Down-modulation Of NKG2D Research Articles

Impact on NK cell functions of acute versus chronic exposure to extracellular vesicle-associated MICA: Dual role in cancer immunosurveillance.

Natural killer (NK) cells are innate cytotoxic lymphocytes that play a key role in cancer immunosurveillance thanks to their ability to recognize and kill cancer cells. NKG2D is an activating receptor that binds to MIC and ULBP molecules typically induced on damaged, transformed or infected cells. The release of NKG2D ligands (NKG2DLs) in the extracellular milieu through protease‐mediated cleavage or by extracellular vesicle (EV) secretion allows cancer cells to evade NKG2D‐mediated immunosurveillance. In this work, we investigated the immunomodulatory properties of the NKG2D ligand MICA*008 associated to distinct populations of EVs (i.e., small extracellular vesicles [sEVs] and medium size extracellular vesicles [mEVs]). By using as model a human MICA*008‐transfected multiple myeloma (MM) cell line, we found that this ligand is present on both vesicle populations. Interestingly, our findings reveal that NKG2D is specifically involved in the uptake of vesicles expressing its cognate ligand. We provide evidence that MICA*008‐expressing sEVs and mEVs are able on one hand to activate NK cells but, following prolonged stimulation induce a sustained NKG2D downmodulation leading to impaired NKG2D‐mediated functions. Moreover, our findings show that MICA*008 can be transferred by vesicles to NK cells causing fratricide. Focusing on MM as a clinically and biologically relevant model of tumour‐NK cell interactions, we found enrichment of EVs expressing MICA in the bone marrow of a cohort of patients. All together our results suggest that the accumulation of NKG2D ligands associated to vesicles in the tumour microenvironment could favour the suppression of NK cell activity either by NKG2D down‐modulation or by fratricide of NK cell dressed with EV‐derived NKG2D ligands.

IL-15 Overcomes Hepatocellular Carcinoma-Induced NK Cell Dysfunction.

NK cells have potent antitumor capacity. They are enriched in the human liver, with a large subset specialized for tissue-residence. The potential for liver-resident versus liver-infiltrating NK cells to populate, and exert antitumor functions in, human liver tumors has not been studied. We examined liver-resident and liver-infiltrating NK cells directly ex vivo from human hepatocellular carcinomas (HCCs) and liver colorectal (CRC) metastases, compared with matched uninvolved liver tissue. We found that NK cells were highly prevalent in both HCC and liver CRC metastases, although at lower frequencies than unaffected liver. Up to 79% of intratumoral NK cells had the CXCR6+CD69+ liver-resident phenotype. Direct ex vivo staining showed that liver-resident NK cells had increased NKG2D expression compared to their non-resident counterparts, but both subsets had NKG2D downregulation within liver tumors compared to uninvolved liver. Proliferation of intratumoral NK cells (identified by Ki67) was selectively impaired in those with the most marked NKG2D downregulation. Human liver tumor NK cells were functionally impaired, with reduced capacity for cytotoxicity and production of cytokines, even when compared to the hypo-functional tissue-resident NK cells in unaffected liver. Coculture of human liver NK cells with the human hepatoma cell line PLC/PRF/5, or with autologous HCC, recapitulated the defects observed in NK cells extracted from tumors, with downmodulation of NKG2D, cytokine production, and target cell cytotoxicity. Transwells and conditioned media confirmed a requirement for cell contact with PLC/PRF/5 to impose NK cell inhibition. IL-15 was able to recover antitumor functionality in NK cells inhibited by in vitro exposure to HCC cell lines or extracted directly from HCC. In summary, our data suggest that the impaired antitumor function of local NK cells reflects a combination of the tolerogenic features inherent to liver-resident NK cells together with additional contact-dependent inhibition imposed by HCC itself. The demonstration that IL-15 can recover hepatic NK cell function following tumor exposure supports its inclusion in immunotherapy strategies.

A possible mechanism of impaired NK cytotoxicity in cancer patients: Down-regulation of DAP10 by TGF-β1

Elevated TGF-BETA1 secretion and down-modulation of NKG2D underlies impaired NK cytotoxicity in cancer patients. However, the molecular mechanism of immunosuppression by TGF-BETA1 is not yet clarified. IL-2-activated human NK cells were cultured with TGF-BETA1. Protein levels of NKG2D and DAP10 were examined by FACS or immunoblot analyses. Real-time RTPCR was performed to quantify the transcription levels. MAPK inhibitors were used to investigate intracellular signaling. TGF-BETA1 down-regulated total and surface NKG2D, which was partially dependent on transcriptional regulation. TGF-BETA1 treatment of human NK cells resulted in significant changes in both transcriptional and translational levels of DAP10. Moreover, treatment with bafilomycin A1 or folimycin restored total NKG2D levels in TGF-BETA1-treated NK cells. The impaired NKG2D down-modulation by TGF-BETA1 was not associated with activation of the MAPK signaling pathway. TGF-BETA1 down-modulates surface NKG2D expression by controlling the transcriptional and translational levels of DAP10.

Chronic In Vivo Interaction of Dendritic Cells Expressing the Ligand Rae-1ε with NK Cells Impacts NKG2D Expression and Function.

To investigate how dendritic cells (DCs) interact with NK cells in vivo, we developed a novel mouse model in which Rae-1ε, a ligand of the NKG2D receptor, is expressed in cells with high levels of CD11c. In these CD11c-Rae1 mice, expression of Rae-1 was confirmed on all subsets of DCs and a small subset of B and T cells, but not on NK cells. DC numbers and activation status were unchanged, and NK cells in these CD11c-Rae1 mice presented the same Ly49 repertoire and maturation levels as their littermate wildtype controls. Early NK cell activation after mouse CMV infection was slightly lower than in wildtype mice, but NK cell expansion and viral control were comparable. Notably, we demonstrate that chronic interaction of NK cells with NKG2D ligand–expressing DCs leads to a reversible NKG2D down-modulation, as well as impaired NKG2D-dependent NK cell functions, including tumor rejection. In addition to generating a useful mouse model, our studies reveal in vivo the functional importance of the NK cell and DC cross-talk.

Abstract 1491: IPH4301, an antibody targeting MICA and MICB exhibits potent cytotoxic activity and immunomodulatory properties for the treatment of cancer

Abstract MICA and MICB, and ULPB1-6, are ligands for NKG2D, an activating receptor expressed on NK cells and subsets of T cells. Expression of MICA and MICB is induced by cellular stress in transformed tumor cells, upon infections or at sites of chronic inflammation. Their expression is tightly regulated by complex mechanisms both at the mRNA and protein levels. As markers of cellular stress and tumorigenesis, MICA and MICB proteins are attractive candidates for targeting by a cytotoxic antibody. Moreover, ionizing radiation and various chemotherapies that cause cellular stress have been shown to induce expression of NKG2D ligands, opening interesting options for combination therapies. MICA and MICB are also compelling targets for immunomodulation. MICA and -B cause internalization of NKG2D, leading to reduced cell surface NKG2D levels and desensitization of cytotoxic effector cells in cancer patients. It was recently reported that blockade of the interactions between NKG2D and its ligands could lead to significant anti-tumor responses in mouse models. Moreover, NKG2D ligand expression was induced on immunosuppressive macrophages in cancer patients and in mouse tumor models, raising the possibility that anti-MICA/B antibodies may be used to counter local immunosuppression by targeting myeloid derived suppressor cells. We have selected the IPH4301 antibody for its ability to bind to all allotypes of MICA and MICB, and for its dual action as an immunomodulatory agent, as well as direct cytotoxicity towards MICA/B-expressing tumor cells. First, IPH4301 induces killing of MICA/B expressing tumor cells through antibody-dependent cell cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP) measured towards MICA expressing cells in vitro. In vivo ADCC/ADCP efficacy was demonstrated in several preventive and curative settings using MICA expressing cell lines or endogenous tumors. Second, IPH4301 blocks the binding of MICA/B to NKG2D. In a tumor context of chronic triggering, NKG2D downmodulation has been described in several studies of cancer patients. This modulation is mainly induced by expression of MICA/B, and less by the ULBPs. By blocking the MICA/NKG2D interaction, IPH4301 effectively restored NKG2D expression and function in vitro on primary NK and T cells. Third, we show that IPH4301 can override immunosuppression induced by suppressive myeloid cells. In vitro differentiated M2 macrophages, but not M1 macrophages, have the capacity to impair cytotoxic functions of autologous NK cells towards MICA expressing tumor cell lines. This suppression could be overcome by IPH4301, which triggered ADCC by these otherwise impaired NK cells. Altogether, IPH4301 is a novel, first-in-class anti-MICA/B mAb with both cytotoxic and immunomodulatory properties. Ongoing work aims to perform regulatory toxicology studies and manufacture a clinical grade product for testing in a clinical trial. Citation Format: Ariane Morel, Nicolas Viaud, Cécile Bonnafous, Sylvia Trichard, Alix Joulin-Giet, Samia Mizari, Gwendoline Grondin, Nadia Anceriz, J. Zhang, J. Jarzen, J. Wu, Gwendoline Grondin, Laetitia Cohen-Tannoudji, Yannis Morel, Benjamin Rossi, Carine Paturel, Renaud Buffet, Laurent Gauthier, Nicolai Wagtmann, Mathieu Blery. IPH4301, an antibody targeting MICA and MICB exhibits potent cytotoxic activity and immunomodulatory properties for the treatment of cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1491.

C-Cbl regulates MICA- but not ULBP2-induced NKG2D down-modulation in human NK cells.

The NKG2D activating receptor on human NK cells mediates "altered self" recognition, as its ligands (NKG2DLs) are upregulated on target cells in a variety of stress conditions. Evidence collected in the past years shows that, even though expression of NKG2DLs acts as a danger signal that renders tumor cells susceptible to cytotoxicity, chronic exposure to soluble or membrane-bound NKG2DLs can lead to down-modulation of receptor expression and impairment of NKG2D-mediated cell functions. Here, we evaluated whether different cell-bound NKG2DLs, namely MICA and ULBP2, are equivalently able to induce NKG2D down-modulation on human NK cells. We found that although both ligands reduce NKG2D surface expression, MICA promotes a stronger receptor down-modulation than ULBP2, leading to a severe impairment of NKG2D-dependent NK-cell cytotoxicity. We also provide evidence that the ubiquitin pathway and c-Cbl direct MICA-induced but not ULBP2-induced NKG2D internalization and degradation, thus identifying a molecular mechanism to explain the differential effects of MICA and ULBP2 on NKG2D expression. A better understanding of the molecular mechanisms employed by the different NKG2DLs to control NKG2D surface expression could be useful for the development of anti-tumor strategies to restore a normal level of NKG2D receptors on human NK cells.

Down-regulation of the NKG2D receptor is differentially controlled by MICA and ULBP2 ligands

Event Abstract Back to Event Down-regulation of the NKG2D receptor is differentially controlled by MICA and ULBP2 ligands Linda Quatrini1*, Rosa Molfetta1, Francesca Gasparrini1, Cristina Capuano2, Alessandra Zingoni1, Ricciarda Galandrini2, Angela Santoni1 and Rossella Paolini1 1 "Sapienza" University of Rome, Molecular Medicine, Italy 2 "Sapienza" University of Rome, Experimental Medicine, Italy The activating NKG2D receptor on human NK cells mediates “induced self recognition” in that its ligands are up-regulated by stressed or diseased cells. Evidence collected in the past years demonstrated that chronic exposure to NKG2DLs induces receptor down-modulation. The aim of this study was to evaluate whether different NKG2D ligands (NKG2DLs), namely MICA and ULBP2, are equivalent in their capacities to down-modulate the surface receptor expression on human NK cells. We analyzed the rate and kinetics of NKG2D down-modulation in primary cultured NK cells and in the NKL NK cell line upon stimulation with the Ba/F3 cell line stably overexpressing comparable levels of MICA or ULBP2 by FACS and fluorescence microscopic analysis. Although both ligands were able to reduce NKG2D expression, exposure to MICA over-expressing target cells resulted in a more rapid and efficient receptor down-modulation and in a more pronounced impairment of NKG2D-dependent cytotoxicity. MICA-experienced NK cells also showed a higher tyrosine phosphorylation of the ubiquitin ligase c-Cbl. Furthermore, ligand-induced receptor down-modulation affected the total NKG2D cellular levels, suggesting that the internalized receptor complexes were mainly subjected to degradation. All together these results demonstrate that NKG2D down-regulation is influenced by the nature of its ligand and suggest a different contribution of the ubiquitin pathway in the control of NKG2D internalization and degradation in MICA- versus ULBP2-experienced cells. Understanding the mechanisms of ligand-induced NKG2D down-modulation will be helpful to prevent evasion from NK cell-mediated immune response. Keywords: innate immunity, NK cells, NK receptor, Endocytosis, degradation Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Quatrini L, Molfetta R, Gasparrini F, Capuano C, Zingoni A, Galandrini R, Santoni A and Paolini R (2013). Down-regulation of the NKG2D receptor is differentially controlled by MICA and ULBP2 ligands. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00013 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 08 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Linda Quatrini, "Sapienza" University of Rome, Molecular Medicine, Rome, Italy, Italy, linda.quatrini@opbg.net Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Linda Quatrini Rosa Molfetta Francesca Gasparrini Cristina Capuano Alessandra Zingoni Ricciarda Galandrini Angela Santoni Rossella Paolini Google Linda Quatrini Rosa Molfetta Francesca Gasparrini Cristina Capuano Alessandra Zingoni Ricciarda Galandrini Angela Santoni Rossella Paolini Google Scholar Linda Quatrini Rosa Molfetta Francesca Gasparrini Cristina Capuano Alessandra Zingoni Ricciarda Galandrini Angela Santoni Rossella Paolini PubMed Linda Quatrini Rosa Molfetta Francesca Gasparrini Cristina Capuano Alessandra Zingoni Ricciarda Galandrini Angela Santoni Rossella Paolini Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Cervical cancer cell lines expressing NKG2D-ligands are able to down-modulate the NKG2D receptor on NKL cells with functional implications

BackgroundCervical cancer represents the third most commonly diagnosed cancer and the fourth leading cause of cancer-related deaths in women worldwide. Natural killer (NK) cells play an important role in the defense against viruses, intracellular bacteria and tumors. NKG2D, an activating receptor on NK cells, recognizes MHC class I chain-related molecules, such as MICA/B and members of the ULBP/RAET1 family. Tumor-derived soluble NKG2D-ligands have been shown to down-modulate the expression of NKG2D on NK cells. In addition to the down-modulation induced by soluble NKG2D-ligands, it has recently been described that persistent cell-cell contact can also down-modulate NKG2D expression. The goal of this study was to determine whether the NKG2D receptor is down-modulated by cell-cell contact with cervical cancer cells and whether this down-modulation might be associated with changes in NK cell activity.ResultsWe demonstrate that NKG2D expressed on NKL cells is down-modulated by direct cell contact with cervical cancer cell lines HeLa, SiHa, and C33A, but not with non-tumorigenic keratinocytes (HaCaT). Moreover, this down-modulation had functional implications. We found expression of NKG2D-ligands in all cervical cancer cell lines, but the patterns of ligand distribution were different in each cell line. Cervical cancer cell lines co-cultured with NKL cells or fresh NK cells induced a marked diminution of NKG2D expression on NKL cells. Additionally, the cytotoxic activity of NKL cells against K562 targets was compromised after co-culture with HeLa and SiHa cells, while co-culture with C33A increased the cytotoxic activity of the NKL cells.ConclusionsOur results suggest that differential expression of NKG2D-ligands in cervical cancer cell lines might be associated with the down-modulation of NKG2D, as well as with changes in the cytotoxic activity of NKL cells after cell-cell contact with the tumor cells.

A possible mechanism of impaired NK cytotoxicity in cancer patients: Down-regulation of DAP10 by TGF-β1

Elevated TGF-BETA1 secretion and down-modulation of NKG2D underlies impaired NK cytotoxicity in cancer patients. However, the molecular mechanism of immunosuppression by TGF-BETA1 is not yet clarified. IL-2-activated human NK cells were cultured with TGF-BETA1. Protein levels of NKG2D and DAP10 were examined by FACS or immunoblot analyses. Real-time RTPCR was performed to quantify the transcription levels. MAPK inhibitors were used to investigate intracellular signaling. TGF-BETA1 down-regulated total and surface NKG2D, which was partially dependent on transcriptional regulation. TGF-BETA1 treatment of human NK cells resulted in significant changes in both transcriptional and translational levels of DAP10. Moreover, treatment with bafilomycin A1 or folimycin restored total NKG2D levels in TGF-BETA1-treated NK cells. The impaired NKG2D down-modulation by TGF-BETA1 was not associated with activation of the MAPK signaling pathway. TGF-BETA1 down-modulates surface NKG2D expression by controlling the transcriptional and translational levels of DAP10.

Hepatitis C Virus (HCV) Evades NKG2D-Dependent NK Cell Responses through NS5A-Mediated Imbalance of Inflammatory Cytokines

Understanding how hepatitis C virus (HCV) induces and circumvents the host's natural killer (NK) cell-mediated immunity is of critical importance in efforts to design effective therapeutics. We report here the decreased expression of the NKG2D activating receptor as a novel strategy adopted by HCV to evade NK-cell mediated responses. We show that chronic HCV infection is associated with expression of ligands for NKG2D, the MHC class I-related Chain (MIC) molecules, on hepatocytes. However, NKG2D expression is downmodulated on circulating NK cells, and consequently NK cell-mediated cytotoxic capacity and interferon-γ production are impaired. Using an endotoxin-free recombinant NS5A protein, we show that NS5A stimulation of monocytes through Toll-like Receptor 4 (TLR4) promotes p38- and PI3 kinase-dependent IL-10 production, while inhibiting IL-12 production. In turn, IL-10 triggers secretion of TGFβ which downmodulates NKG2D expression on NK cells, leading to their impaired effector functions. Moreover, culture supernatants of HCV JFH1 replicating Huh-7.5.1 cells reproduce the effect of recombinant NS5A on NKG2D downmodulation. Exogenous IL-15 can antagonize the TGFβ effect and restore normal NKG2D expression on NK cells. We conclude that NKG2D-dependent NK cell functions are modulated during chronic HCV infection, and demonstrate that this alteration can be prevented by exogenous IL-15, which could represent a meaningful adjuvant for therapeutic intervention.

Detuning CD8+ T lymphocytes by down-regulation of the activating receptor NKG2D: role of NKG2D ligands released by activated T cells

AbstractNKG2D is an activating receptor expressed on CD8+αβ+ T cells, γδ+ T cells, natural killer (NK) cells, and some CD4+ T cells. For a long time, the interaction of NKG2D with its ligands (NKG2DLs) MICA, MICB, and ULBP1-3 has been considered a mechanism for recognition and elimination of tumor, infected, or otherwise “stressed” cells. However, a new role for NKG2D as an immunoregulatory receptor is emerging. Here, we show that NKG2D is strongly down-modulated on antigen-activated CD8+ T cells but only if CD4+ T cells are present. Down-modulation was caused by soluble factors produced by CD4+ T cells, and in particular soluble NKG2DLs were found in the supernatants of antigen-activated T-cell cultures. MICB was the ligand released at higher levels when CD4+ T cells were present in the cell cultures, suggesting that it could be the major player of NKG2D down-modulation. CD8+ T cells expressing low levels of NKG2D had impaired effector functions, as evaluated by proliferation, cytokine production, and cytotoxicity assays after combined triggering of NKG2D and TCR-CD3 complex. These findings show that activated CD4+ T cells expressing NKG2DLs can efficiently prevent NKG2D-mediated CD8+ T-cell functions, and suggest that the NKG2D/NKG2DL interaction can regulate immune responses.

Human Tumor-Derived Exosomes Down-Modulate NKG2D Expression

NKG2D is an activating receptor for NK, NKT, CD8(+), and gammadelta(+) T cells, whose aberrant loss in cancer is a key mechanism of immune evasion. Soluble NKG2D ligands and growth factors, such as TGFbeta1 emanating from tumors, are mechanisms for down-regulating NKG2D expression. Cancers thereby impair the capacity of lymphocytes to recognize and destroy them. In this study, we show that exosomes derived from cancer cells express ligands for NKG2D and express TGFbeta1, and we investigate the impact of such exosomes on CD8(+) T and NK cell NKG2D expression and on NKG2D-dependent functions. Exosomes produced by various cancer cell lines in vitro, or isolated from pleural effusions of mesothelioma patients triggered down-regulation of surface NKG2D expression by NK cells and CD8(+) T cells. This decrease was rapid, sustained, and resulted from direct interactions between exosomes and NK cells or CD8(+) T cells. Other markers (CD4, CD8, CD56, CD16, CD94, or CD69) remained unchanged, indicating the selectivity and nonactivatory nature of the response. Exosomal NKG2D ligands were partially responsible for this effect, as down-modulation of NKG2D was slightly attenuated in the presence of MICA-specific Ab. In contrast, TGFbeta1-neutralizing Ab strongly abrogated NKG2D down-modulation, suggesting exosomally expressed TGFbeta as the principal mechanism. Lymphocyte effector function was impaired by pretreatment with tumor exosomes, as these cells exhibited poor NKG2D-dependent production of IFN-gamma and poor NKG2D-dependent killing function. This hyporesponsiveness was evident even in the presence of IL-15, a strong inducer of NKG2D. Our data show that NKG2D is a likely physiological target for exosome-mediated immune evasion in cancer.

4-1BB regulates NKG2D costimulation in human cord blood CD8+ T cells

AbstractLigation of NKG2D, a potent costimulatory receptor, can be either beneficial or detrimental to CD8+ cytotoxic T cell (CTL) responses. Factors for these diverse NKG2D effects remain elusive. In this study, we demonstrate that 4-1BB, another costimulatory receptor, is an essential regulator of NKG2D in CD8+ T cells. Costimulation of NKG2D caused down-modulation of NKG2D, but induced 4-1BB expression on the cell surface, even in the presence of TGF-β1, which inhibits 4-1BB expression. Resulting NKG2D−4-1BB+ cells were activated but still in an immature state with low cytotoxic activity. However, subsequent 4-1BB costimulation induced cytotoxic activity and restored down-modulated NKG2D. The cytotoxic activity and NKG2D expression induced by 4-1BB on NKG2D+4-1BB+ cells were refractory to TGF-β1 down-modulation. Such 4-1BB effects were enhanced by IL-12. In contrast, in the presence of IL-4, 4-1BB effects were abolished because IL-4 down-modulated NKG2D and 4-1BB expression in cooperation with TGF-β1, generating another CD8+ T-cell type lacking both NKG2D and 4-1BB. These NKG2D−4-1BB− cells were inert and unable to gain cytotoxic activity. Our results suggest that 4-1BB plays a critical role in protecting NKG2D from TGF-β1–mediated down-modulation. Co-expression of NKG2D and 4-1BB may represent an important biomarker for defining competency of tumor infiltrating CD8+ T cells.

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

NKG2D receptor–ligand interaction triggers NK cell-mediated cytolysis and IFN-γ secretion. IFN-γ 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-γ 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-γ-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-γ-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-γ-induced MIC molecules participated in monocyte/NK cell interaction and that this interaction also involved mIL-15.

Myeloid Derived Dendritic Cells Inhibit CD8+ T Cell Responses by Down-Modulating NKG2D and 4–1BB Expression.

NKG2D is constitutively expressed on naïve human CD8+ T cells (CTLs) and is involved in antitumor and antiviral responses as a costimulatory receptor. Down-modulating NKG2D expression from the surface of CTLs may allow tumors to evade anti-tumor host immune reactions. Thus, stable NKG2D expression on CTLs is a potential clinical strategy to counteract tumor-induced immune evasion in cancer patients. Recently, evidence suggests that tolerogenic myeloid derived dendritic cells (MDCs) recruited to the tumor microenvironment are involved in immune evasion against anti-tumor CTL functions. In this study, we investigated the possibility that tolerogenic MDCs may control CTL activities by modulating NKG2D expression. To test this possibility, we developed MDCs in vitro from CD14+ monocytes isolated from human cord blood through a long term culture with M-CSF and IL-4 (Li et al. J Immunol . 174: 4706, 2005 and Blood in press, 2007) and subsequent activation with LPS. We found that the MDCs generated were able to dramatically inhibit CTL killing activity to the target C1R B lymphoma cell line that expressed MHC class I related chains (MIC), a ligand for NKG2D. Most importantly, the CTLs co-cultured with the allogenic MDCs lost expression of NKG2D, which was necessary to induce 4–1BB, another costimulatory receptor known to be important for effector/memory CTL responses. MDCs generated without IL-4 were less potent in down-regulating NKG2D. In contrast to MDCs, prototype DCs developed with GM-CSF and IL-4 rather increased both NKG2D and 4-1BB expression indicating that capacity for down-modulating NKG2D and 4-1BB on CTLs was unique for MDCs. CTLs co-cultured with MDCs became tolerogenic because they failed to respond to CD3 stimulation. A majority of the cells stayed in a Go cell cycle stage. Neither NKG2D nor cytotoxic activity could be restored in the presence of CD3/CD28 stimulation. We next investigated whether tolerization of CTLs induced by MDCs was correlated with lack of NKG2D and 4-1BB expression. To test this possibility, we generated CTLs of which NKG2D and 4–1BB expression were similarly down-modulated in the absence of MDCs by culturing naïve human cord blood CD8+ T cells with TGF-b1 and IL-4. These CTLs were tolerized similarly as seen in those co-cultured with MDCs, suggesting that lack of both NKG2D and 4–1BB expression on CTLs was a major contributor to the tolerization induced by MDCs. To further verify the importance of NKG2D and 4–1BB expression in CTL functions, we generated CTLs expressing both NKG2D and 4–1BB in vitro from naïve CD8+ T cells by costimulating consecutively with agonistic anti-NKG2D and anti-4–1BB in the presence of anti-CD3. These CTLs were not susceptible to down-modulation of NKG2D by MDCs and exhibited high levels of cytotoxic activity. They expressed CD45RO and CD44 and promptly produced IFN-g in response to IL-12, which are characteristic phenotypes of effector/memory CTL. In conclusion, MDCs developed in vitro from human cord blood CD14+ monocytes showed tolerizing capacity for CTLs by down-modulating NKG2D as well as 4-1BB expression. Because expression of NKG2D and 4–1BB was inter-dependent, CTLs in which NKG2D and 4–1BB expression was down-modulated by MDCs were not able to differentiate into NKG2D+4–1BB+ effector/memory CTLs. Our results suggest that tolerogenic MDCs recruited to the tumor microenvironment might allow evasion of immune tumor responses by down-modulating NKG2D and 4–1BB expression on tumor specific CTLs.

IL-2/IL-18 prevent the down-modulation of NKG2D by TGF-β in NK cells via the c-Jun N-terminal kinase (JNK) pathway

TGF-β is known to play a major role for the reduced NKG2D expression seen in cancer patients. However, the mechanisms for reduced TGF-β-induced down-regulation of NKG2D are unclear. In this study, we observed that IL-2/IL-18 increased the NKG2D expression in the TGF-β treated NK cell line in a dose-dependent manner. Incubation with the JNK inhibitor SP600125 inhibited the NKG2D expression induced by IL-2/IL-18 in the TGF-β treated human NK cell line. Moreover, the NK cytotoxicity assay showed that the reduced NK cytotoxicity by TGF-β was recovered by IL-2/IL-18 treatment. The results indicate that IL-2/IL-18 strongly prevented the TGF-β-induced NKG2D down-regulation in NK cells via the JNK pathway. Taken together, the protected expression of NKG2D by IL-2/IL-18 provides insight into the mechanism of NKG2D regulation and it also supplied useful information for creating a novel therapeutic approach to treat TGF-β-secreting cancer cells.

Evasion from NK cell immunity by MHC class I chain-related molecules expressing colon adenocarcinoma.

Evasion of host immune responses is well documented for viruses and may also occur during tumor immunosurveillance. The mechanisms involve alterations in MHC class I expression, Ag processing and presentation, chemokine and cytokine production, and lymphocyte receptor expression. Epithelial tumors overexpress MHC class I chain-related (MIC) molecules, which are ligands for the activating receptor NKG2D on NK and T cells. We report that NK cells from patients with colorectal cancer lack expression of activating NKG2D and chemokine CXCR1 receptors, both of which are internalized. Serum levels of soluble MIC (sMIC) are elevated and are responsible for down-modulation of NKG2D and CXCR1. In contrast, high serum levels of CXC ligands, IL-8, and epithelial-neutrophil-activating peptide (ENA-78) do not down-modulate CXCR1. In vitro, internalization of NKG2D and CXCR1 occurs within 4 and 24 h, respectively, of incubating normal NK cells with sMIC-containing serum. Furthermore, natural cytotoxicity receptor NKp44 and chemokine receptor CCR7 are also down-modulated in IL-2-activated NK cells cocultured in MIC-containing serum-an effect secondary to the down-modulation of NKG2D and not directly caused by physical association with sMIC. The patients' NK cells up-regulate expression of NKG2D, NKp44, CXCR1, and CCR7 when cultured in normal serum or anti-MIC Ab-treated autologous serum. NKG2D(+) but not NKG2D(-) NK cells are tumoricidal in vitro, and in vivo they selectively traffic to the xenografted carcinoma, form immunological synapse with tumor cells, and significantly retard tumor growth in the SCID mice. These results suggest that circulating sMIC in the cancer patients deactivates NK immunity by down-modulating important activating and chemokine receptors.