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[Cancer Research 66, 2520-2526, March 1, 2006]
© 2006 American Association for Cancer Research

Priority Reports

Proteolytic Release of Soluble UL16-Binding Protein 2 from Tumor Cells

Inja Waldhauer and Alexander Steinle

Department of Immunology, Institute for Cell Biology, Eberhard-Karls University Tübingen, Tübingen, Germany

Requests for reprints: Alexander Steinle, Department of Immunology, Institute for Cell Biology, University of Tübingen, Auf der Morgenstelle 15, D-72076 Tübingen, Germany. Phone: 49-7071-29-80992; Fax: 49-7071-29-5653; E-mail: alexander.steinle{at}uni-tuebingen.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The MHC class I–related ligands of the immunoreceptor NKG2D are frequently expressed by tumor cells and stimulate tumor immunity mediated by CD8 T cells and natural killer (NK) cells. In humans, NKG2D ligands (NKG2DL) are encoded by the MHC-encoded MIC and non–MHC-encoded UL16-binding protein (ULBP) families of proteins. Recently, we and others showed that tumor cells release soluble MICA (sMICA), thereby counteracting NKG2D-mediated tumor immunosurveillance. Here, we now report that ULBP2 molecules are likewise released from tumor cells in a processed soluble form, and that soluble ULBP2 (sULBP2) can be detected in sera of some patients with hematopoietic malignancies. Tumor cell–derived sULBP2 as opposed to cell-bound ULBP2 does not down-regulate NKG2D on NK cells. Unexpectedly, the glycosylphosphatidylinositol-anchored ULBP2 molecules are not released by phospholipases but by the action of metalloproteases. Proteolytic shedding of both NKG2D ligands MICA and ULBP2 by tumor cells was strongly enhanced after phorbol 12-myristate 13-acetate treatment and paralleled by a markedly reduced susceptibility to NKG2D-mediated cytotoxicity. Shedding of MICA and ULBP2 can be blocked by the same inhibitors, suggesting the involvement of related metalloproteases. Thus, our data suggest that reducing NKG2DL surface densities is due to a common cleavage process executed by metalloproteases that promotes escape of tumors from NKG2D-mediated immunosurveillance. (Cancer Res 2006; 66(5): 2520-6)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The C-type lectin-like NKG2D receptor is expressed by most natural killer (NK) cells, CD8 {alpha}ß T cells, and {gamma}{delta} T cells in humans (1). In association with the adaptor protein DAP10, NKG2D transduces signals that activate or costimulate effector functions of these cytotoxic lymphocytes (1–3). A peculiarity of NKG2D consists in the multitude of ligands that are not constitutively expressed but rather are induced subsequently to harmful events like genotoxic stress or infection (3–6). Of note, NKG2D ligands (NKG2DL) are also frequently expressed on malignant cells but absent from the respective benign tissues rendering the NKG2D/NKG2DL system an interesting target for tumor immunotherapy (7, 8). In fact, recent studies in mice strongly support a stimulatory role of NKG2D for tumor immunity. NKG2DL expression is induced by carcinogens and genotoxic stress, and tumor cell lines transduced with mouse NKG2DL were readily eliminated in vivo due to NK and CD8 T-cell activity and induced tumor immunity against the parental cell line (6, 9, 10). Human NKG2DL belong to the two families of MHC class I–related MIC and UL16-binding protein (ULBP) molecules, respectively (5). The MHC-encoded MICA and MICB molecules exhibit a highly restricted expression on healthy tissue in vivo but are broadly expressed on epithelial tumors and hematopoietic malignancies (7, 8, 11). Targeting cytotoxic lymphocytes towards MICA-expressing tumors is counteracted by proteolytic shedding of MICA molecules (12, 13). In addition, soluble MICA (sMICA) and transforming growth factor-ß (TGF-ß) have been reported to systemically down-regulate NKG2D expression on cytotoxic lymphocytes providing another route of escape from NKG2D-mediated surveillance (12, 14–16). Considerably less is known about the expression and regulation of ULBP molecules that differ from MIC molecules by the lack of an {alpha}3 domain. Like MIC molecules, ULBPs are expressed by many tumor cell lines and some hematological malignancies (8, 17). However, knowledge of expression of ULBP in vivo remains scarce. In contrast to MIC molecules, ULBP1-3 have been shown to be linked to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor similarly to their mouse counterparts, the RAE-1 molecules (5, 17). Previously, release of soluble ULBP2 (sULBP2)/ALCAN from some tumor cells in vitro has been reported (18), but neither the molecular mechanism of ULBP2 release nor its functional implications have been addressed. Here, we report that ULBP2 molecules are released from tumor cells by metalloproteases and can be detected in sera of patients with hematopoietic malignancies.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and sera. Cell lines C1R, Jurkat, Molt4, HL60, and K562 were cultured in 10% FCS/RPMI 1640, HCT116, SW756 in 10% FCS/DMEM, 293T in 10% FCS/Iscove's modified Dulbecco's medium, and C1R transfectants in 10% FCS/RPMI 1640 with 1.8 mg G418/mL. NKL cells were cultured in 10% FCS/RPMI 1640 with 200 units/mL interleukin 2 (Proleukin, Chiron, CA). Human patient sera were obtained after written informed consent and with approval of the local ethics committee.

Reagents. Anti-NKG2D (clone 139), anti-NKG2D-PE (clone H106.771), anti-ULBP2 (clone 165903), polyclonal anti-ULBP2, and ULBP2-Fc were from R&D Systems (Minneapolis, MN). Anti-mouse IgG1-PE conjugate (clone X40) and anti-CD80-FITC conjugate (clone BB1) were obtained from BD Biosciences (San Jose, CA). Soluble human phycoerythrin-coupled NKG2D tetramers and anti-ULBP2 BUMO1 were produced as described elsewhere (4). Rabbit anti-goat horseradish peroxidase (HRP) conjugate was from Jackson ImmunoResearch Laboratories (West Grove, PA); anti-mouse IgG2a-HRP was from Southern Biotechnology Associates (Birmingham, AL); and goat anti-mouse IgG-coated microspheres were from Bangs Laboratories (Fishers, IN). Hydroxamate-based broad metalloprotease inhibitors matrix metalloproteinase inhibitor III (MMPI III; Merck, Darmstadt, Germany) and Batimastat (BB94; kind gift of Klaus Maskos, Max Planck Institute for Biochemistry, Martinsried, Germany) were used. BB94 was dissolved in dimethylformamide and added as 1:200 volume to cultures. Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus, Brefeldin A, and phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma (St. Louis, MO).

Flow cytometry. Cells were incubated with the anti-ULBP2 BUMO1 or mouse IgG1 at 10 µg/mL and then, after washing, with goat anti-mouse-PE conjugate (1:200) as secondary reagent, or, alternatively, with phycoerythrin-labeled soluble human NKG2D tetramers at 10 µg/mL. Samples were analyzed on a FACScan (BD Biosciences). Specific fluorescence intensities were calculated by subtracting the mean fluorescence intensity (MFI) of the isotype control from the MFI of the specific antibody.

NKG2D down-regulation assay. NKL cells were cocultured for 24 hours with irradiated C1R transfectants at 1:1 ratio or with concentrated supernatants of C1R transfectants. Afterwards, cells were stained for NKG2D expression with NKG2D-PE or the respective isotype control. Cocultured C1R transfectants were excluded from histogram analysis by staining with FITC-conjugated anti-CD80. After washing, samples were analyzed on a FACScan.

NKG2D binding assay. Goat anti-mouse IgG-coated microspheres were incubated with 50 µg/mL anti-ULBP2 monoclonal antibody (mAb) BUMO1 or anti-MHC class I W6/32, respectively (8). After washing, microspheres were resuspended with concentrated supernatants of C1R-ULBP2 transfectants. Then, washed microspheres were stained with phycoerythrin-conjugated NKG2D or H2-Kd tetramers (4), respectively, and fluorescence was assessed by flow cytometry on a FACScan.

ELISA. For the detection of sULBP2, two monoclonal anti-ULBP2 mAb binding nonoverlapping ULBP2 epitopes were implemented. Plates were coated with the anti-ULBP2 mAb BUMO1 at 1 µg/mL in PBS, then blocked by addition of 50 µL of 2% bovine serum albumin (BSA) for 1 hour at 37°C and washed. Afterwards, ULBP2-Fc (R&D Systems) and the samples were added, and the plates were incubated for 2 hours at 37°C. For analysis of patient samples, sera were diluted 1:3 in PBS before addition to the plates. After incubation, plates were washed, and the detection mAb anti-ULBP2 (R&D Systems) at 1 µg/mL in 1% BSA/PBS was added for 2 hours at 37°C. Plates were then washed, and anti-mouse IgG2a-HRP (1:10,000 in 3.25% BSA/PBS) was added for 1 hour at 37°C. Plates were then washed and developed using the Tetramethylbenzidine Peroxidase Substrate System (KPL, Gaithersburg, MD). The absorbance was measured at 450 nm. Results are shown as means with SD of triplicates. The ELISA procedure for sMICA has been previously described (8).

Immunoblot analysis. Samples were separated by 15% SDS-PAGE. Where indicated, samples were treated before with peptide:N-Glycanase F (PNGaseF; New England Biolabs, Beverly, MA) for 1 hour at 37°C according to the manufacturer's instructions. Gels were blotted to Hybond-enhanced chemiluminescence membranes (Amersham, Little Chalfont, United Kingdom), blocked with TBS containing 5% nonfat dried milk, and then analyzed with 0.1 µg/mL anti-ULBP2 serum. Binding of anti-ULBP2 was detected with a rabbit anti-goat HRP-conjugate and chemiluminescence reagent (Pierce Biotechnology, Rockford, IL).

Chromium release assay. Cytotoxicity of NKL cells against 293T cells was assessed in a 2-hour 51Cr release assay. For NKG2D blockade, NKL cells were pretreated with mAb 139, and mAb139 was also added to the assay medium (10 µg/mL). Where indicated, 293T were pretreated with 10 µg Brefeldin A/mL and 100 ng PMA/mL for 12 and 11.5 hours, respectively, before labeling with 50 µCi of 51Cr (Amersham, Freiburg, Germany) for 1 hour at 37°C, and subsequently washed thrice before the assay. Calculation of % lysis = 100 x (experimental release – spontaneous release) / (maximum release – spontaneous release). Data are means of triplicates.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Tumor cell lines release sULBP2. The recent description of proteolytic release of MICA from tumor cells (12, 13) and its implications for tumor immune evasion prompted us to investigate a similar mechanism for the second family of human NKG2DL, the ULBP. We chose to investigate release of ULBP2 as a representative of the GPI-linked ULBP molecules. To this aim, we established a highly sensitive ULBP2 sandwich ELISA detecting sULBP2 down to a concentration of 20 pg/mL (Fig. 1A). Using this ELISA, we analyzed culture supernatants of ULBP2-transfected C1R cells and detected high concentrations (~10 ng/mL) of sULBP2 within 16 hours of culture (Fig. 1B). Supernatants of mock-transfected C1R cells that express about 100-fold less ULBP2 endogenously (8) as well as supernatants of ULBP1-transfected and ULBP3-transfected gave rise to only weak signals underlining the specificity of the ELISA (Fig. 1B). In supernatants of several hematopoietic and nonhematopoietic cells lines expressing endogenous ULBP2, concentrations of sULBP2 roughly paralleled ULBP2 cell surface levels (Fig. 1C).


Figure 1
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  		Figure 1. sULBP2 is released by tumor cells. A, ULBP2 sandwich ELISA. Serial dilutions of recombinant ULBP2-Fc were analyzed in a sandwich of anti-ULBP2–specific mAb BUMO1 and mAb 165903. Points, means of triplicates of a representative experiment from a total of three; bars, SD. B, supernatants of C1R cells stably transfected with ULBP1, ULBP2, ULBP3, MICA*01, and vector alone were analyzed by ULBP2 ELISA after 16 hours of culture in fresh medium. ULBP surface expression of the various C1R-ULBP transfectants has previously been shown (8). Columns, means of triplicates of a representative experiment from a total of three; bars, SD. C, supernatants of various cell lines endogenously expressing ULBP2 (specific fluorescence intensities of ULBP2 surface expression in brackets) were analyzed by ULBP2 ELISA after 36 hours of culture in fresh medium. Columns, means of triplicates of a representative experiment from a total of three; bars, SD. D, ULBP2 immunoblot of fourfold concentrated supernatants or lysates of C1R-ULBP2 cells. Supernatants were from PI-PLC–treated or untreated C1R-ULBP2 cells. Samples were treated with PNGaseF before SDS-PAGE, where indicated.

 
Next, we assayed ULBP2 in lysates and supernatants of C1R-ULBP2 cells by immunoblotting and detected molecular species of about 30 to 35 kDa with a diffuse appearance most likely due to heterogeneous glycosylation. In line with this presumption, pretreatment of the C1R-ULBP2 lysate with PNGaseF resulted in a distinct band of lower molecular weight corresponding to the expected size of mature ULBP2 protein (~24 kDa; Fig. 1D). Deglycosylated sULBP2 detected in the C1R-ULBP2 supernatant was of even lower apparent molecular weight, suggesting enzymatic processing of the membrane-bound protein. ULBP2 is attached to the cell membrane by a GPI anchor, which previously was shown by treatment of ULBP2-transfected cells with PI-PLC (17). Enzymatic release of several GPI-linked proteins by phospholipases has been reported (19). Therefore, we treated C1R-ULBP2 transfectants with PI-PLC to analyze phospholipase-shed ULBP2 molecules. Unexpectedly, PNGaseF-treated supernatants of PI-PLC–treated C1R-ULBP2 cells contained an additional band of higher molecular weight corresponding to the ULBP2 species in cell lysates, suggesting that the naturally released sULBP2 is not generated by the activities of phospholipases (Fig. 1D).

ULBP2 molecules are released by metalloproteases. To address an involvement of metalloproteases in the shedding of ULBP2, we treated C1R-ULBP2 cells with MMPI III and observed a pronounced reduction of ULBP2 release correlating with increasing concentrations of the inhibitor (Fig. 2A). Similar data were obtained using the broad metalloprotease inhibitor batimastat (BB94) that has previously been tested in phase I/II trials in cancer patients. To verify that shedding by metalloproteases is not a peculiarity of C1R-ULBP2 transfectants, we treated Jurkat and 293T cells with MMPI III and BB94, respectively, and also found a dose-dependent reduction of ULBP2 levels in the respective supernatants (Fig. 2B; data not shown). Next, we investigated sULBP2 levels in supernatants of C1R-ULBP2 cells that were treated with bacterial phospholipase PI-PLC and detected ~1.3-fold higher sULBP2 concentrations compared with supernatants of mock-treated cells (Fig. 2C). Whereas physiologic ULBP2 shedding was largely inhibited by BB94, there was only a slight reduction of sULBP2 levels in the supernatants of PI-PLC–treated C1R-ULBP2 cells, suggesting that BB94 does not affect the activity of phospholipases. Altogether, these data show that ULBP2 is released from tumor cells by metalloproteases and not by phospholipases.


Figure 2
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  		Figure 2. ULBP2 is released by metalloproteases. A, C1R-ULBP2 cells were incubated for 16 hours with MMPI III or BB94 at various concentrations; subsequently, supernatants analyzed by ULBP2 ELISA. B, Jurkat cells were incubated for 20 hours with BB94 at various concentrations; subsequently, supernatants analyzed by ULBP2 ELISA. C, sULBP2 was determined in supernatants of C1R-ULBP2 cells treated with PI-PLC and/or BB94 for 2 hours at 37°C by ULBP2 ELISA. A-C, concentrations of sULBP2 in supernatants of untreated cells were set as 100%. Columns, means of triplicates of one representative experiment from a total of three; bars, SD.

 
Proteolytic shedding of ULBP2 is enhanced by PMA. To further characterize the ULBP2 shedding activity, we investigated the short-term effect of PMA treatment on ULBP2 release. Whereas most members of the MMP family are released as soluble enzymes and thus are not expected to respond to PMA-mediated activation of protein kinase C, an enhanced proteolytic activity has been reported for members of the membrane-bound "a disintegrin and metalloprotease" (ADAM) proteins following PMA treatment (20). We detected up to 5-fold increased concentrations of sULBP2 in the supernatants of PMA-treated C1R-ULBP2 cells compared with solvent-treated cells (Fig. 3A). Enhancement of ULBP2 shedding was dependent on the PMA concentration and could largely be blocked by addition of BB94. We wondered whether shedding of MICA succumbs to the same mechanisms as ULBP2 shedding. Therefore, we also treated C1R-MICA transfectants with PMA and analyzed sMICA levels in the culture supernatants with our previously described MICA sandwich ELISA (8). It turned out that also MICA shedding is induced by PMA treatment and inhibited by BB94 (Fig. 3B). We then investigated the kinetics of PMA-induced ULBP2 shedding and observed that sULBP2 in supernatants of PMA-treated C1R-ULBP2 cells was about 4-fold increased as early as 15 minutes after begin of treatment compared with mock-treated cells (Fig. 3C). Conversely, PMA-induced shedding resulted in a decrease of ULBP2 and overall NKG2DL surface levels of Brefeldin A–pretreated 293T cells (Fig. 3C). Reduced NKG2DL surface levels were paralleled by a marked reduction in NKG2D-mediated NK lysis of 293 T cells (Fig. 3D).


Figure 3
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  		Figure 3. PMA stimulates shedding of ULBP2 and MICA. A, C1R-ULBP2 cells (left) or C1R-MICA*01 cells (right) were preincubated with 5 µmol/L BB94. After 20 minutes, cells were incubated with various concentrations of PMA for 6 hours; subsequently, supernatants analyzed by ULBP2 ELISA or MICA ELISA, respectively. B, C1R-ULBP2 cells were incubated with or without 100 ng/mL PMA for different times; subsequently, supernatants analyzed by ULBP2 ELISA. Columns, means of triplicates; bars, SD. C, flow cytometric analysis of 293T stained with BUMO1 and soluble NKG2D tetramers, respectively. 293T cells were pretreated with Brefeldin A and PMA (filled histogram), Brefeldin A alone (black line), or untreated (gray line). Dotted lines, control stainings with irrelevant IgG1 (BUMO1) or MHC class I tetramer. D, cytotoxicity assay of untreated 293T cells (bullet) or 293T cells pretreated with Brefeldin A ({blacktriangledown}) or Brefeldin A and PMA ({triangledown}) using NKL cells. NKG2D-blockade abrogated lysis of 293 T cells by NKL completely ({circ}).

 
sULBP2 in sera of patients with malignant diseases. sMICA has been detected in many sera of patients with hematopoietic and epithelial malignancies (8, 12, 13, 16, 21). Accordingly, we analyzed sera of patients with leukemias and gastrointestinal tumors for elevated levels of sULBP2. Four of 23 sera from leukemic patients contained substantial levels of sULBP2 (range, 0.09-0.72 ng/mL), including patients with T-NHL, acute myelogenous leukemia, and chronic myelogenous leukemia (Fig. 4A). Interestingly, high levels of sULBP2 in a patient with T-cell non-Hodgkin's lymphoma (T-NHL) correlated with the previously described pronounced ULBP2 surface expression on the respective malignant cells (8). In contrast, 19 sera of patients with gastrointestinal malignancies and 14 of 15 sera from healthy donors contained no detectable sULBP2. Previous studies reported NKG2D down-regulation by cell-bound MICA and sMICA (12, 16, 21). We investigated a similar phenomenon for ULBP2. Upon cocultivation of the NKG2D-expressing cell line NKL with C1R-ULBP2, a pronounced down-regulation of surface NKG2D was observed but upon cocultivation with control C1R-neo transfectants (Fig. 4B). When we treated NKL cells with supernatants of both C1R-neo and C1R-ULBP2 cells, we did not observe NKG2D down-regulation, although C1R-ULBP2 supernatants contained sULBP2 at ~100 ng/mL (Fig. 4C). We verified that our tumor cell–derived sULBP2 was capable to interact with NKG2D by immobilizing ULBP2 from the supernatants of C1R-ULBP2 cells on protein A–coated microspheres and staining with fluorochrome-conjugated NKG2D tetramers (Fig. 4D). Accordingly, pretreating NKL cells with sULBP2 (100 ng/mL) for 24 hours did not affect cytotoxicity of NKL cells against 293T cells (data not shown).


Figure 4
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  		Figure 4. sULBP2 in sera of leukemic patients. A, 4 of 23 sera from patients with leukemia and 1 of 14 sera from healthy donors, but none of 19 sera from patients with gastrointestinal malignancies contained sULBP2 at detectable levels (>0.05 ng sULBP2/mL serum). sULBP2-containing sera were from patients UPN1, UPN3, UPN12, and UPN20 with T-NHL, acute myelogenous leukemia, secondary acute myelogenous leukemia, and chronic myelogenous leukemia, respectively (8). Points, means of triplicates. B, NKG2D expression by NKL cells after coculture with C1R-ULBP2 or C1R-neo cells, respectively. NKG2D surface expression of NKL cells was determined by flow cytometry using mAb 139 after 24 hours of incubation with irradiated C1R-neo (filled histograms) or C1R-ULBP2 transfectants (open histograms, dark lines). Isotype control stainings (light lines). C, NKL cells were incubated for 24 hours with concentrated supernatants from C1R-neo cells (filled histograms) or from C1R-ULBP2 cells containing 100 ng/mL sULBP2 (open histograms, dark line). Isotype control stainings (light lines). D, microbeads coated with mAb BUMO1 were incubated with supernatants from C1R-ULBP2 (right) or C1R-neo cells (left), respectively, and stained with NKG2D tetramers (dark lines) or H2-Kd tetramers (light lines). As additional controls, W6/32-coated microbeads were incubated with C1R-neo and C1R-ULBP2 supernatants and stained with NKG2D tetramers (filled histograms).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Shedding of the human NKG2DL MICA has recently been described as a novel immune escape mechanism of tumors (12, 13, 16, 21). Here, we investigated tumor cell shedding of ULBP2 as a representative of GPI-anchored NKG2DL. A previous study reported release of ULBP2 by several tumor cell lines and proposed shedding by phospholipases (18). We however find that ULBP2 shedding is, at least for the most part, executed by metalloproteases and not by phospholipases. Shedding of both MICA and ULBP2 is susceptible to the same set of metalloprotease inhibitors and inducible by PMA suggesting the involvement of the same or related metalloproteases (Fig. 3B; data not shown). PMA-inducible shedding has been reported for a variety of other cell surface proteins like tumor necrosis factor-{alpha}, TGF-{alpha}, and heparin-binding epidermal growth factor that are shed by members of the ADAM family of transmembrane metalloproteases (e.g., ADAM17; ref. 20). Current work in our laboratory investigates involvement of ADAMs in the shedding of NKG2DL.

Proteolytic shedding of membrane proteins may either regulate cell surface expression levels and/or promote release of biological active soluble isoforms. For NKG2DL, regulation of cell surface expression is of crucial importance, because NKG2DL surface levels critically determine the susceptibility to NKG2D-stimulated cytolysis (9, 17). We show that PMA-induced shedding of ULBP2 results in markedly reduced ULBP2 surface levels and paralleled by reduced NKG2DL surface densities as well as impaired NKG2D-mediated NK lysis. Furthermore, sMICA from sera of tumor patients has also been described to cause a systemic impairment of antitumor cytotoxicity by down-regulation of NKG2D on peripheral CD8 T cells and NK cells (12, 16, 21). With regard to sULBP2, we did not observe down-regulation of NKG2D on NK cells using concentrations of tumor cell–derived sULBP2 that were well above the concentrations in sera of patients with leukemia. Interestingly, we did not detect sULBP2 in sera of patients with gastrointestinal tumors, whereas sMICA was broadly detected in sera of patients with gastrointestinal and other epithelial tumors (12, 13, 16, 21). Although this may indicate that ULBP2 is primarily expressed and shed by malignant hematopoietic cells, several tumor cell lines of epithelial origin have also been reported to express ULBP molecules (17, 22). However, expression of ULBP by epithelial tumors in vivo has yet to be shown.

In summary, we here report that sULBP2 molecules originate from tumor cells by metalloproteolytic cleavage and are detectable in sera of some patients with leukemia. Shedding of ULBP2 reduces NKG2DL surface levels and may impair immunogenicity of tumor cells. Further studies have to address the molecular mechanics of ULBP2 cleavage, the relevance of ULBP shedding for tumor immunity, and the potential of sULBP as novel variable for diagnosis and/or prognosis in leukemia.


   Acknowledgments
 
Grant support: Wilhelm Sander-Foundation, Munich, Germany grant 2004.018.1 and Medical Faculty at the Eberhard-Karls-University Tübingen fortüne program grant 1349-0-0.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Wiebke Ruschmeier for excellent technical assistance and Cecile Gouttefangeas (Department of Immunology, Institute for Cell Biology, Eberhard-Karls University Tübingen, Tübingen, Germany), Andrea Peterfi, and Helmut Salih (Department of Internal Medicine II, University Hospital Tübingen, Tübingen, Germany) for kindly providing sera of tumor patients.

Received 7/18/05. Revised 12/15/05. Accepted 1/ 6/06.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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