CAR-NK Cells: From Natural Basis to Design for Kill.

Chimeric antigen receptors (CARs) are fusion proteins with an extracellular antigen recognition domain and numerous intracellular signaling domains that have been genetically modified. CAR-engineered T lymphocyte-based therapies have shown great success against blood cancers; however, potential fatal toxicity, such as in cytokine release syndrome, and high costs are some shortcomings that limit the clinical application of CAR-engineered T lymphocytes and remain to overcome. Natural killer (NK) cells are the focal point of current immunological research owing to their receptors that prove to be promising immunotherapeutic candidates for treating cancer. However, to date, manipulation of NK cells to treat malignancies has been moderately successful. Recent progress in the biology of NK cell receptors has greatly transformed our understanding of how NK cells recognize and kill tumor and infected cells. CAR-NK cells may serve as an alternative candidate for retargeting cancer because of their unique recognition mechanisms, powerful cytotoxic effects especially on cancer cells in both CAR-dependent and CAR-independent manners and clinical safety. Moreover, NK cells can serve as an 'off-the-shelf product' because NK cells from allogeneic sources can also be used in immunotherapies owing to their reduced risk of alloreactivity. Although ongoing fundamental research is in the beginning stages, this review provides an overview of recent developments implemented to design CAR constructs to stimulate NK activation and manipulate NK receptors for improving the efficiency of immunotherapy against cancer, summarizes the preclinical and clinical advances of CAR-NK cells against both hematological malignancies and solid tumors and confronts current challenges and obstacles of their applications. In addition, this review provides insights into prospective novel approaches that further enhance the efficiency of CAR-NK therapies and highlights potential questions that require to be addressed in the future.

Defective Natural Killer Cells in Melanoma: Role of NKG2D in Pathogenesis and Immunotherapy.

Melanoma is the most aggressive and deadliest form of skin cancer, and its prognosis is very poor. Although the early detection is responsive to many treatments, metastatic melanoma is refractory to most of them. In the United States, skin melanoma is the fifth most common type of cancer in men and the sixth in women. Current treatment modalities, depending on the cancer stage, consist primarily of surgical excision, chemotherapy, adjuvant therapy, targeted therapies, and immunotherapy. Despite the wide range of therapeutic options and the steadily increasing response rates, a large subset of the treated patients relapse and develop resistance to further treatments. One novel approach in preclinical and clinical trials in immunotherapy is the adaptation of natural killer (NK) cells against resistant cancer cells. NK cells can kill a variety of cancer cell types, as well as the cancer stem cells, while leaving normal cells intact. In skin melanoma, as in most cancers, NK cells in the tumor microenvironment (TME) are functionally impaired. Several factors underlie the defective cause of NK cells, one of which is the dysregulation of the activating receptor NKG2D. This is the dominant receptor in regulating the cytotoxic activity, cytokine production, and regulation of other receptors expressed on NK cells and other lymphocytes. The defective NK cells in cancer models were associated with tumor growth and metastasis. In this review, we discuss the role of NK cells and their phenotypic variants in skin melanoma. Using bioinformatics, we have further analyzed the expression of NKG2D, confirming its low transcript levels in patients with skin melanoma. Furthermore, we show that the CD133 subset of cancer stem cells expresses low levels of NKG2D. Based on these findings we discuss the potential therapeutic approaches that can be exploited to upregulate NKG2D in patients' NK cells and restore their anti-melanoma effects, resulting in tumor regression and prolonged survival.

Finding new lanes: Chimeric antigen receptor (CAR) T-cells for myeloid leukemia.

BACKGROUND: Myeloid leukemia represents a heterogeneous group of cancers of blood and bone marrow which arise from clonal expansion of hematopoietic myeloid lineage cells. Acute myeloid leukemia (AML) has traditionally been treated with multi-agent chemotherapy, but conventional therapies have not improved the long-term survival for decades. Chronic myeloid leukemia (CML) is an indolent disease which requires lifelong treatment, is associated with significant side effects, and carries a risk of progression to potentially lethal blast crises. RECENT FINDINGS: Recent advances in molecular biology, virology, and immunology have enabled researchers to grow and modify T lymphocytes ex-vivo. Chimeric antigen receptor (CAR) T-cell therapy has been shown to specifically target cells of lymphoid lineage and induce remission in acute lymphoblastic leukemia (ALL) patients. While the success of CAR T-cells against ALL is considered a defining moment in modern oncology, similar efficacy against myeloid leukemia cells remains elusive. Over the past 10 years, numerous CAR T-cells have been developed that can target novel myeloid antigens, and many clinical trials are finally starting to yield encouraging results. In this review, we present the recent advances in this field and discuss strategies for future development of myeloid targeting CAR T-cell therapy. CONCLUSIONS: The field of CAR T-cell therapy has rapidly evolved over the past few years. It represents a radically new approach towards cancers, and with continued refinement it may become a viable therapeutic option for patients of acute and chronic myeloid leukemia.

Recent Advances in Molecular Mechanisms of the NKG2D Pathway in Hepatocellular Carcinoma.

Hepatocellular carcinoma is a common malignant tumor with high mortality. Its malignant proliferation, invasion, and metastasis are closely related to the cellular immune function of the patients. NKG2D is a key activated and type II membrane protein molecule expressed on the surface of almost all NK cells. The human NKG2D gene is 270 kb long, located at 12p12.3-p13.1, and contains 10 exons and 9 introns. The three-dimensional structure of the NKG2D monomeric protein contains two alpha-helices, two beta-lamellae, and four disulfide bonds, and its' signal of activation is transmitted mainly by the adaptor protein (DAP). NKG2D ligands, including MICA, MICB, and ULBPs, can be widely expressed in hepatoma cells. After a combination of NKG2D and DAP10 in the form of homologous two polymers, the YxxM motif in the cytoplasm is phosphorylated and then signaling pathways are also gradually activated, such as PI3K, PLCγ2, JNK-cJunN, and others. Activated NK cells can enhance the sensitivity to hepatoma cells and specifically dissolve by releasing a variety of cytokines (TNF-α and IFN-γ), perforin, and high expression of FasL, CD16, and TRAIL. NK cells may specifically bind to the over-expressed MICA, MICB, and ULBPs of hepatocellular carcinoma cells through the surface activating receptor NKG2D, which can help to accurately identify hepatoma, play a critical role in anti-hepatoma via the pathway of cytotoxic effects, and obviously delay the poor progress of hepatocellular carcinoma.

CD4+CD25+Foxp3+ regulatory T cells suppress NKG2D-mediated NK cell cytotoxicity in peripheral blood.

BACKGROUND: Studies have shown that CD4CD25Foxp3Treg cells suppress NKG2D expression on NK cells via a cell contact-dependent mechanism and increased TGF-ß and IL-10 production in some cancer models. We herein aimed to explore whether CD4CD25Foxp3Tregs suppress NKG2D-mediated NK cell cytotoxicity in peripheral blood and elucidate the exact mechanism underlying this phenomenon. METHODS: To explore the function of NKG2D, NK cell cultures were treated with an NKG2D-blocking antibody to block these receptors. Additionally, TGF-ß- and IL-10-blocking antibodies were added to NK and CD4CD25Foxp3Treg cell cocultures to evaluate whether the latter cells suppress NKG2D expression of NK cells via increasing the production of TGF-ß and IL-10. The expression of NKG2D on NK cells was detected by 3-color flow cytometry, and NK cell activity was assessed by 3 assays: a nonradioactive cytotoxicity assay, an ELISA measuring IFN-γ production and a flow cytometry assay to evaluate CD107a expression. RESULTS: Blocking NKG2D decreased NK cell cytotoxicity, IFN-γ production and CD107a expression. Moreover, blocking TGF-ß and IL-10 substantially increased the NKG2D expression in NK and CD4CD25Foxp3Treg cell cocultures. Similarly, blocking TGF-ß and IL-10 enhanced NK cell cytotoxicity, IFN-γ production and CD107a expression; Transwell insert assays also revealed increased IFN-γ production and CD107a and NKG2D expression. CONCLUSION: CD4CD25Foxp3Tregs suppress NKG2D-mediated NK cell cytotoxicity in peripheral blood via a cell contact-dependent mechanism and increased TGF-ß and IL-10 production.

The mechanistic study behind suppression of GVHD while retaining GVL activities by myeloid-derived suppressor cells.

Graft-versus-host disease (GVHD) is a major barrier to the widespread use of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for treating hematologic malignancies. Myeloid-derived suppressor cells (MDSCs) have been recognized as crucial immunosuppressive cells in various pathologic settings. Here, we investigated whether the unique functional properties of MDSCs could be harnessed to control allo-HSCT-associated GVHD. Using multiple murine GVHD/GVL models including both MHC-mismatched and miHA-mismatched, we demonstrated that treatment with CD115+ MDSCs efficiently suppressed GVHD but did not significantly impair graft-versus-leukemia (GVL) activity, leading to 80 and 67% protection in treated mice in GVHD and GVL models, respectively. The mechanism for this dissociation of GVHD from GVL, specifically the emergence of donor-derived NKG2D+ CD8 T cells with a memory phenotype in MDSC-treated recipient mice, was identified. NKG2D expression on donor T cells was required for eradication of allogeneic lymphoma cells. Furthermore, long-term surviving MDSC recipients that exhibited cytolytic activities against allogeneic leukemia cells had a significantly increased percentage of T regulatory cells and, more importantly, NKG2D+ CD8 T cells. These findings indicate that MDSCs can be used as a novel cell-based therapy to suppress GVHD while maintaining GVL activities through selective induction of NKG2D+ CD8 memory T cells.

Enriching the Housing Environment for Mice Enhances Their NK Cell Antitumor Immunity via Sympathetic Nerve-Dependent Regulation of NKG2D and CCR5.

Mice housed in an enriched environment display a tumor-resistant phenotype due to eustress stimulation. However, the mechanisms underlying enriched environment-induced protection against cancers remain largely unexplained. In this study, we observed a significant antitumor effect induced by enriched environment in murine pancreatic cancer and lung cancer models. This effect remained intact in T/B lymphocyte-deficient Rag1-/- mice, but was nearly eliminated in natural killer (NK) cell-deficient Beige mice or in antibody-mediated NK-cell-depleted mice, suggesting a predominant role of NK cells in enriched environment-induced tumor inhibition. Exposure to enriched environment enhanced NK-cell activity against tumors and promoted tumoral infiltration of NK cells. Enriched environment increased the expression levels of CCR5 and NKG2D (KLRK1) in NK cells; blocking their function effectively blunted the enriched environment-induced enhancement of tumoral infiltration and cytotoxic activity of NK cells. Moreover, blockade of ß-adrenergic signaling or chemical sympathectomy abolished the effects of enriched environment on NK cells and attenuated the antitumor effect of enriched environment. Taken together, our results provide new insight into the mechanism by which eustress exerts a beneficial effect against cancer. Cancer Res; 77(7); 1611-22. ©2017 AACR.

Cytotoxic potential of IL-15-activated cytokine-induced killer cells against human neuroblastoma cells.

BACKGROUND: Neuroblastoma (NB) is the most common solid extracranial tumor in childhood. Despite advances in therapy, the prognosis is poor and optimized therapies are urgently needed. Therefore, we investigated the antitumor potential of interleukin-15 (IL-15)-activated cytokine-induced killer (CIK) cells against different NB cell lines. PROCEDURE: CIK cells were generated from peripheral blood mononuclear cells by the stimulation with interferon-γ (IFN-γ), IL-2, OKT-3 and IL-15 over a period of 10-12 days. The cytotoxic activity against NB cells was analyzed by nonradioactive Europium release assay before and after blocking of different receptor-ligand interactions relevant in CIK cell-mediated cytotoxicity. RESULTS: The final CIK cell products consisted in median of 83% (range: 75.9-91.9%) CD3+ CD56- T cells, 14% (range: 5.2-20.7%) CD3+ CD56+ NK-like T cells and 2% (range: 0.9-4.8%) CD3- CD56+ NK cells. CIK cells expanded significantly upon ex vivo stimulation with median rates of 22.3-fold for T cells, 58.3-fold for NK-like T cells and 2.5-fold for NK cells. Interestingly, CD25 surface expression increased from less than equal to 1% up to median 79.7%. Cytotoxic activity of CIK cells against NB cells was in median 34.7, 25.9 and 34.8% against the cell lines UKF-NB-3, UKF-NB-4 and SK-N-SH, respectively. In comparison with IL-2-stimulated NK cells, CIK cells showed a significantly higher cytotoxicity. Antibody-mediated blocking of the receptors NKG2D, TRAIL, FasL, DNAM-1, NKp30 and lymphocyte function-associated antigen-1 (LFA-1) significantly reduced lytic activity, indicating that diverse cytotoxic mechanisms might be involved in CIK cell-mediated NB killing. CONCLUSIONS: Unlike the mechanism reported in other malignancies, NKG2D-mediated cytotoxicity does not constitute the major killing mechanism of CIK cells against NB.

Enhancement of tumor cell susceptibility to natural killer cell activity through inhibition of the PI3K signaling pathway.

Natural killer (NK) cells are the primary effectors of the innate immune response against virus-infected cells or cells that have undergone malignant transformation. NK cells recognize their targets through a complex array of activating and inhibitory receptors, which regulate the intensity of the effector response against individual target cells. However, many studies have shown that tumor cells can escape immune cell recognition through a variety of mechanisms, developing resistance to NK cell killing. Using a lentiviral shRNA library, we previously demonstrated that several common signaling pathways modulate susceptibility of tumor cells to NK cell activity. In this study, we focused on one of the genes (PI3KCB), identified in this genetic screen. The PI3KCB gene encodes an isoform of the catalytic subunit of PI3K called P110ß. The PI3K pathway has been linked to diverse cellular functions, but has never been associated with susceptibility to NK cell activity. Gene silencing of PI3KCB resulted in increased susceptibility of several tumor cell lines to NK cell lytic activity and induced increased IFN-γ secretion by NK cells. Treatment of primary tumor cells with two different PI3K inhibitors also increased target cell susceptibility to NK cell activity. These effects are due, at least in part, to modulation of several activating and inhibitory ligands and appear to be correlated with PI3K signaling pathway inhibition. These findings identify a new and important role of PI3KCB in modulating tumor cell susceptibility to NK cells and open the way to future combined target immunotherapies.

MICA SNPs and the NKG2D system in virus-induced HCC.

Hepatocellular carcinoma (HCC) is one of the most frequent causes of cancer-related death globally. Above well-known risk factors for HCC development ranging from various toxins to diseases such as diabetes mellitus, chronic infection with hepatitis B virus and hepatitis C virus (HCV) poses the most serious threat, constituting the cause in more than 80 % of cases. In addition to the viral genes intensively investigated, the pathophysiological importance of host genetic factors has also been greatly and increasingly appreciated. Genome-wide association studies (GWAS) comprehensively search the host genome at the single-nucleotide level, and have successfully identified the genomic region associated with a whole variety of diseases. With respect to HCC, there have been reports from several groups on single nucleotide polymorphisms (SNPs) associated with hepatocarcinogenesis, among which was our GWAS discovering MHC class I polypeptide-related sequence A (MICA) as a susceptibility gene for HCV-induced HCC. MICA is a natural killer (NK) group 2D (NKG2D) ligand, whose interaction with NKG2D triggers NK cell-mediated cytotoxicity toward the target cells, and is a key molecule in tumor immune surveillance as its expression is induced on stressed cells such as transformed tumor cells for the detection by NK cells. In this review, the latest understanding of the MICA-NKG2D system in viral HCC, particularly focused on its antitumor properties and the involvement of MICA SNPs, is summarized, followed by a discussion of targets for state-of-the-art cancer immunotherapy with personalized medicine in view.

NKG2D-directed cytokine-activated killer lymphocyte therapy combined with gemcitabine for patients with chemoresistant metastatic solid tumors.

Natural-killer group 2, member D (NKG2D) is an activating receptor found on activated natural killer cells and on activated T-cells, here termed cytokine-activated killer (CAK) cells. NKG2D ligands are expressed on various human cancer types. Gemcitabine is an anticancer drug which is a less immune-destructive agent than others. Herein, we investigated the clinical efficacy and the underlying mechanisms of a combination of CAK cell infusion therapy and gemcitabine. Twenty-three patients with disseminated carcinomas were treated with chemo-immunotherapy consisting of CAK cell infusion therapy following gemcitabine treatment. To investigate the underlying mechanisms by which CAK cells synergize with gemcitabine, we used enzyme-linked immunosorbent assay, Real-time reverse transcription polymerase chain reaction assay, calcein-release assay, and adherent target detachment assay. Using these assays we determined the NKG2D ligands such as major histocompatibility complex-class I-related chain (MIC)A/B expression in carcinoma cells and the level of cellular cytotoxicity generated by treatment with gemcitabine with/without CAK cells. The tumor responses differed among the patients (n=23). In vitro experiments revealed that MICA/B protein and mRNA expression were up-regulated in several carcinoma cell lines after gemcitabine treatment. Pre-treatment with gemcitabine and subsequent exposure to CAK cells induced greater cytotoxicity than either treatment alone. Inclusion of soluble MICB in CAK cell-mediated cytotoxicity assay significantly reduced cytotoxicity. Our clinical results of gemcitabine-CAK combinatorial therapy demonstrated long-term stable disease despite chemoresistance. In conclusion, the combination of gemcitabine and CAK cells may have clinical therapeutic significance for pancreatic, hepato-biliary tract, and urothelial tract cancer. Our study shows that combining CAK therapy with gemcitabine can lead to successful treatment of metastatic cancer.

Mesenchymal stem cells suppress CD8+ T cell-mediated activation by suppressing natural killer group 2, member D protein receptor expression and secretion of prostaglandin E2, indoleamine 2, 3-dioxygenase and transforming growth factor-ß.

Bone marrow mesenchymal stem cells (BMSCs) inhibit immune cell responsiveness, and especially of T lymphocytes. We showed that BMSCs markedly inhibited the proliferation and cytokine production by CD8(+) T cells by a cell-to-cell contact phenomenon and secretion of soluble factors. BMSCs down-regulate the expression of natural killer group 2, member D protein (NKG2D) receptors on CD8(+) T cells when co-cultured with them. Moreover, CD8(+) T cells that express low levels of NKG2D had impaired proliferation after triggering by a mitogen. The major histocompatibility complex (MHC) class I chain-related (MIC) A/B molecule, which is a typical ligand for NKG2D, was expressed on BMSCs, and caused dampening of cell proliferation. Monoclonal antibody blocking experiments targeted to MIC A/B impaired CD8(+) T cell function, as evaluated by proliferation and cytokine production. In addition, the production of prostaglandin E2 (PGE2 ), indoleamine 2, 3-dioxygenase (IDO) and transforming growth factor (TGF)-ß1 were increased when BMSCs were co-cultured with CD8(+) T cells. The addition of specific inhibitors against PGE2 , IDO and TGF-ß partially restored the proliferation of CD8(+) T cells. Our results suggest that BMSCs suppress CD8(+) T cell-mediated activation by suppressing NKG2D expression and secretion of PGE2, IDO and TGF-ß. Our observations further confirm the feasibility of BMSCs as a potential adoptive cellular therapy in immune-mediated diseases such as graft-versus-host disease (GVHD).

Two novel human cytomegalovirus NK cell evasion functions target MICA for lysosomal degradation.

NKG2D plays a major role in controlling immune responses through the regulation of natural killer (NK) cells, αß and γδ T-cell function. This activating receptor recognizes eight distinct ligands (the MHC Class I polypeptide-related sequences (MIC) A andB, and UL16-binding proteins (ULBP)1-6) induced by cellular stress to promote recognition cells perturbed by malignant transformation or microbial infection. Studies into human cytomegalovirus (HCMV) have aided both the identification and characterization of NKG2D ligands (NKG2DLs). HCMV immediate early (IE) gene up regulates NKGDLs, and we now describe the differential activation of ULBP2 and MICA/B by IE1 and IE2 respectively. Despite activation by IE functions, HCMV effectively suppressed cell surface expression of NKGDLs through both the early and late phases of infection. The immune evasion functions UL16, UL142, and microRNA(miR)-UL112 are known to target NKG2DLs. While infection with a UL16 deletion mutant caused the expected increase in MICB and ULBP2 cell surface expression, deletion of UL142 did not have a similar impact on its target, MICA. We therefore performed a systematic screen of the viral genome to search of addition functions that targeted MICA. US18 and US20 were identified as novel NK cell evasion functions capable of acting independently to promote MICA degradation by lysosomal degradation. The most dramatic effect on MICA expression was achieved when US18 and US20 acted in concert. US18 and US20 are the first members of the US12 gene family to have been assigned a function. The US12 family has 10 members encoded sequentially through US12-US21; a genetic arrangement, which is suggestive of an 'accordion' expansion of an ancestral gene in response to a selective pressure. This expansion must have be an ancient event as the whole family is conserved across simian cytomegaloviruses from old world monkeys. The evolutionary benefit bestowed by the combinatorial effect of US18 and US20 on MICA may have contributed to sustaining the US12 gene family.

Epithelial-mesenchymal transition induces an antitumor immune response mediated by NKG2D receptor.

Epithelial-mesenchymal transition (EMT) is a morphogenetic process characterized by the acquisition of mesenchymal properties linked with an invasive phenotype and metastasis of tumor cells. NK group 2, member D (NKG2D) is an NK cell-activating receptor crucially involved in cancer immunosurveillance. In this study, we show that induction of EMT by TGF-ß stimulation of human keratinocytes, by glycogen synthase kinase-3ß inhibition in several epithelial tumor cell lines, and by Snail1 overexpression in colorectal cancer cells strongly upregulated the expression of NKG2D ligands (NKG2DLs), MHC class I chain-related molecules A and B (MICA/B) and ULBP1-3. Overexpression of Snail1 and inhibition of glycogen synthase kinase-3ß in colorectal tumor cells markedly induced the activity of Sp1 transcription factor, which plays a key role in the upregulation of NKG2DL expression during EMT. The stimulation of MICA/B expression by TGF-ß treatment was independent of Sp1, but it involved posttranslational mechanisms mediated by mammalian target of rapamycin pathway. Accordingly, with the increased expression of NKG2DLs, triggering of EMT rendered cancer cells more susceptible to NKG2D-mediated killing by NK cells. In agreement, MICA/B were expressed in vivo in well-differentiated colorectal tumors with retained epithelial characteristics, whereas no expression of MICA/B was detected in poorly differentiated and invasive colorectal tumors that have lost epithelial characteristics. This decrease of MICA/B expression was associated with a dramatic increase of NKG2D(+)-tumor infiltrating lymphocytes. Overall, our findings indicate that EMT is a relevant checkpoint in the control of tumor progression through NKG2D-mediated immune responses.

Hmgb1-IL-23-IL-17-IL-6-Stat3 axis promotes tumor growth in murine models of melanoma.

In order to understand how tumor cells can escape immune surveillance mechanisms and thus develop antitumor therapies, it is critically important to investigate the mechanisms by which the immune system interacts with the tumor microenvironment. In our current study, IL-17 deficiency results in reduced melanoma tumor size, diminished numbers of proliferating cells and blood vessels, and decreased percentage of CD11b(+)Gr-1(+) MDSCs in tumor tissues. IL-17 promotes IL-6 induction and Stat3 activation. Treatment of Stat3 inhibitor WP1066 in B16-F10 tumor cells inoculated wild-type mice inhibits tumor growth. Additional administration of recombinant IL-6 into B16-F10 tumor-bearing IL-17(-/-) mice results in markedly increased tumor size and p-Stat3 expression, whereas additional recombinant IL-17 administration into B16-F10 tumor-bearing wild-type mice treated with anti-IL-6 mAb does not significantly alter the tumor growth and p-Stat3 expression. In our further study, blockade of Hmgb1-RAGE pathway inhibits melanoma tumor growth and reduces production of IL-23 and IL-17. All these data suggest that Hmgb1-IL-23-IL-17-IL-6-Stat3 axis plays a pivotal role in tumor development in murine models of melanoma, and blocking any portion of this axis will attenuate melanoma tumor growth.

Cutting edge: tumor-targeting antibodies enhance NKG2D-mediated NK cell cytotoxicity by stabilizing NK cell-tumor cell interactions.

Monoclonal antibodies represent a promising approach to fight a variety of tumors, but their mode of action remains to be fully understood. NK cells can recognize Ab-coated targets, as well as stress ligands, on tumor cells. In this study, we investigated how NK cells integrate both kinds of activating signals. NK cell-mediated killing was maximal with the combined recognition of NKG2D ligands and Ab; surprisingly, only NKG2D engagement substantially enhanced degranulation. Conversely, Ab recognition by NK cells uniquely increased contact stability with tumor cells. Furthermore, using intravital imaging of solid tumors, we showed that Ab recognition favored prolonged interactions between NK cells and targets. Altogether, our results demonstrate that NK cell-mediated killing can be differentially regulated at the level of degranulation and contact stability by distinct activating receptors. Thus, complementary signals mediated by recognition of stress ligands and tumor-specific Abs may contribute to the efficacy of NK cells during mAb therapy.

Activation of NK cell cytotoxicity by the natural compound 2,3-butanediol.

The natural compound 2,3-BTD has diverse physiological effects in a range of organisms, including acting as a detoxifying product of liver alcohol metabolism in humans and ameliorating endotoxin-induced acute lung injury in rats. In this study, we reveal that 2,3-BTD enhances NK cell cytotoxic activity in human pNK cells and NK92 cells. Treatment of NK cells with 2,3-BTD increased perforin expression in a dose-dependent manner. This was accompanied by elevated JNK and ERK1/2 MAPK activities and enhanced expression of NKG2D/NCRs, upstream signaling molecules of the MAPK pathways. The 2,3-BTD effect was inhibited by pretreatment with inhibitors of JNK (SP) or ERK1/2 (PD) or by depleting NKG2D/NCRs or JNK1 or ERK2 with siRNA. These results indicate that 2,3-BTD activates NK cell cytotoxicity by NKG2D/NCR pathways and represent the first report of the 2,3-BTD effect on activation of innate immunity cells.

Battle of the midgets: innate microRNA networking.

Natural killer (NK) cells play an important role in the direct killing of cancerous and virus-infected cells. One of the important activating receptors which mediates this killing is NKG2D. This receptor recognizes various stress-induced ligands including the major histocompatibility complex class I-related chain A and B (MICA and MICB respectively). The mechanisms controlling the expression of the NKG2D ligands are not completely understood, yet various studies have demonstrated that the expression of the NKG2D ligands is manipulated by viruses and by tumor cells in order to escape the NKG2D detection. Cumulative data have emphasized that various microRNAs (miRNAs) of both human and viral origin control the expression of NKG2D ligands, particularly MICB. Herein we review recent findings regarding the miRNA regulation of the NKG2D ligands. We propose that these miRNAs generate a complex network of interactions that control the expression of the NKG2D ligands under normal conditions and during disease development.

NK cells from HCV-infected patients effectively induce apoptosis of activated primary human hepatic stellate cells in a TRAIL-, FasL- and NKG2D-dependent manner.

In mouse models it has been shown that natural killer (NK) cells can attenuate liver fibrosis via killing of activated hepatic stellate cells (HSCs) in a NKG2D- and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-dependent manner. However, only little data exist regarding interactions of human NK cells with HSCs and their potential role in hepatitis C virus (HCV)-associated fibrogenesis. Therefore, purified NK cells from untreated HCV RNA(+) patients (n=33), interferon-α (IFN-α)-treated patients (n=17) and healthy controls (n=18) were coincubated with activated primary HSCs, and were tested for degranulation (CD107a expression) and secretion of IFN-γ and TNF-α, respectively. Induction of HSC apoptosis was analyzed using an active caspase-3 assay. We found that following coincubation with HSCs a significant increase in CD107a expression could be observed in both NK cells from HCV(+) patients and healthy controls, whereas only negligible secretion of IFN-γ and TNF-α could be detected. More importantly, NK cells from untreated HCV RNA(+) patients were significantly more effective in induction of HSC apoptosis (17.8 ± 9.2%) than NK cells from healthy controls (6.2 ± 2.1%; P<0.0001). Additionally, we observed an inverse correlation of liver fibrosis stage and the ability of NK cells to induce HSC apoptosis. Induction of HSC apoptosis was contact dependent and could partly be blocked by antibodies specific for TRAIL, NKG2D and FasL, respectively. It is noteworthy that NK cells from IFN-α-treated HCV(+) patients displayed the highest capability to kill HSCs (27.6 ± 10.5%). Accordingly, pre-stimulation of NK cells with recombinant IFN-α significantly increased the ability of NK cells to induce cell death in primary HSCs and was dependent on upregulated expression of TRAIL. Here we demonstrate that NK cells from HCV-infected patients are highly efficient in inducing apoptosis of activated HSCs. Thus, NK cells may have an important anti-fibrotic role in chronic hepatitis C.