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The activating immune receptor NKG2D binds to several stress-induced ligands that are structurally different. MHC-class-I-related chain (MIC) A/B molecules have a transmembrane domain, whereas most UL16 binding proteins (ULBPs) are glycosylphosphatidylinositol (GPI)-linked molecules. The significance of this variability in membrane anchors is unclear. Here, we demonstrate that ULBP2, but not ULBP1 or ULBP3, can reach the cell surface without the GPI modification. Several proteins are expressed at the cell surface as both transmembrane and GPI-linked molecules, either via alternative splicing or by the expression of linked genes. However, to our knowledge, ULBP2 is the first single mammalian cDNA that can be expressed as either a transmembrane or a GPI-anchored protein. The rate of maturation and the levels of cell surface expression of the non-GPI-linked form were lower than those of the GPI-linked ULBP2. Nonetheless, non-GPI ULBP2 was recognised by NKG2D and triggered NK cell cytotoxicity. These data show that differences in membrane attachment by NKG2D ligands are more important for regulation of their surface expression than for cytotoxic recognition by NKG2D and emphasise that detailed characterisation of the cell biology of individual NKG2D ligands will be necessary to allow targeted modulation of this system.

Natural-killer group 2, member D (NKG2D) binds to a variety of ligands, including the major histocompatibility complex (MHC) class I chain-related proteins (MIC) and UL16-binding proteins (ULBP). It is regarded as a co-activating receptor on NK cells, having an important role in the cell-mediated immune response to tumours. We studied the influence of interleukin (IL)-10 on the regulation of MIC and ULBP expression on melanoma cells, and its effect on the cytotoxic function of NK cells in vitro. Here, we show that, in the presence of IL-10, FMS mel and BL mel cell lines decreased MICA and ULBP2 surface expression, whereas MHC class I did not change substantially on the cell surface. MICA mRNA levels decreased in IL-10-treated FMS and IL-10-transduced BL cell lines. Interestingly, we observed that MICB surface expression and its mRNA levels increased upon IL-10 treatment in a melanoma cell line. These changes in NKG2D ligands surface expression patterns owing to IL-10 treatment resulted in an effect on lysis susceptibility mediated by lymphocyte-activated killer cells, as tumour cell lines that displayed a higher decrease of MICA on their surface had lower levels of lysis. In addition, expression of CD107a was downregulated on the surface of NK cells following stimulation with IL-10-treated FMS cells. Our results suggest a novel function for IL-10 in the modulation of NKG2D ligand expression and in the control of cytotoxicity mediated by NKG2D/NKG2D ligand axis.

Zentrale Fragestellung der Arbeit war es, eine mögliche funktionelle Verknüpfung zwischen dem Onkoprotein Ras und dem Prozess der DNA-Methylierung in der Suppression bestimmter für die Tumorabwehr wichtiger Gene experimentell zu unterstützen. Eine Verminderung der MHC-I-Expression wurde bereits im Zusammenhang mit Ras beschrieben. Weitere wichtige Erkennungs-Moleküle für NK- Zellen und T-Zellen sind ULBPs, MHC-Klasse-I verwandte Liganden der Natürlichen Killerzellrezeptoren (NKG2D). Die ULBP-Expression ist bis jetzt nur wenig verstanden. Gesundes Gewebe exprimiert kaum ULBPs, wohingegen krankes Gewebe als Stressantwort normalerweise eine deutliche Oberflächenanreicherung verschiedenster NKG2D-Liganden (MIC-A, MIC-B, ULBP1/2/3) zeigt. Im Hinblick auf Tumoren findet sich ein heterogenes Bild. Einige Tumore exprimieren wie vermeintlich gesundes Gewebe gar keine Liganden, andere sehr viele. Aufgrund der vielseitigen therapeutischen Ansatzmöglichkeiten, die sich aus der Aufklärung der Regulationsmechanismen der NKG2D-Liganden ergeben könnten, sind diese ubiquitärer Gegenstand der aktuellen tumorimmunologischen Forschung. Um einen potentiellen Zusammenhang zwischen Ras, DNA-Methylierung und Gensuppression zu untersuchen, wurden Genexpressionsprofile für die humane Kolonkarzinom Zelllinie HCT116 erstellt. Unter den supprimierten Genen, die nach Inhibition der Ras-Raf-Mek-Erk-Kaskade wieder aufreguliert wurden, befanden sich HLA-A1/2 und ULBP2 und -3. In der vorliegenden Arbeit analysierten wir die Methylierungsmuster der Promotoren dieser Gene mittels Bisulfitsequenzierung in der parentalen HCT116 Zelllinie und in drei weiteren von Rhee et al. generierten Knockout-Zelllinien: HCT116 DNMT1-/-, DNMT3b-/- und DKO. Der ULBP2-Promotor zeigte eine signifikante Hypermethylierung, die nach einer Behandlung mit dem Mek/Erk-Inhibitor U0126 nahezu vollständig aufgehoben werden konnte. Weiterhin konnten wir eine deutliche Hypomethylierung in den drei Knockout-Zelllinien DNMT1-/-, DNMT3b-/- und DKO zeigen. HLA-A1/2 waren nicht methyliert, jedoch ergaben Vorexperimente nach Mek/Erk-Inhibierung und Doppelknockout eine vermehrte Oberflächenexpression für beide Allele. Sowohl ULBP2, als auch HLA-A1/2 scheinen durch ein abgestimmtes Zusammenspiel zwischen onkogenem Ras und DNMTs direkt bzw. indirekt reguliert zu werden. Unsere Ergebnisse verknüpfen daher die DNMT-Aktivität mit dem K-Ras induzierten Mek/Erk-Signalweg auf der Ebene der Zielgene. Die Ras-vermittelte und DNMT-abhängige Suppression der Immunerkennung könnte zukünftig in der Behandlung von Tumoren mit Inhibitoren der Mek/Erk-Signalkaskade einen wichtigen Fortschritt in der Immuntherapie bedeuten., Most malignant features of cancer cells are triggered by activated oncogenes and the loss of tumor suppressors due to mutation or epigenetic inactivation. It is still unclear, to what extend the escape of emerging cancer cells from recognition and elimination by the immune system is determined by similar mechanisms. Previously transcriptomes of HCT116 colorectal cancer cells deficient in DNA methyltransferases (DNMTs) and of cells, in which the RAS pathway as the major growth-promoting signaling system is blocked by inhibition of MAPK were compared. Interestingly the MHC Class I genes HLA-A1/A2 and the ULBP2 gene encoding 1 of the 8 known ligands of the activating NK receptor NKG2D were among a cluster of immune genes up-regulated under the conditions of both DNMT-deficiency and MEK-inhibition. Bisulphite sequencing analyses of HCT116 with DNMT deficiency or after MEK-inhibition showed that de-methylation of the ULPB2 promoter correlated with its enhanced surface expression. Cosuppression of HLA Class I and NKG2D ligands and genes encoding peptide transporters or proteasomal genes mediates a strong functional link between RAS activation, DNMT activity and disruption of the antigen presenting system controlling immune recognition in colorectal cancer cells.

Purpose: Cytotoxic lymphocytes interact with human tumor cells via the activating immunoreceptor NKG2D, recognizing a variety of stress-associated MIC and ULBP surface molecules. However, tumors can escape from this immunosurveillance by shedding NKG2D ligands (NKG2DL), rendering the soluble products detectable in patients' sera. Experimental Design: To elucidate the clinical significance of NKG2DL diversity, we studied their expression on melanoma tissues and their presence as soluble molecules in sera from >200 melanoma patients and compared the latter with the well-established serum marker S100B. Results: Immunohistochemistry revealed a heterogeneous expression of MIC and ULBP2 molecules between and within melanoma metastases. Compared with MIC, ULBP2 was less frequently expressed. Accordingly, elevated levels of soluble ULBP2 (sULBP2) were detected in sera of melanoma patients less frequently than elevated levels of soluble MICA (sMICA), although both soluble NKG2DL (sNKG2DL) were significantly increased compared with sera of healthy controls (P < 0.0001). Strikingly, elevated concentrations of sULBP2, but not of sMICA, were strongly associated with disease progression (P < 0.0001) and tumor load (P = 0.0003). Elevated serum levels of either sNKG2DL correlated with reduced overall survival, albeit considerably stronger for sULBP2 (P < 0.0001) than for sMICA (P = 0.011). In early-stage (I-III) melanoma patients, only sULBP2 (P < 0.0001) but neither sMICA nor S100B revealed prognostic significance. Multivariate analysis identified sULBP2 (P = 0.0015) and S100B (P = 0.013) but not sMICA as independent predictors of prognosis. Conclusion: Our data reveal marked differences in the clinical significance of individual sNKG2DL. Only sULBP2 is an independent predictor of prognosis, the significance of which is superior to the well-established and widely used melanoma serum marker S100B. (Clin Cancer Res 2009;15(16):5208–15)

Most malignant features of cancer cells are triggered by activated oncogenes and the loss of tumor suppressors due to mutation or epigenetic inactivation. It is still unclear, to what extend the escape of emerging cancer cells from recognition and elimination by the immune system is determined by similar mechanisms. We compared the transcriptomes of HCT116 colorectal cancer cells deficient in DNA methyltransferases (DNMTs) and of cells, in which the RAS pathway as the major growth-promoting signaling system is blocked by inhibition of MAPK. We identified the MHC Class I genes HLA-A1/A2 and the ULBP2 gene encoding 1 of the 8 known ligands of the activating NK receptor NKG2D among a cluster of immune genes up-regulated under the conditions of both DNMT-deficiency and MEK-inhibition. Bisulphite sequencing analyses of HCT116 with DNMT deficiency or after MEK-inhibition showed that de-methylation of the ULPB2 promoter correlated with its enhanced surface expression. The HLA-A promoters were not methylated indicating that components of the HLA assembly machinery were also suppressed in DNMT-deficient and MEK-inhibited cells. Increased HLA-A2 surface expression was correlated with enhanced recognition and lysis by A2-specific CTL. On the contrary, elevated ULBP2 expression was not reflected by enhanced recognition and lysis by NK cells. Cosuppression of HLA Class I and NKG2D ligands and genes encoding peptide transporters or proteasomal genes mediates a strong functional link between RAS activation, DNMT activity and disruption of the antigen presenting system controlling immune recognition in colorectal cancer cells.

The activating NK cell receptor NKG2D binds to numerous stress-induced cell surface glycoproteins with MHC class I-related ectodomains. In humans, NKG2D ligands (NKG2DL) are members of the MHC-encoded MIC and non-MHC-encoded ULBP families of proteins. The redundancy of NKG2DL raises questions about unique features associated with individual NKG2DL. The ULBP family member RAET1G contains an ectodomain highly related to ULBP2, but is unique by virtue of an extended cytoplasmic domain. Since RAET1G is poorly characterized, we studied expression and functional interactions of RAET1G in comparison to ULBP2. RAET1G transcripts were detected in most human tissues with an overall expression pattern similar to ULBP2. However, although ULBP2 strongly binds both NKG2D and the immunoevasive human cytomegalovirus glycoprotein UL16, RAET1G only weakly interacts with NKG2D and does not bind UL16. Differential binding capacities of the two highly related ectodomains are mainly due to a substitution of a conserved amino acid in the alpha2 domain of RAET1G. In functional terms, the reduced apparent avidity of RAET1G for NKG2D results in a less-efficient NKG2D down-regulation and NK degranulation. Altogether, RAET1G, like ULBP2, appears broadly expressed, but exhibits a lower apparent avidity for NKG2D due to a mutation in the center of the MHC-like fold.

Human papillomavirus-induced cervical carcinomas often show impaired expression of MHC class I molecules resulting in the inability of tumor cells to directly present viral peptides to cytotoxic T lymphocytes. Loss of MHC class I expression combined with the expression of activating NK cell receptor ligands renders tumor cells potentially susceptible to NK cell attack. Thus, in this study, we analyzed the expression of activating NK cell receptor ligands, NK cell accumulation and activation status in situ in normal ectocervical tissue (NCT), cervical intraepithelial neoplasia (CIN) and squamous cervical carcinoma (CxCa). We observed that expression of the DNAM-1 ligand CD155 was frequently upregulated in CxCa, but not in CIN. The NKG2D ligand MICA was upregulated in fewer CxCa biopsies. In contrast, another NKG2D ligand ULBP2 was preferentially expressed in differentiated epithelial cells of NCT. Increased numbers of NK cells were detected in CIN as compared to NCT and CxCa. Expression of activating NK cell receptor ligands combined with loss of MHC class I was not correlated with enhanced NK cell accumulation or activation status. Furthermore, we demonstrate that cervical cancer cell lines are killed by the NK cell line, NKL, in a NKG2D- and DNAM-1-dependent manner in vitro. Since a significant number of CxCa biopsies showed low MHC class I expression combined with high expression of one or more of the tested activating NK cell receptor ligands, we conclude that CxCa might be a promising target for NK cell-based adoptive immunotherapy.

The interaction of the activating receptor NKG2D with its ligands plays an important role in immunosurveillance of tumors and infectious pathogens, but dysregulation of this system may lead to autoimmunity. The expression of NKG2D ligands is induced by cellular “stress.” However, the regulation of expression of these molecules is not well understood. Here, we show that cells treated with proteasome inhibitors can become more susceptible to cytotoxicity mediated by natural killer cells because of the induction of expression of ligands for NKG2D, specifically ULBP2, but not down-regulation of MHC class I. Treatment with proteasome inhibitors led to up-regulation of ULBP2 expression in multiple, but not all, cell lines tested. This increase in expression of ULBP2 at the cell surface correlated with induction of transcription of the ULBP2 gene and synthesis of ULBP2 protein. In contrast, treatment with inhibitors of histone deacetylases led to increased levels of mRNA and protein, for both ULBP2 and MHC class I–related chain A/B molecules. Thus, different types of stress can trigger up-regulated expression of different sets of NKG2D ligands. Proteasome inhibitors are proving to be of significant value in the treatment of hematologic malignancies and these observations may help to better understand the biology of therapy with these compounds. [Cancer Res 2008;68(5):1546–54]

Natural killer (NK) cells can defend an organism against a variety of threats, probably using several different strategies to discriminate between normal and aberrant cells. According to the 'missing self' hypothesis, one function of NK cells is to recognize and eliminate cells that fail to express self major histocompatibility complex (MHC) class I molecules. In this article Hans-Gustaf Ljunggren and Klas Kärre review in vivo studies with H-2-deficient targets that support this hypothesis. In vitro studies, some of which have given conflicting results, are interpreted within a multiple choice model for NK cell recognition. The authors derive testable predictions for how MHC class I molecules act in cases where they control a rate-limiting step in the NK cell-target interaction.

Monocyte-derived dendritic cells (mDCs) and NK cells are reciprocally activate via cytokines and cell-cell contact. Although seven human NKG2D ligands (NKG2DLs), UL16-binding proteins (ULBP) 1, 2, 3 and 4, retinoic acid early transcript 1G (RAET1G) and MHC class I-related chains A and B, have been reported, the differential distribution and roles of these ligands in the maturation of human mDCs have not been elucidated. In the present study, we produced polyclonal antibodies (pAbs) directed against human ULBP1, 2 and 3. All these ULBPs were detected on human mDCs when probed by the pAbs, although their expression profiles were different. We next investigated what kinds of Toll-like receptor agonists and RNA viruses [influenza virus, human respiratory syncytial virus (RSV), measles virus and hepatitis C virus (HCV)] induced the expression of NKG2DLs on mDCs. ULBP1 was up-regulated on mDCs in response to LPS or infection with RSV. The expression of ULBP2 was induced by LPS and poly I:C, indicating that the TIR-containing adapter molecule-1 (TIR domain-containing adaptor-inducing IFN) pathway is associated with ULBP2 induction. Although infection with HCV did not cause up-regulation of NKG2DLs, other RNA virus infections and poly I:C promoted expression of ULBP2 and RAET1G in an IFN-alpha/beta-independent manner. Finally, the over-expression of ULBP1 and 2 on mDCs facilitated NK cell proliferation and IFN-gamma production through a mDC-NK cell interaction in the presence of IL-2. Hence, the results reflect the important role of NKG2DLs on human mDCs in mDC-mediated NK cell activation.

Data on 23 patients with low-grade non-Hodgkin lymphomas (NHLs), 4 mantle (MT), 4 marginal zone (MZ), and 15 follicular (FL), were analyzed and compared with 10 high-risk (HR) B-cell chronic lymphocytic leukemias (B-CLLs) with lymph node involvement and 4 diffuse large-cell lymphomas (DLCLs). A significant increase in circulating Vδ1 T lymphocytes producing interleukin-4 (IL-4) was found in patients with FL, MT, and MZ NHL, at variance with DLCL and HR B-CLL. IL-4 was also detectable in the sera and lymph nodes of the same patients. In 19 of the 23 patients with NHL with increased circulating Vδ1 T lymphocytes, B cells expressing the UL-16–binding proteins (ULBPs) ULBP2 or ULBP3 or both were found in peripheral blood, bone marrow, or lymph nodes. Of note, in HR B-CLL or in DLCL, where leukemic cells were negative for ULBPs, no Vδ1 T-cell increase was found. Moreover, Vδ1 T lymphocytes from patients with FL NHL proliferate in response to ULBP2+ and ULBP3+ lymphoma cells. Finally, patients with high expression of ULBPs, increased circulating Vδ1 T lymphocytes, and high levels of serum IL-4 showed stable disease in a 1-year follow-up in contrast to patients with low circulating Vδ1 T cells and undetectable IL-4 or ULBPs.

The interaction of the MHC class I-related chain molecules A and B (MICA and MICB) and UL-16 binding protein (ULBP) family members expressed on tumor cells with the corresponding NKG2D receptor triggers cytotoxic effector functions in NK cells and γδ T cells. However, as a mechanism of tumor immune escape, NKG2D ligands (NKG2DLs) can be released from the cell surface. In this study, we investigated the NKG2DL system in different human glioblastoma (GBM) cell lines, the most lethal brain tumor in adults. Flow cytometric analysis and ELISA revealed that despite the expression of various NKG2DLs only ULBP2 is released as a soluble protein via the proteolytic activity of “a disintegrin and metalloproteases” (ADAM) 10 and 17. Moreover, we report that temozolomide (TMZ), a chemotherapeutic agent in clinical use for the treatment of GBM, increases the cell surface expression of NKG2DLs and sensitizes GBM cells to γδ T cell-mediated lysis. Both NKG2D and the T-cell receptor (TCR) are involved. The cytotoxic activity of γδ T cells toward GBM cells is strongly enhanced in a TCR-dependent manner by stimulation with pyrophosphate antigens. These data clearly demonstrate the complexity of mechanisms regulating NKG2DL expression in GBM cells and further show that treatment with TMZ can increase the immunogenicity of GBM. Thus, TMZ might enhance the potential of the adoptive transfer of ex vivo expanded γδ T cells for the treatment of malignant glioblastoma.

Human CMV infection results in MHC class I down-regulation and induction of NKG2D ligand expression favoring NK recognition of infected cells. However, human CMV-encoded UL16 counteracts surface expression of several NKG2D ligands by intracellular retention. Interestingly, UL16 interacts with MICB, but not with the closely related MICA, and with UL16-binding proteins (ULBP) ULBP1 and ULBP2, which are only distantly related to MICB, but not with ULPB3 or ULBP4, although all constitute ligands for NKG2D. Here, we dissected the molecular basis of MICA-MICB discrimination by UL16 to elucidate its puzzling binding behavior. We report that the UL16-MICB interaction is independent of glycosylation and demonstrate that selective MICB recognition by UL16 is governed by helical structures of the MICB α2 domain. Transplantation of the MICB α2 domain confers UL16 binding capacity to MICA, and thus, diversification of the MICA α2 domain may have been driven by the selective pressure exerted by UL16.

NKG2D ligands (NKG2DL) are expressed by infected and transformed cells. They transmit danger signals to NKG2D-expressing immune cells, leading to lysis of NKG2DL-expressing cells. We here report that the NKG2DL MHC class I-chain-related molecules A and B (MICA/B) and UL16-binding proteins (ULBP) 1-3 are expressed in human brain tumours in vivo, while expression levels are low or undetectable in normal brain. MICA and ULBP2 expression decrease with increasing WHO grade of malignancy, while MICB and ULBP1 are expressed independently of tumour grade. We further delineate two independent mechanisms that can explain these expression patterns: (i) transforming growth factor-beta (TGF-beta) is upregulated during malignant progression and selectively downregulates MICA, ULBP2 and ULBP4 expression, while MICB, ULBP1 and ULBP3 are unaffected. (ii) Cleavage of MICA and ULBP2 is reduced by inhibition of metalloproteinases (MP), whereas no changes in the expression levels of other NKG2DL were detected. Consequently, NKG2DL-dependent NK cell-mediated lysis is enhanced by depletion of TGF-beta or inhibition of MP. Thus, escape from NKG2D-mediated immune surveillance of malignant gliomas in vivo may be promoted by the inhibition of MICA and ULBP2 expression via an autocrine TGF-beta loop and by MP-dependent shedding from the cell surface. Loss of MICA and ULBP2, in contrast to other NKG2DL, may be particularly important in glioma immune escape, and differential regulation of human NKG2DL expression is part of the immunosuppressive properties of human malignant glioma cells.

Human cytomegalovirus (HCMV) employs a variety of strategies to modify or evade the host immune response, and natural killer (NK) cells play a crucial role in controlling cytomegalovirus infections in mice and humans. Activation of NK cells through the receptor NKG2D/DAP10 leads to killing of NKG2D ligand-expressing cells. We have previously shown that HCMV is able to down-regulate the surface expression of some NKG2D ligands, ULBP1, ULBP2, and MICB via the viral glycoprotein UL16. Here, we show that the viral gene product UL142 is able to down-regulate another NKG2D ligand, MICA, leading to protection from NK cytotoxicity. UL142 is not able to affect surface expression of all MICA alleles, however, which may reflect selective pressure on the host to thwart viral immune evasion, further supporting an important role for the MICA-NKG2D interaction in immune surveillance.

We studied the role of NK cell-activating receptors and their ligands in the lysis of mononuclear phagocytes infected with the intracellular pathogen Mycobacterium tuberculosis. Expression of the activating receptors NKp30, NKp46, and NKG2D were enhanced on NK cells by exposure to M. tuberculosis-infected monocytes, whereas expression of DNAX accessory molecule-1 and 2B4 was not. Anti-NKG2D and anti-NKp46 inhibited NK cell lysis of M. tuberculosis-infected monocytes, but Abs to NKp30, DNAX accessory molecule-1, and 2B4 had no effect. Infection of monocytes up-regulated expression of the NKG2D ligand, UL-16 binding protein (ULBP)1, but not expression of ULBP2, ULBP3, or MHC class I-related chain A or chain B. Up-regulation of ULBP1 on infected monocytes was dependent on TLR2, and anti-ULBP1 abrogated NK cell lysis of infected monocytes. The dominant roles of NKp46, NKG2D, and ULBP1 were confirmed for NK cell lysis of M. tuberculosis-infected alveolar macrophages. We conclude that NKp46 and NKG2D are the principal receptors involved in lysis of M. tuberculosis-infected mononuclear phagocytes, and that ULBP1 on infected cells is the major ligand for NKG2D. Furthermore, TLR2 contributes to up-regulation of ULBP1 expression.

The NKG2D receptor is expressed by human NK, gammadelta T and alpha/beta T lymphocytes and its engagement results in the stimulation of effector cells. We evaluated the role of NKG2D receptor in anti-colorectal cancer (CRC) immune response. The cell surface expression of stress-inducible NKG2D ligands MICA/B (MHC class I-related chain molecules A/B) and ULBP (UL16 binding protein) by a panel of CRC lines was evaluated by flow cytometry. MICA and ULBP2/3 were widely expressed by the analyzed lines, with a minority of them being also ULBP-1+, whereas MICB was undetectable. CD8+ and CD4+ HLA-restricted anti-tumor T cell clones of a CRC patient were used to evaluate whether NKG2D engagement could mediate tumor recognition. Three out of four CD8+ T cell clones recognized the autologous tumor with a marginal NKG2D engagement, a finding that was correlated with the weak expression of NKG2D ligands by the autologous tumor. On the contrary, NKG2D triggering of these CD8+ T cell clones induced recognition of allogeneic CRC lines showing high expression of MICA and ULBP. A costimulatory role of NKG2D was observed with one CD4+/NKG2D+ T cell clone when stimulated by tumors sharing the HLA class II alleles and expressing NKG2D ligands. Taken together these data indicate that the engagement of NKG2D, depending on the expression of its ligands by target cells, can influence the pattern of anti-tumor reactivity by T lymphocytes.

Human CMV (HCMV) interferes with NK cell functions at various levels. The HCMV glycoprotein UL16 binds some of the ligands recognized by the NK-activating receptor NKG2D, namely UL16-binding proteins (ULBP) 1 and 2 and MHC class I-related chain B, possibly representing another mechanism of viral immune escape. This study addressed the expression and function of these proteins in infected cells. HCMV induced the expression of all three ULBPs, which were predominantly localized in the endoplasmic reticulum of infected fibroblasts together with UL16. However, while at a lower viral dose ULBP1 and 2 surface expression was completely inhibited compared to ULBP3, at a higher viral dose cell surface expression of ULBP1 and ULBP2 was delayed. The induction of ULBPs correlated with an increased dependency on NKG2D for recognition; however, the overall NK sensitivity did not change (suggesting that additional viral mechanisms interfere with NKG2D-independent pathways for recognition). Infection with a UL16 deletion mutant virus resulted in a different pattern compared to the wild type: all three ULBP molecules were induced with similar kinetics at the cell surface, accompanied by a pronounced, entirely NKG2D-dependent increase in NK sensitivity. Together our findings show that upon infection with HCMV, the host cell responds by expression of ULBPs and increased susceptibility to the NKG2D-mediated component of NK cell recognition, but UL16 limits these effects by interfering with the surface expression of ULBP1 and ULBP2.

The secretion of the soluble NKG2D-ligand ULBP2 is a marker for poor prognosis in several types of cancer. NKG2D-ligands are normally directed to the cell surface of stressed, inflamed or pre-cancer cells and serve as targets for immune effector cells expressing the NKG2D receptor. The soluble NKG2D-ligands are devastating due to their chronic activation and thereby desensitization of NKG2D expressing immune effector cells. In fact this leads to severely compromised immune activation governing cancer development. The present study describes that methylselenic acid (MSA) is able to inhibit soluble ULBP2 in different primary melanoma cell lines. Moreover, MSA actively suppressed ULBP2 both from constitutively expressing tumors and after induction by HDAC-inhibitors. In the attempt to elucidate the mechanism of inhibition, real-time qPCR experiments were performed. MSA activated the promoter and the transcription of the ULBP2 gene at a level similar to HDAC-inhibitors. These findings strongly indicate that ULBP2 is post-transcriptional regulated. Selective blocking of ULBP2 from melanomas or other cancers that produces soluble immune suppressive ULBP2 is thus a new treatment strategy that also has the potential to be used in combination with existing treatment regimens. Citation Format: Franziska K. Uhlenbrock, Michael Hagemann Jensen, Stephanie Kehlet, Lars Andresen, Søren Skov. Methylselenic acid inhibits the soluble and cell-surface bound NKG2D-ligand ULBP2 in melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4088. doi:10.1158/1538-7445.AM2014-4088

Aim: The role of immune escape of tumor cells is poorly understood. We investigated anticancer drugs induced NK group 2 member D (NKG2D) ligand expression in NSCLC, since immunosurveillance is maintained by mainly NK cells via the NKG2D-NKG2D ligand interaction. Here we demonstrate cytotoxic regents enhance NKG2D ligand MHC class I-related chain A and B (MICA/B) and UL16 binding protein (ULBP), resulting in enhancement of NK cell-mediated cytotoxicity in NSCLC cells in vitro, while contrasting effects are seen in clinical samples from NSCLC patients who received induction chemotherapy. Methods: A possible influence of anticancer drugs (gemcitabine, pemetrexed, docetaxel and vinorelbine) on the expressions of NKG2D ligands MICA/B and ULBP1-3 in 5 NSCLC cell lines was investigated by flow cytometry. To access the influence of DNA stress-induced ATM-ATR signaling, A549 cells were pretreated with ATM-ATR inhibitor KU55399 or caffeine then treated with gemcitabine, then the expressions of NKG2D ligands were assessed. NK cell-mediated cytotoxicity against A549 cells treated with gemcitabine was assessed by LDH release assay or CD107a degranulation assay. Formaldehyde-fixed, paraffin-embedded NSCLC specimens before and after chemotherapy were collected from 3 patients, then both MICA/B and ULBP-2 expressions were analyzed by immunohistochemical reaction (IHC). Results: Both gemcitabine and docetaxel promoted NKG2D ligands in NSCLC cell lines. Gemcitabine-induced NKG2D ligands expression was blocked by ATM-ATR inhibitors, and, as expected, gemcitabine enhanced NKG2D ligands induced NK cell-mediated cytotoxicity, and anti-NKG2D blocking antibody attenuated gemcitabine-induced NK killing in A549 cells. On the other hand, IHC data showed the expression of MICA/B and ULBP2 were regressed upon gemcitabine treatment in tumors from NSCLC patients. Conclusions: Cytotoxic anticancer drugs induce NKG2D ligands in cancer cell lines, while regressing them in clinical samples, suggesting that the cells expressing low level of NKG2D ligands can escape from NK cells in a host. Keeping the expression of NKG2D ligands in tumor around chemotherapy may be a good strategy for NSCLC treatment. Disclosure: M. Nakata: The author have received research grant from AstraZeneca. All other authors have declared no conflicts of interest.

UL16 binding proteins (ULBPs) are a family of cell surface proteins that are present in transformed and stressed cells and ligands for NKG2D. Soluble NKG2D ligands have been found in sera from cancer patients with their protein concentrations correlated with poor cancer prognosis. Here we show, for the first time, that human tumor cells lost their surface expression of ULBP2, but not ULBP1 and ULBP3, during NK cell-mediated cytolysis. In contrast to spontaneous shedding of NKG2D ligands, NK cytolysis-mediated shedding of ULBP2 was linked to target cell apoptosis, although both resulted from metalloproteinase cleavages. Inhibition of ULBP2 shedding by a metalloproteinase inhibitor BB-94 lead to reduced NK cell-mediated cytotoxicity and cytokine production. These results illustrate a regulation of NK cell effector functions through cytolysis-induced NKG2D ligand shedding. Consequently, compounds inhibiting NKG2D ligand shedding may offer therapeutic means to reduce excessive pathogenic NK cell activities.

The major histocompatibility complex (MHC) class I homolog, MICA, is a stress-inducible ligand for NKG2D, a C-type lectin-like activating immunoreceptor. The crystal structure of this ligand-receptor complex that we report here reveals an NKG2D homodimer bound to a MICA monomer in an interaction that is analogous to that seen in T cell receptor-MHC class I protein complexes. Similar surfaces on each NKG2D monomer interact with different surfaces on either the alpha1 or alpha2 domains of MICA. The binding interactions are large in area and highly complementary. The central section of the alpha2-domain helix, disordered in the structure of MICA alone, is ordered in the complex and forms part of the NKG2D interface. The extensive flexibility of the interdomain linker of MICA is shown by its altered conformation when crystallized alone or in complex with NKG2D.

Human MHC class I-related molecules, MICA and MICB, are stress-induced antigens that are recognized by a subset of γδ T cells expressing the variable region Vδ1. This functional association has been found to be limited to intestinal epithelium, where these T cells are prevalent and where MICA and, presumably, MICB are mainly expressed. However, increased frequencies of Vδ1 γδ T cells have been observed in various epithelial tumors; moreover, MICA/B are expressed on diverse cultured epithelial tumor cells. With freshly isolated tumor specimens, expression of MICA/B was documented in many, but not all, carcinomas of the lung, breast, kidney, ovary, prostate, and colon. In tumors that were positive for MICA/B, the frequencies of Vδ1 γδ T cells were significantly higher than in those that were negative. Vδ1 γδ T cell lines and clones derived from different tumors recognized MICA/B on autologous and heterologous tumor cells. In accord with previous evidence, no constraints were observed in these interactions, such as those imposed by specific peptide ligands. Thus, MICA/B are tumor-associated antigens that can be recognized, in an apparently unconditional manner, by a subset of tumor-infiltrating γδ T cells. These results raise the possibility that an induced expression of MICA/B, by conditions that may be related to tumor homeostasis and growth, could play a role in immune responses against tumors.

T cells with variable region Vδ1 γδ T cell receptors (TCRs) are distributed throughout the human intestinal epithelium and may function as sentinels that respond to self antigens. The expression of a major histocompatibility complex (MHC) class I–related molecule, MICA, matches this localization. MICA and the closely related MICB were recognized by intestinal epithelial T cells expressing diverse Vδ1 γδ TCRs. These interactions involved the α1α2 domains of MICA and MICB but were independent of antigen processing. With intestinal epithelial cell lines, the expression and recognition of MICA and MICB could be stress-induced. Thus, these molecules may broadly regulate protective responses by the Vδ1 γδ T cells in the epithelium of the intestinal tract.

NKG2D is an activating receptor that is expressed in immune effector cells, and the ligands for which are expressed in transformed cells. NKG2D-NKG2D ligand system might play an essential role in the tumor immunity of host cells. We have previously reported that a member of NKG2D ligand family, UL-16-binding protein 2 (ULBP2), is predominantly expressed on the surface of lung cancer cells, and is shed and solubilized into the sera of the lung cancer patients. In addition, we showed that the soluble form of ULBP2 (sULBP2) aggravates the cytotoxicity of host immune effector cells. Therefore, shedding of ULBP2 from the surface of cancer cells might be a critical component of the immune escape mechanisms employed in lung cancer. In this study, we investigated the mechanism of ULBP2 shedding from the cell surfaces in human lung cancer cell lines. First, we compared the amount of ULBP2 expression on the surface of normal cells and that in the culture medium of various lung cancer cell lines. We observed a significant correlation among non-small-cell lung cancer (NSCLC) cell lines (r=0.835, P Citation Format: Kosuke Yamaguchi, Hiroki Hikumi, Shinji Matsumaga, Miyako Takata, Naoki Kinoshita, Kiyoshi Hashimoto, Masaki Nakamoto, Jun Kurai, Masanari Watanabe, Akira Yamasaki, Tadashi Igishi, Naoto Burioka, Eiji Shimizu. ADAM proteases shed UL-16-binding protein 2 in human lung cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 454. doi:10.1158/1538-7445.AM2013-454

s / Pancreatology 13 (2013) e1–e20 e10 vs. 79.1%). In HC, CP and PC, the combination of threemarkers (iC3b, ULBP2 and CA 19.9) has better predictive value than CA19.9 alone or combination of ULBP2 and CA19.9 or combination of iC3b and CA19.9 (79.6% vs. 78% vs. 77.8% vs. 79.4%). Conclusion: Our preliminary results show that there is possibility of better predictive value in sensitivity and specificity with a combination of markers rather than a single marker alone. More samples are needed to qualify this finding. Take-home message: A combination of markers may have better predictive value than a single marker alone. Abstract previously presented? no () Any disclosures? no ()

Major histocompatibility complex (MHC) class I genes typically encode polymorphic peptide-binding chains which are ubiquitously expressed and mediate the recognition of intracellular antigens by cytotoxic T cells. They constitute diverse gene families in different species and include the numerous so-called nonclassical genes in the mouse H-2 complex, of which some have been adapted to variously modified functions. We have identified a distinct family of five related sequences in the human MHC which are distantly homologous to class I chains. These MIC genes (MHC class I chain-related genes) evolved in parallel with the human class I genes and with those of most if not all mammalian orders. The MICA gene in this family is located near HLA-B and is by far the most divergent mammalian MHC class I gene known. It is further distinguished by its unusual exon-intron organization and preferential expression in fibroblasts and epithelial cells. However, the presence of diagnostic residues in the MICA amino acid sequence translated from cDNA suggests that the putative MICA chain folds similarly to typical class I chains and may have the capacity to bind peptide or other short ligands. These results define a second lineage of evolutionarily conserved MHC class I genes. This implies that MICA and possibly other members in this family have been selected for specialized functions that are either ancient or derived from those of typical MHC class I genes, in analogy to some of the nonclassical mouse H-2 genes.

NKG2D ligands are the surface molecules which induce expression on transformed cells. Specifically they bind to NKG2D on NK cells, and this binding induces activation of NK cells. In humans, NKG2D ligands are described as MICA, MICB and ULBP1, 2, 3, and 4. In this study, we explored the clinical and immunological significance of NKG2D ligands on lung cancer. We evaluated the expression of each NKG2D ligand on the cell surface of various lung cancer cell lines by flow cytometory. The soluble form of ULBP2 was measured in culture supernatant and in sera of lung cancer patients by an original developed ELISA system. Immunological effects of cell surface ULBP2 and soluble form of ULBP2 were examined by 3.5-h 51Cr release assay. We found that ULBP2 was the most widely and strongly expressed NKG2D ligand in lung cancer cell lines. Soluble form of ULBP2 was increased in culture supernatant of the lung cancer cell lines with high expression of cell surface ULBP2. In addition, soluble form of ULBP2 was detectable in the sera of lung cancer patients and increased amount of soluble ULBP2 in sera significantly correlated with poor prognosis lung cancer patients. We evaluated the the immunological effect of soluble ULBP2 separately using human peripheral blood mononuclear cells (PBMCs), and found that soluble ULBP2 suppressed NK activity. In conclusion, ULBP2 is the most significant NKG2D ligand in lung cancer and its soluble form was detectable and elevated in sera of lung cancer patients. Because soluble form of ULBP2 is one of the determinant factor of the anti-tumor immunity of the cancer patients, the development of intervention in this effect would be a candidate for the promising immuno-modulatory therapy Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-313.

The activating immunoreceptor NKG2D promotes elimination of infected or malignant cells by cytotoxic lymphocytes through engagement of stress-induced MHC class I-related ligands. The human cytomegalovirus (HCMV)-encoded immunoevasin UL16 subverts NKG2D-mediated immune responses by retaining a select group of diverse NKG2D ligands inside the cell. We report here the crystal structure of UL16 in complex with the NKG2D ligand MICB at 1.8 Å resolution, revealing the molecular basis for the promiscuous, but highly selective, binding of UL16 to unrelated NKG2D ligands. The immunoglobulin-like UL16 protein utilizes a three-stranded β-sheet to engage the α-helical surface of the MHC class I-like MICB platform domain. Intriguingly, residues at the center of this β-sheet mimic a central binding motif employed by the structurally unrelated C-type lectin-like NKG2D to facilitate engagement of diverse NKG2D ligands. Using surface plasmon resonance, we find that UL16 binds MICB, ULBP1, and ULBP2 with similar affinities that lie in the nanomolar range (12–66 nM). The ability of UL16 to bind its ligands depends critically on the presence of a glutamine (MICB) or closely related glutamate (ULBP1 and ULBP2) at position 169. An arginine residue at this position however, as found for example in MICA or ULBP3, would cause steric clashes with UL16 residues. The inability of UL16 to bind MICA and ULBP3 can therefore be attributed to single substitutions at key NKG2D ligand locations. This indicates that selective pressure exerted by viral immunoevasins such as UL16 contributed to the diversification of NKG2D ligands., Author Summary Cytotoxic lymphocytes such as natural killer (NK) cells or CD8 T cells have the ability to detect and destroy cells infected by viruses. They therefore are tools on which the human immune system critically depends in order to control viral infections. To avoid discovery by cytotoxic lymphocytes and to allow for longtime persistence in the human host, the human cytomegalovirus (HCMV) has developed a multitude of immune evasive strategies that are mediated by so-called immunoevasins. We present here a structure-function analysis of one of the best-known HCMV immunevasins, UL16, and its interaction with a cellular ligand for NK cells, MICB. The normal function of MICB is to activate NK cells by engaging the most well-known NK receptor, NKG2D. Our results provide molecular evidence for the strategy used by UL16 to disable NK cell activation. In a rare example of structural mimicry that has likely arisen through convergent evolution, UL16 mimics a central binding motif of the structurally unrelated NKG2D protein. This allows UL16 to engage and disable several diverse NKG2D ligands, while others have apparently evolved to escape recognition by UL16 through alteration of key residues at strategic locations.

Abstract 4745 To observe how did the allogeneic NK cells kill human multiple myeloma cell RPMI-8266 and the immune mechanisms why RPMI-8266 cells escape from the NK cytotoxicity. Methods Detect the cytotoxicity of NK cells against RPMI 8226 cells by LDH release assay at different effect-to-target cell ratios in vitro, K562, high-sensitive with NK cells,was control cell. Test the expression of MHC-± chain-related molecules (MICA/B), human cytomegalovirus glycoprotein UL16-binding protein (ULBP1-3) and HLA -± MHC molecules on K562 and RPMI-8266 cell by flow cytometry. Detect mRNA level of MICA / B and ULBP1 ∼ 3 in K562, RPMI-266, and KIR genotyping of NK cell from 9 cases of healthy volunteers' by PCR method. As the E:T was 20:1, we used AMO-1, BMO-1, M295, M310, M551 and W6/32 mAb to block the effect of cell-surface protein MICA, MICB, ULBP1, ULBP2, ULBP3 and HLA-± in K562 and RPMI-8266 cell respectively, then observe the change of cytotoxicity of NK cells on K562 and RPMI-8266 cell. Results When the E:T was 5:1, cytotoxicity of NK cells on K562 and RPMI-8266 were (29.52 ± 0.27)% and (2.15 ± 0.32)% respectively; As the E:T was 10:1, the cytotoxicity were (36.37 ± 0.78) % and (5.26 ± 0.84)%; As the E:T was 20:1, they were (59.57 ± 1.05)% and (7.63 ± 1.05)%; As the E:T was 40:1, they were (70.64 ± 1.34)% and (10.18 ± 1.53)%. There wre significantly difference of the cytotoxicity of NK cell between on RPMI-8266 and K562 at all E:T ratio(P 0.05); But as we bloked HLA -± molecules with W6/32 monoclonal antibody, the cytotoxicity of NK cell on RPMI-8266 increased to (48.77 ± 4.61)%, there were significant differences between before and after bloking (P Conclusion For 8266 cell, the mechanisms of immune escape from NK killing effect may be related to high expression of HLA -± molecule, and did not express the NKG2D ligands MICA / B and ULBP1 ∼ 3 molecules. Disclosures: No relevant conflicts of interest to declare.

Activating and inhibitory cell surface receptors regulate natural killer (NK) cell effector functions. The extent of expression of their activating ligands on target cells probably plays a critical role in tumor immune surveillance, but the data of this expression on blasts of leukemia patients are still poor. We examined blasts of children with ALL for the activating human NKG2D ligands MICA, MICB, ULBP1, ULBP2 and ULBP3, and for the ligands of the activating human natural cytotoxic receptors (NCRs) NKp30, NKp44 and NKp46. Using ligand specific mouse monoclonal antibodies and flow cytometry, we screened 24 children (23 common-ALL, one pre-T-ALL) for the expression of NKG2D ligands and 15 children (all common-ALL) for NCR ligands. In 13 patients (all common-ALL), also the density of the NKG2D ligands was determined by quantitative flow cytometry. Considering cells positive for a particular ligand in case of a two fold increase of median fluorescence above negative control, 38 percent of the patients were positive for one or more NKG2D ligands, while only 13 percent expressed one or more NCR ligands at significant levels. ULBP1 was most frequently expressed (29 percent of patients positive), while no patients were positive for ULBP2 and NKp44 ligands. ULBP3 was positive in 17 percent of the patients, NKp30 and NKp46 ligands in 13 percent, MICA and MICB in 4 percent. The patient with pre-T-ALL was positive only for ULBP1. So ULBP1 was expressed more frequently, while the other NKG2D ligands were expressed less frequently in children than reported for adult leukemia patients before (Salih et al. Blood.2003;102:1389–1396.). The density of detected NKG2D ligand molecules was always rather low. For MICA the maximum were 1700 molecules per cell in a single patient, for MICB 900, for ULBP1 1100, and for ULBP3 1000 molecules per cell. In summary, blasts of pediatric ALL patients displayed low or negative surface levels of ligands for the human activating NK cell receptors NKG2D, NKp30, NKp44 and NKp46. QUALITATIVE LIGAND EXPRESSION QUALITATIVE LIGAND EXPRESSION QUANTITATIVE LIGAND EXPRESSION QUANTITATIVE LIGAND EXPRESSION

The mechanism by which paroxysmal nocturnal hemoglobinuria (PNH) clones expand to manifest symptoms is still unknown. PNH cells with PIG-A mutations do not synthesize glycosylphosphatidylinositol (GPI), resulting in deficiency of a series of GPI-linked membrane proteins. Blood cells with PNH phenotype (GPI− cells) expand in patients with bone marrow (BM) failure syndromes including PNH, aplastic anemia, and myelodysplastic syndromes. The diseases share an immune-mediated BM injury possibly by cytotoxic lymphocytes such as natural killer (NK) cells and cytotoxic T lymphocytes (CTL). It is suggested that the BM injury allows PNH clones survive selectively. Indeed, we have shown that human leukemic cells (K562) decrease their sensitivity to NK cells in vitro when K562 cells acquire PIG-A mutations (Nagakura et al, Blood 2002). In the present study, we show that the decrease in NK sensitivity of PIG-A mutant cells is ascribable to the deficiency of GPI-linked membrane proteins, ULBPs. ULBPs bind cytomegalovirus protein UL-16, consist of ULBP1-3, and emerge in cell membrane when cells are infected or transformed. They trigger off a NK activation signal, which overrides an inhibitory signal from MHC class I (Cosman et al, Immunity 2001). ULBPs also activate CTL besides NK cells. As target cells, a pair of GPI+ control and GPI− mutant cell lines was prepared from a GPI− K562 cell line bearing a PIG-A mutation by transfection with a PIG-A cDNA and a vector alone, respectively. Flow cytometry detected ULBP1-3 on the surface of GPI+ but none of GPI− K562 cells. As effector cells, we used cultured human NK cells (KHYG-1) deficient in Fc γR type III (CD16). GPI− cells were more resistant than GPI+ cells to the killing by NK cells in the 51Cr-release assay. GPI+ cells decreased their sensitivity to NK cells to the level of GPI− cells in the presence of antibodies to both ULBP1 and ULBP2, while antibodies to ULBP3 exerted no effects. None of the antibodies to ULBPs showed any effects on the killing of GPI− cells, while antibodies to NKG2D, which is a NK receptor for both ULBPs and MICA/B, inhibited the killing of both GPI+ and GPI− cells. Thus, GPI− cells that lack membrane ULBPs show a survival advantage in the setting of immune attack in vitro. Of clinical interests, ULBPs were detected on the cell surface of GPI+ but none of GPI− granulocytes of patients with PNH. GPI+ granulocytes of healthy individuals were negative for ULBPs. There appears pressure to induce membrane ULBPs in patients with PNH, leading to BM injury. These findings suggest that the failure of membrane expression of ULBPs permits selective expansion of PNH clones in patients with PNH and other BM failure syndromes.