IDO metabolite produced by EBV-transformed B cells inhibits surface expression of NKG2D in NK cells via the c-Jun N-terminal kinase (JNK) pathway

Natural Killer cells are known to play a major role in the innate immune response against viral infections and tumor cells. Several viruses, such as CMV, EBV and HIV-1, have acquired strategies to escape elimination by NK cells. In this study, we observed that EBV infection increased expression of IDO on B cells. To evaluate the function of IDO associated with EBV infection, we investigated whether EBV-induced IDO could modulate expression of NK cell-activation receptor, NKG2D. When NK cells were co-incubated with EBV transformed B cells, surface expression of NKG2D was significantly reduced in NK cells. Incubation with L-kynurenine, an IDO metabolite, down-modulated NKG2D expression in NK cells in a dose- and time-dependent manner. Incubation with the JNK inhibitor SP600125 also inhibited NKG2D expression in NK cells. In addition, we observed that the effect of L-kynurenine was blocked by JNK agonist, anisomycin, suggesting the involvement of the JNK pathway in the signal transduction of L-kynurenine-reduced NKG2D expression. Furthermore, IL-18 significantly reduced L-kynurenine-induced down-regulation of NKG2D expression in NK cells. Taken together, these data indicate that down-regulation of NKG2D by EBV-induced IDO metabolite provides a potential mechanism by which EBV escapes NKG2D-mediated attack by immune cells.     

Human natural killer cells expressing the memory-associated marker CD45RO from tuberculous pleurisy respond more strongly and rapidly than CD45RO- natural killer cells following stimulation with interleukin-12

Natural killer (NK) cells are known as innate immune lymphocytes that respond rapidly when challenged by pathogens but little is known about adaptive immune features including memory related to NK cells from human beings. In the present study, we demonstrate for the first time that human NK cells expressing the memory-associated marker CD45RO were persistent in pleural fluid cells (PFCs) from tuberculous patients. CD45RO(+) NK cells produced significantly more interferon-γ and were more cytotoxic compared with CD45RO(-) NK cells from PFCs when stimulated with interleukin-12 (IL-12). Consistently, IL-12 enhanced the expression of granzyme B, CD69, CD25, NKG2D, IL-12 receptors β1 and β2 on CD45RO(+) NK cells from PFCs. Our experiments contribute to a better understanding of the NK cells from PFCs and indicate that human CD45RO(+) NK cells from PFCs expressing a 'memory-like' phenotype may have an important role in defending against infection by Mycobacterium tuberculosis.     

Potentiation of NK cell-mediated cytotoxicity in human lung adenocarcinoma: role of NKG2D-dependent pathway

Natural cytotoxicity receptors and NKG2D correspond to major activating receptors involved in triggering of tumor cell lysis by human NK cells. In this report, we investigated the expression of NKG2D ligands (NKG2DLs), MHC class I-related chain (MIC) A, MICB and UL16-binding proteins 1, 2 and 3, on a panel of human non-small-cell lung carcinoma cell lines, and we analyzed their role in tumor cell susceptibility to NK cell lysis. Although adenocarcinoma (ADC) cells expressed heterogeneous levels of NKG2DLs, they were often resistant to NK cell-mediated killing. Resistance of a selected cell line, ADC-Coco, to allogeneic polyclonal NK cells and autologous NK cell clones correlated with shedding of NKG2DLs resulting from a matrix metalloproteinase (MMP) production. Treatment of ADC-Coco cells with a MMP inhibitor (MMPI) combined with IL-15 stimulation of autologous NK cell clones lead to a potentiation of NK cell-mediated cytotoxicity. This lysis is mainly NKG2D mediated, since it is abrogated by anti-NKG2D-neutralizing mAb. These results suggest that MMPIs, in combination with IL-15, may be useful for overcoming tumor cell escape from the innate immune response.     

NKG2D ligands expression and NKG2D-mediated cytotoxicity in human laryngeal squamous carcinoma cells

The NKG2D is an activating immunoreceptor expressed by NK cells and CD8⁺ T cells. Engagement of NKG2D by its ligands is critical for both innate and adoptive immunity. While the overexpression of NKG2D ligands on certain tumour cells has previously been demonstrated, little is known about NKG2D ligand expression on human laryngeal tumour cells. In this study, we first verified that the interaction between NKG2D and its ligands was critical for NK cell-based immune response to human laryngeal squamous carcinoma cells Hep-2. This NKG2D-mediated effect was observed by transfecting the recombinant eukaryotic expression vector pEGFP-N1/NKG2D as well as the NKG2D blockade. The mRNA and protein expression of NKG2D ligands, MHC class I-related chain molecules A (MICA) and UL16-binding proteins (ULBPs), in human laryngeal carcinoma cell line Hep-2 and fresh tumour tissues were evaluated. Compared with non-tumour tissues of vocal cords polyps, MICA and ULBP-3 were strongly overexpressed on both the human laryngeal carcinoma cell line Hep-2 and fresh human laryngeal carcinoma tissues. The mechanism and impact of NKG2D ligands overexpression on NK cell-mediated anti-laryngeal cancer immune response would require further investigation.     

MHC class I chain-related molecules induced on monocytes by IFN-gamma promote NK cell activation

NKG2D receptor-ligand interaction triggers NK cell-mediated cytolysis and IFN-gamma secretion. IFN-gamma produced by NK cells has been found to promote the interaction between NK cells and monocytes; however, the underlying mechanism remains elusive. We demonstrate here that IFN-gamma exclusively induced or upregulated the expression of MHC class I chain-related (MIC) molecules, which are ligands of the NKG2D receptor, on the surface of human monocytes of the PBMC population. The IFN-gamma-induced MIC molecules on monocytes played an essential role in triggering the activation of NK cells because mAb-mediated masking of the MIC molecules and the inhibition of cell-to-cell contact using transwell inserts significantly abolished NK cell activation. Meanwhile, membrane-bound IL-15 (mIL-15) was concomitantly induced with MIC molecules on IFN-gamma-treated monocytes and played an essential role in protecting NK cells cocultured with monocytes from MIC-induced NKG2D down-modulation. Therefore, we conclude that the IFN-gamma-induced MIC molecules participated in monocyte/NK cell interaction and that this interaction also involved mIL-15.     

Loss of NKG2D in murine NK cells leads to increased perforin production upon long-term stimulation with IL-2

NK cells are innate lymphocytes responsible for lysis of pathogen-infected and transformed cells. One of the major activating receptors required for target cell recognition is the NK group 2D (NKG2D) receptor. Numerous reports show the necessity of NKG2D for effective tumor immune surveillance. Further studies identified NKG2D as a key element allowing tumor immune escape. We here use a mouse model with restricted deletion of NKG2D in mature NKp46⁺ cells (NKG2D^ΔNK). NKG2D^ΔNK NK cells develop normally, have an unaltered IFN-γ production but kill tumor cell lines expressing NKG2D ligands (NKG2DLs) less efficiently. However, upon long-term stimulation with IL-2, NKG2D-deficient NK cells show increased levels of the lytic molecule perforin. Thus, our findings demonstrate a dual function of NKG2D for NK cell cytotoxicity; while NKG2D is a crucial trigger for cytotoxicity of tumor cells expressing activating ligands it is also capable to limit perforin production in IL-2 activated NK cells.     

Interleukin-10 promotes NK cell killing of autologous macrophages by stimulating expression of NKG2D ligands

Under inflammatory conditions, the pleiotropic cytokine interleukin-10 (IL-10) is released in many tissues. It mediates anti-inflammatory effects in particular by inhibiting the release of T helper type 1 (Th1) cytokines. In contrast, we show here that NK cell cytotoxicity against autologous macrophages is elevated if both cell types are cultured with IL-10. The expression of most activatory NK receptors is increased after culture in the presence of IL-10. On the other hand, macrophages cultured in the presence of IL-10 show elevated expression of the NKG2D ligands major histocompatibility complex (MHC) class 1-like molecules (MIC) - A and - B, as well as UL-16 binding proteins (ULBP) - ULBP-1, ULBP-2 and ULBP-3. By masking the interaction of NK cells with macrophages through interruption of the NKG2D receptor with its ligands, we could reverse the IL-10-induced lysis of macrophages. Our data therefore reveal that IL-10 may exert a novel immunomodulatory role by stimulating NKG2D ligand expression on macrophages, thereby rendering them susceptible to NK cell elimination. This suggests that NK cells would delete macrophages and potentially other immature antigen-presenting cells (APC) or their precursors under inflammatory conditions as a feedback mechanism to shut off uncontrolled immune responses.     

Effects of NKG2D haplotypes on the cell-surface expression of NKG2D protein on natural killer and CD8 T cells of peripheral blood among atomic-bomb survivors

NKG2D is a primary activating receptor that triggers cell-mediated cytotoxicity in NK cells against tumor and virus-infected cells. We previously identified the NKG2D haplotypes in the natural killer gene complex region on chromosome 12p. Two major haplotype alleles, LNK1 and HNK1, were closely related to low and high natural cytotoxic activity phenotypes, respectively. Furthermore, the haplotype of HNK1/HNK1 has revealed a decreased risk of cancer compared with LNK1/LNK1. In the present study, using flow cytometry, we evaluated the functional effects of NKG2D haplotypes and five htSNPs in terms of the cell-surface expression of NKG2D protein on NK and CD8 T cells of peripheral blood among 732 atomic-bomb survivors. NKG2D expression on NK cells showed significant increases, in the order of LNK1/LNK1, LNK1/HNK1 and HNK1/HNK1 haplotypes (p for trend=0.003), or with major homozygous, heterozygous, and minor homozygous genotypes for individual htSNPs (p for trend=0.02-0.003). The same trend was observed for NKG2D expression on CD8 T cells. Our findings indicate that the NKG2D haplotypes are associated with the expression levels of NKG2D protein on NK and CD8 T cells, resulting in inter-individual variations in human cytotoxic response.     

Human Natural Killer cell expression of ULBP2 is associated with a mature functional phenotype

NKG2D is an important activating receptor expressed on NK cells. Ligands (termed NKG2DL) for this receptor include ULBP1-6, MICA and MICB in humans; they are upregulated in stressed, cancerous or infected cells where they engage NKG2D to induce NK cell cytotoxicity and cytokine production.Expression of NKG2DL on effector cells has been described in mice and more recently in human cells. We confirm that NK cell lines and IL-2 stimulated primary human NK cells also express the NKG2DL, ULBP2. However, expression of ULBP2 was not a result of transfer from a non-NK cell to an NK cell and in contrast to recent reports we saw no evidence that ULBP2 expression targeted these NK cells for fratricide or for cytotoxicity by NKG2D-expressing, non-NK effector cells.ULBP2 expression was however linked to expression of mature CD57⁺ NK cells. In particular, expression of ULBP2 was strongest on those NK cells that had evidence of recent activation and proliferation. We suggest that ULBP2 could be used to identify recently activated “mature” NK cells. Defining this phenotype would be useful for understanding the ontogeny on human NK cells.     

膜型/分泌型MICA对NK细胞受体NKG2D的相反调节效应及其对NK细胞受体谱的影响

目的　观察膜型和分泌型MICA对NK细胞受体表达的影响 ,以探讨NK细胞抗肿瘤活化机制及肿瘤细胞表达MICA分子的意义。方法　用MTT法测定人NK细胞系 (NK92 )的细胞毒活性 ;用RT PCR或FACS检测NK细胞受体 (NKG2D ,NKG2A B ,KIR2DL1,KIR2DS1)及NKG2D的识别配体MICA的表达。结果　肿瘤细胞表面的MICA分子可上调NKG2D的表达 ,下调抑制性受体NKG2A B和KIR2DL1的表达 ;而分泌型MICA (sMICA)分子对NKG2D及抑制性受体的表达均有抑制作用。结论　膜型MICA分子可上调NKG2D的表达 ,激发NK细胞对肿瘤细胞的细胞毒效应 ;分泌型MICA分子则通过降低NKG2D的表达下调机体的抗肿瘤免疫效应 ,肿瘤细胞分泌sMICA分子为肿瘤发生免疫逃逸的机制之一。     

Suppressive Effect of a Standardized Mistletoe Extract on the Expression of Activatory NK Receptors and Function of Human NK Cells

Despite long-term use of mistletoe extracts for cancer treatment, their mode of action remains elusive. In this study, it was studied in vitro if mistletoe extract is able to modulate the expression of natural cytotoxic receptors (NCRs) and NKG2D receptor, which stimulate natural killer cell-mediated cytotoxicity. Unexpectedly, a mistletoe extract, ABNOBA viscum Fraxini, inhibited the expression level of NKp46 and NKG2D receptors in dose- and time-dependent manners. The levels of NKp30 and NKG2D receptors were remarkably induced and NKp44 was slightly induced after 48 h treatment with IL-2 and IL-15 in both mRNA and surface expression. The activatory NK receptors were not induced significantly after treatment with IL-12, IL-18, and IL-21 for 48 h. Induction of activatory NK receptors by IL-2 and IL-15 was suppressed almost to the untreated levels by treatment with mistletoe extract, which appeared to induce apoptosis of NK cells in a dose-dependent manner. However, the treatment with IL-2 and IL-15 did not prevent the mistletoe-induced NK-cell death. Mistletoe extract inhibited significantly the cytotoxic activity of resting and IL-2- or IL-15-stimulated NK cells. These results suggest that inhibition of survival and function of NK cells by mistletoe extract may curtail in part the therapeutic effects of mistletoe. Soo Jung Lee, Young-Ok Son, and Hyunjin Kim contributed equally to this article     

Low expression of CD161 and NKG2D activating NK receptor is associated with impaired NK cell cytotoxicity in metastatic melanoma patients

Natural killer (NK) cells play a role in the innate and adaptive antitumor immune responses. The activity of NK cells is regulated by functionally opposing, activating and inhibitory receptors whose balance ultimately determines whether target cells will be susceptible to NK cell mediated lysis. As melanoma is an immunogenic tumor, the effect of immunomodulating agents is consistently investigated. In this study in 79 metastatic melanoma (MM) patients and 52 controls NK activity, expression of activating NKG2D and CD161 receptors and KIR receptors, CD158a and CD158b, on freshly isolated PBL and NK cells were evaluated. Native NK cell activity of melanoma patients in clinical stage I–III and MM patients was determined against NK sensitive K562, NK resistant Daudi, human melanoma FemX, HeLa and HL 60 target tumor cell lines. In addition, predictive pretherapy immunomodulating effect after 18 h in vitro treatments of PBL of MM patients with rh IL-2, IFN-α (IFN), 13-cis retinoic acid (RA) and combination IFN-α and RA was evaluated with respect to NK cell lyses against K562 and FemX cell lines. In this study we show for the first time that low expression of CD161 and activating NKG2D receptors, without increased expression of KIR receptors CD158a and CD158b, as well as a decrease in the cytotoxic, CD16^bright NK cell subset, is associated with a significant impairment in NK cell activity in MM patients. Furthermore, the predictive pretherapy finding that IL-2, IFN, IFN and RA, unlike RA alone, can enhance NK cell activity of MM patients against FemX melanoma tumor cell line can be of help in the design and development of therapeutic regimens, considering that it has recently been shown that low-dose combination of different immunomodulators represents the most promising approach in the therapy of MM.     

NK cell‐mediated anti‐leukemia cytotoxicity is enhanced using a NKG2D ligand MICA and anti‐CD20 scfv chimeric protein

NK cells are important innate cytotoxic lymphocytes that have potential in treatment of leukemia. Engagement of NKG2D receptor on NK cells enhances the target cytotoxicity. Here, we produced a fusion protein consisting of the extracellular domain of the NKG2D ligand MICA and the anti‐CD20 single‐chain variable fragment (scfv). This recombinant protein is capable of binding both NK cells and CD20⁺ tumor cells. Using a human NKG2D reporter cell system we developed, we showed that this fusion protein could decorate CD20⁺ tumor cells with MICA extracellular domain and activate NK through NKG2D. We further demonstrated that this protein could specifically induce the ability of a NK cell line (NKL) and primary NK cells to lyse CD20⁺ leukemia cells. Moreover, we found that downregulation of surface HLA class I expression in the target cells improved NKL‐mediated killing. Our results demonstrated that this recombinant protein specifically lyses leukemia cells by NK cells, which may lead to development of a novel strategy for treating leukemia and other tumors.     

Intracellular expression of MICA in activated CD4 T lymphocytes and protection from NK cell-mediated MICA-dependent cytotoxicity

MICA is a stress-regulated molecule recognized by the NK cell-activating receptor NKG2D. Previously, we demonstrated that MICA is induced on activated T cells but regulation by mitogenic cytokines and its biological consequences remain unexplored. Here, we show that IL-2, IL-4, and IL-15 but not TNF-alpha or IFN-alpha induced MICA expression in T lymphocytes present in peripheral blood mononuclear cells (PBMCs), as assessed by Western blot. IL-2 effect involved Jak3/STAT5, p38 MAPK, p70(56) kinase, Lck/fyn kinases, and NF-kappaB. MICA expression was also observed in Th1 and Th2 cells. However, surface expression was not detected. T lymphocytes present in PBMCs and isolated CD4+ T lymphocytes stimulated with phorbol-12-myristate-13-acetate and ionomycin also induced MICA expression as assessed by Western blot, but only low levels were expressed at the cell surface. Activated but not resting CD4+ T lymphocytes were lysed by IL-15- or IL-2-stimulated NK cells, and susceptibility was increased when HLA class I molecules were blocked. Also, cytokine-stimulated NK cells produced more IFN-gamma after culture with activated CD4+ T lymphocytes. However, the participation of MICA in these responses, if any, was marginal. Confocal microscopy revealed that MICA is retained mostly inside activated CD4+ T cells. Our results suggest that low surface expression of MICA on activated CD4+ T lymphocytes might be a safeguard mechanism to protect them from NK cells in an inflammatory, virus-infected, or tumor microenvironment, where NK and activated CD4+ T cells are recruited.     

Regulation of NK cell repertoire and function in the liver

NK cells represent a large proportion of the lymphocyte population in the liver and are involved in early innate immunity to pathogen infection. As a result of liver endothelial cell fenestrations, parenchymal cells are not separated by a basal membrane, and thereby pathogen-infected hepatocytes are extensively capable of interacting with innate immune cells including NK cells. In addition, hepatic NK cells interact with surrounding DC and alter their differentiation and function. Recent studies reveal that NK cells exhibit a regulatory function that modulates T cell responses through their interaction with DC and/or direct effect on T cells. Thus, NK cells play a central role, not only in innate immunity, but also in shaping the adaptive immune response. During pathogen infection, there is a remarkable increase of hepatic NK cells, possibly due to the expansion of resident liver NK cells and/or recruitement of NK cells from the blood. The liver microenvironment is believed to modulate hepatic NK cell function through the induction of activating/inhibitory receptor expression and inflammatory cytokine secretion. Particularly, the liver maintains intrahepatic NK cells in a functionally hyporesponsive state compared to splenic NK cells: liver NK cells displayed a dampened IFN-γ response to IL-12/IL-18 stimulation. Notably, the liver contains a significant population of functionally hyporesponsive NK cells that express high levels of the inhibitory receptor NKG2A and lack expression of MHC class I-binding Ly49 receptors. Importantly, adoptively transferred splenic NK cells that migrate to the liver displayed phenotypic and functional changes, supporting a view that the liver environment modifies NK cell receptor expression and functional responsiveness. In this article, we will review studies on the regulation of NK cell repertoire and function in the hepatic environment and the impact of liver NK cell immunoregulatory function on influencing adaptive immunity.     

Exposure to cigarette smoke suppresses IL-15 generation and its regulatory NK cell functions in poly I:C-augmented human PBMCs

Both NK cells and IL-15 play crucial roles in innate immunity against viral infections and cancer. Cigarette smoke is known to increase susceptibility to infections and certain cancers. Interleukin (IL)-15 plays an important role in immune responses by regulating proliferation, survival and functions of NK cells. Here, we examined the impact of cigarette smoke on IL-15 production and IL-15 mediated NK cell functions in human PBMCs. We report that cigarette smoke significantly suppresses the induction of IL-15 by poly I:C in human PBMCs. Serum IL-15 levels among smokers was significantly lower than non-smokers. In contrast to a profound increases in intracellular IL-15/IL-15Rα in poly I:C-treated PBMCs, exposure of PBMCs to smoke-conditioned media (SCM) diminished the IL-15/IL-15Rα production. We examined if inhibition of IL-15 production could lead to less NK cell activation. Interestingly, SCM-treated PBMCs had diminished up-regulation of NK cell activation marker, CD69, but not NKG2D compared with controls after poly I:C stimulation. We then confirmed by using IL-15 neutralizing antibody as well as exogenous IL-15 that the ploy I:C-induced NK cells activation was IL-15 mediated. More importantly, cigarette smoke significantly impaired NK cell cytolytic potential to kill K562 cancer cells which was found to be IL-15 mediated. The inhibition of IL-15 and its regulatory NK cell activities were linked to attenuated STAT3 and STAT5, but not ERK1/2 phosphorylations. We demonstrate, for the first time, that cigarette smoke compromises IL-15 production and as a result NK cell function which could link to the higher incidence of cancers or viral infections observed among smokers.