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[CANCER RESEARCH 62, 3453–3458, June 15, 2002] Interferon Stimulated Gene 15 Constitutively Produced by Melanoma Cells Induces E-Cadherin Expression on Human Dendritic Cells1

Elisabetta Padovan,2 Luigi Terracciano, Ulrich Certa, Barbara Jacobs, Anca Reschner, Martin Bolli, Giulio C. Spagnoli, Ernest C. Borden, and Michael Heberer Department of Surgical Research [E. P., A. R., M. B., G. C. S., M. H.] and Institute of Pathology [L. T.], University of Basel, 4031 Basel, Switzerland; Roche Genetics, F. Hoffmann-La Roche Ltd., 4070 Basel, Switzerland [U. C.]; and The Cleveland Clinic Foundation, Cleveland, Ohio 44195 [B. J., E. C. B.]

ABSTRACT promise the antigen-presenting cell functions of infiltrating DCs, thereby favoring tumor immune escape. In a consistent number of The immunobiology of tumor-infiltrating dendritic cells (DCs) can be reports, discrete tumor-derived factors have been shown to prevent strongly influenced by the cytokine environment present in the malignant DC differentiation and maturation and hamper the induction of tissue. We have previously identified discrete melanoma lines, inducing E-cadherin expression on monocyte-derived DCs in vitro. We demonstrate antitumor immunity (14–17). Along with these observations, we here that this effect, independent of cell contact, is not inducible in the have previously identified discrete melanoma cell lines inducing presence of tumor lysates and requires the constitutive expression of IFN E-cadherin expression on monocyte-derived DCs in vitro, potentially stimulated gene 15 (ISG15) by malignant cells. impairing their migratory behavior (9). High-density oligonucleotide arrays were used to investigate the expres- In the present study, melanoma cell clones endowed with different sion pattern of 7000 genes in RNA from two melanoma cell clones com- functional capacities were subjected to gene profiling to identify petent for E-cadherin induction and two clones devoid of DC-modulating differentially expressed genes. We provide evidence that defined capacity. A total of 13 genes encoding soluble proteins were expressed at soluble factors are responsible for the melanoma-induced modulation higher magnitude in melanomas able to induce E-cadherin expression on of DC phenotype. DCs. Combining those data with quantitative protein assays, we could narrow our investigation down to three factors: the chemokine CCL5 and the cytokines ISG15 and type I IFNs. Strikingly, >7 ng/ml of ISG15 could MATERIALS AND METHODS be detected in the corresponding melanoma-conditioned medium and induction of E-cadherin on DCs failed in the presence of antibodies Media and Reagents for Cell Culture. Cells were cultured in RPMI 1640 neutralizing ISG15 protein. Most importantly, strong cytoplasmic expres- supplemented with 1% Ciproxin (Bayer, Zu¨rich, CH), 2 mML-glutamine, 1% Ϫ sion of ISG15 was detected by immunohistochemistry in the original nonessential amino acids, 1% sodium pyruvate, 5 ϫ 10 5 M 2-mercaptoethanol tumor specimen from which the melanoma cell lines under investigation and 10% FCS (Life Technologies, Inc., Paisley, Great Britain), thereafter were derived. referred to as complete medium. LPS content in serum was tested by Limulus These data describe a novel property of ISG15 targeting induction of LAL assay, and only LPS-free batches were used. Human recombinant IL-4 E-cadherin on DCs and possibly influencing their migratory behavior. was produced in our laboratory. Granulocyte/macrophage-colony stimulating factor was provided by Novartis (Basel, CH). INTRODUCTION Cell Cultures. The original cell line Me67 was generated in our laboratory from a metastatic melanoma. Lines Me67.3, Me67.5, Me67.9, and Me67.10 3 DCs are potent inducers of immunity, the properties of which can were derived by culturing the parental cell line in limiting dilution at 0.3 be strongly influenced by the nature of the microenvironment they cells/well in 96-well plates. All cell lines were grown in complete medium and infiltrate (1). Experimental data indicate that the migratory behavior were free from Mycoplasma infection, as monitored by specific reverse tran- of mature DCs can be profoundly different, even when their apparent scription-PCR. Cell lysates were prepared by three cycles of freeze and antigen-presenting capacity is similar (2). In particular, homing of thawing. mature DCs to secondary lymphoid organs, following the induction of Generation of DCs from Peripheral Blood Monocytes. Immature DCs were generated from human peripheral blood mononuclear cells according to specific chemokine receptors (3, 4), allows priming of naı¨ve T cells. published methods (18). Briefly, monocytes were purified by positive sorting On the other hand, induction of chemokines specifically attracting using anti-CD14 conjugated microbeads (Milteny I., Bergisch-Gladbach, D). The antigen-specific T cells (5, 6) will enhance effector functions in the sorted cells were cultured for 6–7 days in complete medium supplemented with 50 periphery and recruitment of memory lymphocytes (7). Derangements ng/ml granulocyte/macrophage-colony stimulating factor and 1000 units/ml IL-4. of these migratory patterns could crucially influence the outcome of Induction of DC Phenotypic Modulation. Immature DCs were cocultured immune responses, including those that are tumor specific (8, 9). with either tumor cells at a 5:1 ratio, in the presence of tumor cell culture The presence of DCs in malignant tissues has been extensively supernatant or their lysates (1:2 dilution). In some experiments, purified rabbit IgG studied, with conflicting results. For some types of cancers, such as anti-ISG15 (19), goat anti-IFNARI, mouse anti-CCR5 IgG1 antibodies, or their oral (10), ovarian (11), colorectal cancers (12) and renal carcinoma isotype matched controls (R&D Systems, Oxon, United Kingdom and BD Bio- ␮ (13), DC infiltration has been associated with prolonged patients sciences, Heidelberg, Germany), were added to the cultures at a 50 g/ml final concentration. Cell cultures were analyzed, after 24-h incubation time, by FACS survival and reduced metastatic disease, specially if associated with staining. T-cell infiltration (10, 12). FACS Analysis. The DC phenotype was monitored by cell surface staining On the other hand, factors secreted by neoplastic cells can com- using FITC-conjugated mouse antibodies from BD Biosciences (Heidelberg, Germany) to human CD86 (clone IT2.2) and CD15 (clone MMA). The mouse Received 1/28/02; accepted 4/18/02. antihuman E-cadherin (clone SHE78–7; R&D Systems) antibody was used in The costs of publication of this article were defrayed in part by the payment of page combination with a goat antimouse IgG2a FITC-conjugated (Southern Biotech- charges. This article must therefore be hereby marked advertisement in accordance with nology Associates, Birmingham, AL) antibody. Samples were analyzed on a 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by the Basel Regional Cancer League Grant 6/00 (to E. P.) and National FACSCalibur (Becton Dickinson, Mountain View, CA) using propidium io- Cancer Institute CA90914 (to E. C. B.). dide to exclude dead cells. 2 To whom requests for reprints should be addressed, at Department of Surgery, Oligonucleotide Array Analysis. Cultured melanoma cells were harvested Research Division, University of Basel, Hebelstrasse 20, CH-4031 Basel, Switzerland. by scraping, and total cellular RNA was extracted (20). Ten ␮g from each sample Phone: 41-61-2652376; Fax: 41-61-2653990; E-mail: [email protected]. 3 The abbreviations used are: DC, dendritic cell; IL, interleukin; FACS, fluorescence- were reverse transcribed, labeled, and processed by using a commercial kit activated cell sorter; ISG, interferon stimulated gene. (Affymetrix, Santa Clara, CA) according to the supplier’s instructions. Upon 3453

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2002 American Association for Cancer Research. ISG15 AND DC PHENOTYPE alkaline heat fragmentation, cDNA were hybridized to the arrays following stand- ard procedures as supplied with the microchips (Affymetrix). Raw data were collected with a confocal laser scanner (Hewlett Packard, Palo Alto, CA), and pixel levels were analyzed using a commercial software (GeneChip v3.1; Af- fymetrix). Three repeats for each array were performed. Expression levels for each gene were calculated as normalized average difference of fluorescence intensity as compared with hybridization to mismatched oligonucleotides, expressed in arbi- trary units. On average, Ͼ25% of the genes under investigation were positive in the cell lines tested. A threshold of 20 normalized average difference units was assigned to any gene with a calculated expression level Ͻ20, because mRNA levels in this low range could not be reliably assessed. Chemokines and Cytokines Detection. CXCL1, CCL5, IL-1␤, IL-6, and IGF-II production was determined by quantitative ELISA assays (sensitivity, Ն10 pg/ml) using cell supernatants of confluent cultures. Antibody pairs and standards were provided by BD Biosciences or R&D Systems. ISG15 was detected by ELISA assay (19). Release of type I IFNs was quantified on HeLa cells as described elsewhere (21). Assays were performed on coded samples. Immunohistochemistry. The original tumor specimen from which Me67 melanoma lines were derived, conserved as paraffin-embedded material, was retrieved and analyzed as follows. Serial sections were incubated overnight at 4°C with anti-ISG15 or isotype-matched control antibodies, followed by avidin-biotin-peroxidase complex (ABC; Vectastain, Burlingame, CA).

RESULTS

E-Cadherin Induction on Melanoma-conditioned DCs Is Inde- pendent from Cell Contact. We have shown previously that melanoma cell lines can activate monocyte-derived DCs in vitro (9). To obtain an insight into the molecular mechanisms underlining these modifica- tions, we first analyzed the effects of coculturing Me67 melanoma cells, their conditioned culture medium, or their lysates with human DCs. Induction of E-cadherin, as well as the increased expression of CD15 and Fig. 1. DC phenotypic modulation induced by melanoma cells is cell contact independent. A, phenotypic maturation of DCs upon 24-h coculture with Me67 melanoma CD86 on DCs was observed in the presence of tumor cells or their culture cells, their conditioned medium or lysates was monitored by measuring the expression of supernatant, whereas cell lysates were unable to induce these effects (Fig. E-cadherin, CD15, and CD86, as indicated. In each plot, dotted lines represent the background staining with isotype-matched control antibodies; thin lines show the staining 1A). Thus, phenotypic DC modulation induced by Me67 was cell contact profile of immature DC; overlayed black lines indicate the phenotype of mature DCs. B, independent and relied on the production of active soluble factors present modulatory capacity of clones derived from Me67 parental cell line. In each plot, data are in the cell culture medium. expressed as median fluorescence intensity (MFI). To identify the molecular entity(s) responsible for these effects, specific cell clones were derived from Me67 parental cell line. Supernatants clones Me67.3 and Me67.9 but barely detectable in clones Me67.5 from clones Me67.3 and Me67.9 induced E-cadherin expression, whereas and Me67.10 (Table 1). clones Me67.5 and Me67.10 were devoid of any modulatory effect on When the corresponding conditioned melanoma cell culture media DC phenotype (Fig. 1B). These clonal cell populations, all expressing the were assessed for proteins detection, the chemokine CXCL1 and the same genetic background, were subjected to gene profiling to identify cytokine IL-1␤ were found to be produced to different extents by differentially expressed gene sequences. three of the melanoma lines (Fig. 2, A and C, respectively). No Pattern of Soluble Factors Expressed in Me67 Melanoma quantitative differences existed for IL-6 and IGF-II secretion among Clones. Total cellular RNA extracted from Me67.3, Me67.9, Me67.5, the different melanoma clones (Fig. 2, D and E). Considering these and Me67.10 melanoma clones was processed for hybridization to patterns, the involvement of these factors in the modulation of DC oligonucleotide arrays containing probe sets from ϳ7000 full-length phenotype was unlikely. On the contrary, production of the chemo- human genes. A total of 52 expressed genes were selected based on a kine CCL5 and the cytokine ISG15 was consistently different among Ͼ3-fold changes between E-cadherin inducing (Me67.3 ϩ Me67.9) the two groups of melanoma clones. More than 4000 pg/ml of CCL5 and noninducing (Me67.5 ϩ Me67.10) clones and grouped according and Ͼ7000 pg/ml of ISG15 were measured in the supernatants of to their putative physiological function (Table 1). Most of the genes clones Me67.3 and Me67.9 but were undetectable in supernatants identified were specific for membrane-associated, intracellular, or from clones Me67.5 and Me67.10 that were devoid of any DC- nuclear proteins and therefore excluded from further analysis. conditioning capacity. A total of 11 genes encoding secreted proteins were found to be Moreover, genes encoding IFN-inducible proteins (i.e., rig-g, the expressed at higher levels in clones Me67.3 and Me67.9 than clones IFN-induced Mr 56,000 protein encoding sequence, ISG20, trip14, Me67.5 and Me67.10 (Table 1). Among these, 5 were specific for 27-sep, and 16-jun) were consistently expressed in clones Me67.3 and proteins present in the extracellular matrix such as peptidoglycan, Me67.9 but poorly amplified in clones Me67.5 and Me67.10. Indeed, collagen, fibronectin, and mucin (Table 1). Because these proteins are the two groups of clones differed for the capacity to secrete type I involved in the formation of three-dimensional structures and poorly IFNs; clones Me67.9 and Me67.3 produced 64 and 128 IU/ml of type detectable in solution, they were excluded from further investigations. I IFNs, respectively, whereas Me67.5 and Me67.10 clone supernatants The other genes were analyzed in detail. Two genes for chemotactic were completely negative (Fig. 2G). Considering the immunomodu- factors (GRO-␣/CXCL1 and RANTES/CCL5) and 4 encoding cyto- latory properties of type I IFNs, these data might be of relevance also kines (IL-1␤, IL-6, IGF-II, and ISG15) were strongly expressed in in the experimental system described here. 3454

Table 1 Genes differentially regulated in melanoma clones endowed with (Me67.3 and 67.9) or devoid of (Me67.5 and 67.10) DC phenotypic modulation capacity. Sequences are grouped based on putative function and localization of specific gene products. Genes are listed by accession number and description. Data are expressed as average difference as compared with mismatched oligonucleotide controls; only genes showing a Ͼ3-fold change factor between competent (Me67.3 ϩ Me67.9) and incompetent (Me67.5 ϩ Me67.10) clones were considered. Average difference

Accession no. Product Me67.3 Me67.9 Me67.5 Me67.10 Secreted protein Chemokines X54489 Melanoma growth stimulatory activity (mgsa; GRO-␣/CXCL1) 961 579 20 61 M21121 T cell-specific protein (RANTES/CCL5) mrna, complete cds 2673 245 38 20 Cytokines X04500 Prointerleukin 1␤ (IL-1␤) 8958 6126 1791 1123 X04602 IL bsf-2 (IL-6) 1865 749 119 51 J03242 Insulin-like growth factor 2 (IGF-II) 487 378 109 83 M13755 Human interferon-induced 17-KDa/15-KDa protein (ISG17/15) 7204 5916 750 286 Extracellular matrix J04599 hpgi mrna encoding bone small proteoglycan i (biglycan) 417 655 20 20 X02761 Fibronectin (fn precursor) 14710 15016 2378 2942 M55998 ␣-1 collagen type I gene, 3Ј end 1687 1873 272 192 HG3044-HT3742 Fibronectin, alt. splice 1 6318 6711 827 1082 HG880-HT880 Mucin 6 157 567 20 20 IFN-induced Unknown function U52513 rig-g mrna 395 200 98 42 M24594 IFN-inducible 56 KD protein 912 1058 47 20 Nuclear bodies U88964 hem45 (ISG20) mrna 718 742 165 163 Membrane protein L40387 Thyroid receptor interactor (trip14) gene, 3Ј end of cds. 1285 596 203 21 J04164 IFN-inducible protein 27-sep 4817 6161 20 20 U22970 16-jun gene (IFN-inducible peptide precursor) 4785 4284 22 20 Membrane proteins U96094 Sarcolipin (sln) 20 20 464 303 D16593 bdr-2 mrna for hippocalcin 63 124 201 379 Z68228 Plakoglobin 171 314 52 20 U59302 Steroid receptor coactivator-1 f-src-1 261 372 20 20 M24439 Liver/bone/kidney-type alkaline phosphatase (alpl) 306 336 20 20 Type I U48705 Receptor tyrosine kinase 80 96 426 344 M24283 Major group rhinovirus receptor (hrv) 314 386 52 73 S76475 trkc 431 551 65 88 Z48481 Matrix metalloproteinase 1 244 306 20 97 Type II X90846 Mixed lineage kinase 2 20 20 314 228 X94612 Type II cgmp-dependent protein kinase 20 20 208 547 L16862 G protein-coupled receptor kinase (grk6) 478 534 88 148 Type III M36284 Glycophorin C 341 455 68 129 Integral membrane proteins U40223 Uridine nucleotide receptor (unr) 227 374 28 20 X16662 Vascular anticoagulant-beta (Annexin 8) 252 304 20 20 U50136 Leukotriene c4 synthase (ltc4s) 500 365 20 77 Intracellular proteins 17793 kiaa0119 53 20 347 253 U14417 ral guanine nucleotide dissociation stimulator 173 301 54 34 X53414 Peroxisomal 1-alanine:glyoxylate aminotransferase 20 20 328 237 AF006041 fas-binding protein (daxx) 299 663 78 103 X15331 Phosphoribosylpyrophosphate synthetase subunit one 514 546 129 105 X68277 Protein tyrosine phosphatase 586 526 144 124 U80226 ␥-Aminobutyric acid transaminase 336 266 81 79 M19267 Tropomyosin 1428 2158 332 303 Z24727 Tropomyosin isoform 3037 2631 687 735 L48546 Tuberin (tsc2) 187 303 37 20 U26266 Deoxyhypusine synthase 504 415 70 115 X81420 hhkb1 1308 791 82 181 Nuclear proteins X12517 u1 small nuclear rnp-specific c protein 20 20 441 430 Z49825 Hepatocyte nuclear factor 4 ␣ 68 20 450 336 M96739 nscl-1 424 598 109 103 M76378 Cysteine-rich protein (crp) 2043 1656 483 365 X61755 Homeoprotein hox3d 155 444 20 20 M59465 Tumor necrosis factor ␣ inducible protein a20 701 325 60 63 X68688 znf33b gene 410 482 20 20

Altogether, by combining the gene chip technology with quantita- Anti-ISG15 Antibodies Inhibit the Expression of E-Cadherin tive protein detection assays, we were able to identify three soluble and Hamper the Up-Regulation of CD15 and CD86. To define to factors (i.e., RANTES, ISG15, and type I IFNs most likely involved what extent RANTES, ISG15, and/or type I IFNs induced DC phe- in the DC phenotypic modulation induced by Me 67.3 and Me67.9 notypic modulation, the cellular in vitro assays were repeated in the conditioned media. presence of specific neutralizing antibodies. The following experi- 3455

Fig. 2. Pattern of soluble factors expressed by Me67 melanoma clones. Concentration of CXCL1, CCL5, IL-1␤, IL-6, IGF-II, and ISG15 proteins in conditioned media from Me67.3, Me67.9, Me67.5, and Me67.10 clones was determined by specific ELISAs and amounts expressed in pg/ml. Type I IFNs were quantified with a biological assay using HeLa cells, and concentrations were expressed in IU/ml. Mean values of triplicates are shown; bars, SD.

ments crucially depended on the use of reagents unable to induce any Detection of ISG15 in Tumor Specimens. In previous work, modification of DC phenotype per se, e.g., devoid of endotoxin tumor-infiltrating CD15ϩ cells with a typical DC morphology were contamination. Commercially available anti-CCL5 antibodies were identified within malignant melanoma tissues. The potential in vivo excluded from our tools because they were found to induce up- relevance of our in vitro findings relied on the expression of ISG15 regulation of CD83 and CD86 on immature DCs. Anti-ISG15, anti- in original tumor specimens. Indeed, immunohistochemical stain- IFNARI, and anti-CCR5 antibodies were suitable for our assays. ing of Me67 metastatic melanoma tissue on paraffin sections Neutralization of ISG15 markedly suppressed the melanoma- revealed that tumor cells were strongly positive for ISG15, in conditioned modulation of DC phenotype (Table 2). The results of contrast to tumor-infiltrating lymphocytes that were completely three different experiments, independently performed, confirmed that negative (Fig. 3A). In malignant cells, ISG15 protein was localized ISG15 played a crucial role in inducing expression of E-cadherin and within the cytoplasm (Fig. 3B). WM9 melanoma human cells strongly influenced up-regulation of CD15 induced by both Me67.3 grown in nude mice as xenografts were examined, in the same and Me67.9 conditioned media (Table 2, Exp. I, II, and III). In settings, as specific control. These cells were strongly positive in contrast, the up-regulation of CD86 expression on melanoma-condi- immunocytochemistry only upon treatment with IFN-␤, a potent tioned DCs was not reproducibly dependent on ISG15. Antibodies to IFN receptors did not inhibit E-cadherin induction and CD15 expres- inducer of ISG15 (Fig. 3, C and D, respectively). Altogether, eight sion but slightly affected the up-regulation of CD86 induced by both tumor specimens were analyzed in immunohistochemistry. Among Me67.3 and Me67.9 conditioned media (Table 2, Exp. IV). Finally, these, four samples were found strongly positive, three showed a because anti-CCR5 antibodies did not interfere with the induction of moderate positive staining, and one specimen was negative (data E-cadherin induced by Me67.3 conditioned medium (Table 2, Exp. not shown). These findings suggest that constitutive expression of V), involvement of CCL5 in conditioned media DC phenotypic mod- ISG15 by malignant cells might frequently occur in vivo, at least in ulation can be unlikely. melanomas.

Table 2 Neutralization of ISG15 impairs E-cadherin induction on DCsa Exp. I Exp. II Exp. III

E-cadh.b CD15 CD86 E-cadh. CD15 CD86 E-cadh. CD15 CD86 iDC ϩ RIg 3.3 20.1 7.5 5.0 22.2 23.2 4.9 24.1 24.1 Me67.3 ϩ RIg 18.1 643.5 33.6 34.2 609.7 55.4 Me67.3 ϩ anti-ISG15 3.5 342.8 13.8 3.8 387.1 69.1 Me67.9 ϩ RIg 13.5 577.7 21.2 29.1 729.9 62.8 Me67.9 ϩ anti-ISG15 3.4 321.9 9.9 3.7 425.5 80.5 Exp. IV Exp. V

E-cadh. CD15 CD86 E-cadh. CD15 CD86 ϩ ϩ iDC GIg 4.3 25.2 24.1 iDC IgG1 2.3 6.2 7.1 ϩ ϩ Me67.3 GIg 39.0 409.5 61.0 Me67.9 IgG1 13.5 106.5 23.1 Me67.3 ϩ anti-IFNARI 34.0 549.0 55.1 Me67.9 ϩ anti-CCR5 14.9 143.3 18.4 Me67.9 ϩ GIg 23.5 827.0 78.2 Me67.9 ϩ anti-IFNARI 25.0 743.1 63.0 a Immature DCs were cultured for 24 h in the presence or absence of melanoma culture supernatants together with the indicated reagents. Cells were then collected and stained with specific mAbs. b Data on cell surphace expression of E-cadherin, CD15, CD86 are expressed as median of fluorescence intensity (MFI). 3456

Fig. 3. Immunohistochemical detection of ISG15 expressing cells on surgically excised Me67 metastatic melanoma tissue. Sections derived from paraffin-embedded Me67 specimen were stained with polyclonal rabbit anti-ISG15 antibodies. Two different magnifications of a representative field are shown in A and B, respectively. Results obtained in the same settings with WM9 melanoma cells grown in nude mice, untreated or treated with IFN-␤, are shown in C and D, respectively.

DISCUSSION higher magnitude in tumor cells that induced DC phenotypic modulation than in cells devoid of this capacity; (b) high amounts of ISG15 In this study, we explored the capacity of melanoma cell-derived protein were detected in the corresponding conditioned media and, proteins to induce phenotypic modulation of human monocyte- remarkably, in original tumor specimens; and (c) most importantly, de derived DCs (9). We performed microchip analysis on cellular novo expression of E-cadherin on monocyte-derived DCs in vitro was reagents, all from the same genetic background, combined with stand- not inducible in the presence of anti-ISG15 antibodies, and up-regu- ard ELISA assays, and this narrowed our search from 52 genes lation of CD15 and CD86 were strongly hampered in these conditions. most likely involved in the DC phenotypic modulation induced by In contrast, neutralization of type I IFN receptors partially inhibited melanoma-conditioned media down to three soluble products, ISG15, CD86 up-regulation, confirming previous results (6), but did not type I IFNs, and CCL5. ISG15 cytokine is strongly induced by IFN-␣/␤ stimulation in affect E-cadherin induction; anti-CCR5 antibodies did not have any different types of cells, including epithelial tumor cell lines in vitro effect. Finally, in vitro exposure to recombinant CCL5 (used in the ␣ (19, 22), and requires a functional proteasome (23). Its expression in range 10–1000 ng/ml) and IFN- 2a (from 10 to 1000 IU/ml) never malignant tissues was, thus far, never investigated. ISG15 is synthe- resulted in E-cadherin expression on immature DCs (data not shown). Thus, the most straightforward explanation of these results is that sized as a Mr 17,000 precursor protein and processed to a mature Mr 15,000 product by cleavage of the COOH-terminal amino acidic tail E-cadherin induction required ISG15 expression, whereas CD86 up- (24). Mature ISG15 may be released as monomer or in the form of regulation relied on the contribution of both ISG15 and tumor-derived high molecular weight conjugates (25). At present, the nature and the type I IFNs. biological functions of these conjugates are not fully understood (26), Impaired DC mobility, eventually caused by E-cadherin expression, although the immunoregulatory properties of ISG15 have been dem- and sequestration of DCs into malignant tissues have been described onstrated on T lymphocytes and natural killer cells. In particular, previously as potential mechanisms of tumor immune escape not exposure to ISG15 in vitro induces IFN-␥ production by T lympho- affecting antigen-presenting capacities (8, 9). These data could have cytes and proliferation of natural killer cells (27). important implications for cancer immunotherapy. As interventional Our experimental data support the hypothesis that ISG15 is cru- strategy, neutralization of tumor-derived ISG15 might be of difficult cially involved in the modulation of DC phenotype induced by mel- application. On the contrary, targeting the migratory pattern of im- anoma cell conditioned medium: (a) the ISG15 gene was expressed at munocompetent cells might represent a possibility of immune inter- 3457

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2002 American Association for Cancer Research. Interferon Stimulated Gene 15 Constitutively Produced by Melanoma Cells Induces E-Cadherin Expression on Human Dendritic Cells

Elisabetta Padovan, Luigi Terracciano, Ulrich Certa, et al.

Cancer Res 2002;62:3453-3458.

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