Brain Advance Access originally published online on August 3, 2006
Brain 2006 129(9):2416-2425; doi:10.1093/brain/awl205
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TGF-ß and metalloproteinases differentially suppress NKG2D ligand surface expression on malignant glioma cells
1 Department of General Neurology, Hertie Institute for Clinical Brain Research, University of Tübingen Tübingen, Germany 2 Institute for Brain Research, Department of Immunology, University of Tübingen Tübingen, Germany 3 Institute for Cell Biology, Department of Immunology, University of Tübingen Tübingen, Germany 4 Present address: Weatherall Institute of Molecular Medicine, MRC Human Immunology Unit, John Radcliffe Hospital Oxford, UK
Correspondence to: Günter Eisele, MD, Department of General Neurology, Hertie Institute for Clinical Brain Research, University of Tübingen, Hoppe-Seyler-Strasse 3, D-72076 Tübingen, Germany E-mail: guenter.eisele{at}uni-tuebingen.de
 |
Summary |
|---|
 Top Summary IntroductionMaterial and methodsResultsDiscussionReferences |
|---|
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-ß (TGF-ß) 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-ß 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-ß 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.
Key Words: glioma; metalloproteinases; NK cells; NKG2DL; TGF-ß
Abbreviations: ADAM, a disintegrin and metalloproteinase; ADAM-TS, ADAM with thrombospondin; E : T, effector : target; (s)MICA/B, (soluble) MHC class I-chain-related molecule A/B; MMP, matrix metalloproteinases; MP, metalloproteinases; NKG2DL, NKG2D ligands; TGF-ß, transforming growth factor-ß; sMICA, soluble MICA; sULBP, soluble UL16-binding protein(s)
Received January 26, 2006. Revised June 27, 2006. Accepted July 5, 2006.
|
Introduction |
|---|
|
TopSummaryIntroductionMaterial and methodsResultsDiscussionReferences |
|---|
NKG2D was identified as an activating receptor on natural killer (NK) cells mediating responses to stress-induced ligands (Bauer et al., 1999
). NKG2D exerts additional roles in innate immunity by activating 
T cells (Das et al., 2001) and in adaptive immunity by providing co-stimulatory signals to CD8+ 
ß T cells (Groh et al., 2001; Friese et al., 2003). Triggering of NKG2D initiates a perforin-mediated cytolytic response against virally infected and tumour cells (Hayakawa et al., 2002). To accomplish its specific targeting, NKG2D interacts with different MHC class I-homologous ligands. In humans, these are MHC class I-chain-related molecules A (MICA) and MICB (Li et al., 2001), UL16-binding proteins (ULBP) 1–3, ULBP4/RAET1E and RAET1G (Cosman et al., 2001; Jan Chalupny et al., 2003; Bacon et al., 2004). Distinct expression patterns indicate that the various NKG2D ligands (NKG2DL) are not simply redundant in function (Bahram et al., 2005). With the exception of intestinal epithelia, normal cells in adults show absent or low-level NKG2DL expression (Groh et al., 1996). In pathological conditions, however, NKG2DL expression is often upregulated, as described for epithelial tumours, glioma and infected cells (Bauer et al., 1999; Groh et al., 1999; Das et al., 2001; Pende et al., 2002; Friese et al., 2003; Welte et al., 2003). NKG2DL are induced by genotoxic stress and blocked DNA replication, conditions known to activate a DNA damage response pathway (Gasser et al., 2005). NKG2D is downregulated by transforming growth factor-ß (TGF-ß) (Castriconi et al., 2003; Friese et al., 2004; Lee et al., 2004). TGF-ß production has been associated with the growth and malignant progression of a large variety of tumours including gliomas (Bodmer et al., 1989; Leitlein et al., 2001; Pasche, 2001) and interferes with several mechanisms of anti-tumour immune responses (Thomas and Massague, 2005).
Tumour cells may also evade NKG2D-mediated immune surveillance by shedding MIC und ULBP molecules via metalloproteinases (MP) (Groh et al., 2002; Salih et al., 2002, 2003; Waldhauer and Steinle, 2006). This is of particular interest since MP inhibitors are clinically available. Currently, there are three known classes of MP: matrix MP (MMP), a disintegrin and MP (ADAM) and ADAM with thrombospondin (ADAM-TS). Glioma cells release high levels of MMP (Wild-Bode et al., 2001; Nakada et al., 2003; Nuttall et al., 2003; Thorns et al., 2003). The MMP family is composed of 25 structurally related proteinases secreted into the extracellular milieu or tethered to the cellular surface (Overall and Lopez-Otin, 2002). MMP expression and activation are increased in almost all human cancers (Egeblad and Werb, 2002) and have been implicated in tumour invasion and metastasis owing to their ability to degrade extracellular matrix (Chambers et al., 2002; Coussens et al., 2002). The related ADAMs protein family consists of 40 different members (Huovila et al., 2005). ADAMs regulate the tumour-related EGFR and ErbB2 signalling pathways (Blobel, 2005; Zhou et al., 2005) and are also over-expressed in a variety of cancers. In gliomas, expression of ADAM 12 is upregulated with increasing grade of malignancy (Kodama et al., 2004). Whereas ADAMs are transmembrane proteins, ADAM-TS are secreted. Currently, 19 different ADAM-TS are known, all of which are characterized by an MP, a disintegrin and a C-terminal thrombospondin-like domain (Porter et al., 2005). ADAM-TS4 has been found to be responsible for brevican cleavage in glioma cells and may therefore be critical in mediating invasiveness (Matthews et al., 2000). However, MP do not only clear the path for invading tumour cells but also regulate the availability of a large variety of cell surface molecules including proteinase inhibitors, adhesion molecules, growth factor binding proteins, cell surface receptors and immunoregulatory proteins (Egeblad and Werb, 2002; Porter et al., 2005; Zhou et al., 2005). Of note, both TGF-ß and MP expression are upregulated with increasing grade of glioma malignancy (Stiles et al., 1997; Kawataki et al., 2000; Kjellman et al., 2000; Wang et al., 2003; Kodama et al., 2004), indicating a prominent role in the malignant properties of this type of cancer.
Here, we show that gliomas of different grades of malignancy express the NKG2DL MICA, MICB and ULBP1–3 in vivo. In the LNT-229 glioma cell line, they are differentially suppressed by TGF-ß at the mRNA level and selectively cleaved from the cell surface by MP. The elucidation of these mechanisms may disclose novel avenues for the therapeutic induction of anti-tumour immune responses.
|
Material and methods |
|---|
|
TopSummaryIntroductionMaterial and methodsResultsDiscussionReferences |
|---|
Human tissue specimens
To study the expression of MICA/B and ULBP1–3 in the normal human brain, 18 white matter tissue samples from a normal brain bank were investigated (Mittelbronn et al., 2001
). Further, 20 cases each of gliomas of the WHO grades II (diffuse astrocytoma), III (anaplastic astrocytoma) and IV (glioblastoma) were studied (Kleihues and Cavenee, 2000).
Cell lines and transfectants
The human malignant glioma cell line LN-229 was originally provided by Dr N. de Tribolet (Lausanne, Switzerland) and renamed LNT-229 for clarification (T for Tübingen). The cells were maintained in DMEM supplemented with 2 mM L-glutamine (Gibco Life Technologies, Paisley, UK), 10% FCS (Biochrom KG) and penicillin (100 IU/ml)/streptomycin (100 µg/ml) (Gibco). The generation of LNT-229 TGF-ß1/2 siRNA cells has been described (Friese et al., 2004).
Antibodies and flow cytometry
The following mAbs were used for the assessment of cell surface expression or the blocking of NKG2D, NKG2DL, CD3 and CD56: MAB 139 IgG1 anti-NKG2D and ILU01 polyclonal goat anti-human ULBP2 were from R&D Systems (Wiesbaden, Germany); HIT3a IgG2a anti-CD3-FITC and B159 IgG1 anti-CD56-PE were from BD Pharmingen (Heidelberg, Germany); AMO1 IgG1 anti-MICA, BAMO1 IgG1 anti-MICA/B, BAMO3 IgG2a anti-MICA/B, BMO2 IgG1 anti-MICB, AUMO1 IgG1 anti-ULBP1, BUMO1 IgG1 anti-ULBP2, CUMO1 IgM anti-ULBP3 have been described (Welte et al., 2003); and CUMO3 IgG1 anti-ULBP3 was newly generated. Cells were blocked with 2% BSA and incubated with the specific mAb or matched isotype antibodies (5 µg/ml) for 30 min on ice. For unlabelled primary antibodies, specific binding was visualized with PE-conjugated goat anti-mouse IgG (Sigma, Deisenhofen, Germany). Fluorescence was measured in a Becton Dickinson FACScalibur. Specific fluorescence indexes (SFI) were calculated by dividing mean fluorescence obtained with specific antibody by mean fluorescence obtained with control antibody.
Immunohistochemistry
Immunohistochemistry was performed on paraffin-embedded samples using the Benchmark system (Ventana, Strasbourg, France). After blocking of endogenous peroxidase, anti-MICA (AMO1, IgG1, 1 : 25), anti-MICB (BMO2, IgG1, 1 : 50), anti-ULBP1 (AUMO1, IgG1, 1 : 100) or anti-ULBP3 (CUMO3, IgG1, 1 : 25) antibodies were applied. Binding specificity was controlled by IgG1 isotype controls (DakoCytomation, Hamburg, Germany). For visualization, I-View horseradish peroxidase-conjugated streptavidin was applied, followed by diaminobenzidine/H2O2. For ULBP2, samples were immersed in citrate buffer and irradiated in a microwave oven. Endogenous peroxidase was blocked and standard porcine serum was applied to prevent non-specific binding. Polyclonal goat anti-human ULBP2 antibody (clone ILU01, R&D Systems) was followed by biotinylated rabbit anti-goat immunoglobulins (DakoCytomation). Diaminobenzidine was used as chromogen. All sections were counterstained with haematoxylin. Using an Olympus BX50 microscope, two raters (M.M., G.E.) quantified expression levels by the following scale: 0 signifies absence of detectable staining, 1 corresponds to single positive cells in a focal pattern, 2 denotes positive cells in diffuse patterns, 3 indicates up to 20% of positive cells, 4 was allotted when the percentage was between 20 and 50%, 5 was given for >50% of positive cells.
Purification of peripheral blood lymphocytes (PBL) and isolation of NK cells
PBL were prepared by density gradient centrifugation (Biocoll, Biochrom KG, Berlin, Germany) and depletion of plastic-adherent monocytic cells. PBL were cultured on irradiated RPMI 8866 feeder cells to obtain polyclonal NK cell populations (Valiante et al., 1992).
Real-time polymerase chain reaction (PCR)
Total RNA was prepared using RNAeasy (Qiagen, Hilden, Germany) and transcribed according to standard protocols. cDNA amplification was monitored using SYBRGreen chemistry on the ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Weiterstadt, Germany). Primers and conditions for 18S rRNA, MICA/B and ULBP1–3 have been published (Schreiner et al., 2005). Primer sequences for ULBP4 were forward: 5'-CTGGCTCAGGGAATTCTTAGG-3' (573–593), reverse: 5'-CTAGAAGAAGACCAGTGG ATATC-3' (665–643). Relative induction levels (rI) of NKG2DL were calculated by the formula
 |
Soluble MICA and ULBP2 enzyme-linked immunosorbent assay (ELISA)
Glioma cell supernatants were analysed in sandwich ELISAs for soluble MICA (sMICA) using anti-MICA mAb AMO1 and BAMO3 or for soluble ULBP2 (sULBP2) using anti-ULBP2 mAb BUMO1 and 165903 (R&D) as described previously (Salih et al., 2003; Waldhauer and Steinle, 2006).
Inhibition of metalloproteinase activity
The broad spectrum MP inhibitors GM6001 (N-[(2R)-2 hydroxyamido-carbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide) (Chemicon, Hampshire, UK) and MMP inhibitor III (Merck, Darmstadt, Germany) were dissolved at 50 mM into DMSO and used at 10 µM working concentrations.
Cytotoxicity assay
Cytotoxicity was assessed in 4 h 51Cr release assays in the absence or presence of various mAb. The antibody concentrations for the blocking experiments were 10 µg/ml. NK cells were pre-treated with normal human IgG to prevent antibody-dependent cellular cytotoxicity before they were co-incubated for 4 h with 104 51Cr-labelled target cells per well at various effector : target (E : T) ratios. Spontaneous 51Cr release was determined by incubating the target cells with medium alone. Maximum release was determined by adding NP-40 (2%). The percentage of 51Cr release was calculated as follows: 100 x [(experimental release – spontaneous release)/(maximum release – spontaneous release)].
Statistics
The experiments shown were repeated at least three times with similar results. Analysis of significance was performed using the two-tailed Student's t-test or, where indicated, ANOVA (analysis of variance) (Excel, Microsoft, Redmond, WA). For the assessment of in vivo expression levels, the scores for the relative staining intensities were compared between the various tumour entities using the Kruskal–Wallis test (P < 0.05*, P < 0.01** and P < 0.001***).
|
Results |
|---|
|
TopSummaryIntroductionMaterial and methodsResultsDiscussionReferences |
|---|
NKG2DL expression in human gliomas in vivo
We examined gliomas of different WHO grades of malignancy (Kleihues and Cavenee, 2000
) for the expression levels of NKG2DL using immunohistochemistry (Fig. 1). Normal central nervous system white matter showed in general undetectable or very low NKG2DL expression levels on cells exhibiting features of reactive astrocytes. In contrast, WHO grade II astrocytomas displayed an almost uniformly upregulated expression of MICA/B and ULBP1–3 on tumour cells compared with normal brain. MICA expression levels decreased in gliomas of higher grades of malignancy. In contrast, MICB and ULBP1 showed no significant downregulation. ULBP2 expression was strongest again on WHO grade II astrocytomas, whereas expression levels were lower in grade III anaplastic astrocytomas and very low in grade IV. The percentage of ULBP3-positive cells was generally lower than that for the other NKG2DL in gliomas of all grades. However, the general pattern was similar with ULBP3 expression being very low or even absent on normal brain and upregulated in WHO grade II astrocytomas. ULBP3 expression was also high in grade III gliomas and was only slightly decreased in grade IV gliomas (Table 1).
|
|
TGF-ß-mediated suppression of MICA and ULBP2 inhibits NKG2D-mediated tumour cell lysis
We have previously reported an inhibitory effect of TGF-ß on MICA expression (Friese et al., 2004
) and a positive effect of TGF-ß on MMP expression and activity (Wick et al., 2001). To further characterize the role of endogenous glioma cell-derived TGF-ß in the regulation of NKG2DL expression, we used LNT-229 cells transfected with siRNA targeted against TGF-ß1 and TGF-ß2 (Friese et al., 2004). Real-time PCR revealed a marked induction of MICA, ULBP2 and ULBP4 mRNA transcripts in TGF-ß1/2 siRNA cells, whereas the levels of MICB, ULBP1 and ULBP3 remained unaltered (Fig. 2A). To correlate the changes in NKG2DL mRNA expression induced by TGF-ß with changes at the protein level, we performed flow cytometry using antibodies specific for the respective NKG2DL. Consistent with the mRNA data, MICA and ULBP2 expression on the cell surface of TGF-ß1/2 siRNA cells was increased by factors of 5.0 ± 1.0 and 2.4 ± 0.2, whereas no change was observed with MICB, ULBP1 or ULBP3. One representative out of three experiments is shown in Fig. 2B. For ULBP4, there is currently no specific antibody available. The re-exposure of TGF-ß1/2 siRNA cells to exogenous TGF-ß reversed the induction of MICA, ULBP2 and ULBP4 mRNA (Fig. 2C) and also decreased the cell surface expression of MICA and ULBP2 by 40 ± 8% and 20 ± 1% (Fig. 2D; n = 3). The changes in the expression levels of MICA, ULBP2 and ULBP4 were paralleled by an increase in NK cell-mediated lysis of TGF-ß1/2 siRNA cells (ANOVA; P < 0.01 for all E : T ratios; Fig. 3). The increased lysis of TGF-ß1/2 siRNA cells was partially blocked by either neutralizing MICA (P < 0.05 for all E : T ratios) or ULBP2 mAb (P < 0.01 for all E : T ratios). No significant differences were found between the effects of anti-MICA and anti-ULBP2, suggesting that both NKG2DLs contribute to a similar extent to glioma cell lysis. In the presence of either antibody, lysis of TGF-ß1/2 siRNA cells still exceeded that of pSUPERpuro control transfectants (P < 0.05 for all E : T ratios). When both antibodies were added, target cell lysis was further reduced (anti-MICA + anti-ULBP2 versus anti-MICA: P < 0.05 for all E : T ratios, anti-MICA + anti-ULBP2 versus anti-ULBP2: P < 0.05 for all E : T ratios) and equalled now the level observed with control transfectants (P > 0.05 for all E : T ratios). Since incubation with both mAbs was equivalent to a blocking NKG2D mAb (P > 0.05 for all E : T ratios) other NKG2DL may only play a minor role in glioma biology.
|
|
Selective and TGF-ß-independent shedding of MICA and ULBP2 by MP inhibits NK cell-mediated lysis
As shown previously (Wick et al., 2001
), TGF-ß promotes MP expression in glioma cells. Since NKG2DL surface expression can be modulated on a post-translational level by proteolytic cleavage (Groh et al., 2002; Salih et al., 2002, 2003; Raffaghello et al., 2004; Wu et al., 2004; Waldhauer and Steinle, 2006), we asked whether shedding of sMICA and sULBP2 was reduced in TGF-ß1/2 siRNA cells, thus contributing to the enhanced surface expression of MICA and ULBP2 on these cells. In control cells, inhibition of MP activity by exposure to the broad spectrum MP inhibitors GM6001 and MMP inhibitor III enhanced the expression levels of MICA at the cell surface 2.4-fold and of ULBP2 1.4-fold, whereas no significant changes were detected for MICB, ULBP1 and ULBP3 (Fig. 4A, upper panel, Table 2 and data not shown; n = 3). A transcriptional effect of the inhibitor on NKG2DL mRNA levels was ruled out by real-time PCR (data not shown). Parallel experiments were also performed to study the effects of MP inhibition in the TGF-ß1/2 siRNA cells. Despite the reduced levels of MMP-2 and MMP-9 activity in TGF-ß1/2 siRNA cells (Friese et al., 2004), MP inhibition super-induced the expression levels of MICA 1.3-fold and of ULBP2 1.5-fold (Fig. 4A, lower panel, Table 2; n = 3). The degree of induction on control transfectants and TGF-ß1/2 siRNA cells was similar for ULBP2 (P = 1.0, n.s.) but significantly higher on control cells for MICA (P < 0.05).
|
|
Further, immunoblotting of cell culture supernatant showed that the basal level of sMICA released by TGF-ß1/2 siRNA cells was markedly enhanced compared with the control cells paralleling higher surface expression (data not shown). While these findings demonstrate that the cleavage of NKG2DL is not mediated by TGF-ß-dependent proteases, the release of sMICA and sULBP2 from LNT-229 control and TGF-ß1/2 siRNA cells in cell culture supernatant could nevertheless be reduced by
50% for sMICA and 80% for sULBP2 when GM6001 was added, as shown by MICA- and ULBP2-specific ELISA (Fig. 4B).
Next, we assessed the functional consequences of reduced NKG2DL shedding in a 51Cr release assay using target glioma cells pre-treated with GM6001 for 48 h. As expected, MP inhibition enhanced the NK cell-dependent killing of LNT-229 control glioma cells (P < 0.05 for E : T ratios 10 : 1–80 : 1). Further, the high constitutive NKG2DL expression in TGF-ß1/2 siRNA cells resulted in massive target cell lysis that was super-induced by GM6001 when E : T ratios were low and lysis conditions presumably suboptimal (P < 0.05 for E : T ratios 5 : 1 and 10 : 1, P > 0.05 for E : T ratios 20 : 1–80 : 1, Fig. 5A). The co-exposure of LNT-229 control or TGF-ß1/2 siRNA cells to blocking NKG2D mAb attenuated the lysis of glioma cell targets (Fig. 5B). Other activating molecules that may also be regulated by MP activity do not seem to play a decisive role in this context since the enhanced lysis of GM6001-pre-treated glioma cells was nullified by NKG2D mAb (P > 0.05 for E : T ratios 5 : 1–80 : 1 in control transfectants and for E : T ratios 10 : 1–80 : 1 in TGF-ß1/2 siRNA cells; data not shown). However, a difference in susceptibility towards NK cell-mediated lysis between LNT-229 control and TGF-ß1/2 siRNA cells persisted in the presence of NKG2D mAb (P < 0.05 for all E : T ratios), suggesting that other mechanisms interfering with NK cell-mediated killing are also regulated by TGF-ß (Castriconi et al., 2003).
|
|
Discussion |
|---|
|
TopSummaryIntroductionMaterial and methodsResultsDiscussionReferences |
|---|
NKG2DL are expressed almost de novo in gliomas in vivo and may thus label tumour cells for recognition by NKG2D-expressing immune effector cells (Fig. 1, Table 1). However, MICA and ULBP2 expression levels are downregulated close to baseline with malignant progression to grade IV tumours. This implies that the immunogenicity of gliomas decreases with increasing grade of malignancy, possibly owing to a selection process favouring the survival of less immunogenic glioma cells. Surprisingly, elevated MICB and ULBP1 expression levels do not change during the course of malignant progression and ULBP3 was only slightly downregulated in grade IV gliomas. Owing to the lack of a suitable antibody, ULBP4 expression could not be assessed in vivo. Interestingly, all examined NKG2DL were expressed at low levels also on single astrocytes in normal brain, with scores ranging from 0.8 for ULBP3 to 1.6 for MICB. These astrocytes showed reactive changes that were considered as morphological alterations induced by mortal agony. Since NKG2DL are known to be stress-inducible, their expression on astrocytes undergoing reactive changes might be due to terminal hypoxia-induced stress in these patients.
It remains unclear whether high cellular NKG2DL expression confers any survival advantage during the development of gliomas. Most likely, the blood–brain barrier that becomes disrupted in high-grade gliomas but is still intact in lower grade astrocytomas (Bronger et al., 2005) will hamper immune cell infiltration into the brain and thus prevent the NKG2D-mediated elimination of low-grade astrocytomas.
Our study identifies two largely independent mechanisms that can explain the different NKG2DL staining patterns in malignant gliomas and their role in escaping NKG2D-mediated anti-tumour immunity. The association between increasing grade of malignancy and elevated expression of immunosuppressive molecules such as TGF-ß has already been documented (Stiles et al., 1997; Kawataki et al., 2000; Kjellman et al., 2000). The mechanisms by which TGF-ß undermines anti-tumour immune surveillance (Gorelik and Flavell, 2001; Thomas and Massagué, 2005) might involve effects on co-stimulatory signals using NKG2D as the target molecule (Friese et al., 2004; Lee et al., 2004). Our previous studies had indicated that there is an activation potential for immune cells when NKG2DL are highly expressed on glioma cells (Friese et al., 2003). However, TGF-ß counteracts these mechanisms by enhancing the expression of the inhibitory receptor CD94/NKG2A and by downregulating the activating receptor NKG2D on CD8+ T and NK cells (Bertone et al., 1999; Castriconi et al., 2003; Friese et al., 2004).
Here, we show that TGF-ß also downregulates the transcription of the human NKG2DL MICA, ULBP2 and ULBP4 (Fig. 2). Interestingly, TGF-ß does not interfere with MICB, ULBP1 and ULBP3 mRNA and cell surface expression, suggesting that the yet uncharacterized promoters may not respond to TGF-ß-dependent transcription factors such as SMADs or that their mRNAs are selectively stabilized. Blocking experiments revealed additive functions of MICA and ULBP2 in triggering NKG2D, indicating that NKG2DL at the surface of transformed cells may contribute to anti-tumour immune responses in an additive manner (Fig. 3). Indeed, the effect of blocking both MICA and ULBP2 equalled that of blocking the receptor. These observations suggest a prominent role for the NKG2DL MICA and ULBP2 in glioma immune surveillance while the other ligands may exert their immune stimulatory functions under different conditions. By promoting the reduction of NKG2DL on the cell surface, tumour cells may efficiently escape innate immune recognition by reducing an induced self-danger signal (Barton and Medzhitov, 2002; Diefenbach and Raulet, 2002). Collectively, these observations confirm that TGF-ß is central to the malignant progression of glial tumours and a principal target for the treatment of gliomas (Weller and Fontana, 1995). Anti-TGF-ß therapies may therefore not only relieve the immune dysfunction in human glioblastoma patients but also act on the tumour cells, restoring MICA, ULBP2 and ULBP4 expression to the levels required for an effective anti-glioma immune response.
Furthermore, TGF-ß-dependent MP are essential for the migratory and pro-invasive phenotype of glioma cells (Friese et al., 2004). A direct targeting of MP would also appear to be justified, not only to impede glioma cell migration and invasion but also to reduce the proteolytic shedding of NKG2DL. Yet again, MP inhibition by the broad spectrum inhibitors GM 6001 or MMP inhibitor III selectively enhanced the expression of MICA and ULBP2 on the surface of LNT-229 cells but not of other NKG2DL (Fig. 4A). For MICA, this effect was significantly more prominent on control than on TGF-ß1/2 siRNA cells. In parallel, the levels of sMICA in glioma cell supernatant were reduced by 50% (Fig. 4B), while mRNA levels were unchanged (data not shown). The fact that MP inhibition predominantly enhanced the cell surface expression of MICA and ULBP2 (Fig. 4A) may be due to the MP equipment of LNT-229 cells and to the low expression level of MICB in these cells. A complete prevention of MICA shedding was not achieved, implicating the involvement of proteases that are unaffected by the inhibitors used. Both GM6001 and MMP inhibitor III inhibit MMP-1, MMP-2 and MMP-3 with GM6001 acting on MMP-8 and MMP-9 as well, while MMP inhibitor III further blocks MMP-7 and MMP-13. Effects of these inhibitors on ADAMs or ADAM-TS are likely, but were not investigated here.
Of note, the relative reduction in sMICA and sULBP2 levels upon treatment with an MP inhibitor did not differ between LNT-229 control and TGF-ß1/2 siRNA cells (Fig. 4B). Since we have previously shown that downregulation of TGF-ß1/2 by siRNA results in a striking reduction in MMP-2 and MMP-9 expression and activity, the modulation of NKG2DL surface expression is likely to depend on different MP (Friese et al., 2004).
We also show that the enhanced NKG2DL expression upon exposure to MP inhibitors translates into an increased immunogenicity of glioma cells (Fig. 5A and B). Of note, at high E : T ratios, a reduction of NKG2DL shedding did not enhance the lysis of TGF-ß1/2 siRNA cells any further, implying that the levels of NKG2DL expression achieved in these cells have already been sufficient to fully activate the NKG2D pathway. Still, the inhibition of MP is a promising option for TGF-ß-independent tumours and may also lead to synergy with anti-TGF-ß strategies.
Taken together, our in vitro data imply that the TGF-ß-mediated transcriptional repression of MICA and ULBP2 together with the MP-mediated selective shedding of these NKG2DL may be a major factor contributing to the immune escape of higher grade gliomas. This suggested mechanism is strongly supported by the immunohistochemical finding of tumour-specific MICA and ULBP2 becoming downregulated with increasing grade of malignancy. The virtually complete blocking of NKG2D-mediated NK cell lysis of glioma cells by the combination of anti-MICA and anti-ULBP2 antibodies (Fig. 3) strongly suggests that further NKG2DL only play a subordinate role in glioma biology. The two mechanisms outlined here are particularly attractive since clinically applicable concepts for the inhibition of TGF-ß (Uhl et al., 2004) and of MP have already been developed and could thus be applied here in a novel context.
|
Acknowledgements |
|---|
This study was supported by a grant from the Wilhelm Sander-Stiftung to M.W.
|
References |
|---|
|
TopSummaryIntroductionMaterial and methodsResultsDiscussionReferences |
|---|
Bacon L, Eagle RA, Meyer M, Easom N, Young NT, Trowsdale J. (2004) Two human ULBP/RAET1 molecules with transmembrane regions are ligands for NKG2D. J Immunol 173:1078–84.
Bahram S, Inoko H, Shiina T, Radosavljevic M. (2005) MIC and other NKG2D ligands: from none to too many. Curr Opin Immunol 17:505–9.[ISI][Medline]
Barton GM and Medzhitov R. (2002) Control of adaptive immune responses by Toll-like receptors. Curr Opin Immunol 14:380–3.[CrossRef][ISI][Medline]
Bauer S, Groh V, Wu J, Steinle A, Phillips JH, Lanier LL, et al. (1999) Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science 285:727–9.
Bertone S, Schiavetti F, Bellomo R, Vitale C, Ponte M, Moretta L, et al. (1999) Transforming growth factor-beta-induced expression of CD94/NKG2A inhibitory receptors in human T lymphocytes. Eur J Immunol 29:23–9.[CrossRef][ISI][Medline]
Blobel CP. (2005) ADAMs: key components in EGFR signalling and development. Nat Rev Mol Cell Biol 6:32–43.[CrossRef][ISI][Medline]
Bodmer S, Strommer K, Frei K, Siepl C, de Tribolet N, Heid I, et al. (1989) Immunosuppression and transforming growth factor-beta in glioblastoma. Preferential production of transforming growth factor-beta 2. J Immunol 143:3222–9.[Abstract]
Bronger H, König J, Kopplow K, Steiner HH, Ahmadi R, Herold-Mende C, et al. (2005) ABCC drug efflux pumps and organic anion uptake transporters in human gliomas and the blood-tumor barrier. Cancer Res 65:11419–28.
Castriconi R, Cantoni C, Della Chiesa M, Vitale M, Marcenaro E, Conte R, et al. (2003) Transforming growth factor beta 1 inhibits expression of NKp30 and NKG2D receptors: consequences for the NK-mediated killing of dendritic cells. Proc Natl Acad Sci USA 100:4120–5.
Chambers AF, Groom AC, MacDonald IC. (2002) Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer 2:563–72.[CrossRef][ISI][Medline]
Cosman D, Mullberg J, Sutherland CL, Chin W, Armitage R, Fanslow W, et al. (2001) ULBPs, novel MHC class I-related molecules, bind to CMV glycoprotein UL16 and stimulate NK cytotoxicity through the NKG2D receptor. Immunity 14:123–33.[CrossRef][ISI][Medline]
Coussens LM, Fingleton B, Matrisian LM. (2002) Matrix metalloproteinase inhibitors and cancer: trials and tribulations. Science 295:2387–92.
Das H, Groh V, Kuijl C, Sugita M, Morita CT, Spies T, et al. (2001) MICA engagement by human Vgamma2Vdelta2 T cells enhances their antigen-dependent effector function. Immunity 15:83–93.[CrossRef][ISI][Medline]
Diefenbach A and Raulet DH. (2002) The innate immune response to tumors and its role in the induction of T-cell immunity. Immunol Rev 188:9–21.[CrossRef][ISI][Medline]
Egeblad M and Werb Z. (2002) New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2:161–74.[ISI][Medline]
Friese MA, Platten M, Lutz SZ, Naumann U, Aulwurm S, Bischof F, et al. (2003) MICA/NKG2D-mediated immunogene therapy of experimental gliomas. Cancer Res 63:8996–9006.
Friese MA, Wischhusen J, Wick W, Weiler M, Eisele G, Steinle A, et al. (2004) RNA interference targeting transforming growth factor-ß enhances NKG2D-mediated antiglioma immune response, inhibits glioma cell migration and invasiveness, and abrogates tumorigenicity in vivo. Cancer Res 64:7596–603.
Gasser S, Orsulic S, Brown EJ, Raulet DH. (2005) The DNA damage pathway regulates innate immune system ligands of the NKG2D receptor. Nature 436:1186–90.[CrossRef][Medline]
Gorelik L and Flavell RA. (2001) Immune-mediated eradication of tumors through the blockade of transforming growth factor-beta signaling in T cells. Nat Med 7:1118–22.[CrossRef][ISI][Medline]
Groh V, Bahram S, Bauer S, Herman A, Beauchamp M, Spies T. (1996) Cell stress-regulated human major histocompatibility complex class I gene expressed in gastrointestinal epithelium. Proc Natl Acad Sci USA 93:12445–50.
Groh V, Rhinehart R, Randolph-Habecker J, Topp MS, Riddell SR, Spies T. (2001) Costimulation of CD8alphabeta T cells by NKG2D via engagement by MIC induced on virus-infected cells. Nat Immunol 2:255–60.[CrossRef][ISI][Medline]
Groh V, Rhinehart R, Secrist H, Bauer S, Grabstein KH, Spies T. (1999) Broad tumor-associated expression and recognition by tumor-derived gamma delta T cells of MICA and MICB. Proc Natl Acad Sci USA 96:6879–84.
Groh V, Wu J, Yee C, Spies T. (2002) Tumour-derived soluble MIC ligands impair expression of NKG2D and T-cell activation. Nature 419:734–8.[CrossRef][Medline]
Hayakawa Y, Kelly JM, Westwood JA, Darcy PK, Diefenbach A, Raulet D, et al. (2002) Cutting edge: tumor rejection mediated by NKG2D receptor-ligand interaction is dependent upon perforin. J Immunol 169:5377–81.
Huovila AP, Turner AJ, Pelto-Huikko M, Karkkainen I, Ortiz RM. (2005) Shedding light on ADAM metalloproteinases. Trends Biochem Sci 30:413–22.[CrossRef][ISI][Medline]
Jan Chalupny N, Sutherland CL, Lawrence WA, Rein-Weston A, Cosman D. (2003) ULBP4 is a novel ligand for human NKG2D. Biochem Biophys Res Commun 305:129–35.[CrossRef][ISI][Medline]
Kawataki T, Naganuma H, Sasaki A, Yoshikawa H, Tasaka K, Nukui H. (2000) Correlation of thrombospondin-1 and transforming growth factor-beta expression with malignancy of glioma. Neuropathology 20:161–9.[CrossRef][ISI][Medline]
Kjellman C, Olofsson SP, Hansson O, Von Schantz T, Lindvall M, Nilsson I, et al. (2000) Expression of TGF-beta isoforms, TGF-beta receptors, and SMAD molecules at different stages of human glioma. Int J Cancer 89:251–8.[CrossRef][ISI][Medline]
Kleihues P and Cavenee WK. (2000) Pathology and genetics of tumours of the nervous system (IARC Press, Lyon).
Kodama T, Ikeda E, Okada A, Ohtsuka T, Shimoda M, Shiomi T, et al. (2004) ADAM12 is selectively overexpressed in human glioblastomas and is associated with glioblastoma cell proliferation and shedding of heparin-binding epidermal growth factor. Am J Pathol 165:1743–53.
Lee J-C, Lee K-M, Kim D-W, Heo DS. (2004) Elevated TGF-{beta}1 secretion and down-modulation of NKG2D underlies impaired NK cytotoxicity in cancer patients. J Immunol 172:7335–40.
Leitlein J, Aulwurm S, Waltereit R, Naumann U, Wagenknecht B, Garten W, et al. (2001) Processing of immunosuppressive pro-tgf-beta1,2 by human glioblastoma cells involves cytoplasmic and secreted furin-like proteases. J Immunol 166:7238–43.
Li P, Morris DL, Willcox BE, Steinle A, Spies T, Strong RK. (2001) Complex structure of the activating immunoreceptor NKG2D and its MHC class I-like ligand MICA. Nat Immunol 2:443–51.[ISI][Medline]
Matthews RT, Gary SC, Zerillo C, Pratta M, Solomon K, Arner EC, et al. (2000) Brain-enriched hyaluronan binding (BEHAB)/brevican cleavage in a glioma cell line is mediated by a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family member. J Biol Chem 275:22695–703.
Mittelbronn M, Dietz K, Schluesener HJ, Meyermann R. (2001) Local distribution of microglia in the normal adult human central nervous system differs by up to one order of magnitude. Acta Neuropathol (Berl) 101:249–55.[Medline]
Nakada M, Okada Y, Yamashita J. (2003) The role of matrix metalloproteinases in glioma invasion. Front Biosci 8:e261–9.[ISI][Medline]
Nuttall RK, Pennington CJ, Taplin J, Wheal A, Yong VW, Forsyth PA, et al. (2003) Elevated membrane-type matrix metalloproteinases in gliomas revealed by profiling proteases and inhibitors in human cancer cells. Mol Cancer Res 1:333–45.
Overall CM and Lopez-Otin C. (2002) Strategies for MMP inhibition in cancer: innovations for the post-trial era. Nat Rev Cancer 2:657–72.[CrossRef][ISI][Medline]
Pasche B. (2001) Role of transforming growth factor beta in cancer. J Cell Physiol 186:153–68.[CrossRef][ISI][Medline]
Pende D, Rivera P, Marcenaro S, Chang CC, Biassoni R, Conte R, et al. (2002) Major histocompatibility complex class I-related chain A and UL16-binding protein expression on tumor cell lines of different histotypes: analysis of tumor susceptibility to NKG2D-dependent natural killer cell cytotoxicity. Cancer Res 62:6178–86.
Porter S, Clark IM, Kevorkian L, Edwards DR. (2005) The ADAMTS metalloproteinases. Biochem J 386:15–27.[CrossRef][ISI][Medline]
Raffaghello L, Prigione I, Airoldi I, Camoriano M, Levreri I, Gambini C, et al. (2004) Downregulation and/or release of NKG2D ligands as immune evasion strategy of human neuroblastoma. Neoplasia 6:558–68.[CrossRef][ISI][Medline]
Salih HR, Antropius H, Gieseke F, Lutz SZ, Kanz L, Rammensee HG, et al. (2003) Functional expression and release of ligands for the activating immunoreceptor NKG2D in leukemia. Blood 102:1389–96.
Salih HR, Rammensee HG, Steinle A. (2002) Cutting edge: down-regulation of MICA on human tumors by proteolytic shedding. J Immunol 169:4098–102.
Schreiner B, Voss J, Wischhusen J, Dombrowski Y, Steinle A, Lochmüller H, et al. (2006) Expression of toll-like receptors by human muscle cells in vitro and in vivo: TLR3 is highly expressed in inflammatory and HIV myopathies, mediates IL-8 release, and up-regulation of NKG2D-ligands. FASEB J 20:118–20.
Stiles JD, Ostrow PT, Balos LL, Greenberg SJ, Plunkett R, Grand W, et al. (1997) Correlation of endothelin-1 and transforming growth factor beta 1 with malignancy and vascularity in human gliomas. J Neuropathol Exp Neurol 56:435–9.[ISI][Medline]
Thomas DA and Massagué J. (2005) TGF-ß directly targets cytotoxic T cell functions during tumor evasion of immune surveillance. Cancer Cell 8:369–80.[CrossRef][ISI][Medline]
Thorns V, Walter GF, Thorns C. (2003) Expression of MMP-2, MMP-7, MMP-9, MMP-10 and MMP-11 in human astrocytic and oligodendroglial gliomas. Anticancer Res 23:3937–44.[ISI][Medline]
Uhl M, Aulwurm S, Wischhusen J, Weiler M, Ma JY, Almirez R, et al. (2004) SD-208, a novel transforming growth factor beta receptor I kinase inhibitor, inhibits growth and invasiveness and enhances immunogenicity of murine and human glioma cells in vitro and in vivo. Cancer Res 64:7954–61.
Valiante NM, Rengaraju M, Trinchieri G. (1992) Role of the production of natural killer cell stimulatory factor (NKSF/IL-12) in the ability of B cell lines to stimulate T and NK cell proliferation. Cell Immunol 145:187–98.[CrossRef][ISI][Medline]
Waldhauer I and Steinle A. (2006) Proteolytic release of soluble UL16-binding protein 2 (ULBP2) from tumor cells. Cancer Res 66:2520–6.
Wang M, Wang T, Liu S, Yoshida D, Teramoto A. (2003) The expression of matrix metalloproteinase-2 and -9 in human gliomas of different pathological grades. Brain Tumor Pathol 20:65–72.[Medline]
Weller M and Fontana A. (1995) The failure of current immunotherapy for malignant glioma. Tumor-derived TGF-beta, T-cell apoptosis, and the immune privilege of the brain. Brain Res Brain Res Rev 21:128–51.[CrossRef][Medline]
Welte SA, Sinzger C, Lutz SZ, Singh-Jasuja H, Sampaio KL, Eknigk U, et al. (2003) Selective intracellular retention of virally induced NKG2D ligands by the human cytomegalovirus UL16 glycoprotein. Eur J Immunol 33:194–203.[CrossRef][ISI][Medline]
Wick W, Platten M, Weller M. (2001) Glioma cell invasion: regulation of metalloproteinase activity by TGF-beta. J Neurooncol 53:177–85.[CrossRef][Medline]
Wild-Bode C, Weller M, Wick W. (2001) Molecular determinants of glioma cell migration and invasion. J Neurosurg 94:978–84.[Medline]
Wu JD, Higgins LM, Steinle A, Cosman D, Haugk K, Plymate SR. (2004) Prevalent expression of the immunostimulatory MHC class I chain-related molecule is counteracted by shedding in prostate cancer. J Clin Invest 114:560–8.[CrossRef][ISI][Medline]
Zhou BB, Fridman JS, Liu X, Friedman SM, Newton RC, Scherle PA. (2005) ADAM proteases, ErbB pathways and cancer. Expert Opin Investig Drugs 14:591–606.[CrossRef][ISI][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Krockenberger, Y. Dombrowski, C. Weidler, M. Ossadnik, A. Honig, S. Hausler, H. Voigt, J. C. Becker, L. Leng, A. Steinle, et al. Macrophage Migration Inhibitory Factor Contributes to the Immune Escape of Ovarian Cancer by Down-Regulating NKG2D J. Immunol., June 1, 2008; 180(11): 7338 - 7348. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Cao, X. Xi, Z. Hao, W. Li, Y. Kong, L. Cui, C. Ma, D. Ba, and W. He RAET1E2, a Soluble Isoform of the UL16-binding Protein RAET1E Produced by Tumor Cells, Inhibits NKG2D-mediated NK Cytotoxicity J. Biol. Chem., June 29, 2007; 282(26): 18922 - 18928. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Roda-Navarro and H. T. Reyburn Intercellular protein transfer at the NK cell immune synapse: mechanisms and physiological significance FASEB J, June 1, 2007; 21(8): 1636 - 1646. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Saikali, J. P. Antel, J. Newcombe, Z. Chen, M. Freedman, M. Blain, R. Cayrol, A. Prat, J. A. Hall, and N. Arbour NKG2D-Mediated Cytotoxicity toward Oligodendrocytes Suggests a Mechanism for Tissue Injury in Multiple Sclerosis J. Neurosci., January 31, 2007; 27(5): 1220 - 1228. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||