to a break in HBV-induced tolerance and subsequent recovery of hepatic HBV-specific CD8+ T cells. Of interest, IL-17−/− mice phenocopied TCRδ−/− mice in terms of losing HBV persistence, and adoptive transfer of γδT cells restored HBV-persistent expression in TCRδ−/− mice. We further observed that hepatic CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs) play a major role in this mechanism, as they were significantly reduced in both HBV-carrier TCRδ−/− and IL-17−/− mice. MDSC numbers also recovered after adoptive transfer of γδT cells, particularly Vγ4+ T cells. Furthermore, anti-Gr1–mediated MDSC depletion in HBV-carrier mice accelerated HBV elimination from the host, whereas MDSCs transferred to γδT cell-deficient mice restored HBV-induced tolerance. Accordingly, inhibition of MDSCs by the arginase-1 inhibitor norNOHA enhanced the number of HBV-specific CD8+ T cells and promoted HBV clearance. We also observed enhanced CD8+ T cell number with a notable decline of MDSCs in TCRδ−/− mice compared with wild-type mice during the recombinant adeno-associated virus/HBV1.3 virus infection. Importantly, HBV-carrier TCRδ−/− mice not only exhibited increased anti-HBV CD8+ T cells but also markedly reduced MDSCs. Overall, the current study reveals that γδT cells play a previously unrecognized regulatory role in liver tolerance by mobilizing MDSC infiltration to the liver, leading to MDSC-mediated CD8+ T cell exhaustion.

Toll-like receptors in acute liver injury and regeneration.

Liver is the lymphoid organ with an overwhelming innate immune system, which functions as a filter organ at the first line between the digestive tract and the rest of the body, with receiving 80% of the blood supply through portal vein. TLRs are widely expressed on parenchymal and non-parenchymal cells in the liver, which play critical roles for the liver health. Recent studies indicate that TLR-medicated signals have been involved in almost all liver diseases such as acute and chronic hepatitis, liver fibrosis and cirrhosis, alcoholic and non-alcoholic liver disease, ischemia/reperfusion liver injury, liver regeneration and hepatocellular carcinoma. In this review, the expressions of TLRs in hepatic cell populations including hepatocytes, LSECs, Kupffer cells, lymphocytes, DCs, biliary epithelial cells and HSCs, and TLR ligands and signaling in the liver are summarized. Further, recent advances in the roles of TLRs in acute liver injury and regeneration as mediator and regulator, and their potential therapeutic targets are discussed.

Diagnostic utility of LunX mRNA in peripheral blood and pleural fluid in patients with primary non-small cell lung cancer

BackgroundProgress in lung cancer is hampered by the lack of clinically useful diagnostic markers. The goal of this study was to provide a detailed evaluation of lung cancer tumor markers indicative of molecular abnormalities and to assess their diagnostic utility in non-small cell lung cancer (NSCLC) patients.MethodsQuantitative real-time RT-PCR was used to determine LunX, CK19, CEA, VEGF-C and hnRNP A2/B1 mRNA levels in peripheral blood and pleural fluid from NSCLC patients, compared with those from patients with other epithelial cancer (esophagus cancer and breast cancer), benign lung disease (pneumonia and tuberculo pleurisy) and from healthy volunteers.ResultsIn peripheral blood LunX mRNA was detectable in 75.0% (33/44) of patients with NSCLC, but not in patients with other epithelial cancer (0/28), benign lung disease (0/10) or in healthy volunteers (0/15). In contrast, all other genetic markers were detected in patients with either NSCLC, other epithelia cancer or benign lung disease, and in healthy volunteers. The expression level and positive rate of LunX mRNA in peripheral blood correlated with the pathologic stage of NSCLC (P < 0.001 and P = 0.010 respectively). Furthermore, LunX mRNA was detected in 92.9% (13/14) of malignant pleural fluid samples and was the only marker whose expression level was significantly different between malignant and benign pleural fluid (P < 0.001). Additionally, expression of LunX mRNA in the peripheral blood of NSCLC patients decreased shortly after clinical treatment (P = 0.005).ConclusionOf several commonly used genetic markers, LunX mRNA is the most specific gene marker for lung cancer and has potential diagnostic utility when measured in the peripheral blood and pleural fluid of NSCLC patients.

Invariant NKT cells promote alcohol-induced steatohepatitis through interleukin-1β in mice

BACKGROUND & AIMS It was reported that alcohol consumption activated the NLRP3 inflammasome in Kupffer cells, leading to mature interleukin (IL)-1β release in alcoholic liver injury; however, how IL-1β promotes liver injury remains unclear. METHODS We investigated the role of IL-1β in alcoholic steatohepatitis by using a chronic plus single-binge ethanol consumption mouse model. RESULTS Here, liver steatosis was accompanied by notably increased invariant natural killer T (iNKT) cell numbers and activation, and iNKT-deficient Jα18(-/-) mice developed less alcohol-induced steatosis, with reduced liver inflammation and neutrophil infiltration. Kupffer cells and IL-1β were required for the hepatic iNKT accumulation, as either blocking IL-1β signaling with a recombinant IL-1 receptor antagonist (IL-1Ra), depleting Kupffer cells by clodronate liposomes, or specifically silencing IL-1β in Kupffer cells by nanoparticle-encapsulated siRNA, resulted in inhibited hepatic iNKT cell accumulation and activation, as well as amelioration of alcoholic fatty liver. In addition, IL-1β overexpression in hepatocytes was sufficient to compensate for Kupffer cell depletion. Increased gene and protein expression of mature IL-1β correlated with elevated expression of the NLRP3 inflammasome components NLRP3, ASC, and cleaved caspase-1 in Kupffer cells from ethanol-exposed wild-type mice. NLRP3 deficiency led to the attenuation of alcoholic steatosis, similarly as Kupffer cell depletion, almost without hepatic NKT cells. CONCLUSIONS After alcohol-exposure Kupffer cell-derived IL-1β triggered by NLRP3 activation, recruits and activates hepatic iNKT cells, subsequently promoting liver inflammation and neutrophil infiltration, and inducing alcoholic liver injury.