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Immunology

Constitutive Expression and Costimulatory Function of LIGHT/TNFSF14 on Human Melanoma Cells and Melanoma-Derived Microvesicles

Roberta Mortarini, Alessia Scarito, Daisuke Nonaka, Marina Zanon, Ilaria Bersani, Elisabetta Montaldi, Elisabetta Pennacchioli, Roberto Patuzzo, Mario Santinami and Andrea Anichini
DOI: 10.1158/0008-5472.CAN-04-3239 Published April 2005

Abstract

Neoplastic cells are thought to have defective expression of costimulatory molecules. However, in this study, we show that human melanoma cells express LIGHT/TNFSF14, a ligand of herpesvirus entry mediator on T cells and of lymphotoxin β receptor on stromal cells. In vitro, melanoma cells stained for LIGHT in the intracellular compartment, with weak or negative cell surface expression. However, LIGHT was expressed on tumor-derived microvesicles released from melanoma cells. In vivo, LIGHT was found in metastatic lesions, and the extent of lymphotoxin β receptor expression on the stromal cells was significantly associated with a “brisk” T-cell infiltrate in the neoplastic tissue. In the lesions with a brisk T-cell infiltrate, stromal cells surrounding the tumor also stained for the T-cell attractant chemokine CCL21. The intratumoral T lymphocytes frequently expressed herpesvirus entry mediator and were characterized by a differentiated phenotype. Coculture of lymphocytes with LIGHT+ melanoma-derived microvesicles or even with LIGHT+ melanoma cells in the presence of interleukin-2 costimulated LIGHT-dependent CD3+CD8+ T-cell proliferation. However, lymphocyte coculture with LIGHT+ microvesicles in the presence of interleukin-2 was also associated with an apoptotic response as documented by increased binding of Annexin V by CD3+CD8+ T cells. These data suggest that LIGHT constitutively expressed in human melanoma cells and microvesicles may contribute to regulate T-cell responses to tumor cells.

  • Melanoma
  • costimulatory molecules
  • T Lymphocytes
  • proliferation
  • apoptosis

Introduction

Current views on the mechanisms that prevent effective activation of antitumor T-cell-mediated response in cancer patients are in part based on the evidence that neoplastic cells are poor antigen-presenting cells (APC) due to defective expression of costimulatory molecules ( 1). In the absence of costimulation or in the absence of tumor-antigen cross-presentation by professional APCs, the interaction of naive T cells with neoplastic cells may lead to either tolerance or anergy ( 2). In addition, tumor-associated antigens expressed by human tumors can be coded for by genes expressed even in nonneoplastic tissues as exemplified by the melanocyte differentiation antigens (MDA) identified in human melanoma ( 3). These self-antigens may provide an additional tolerance barrier hampering activation of antitumor immunity even after dendritic cell–mediated cross-presentation of such antigens ( 3, 4). However, at least in human melanoma, these reasonable views on mechanisms that hamper antitumor T-cell-mediated immunity are challenged by the evidence that progression to metastatic disease to regional lymph nodes (American Joint Committee on Cancer stage III) can be associated with activation of CD8+ T-cell-mediated immunity against MDA epitopes at the systemic level and tumor site ( 5). Moreover, the high frequency of MDA-specific T cells, with a differentiated phenotype, found in the tumor-invaded lymph nodes ( 5, 6) is often characterized by specificity for an antigen (Melan-A/Mart-126-35) that is thought to be poorly processed and presented by professional APCs ( 7). Thus, these data point toward the possibility that activation of antitumor response in the invaded lymph nodes may depend even on direct antigen presentation by melanoma cells. If this hypothesis is correct, then one could also predict that at least in some patients melanoma cells should express not only the tumor antigens (the HLA-peptide complex) but also some costimulatory molecules that may play a relevant role in the regulation of antitumor response at tumor site.

Costimulation plays a central role in the activation of T-cell-mediated immunity and increasing evidence indicates that ligand-receptor pairs belonging to the tumor necrosis factor (TNF)/TNF receptor families are important regulators of this process ( 8). At least five ligand-receptor pairs of the TNF-TNF receptor families [OX40-OX40L, 4-1BB-4-1BBL, CD27-CD70, herpesvirus entry mediator (HVEM)-LIGHT, and CD30-CD30L] are known to affect T-cell activation at early or late stages after antigen encounter ( 8). Among these costimulatory molecules, LIGHT/TNFSF14 (thereafter LIGHT) has attracted considerable interest for its emerging roles in regulating T-cell functions and in promotion of T-cell-mediated antitumor immunity ( 9– 11). LIGHT, which binds to three different receptors [lymphotoxin β receptor (LTβR), HVEM, and DcR3] with distinct patterns of tissue expression ( 9), was initially identified as a ∼29-kDa, type II transmembrane protein transiently induced on activated T lymphocytes ( 12). More recently, additional forms with different molecular weights have been identified ( 13– 15). These include a cytosolic isoform and a soluble form generated by cleavage of the membrane-bound protein by matrix metalloproteinases ( 13– 15). The first known function of LIGHT is T-cell costimulation. In fact, triggering of HVEM on T cells by LIGHT expressed on dendritic cells or on other T cells costimulates T-cell proliferation and IFN-γ secretion in response to T-cell receptor (TCR) engagement and/or interleukin (IL)-2 ( 15– 18). In addition, on engagement of LTβR, LIGHT can also promote cytokine production (such as IL-4, IL-6, and TNF) in mast cells and release of the naive T-cell attractant chemokine CCL21 in stromal cells ( 11, 19). Interestingly, these two functions of LIGHT have been shown recently to cooperate in inducing rejection of an established tumor in a murine model on forced expression of LIGHT in neoplastic cells ( 11). Taken together, these findings suggest that this molecule, if constitutively expressed by a human tumor, could even regulate antitumor T-cell-mediated immunity.

In agreement with this hypothesis, we found that LIGHT can be constitutively expressed in human melanoma cells and melanoma-derived microvesicles. Furthermore, analysis of LIGHT and LIGHT receptor expression in metastatic lesions and functional assays in vitro suggested that LIGHT might contribute to the regulation of the host-tumor interaction.

Materials and Methods

Lymphocytes and melanoma cells. Isolation of lymphocytes from peripheral blood of healthy donors, establishment of melanoma cell lines, and HLA typing and subtyping were as described previously ( 20). All cell lines were maintained in RPMI 1640 (BioWhittaker, Verviers, Belgium) supplemented with 10% FCS (BioWhittaker), 2 mmol/L l-glutamine (BioWhittaker), 20 mmol/L HEPES buffer (BioWhittaker), and antibiotics.

Gene expression profile by cDNA macroarray analysis. Melanoma cells in the log phase of growth were cultured for 24 hours in serum-free medium. Levels of expression of 96 genes were evaluated by the Human Common Cytokine GEArray Q Series (SuperArray, Bethesda, MD) as described ( 21). Gene expression levels as determined in each tumor by the GEArray were compared with the profile obtained by analysis of a reference set produced by mixing equal amounts (3 μg) of mRNA isolated from 25 melanoma cell lines. The gene expression profiles of all tumor samples were analyzed using the J-Express Pro software (Molmine, Bergen, Norway) as described ( 22).

Western blot analysis. Western blot analysis for LIGHT was done as described recently ( 21) using a rabbit polyclonal anti-LIGHT antibody (Peprotech, Inc., Rocky Hill, NJ).

Isolation of melanoma-derived microvesicles. Melanoma cells were cultured in medium supplemented with ultracentrifuged (overnight at 100,000 × g) FCS. Supernatants (72 hours) of 80% confluent melanoma cell lines (2 × 107 cells) were collected and processed according to described procedures ( 23– 25) with slight modifications. Briefly, supernatants were sequentially centrifuged at 4°C at 300 × g for 10 minutes, 1,000 × g for 15 minutes, and 10,000 × g for 30 minutes in a Sorvall centrifuge (Kendro Laboratory Products, Asheville, NC). Microvesicles were then pelleted at 100,000 × g for 2 hours at 4°C using a SW28 rotor on a Beckman ultracentrifuge (Beckman Coulter, Fullerton, CA). The vesicle-containing pellets were resuspended in 2 mL of 0.1 μm prefiltered PBS and used for the different assays. The mean protein content of the microvesicle preparations as estimated by the Bio-Rad Protein Assay (Bio-Rad Laboratories) was 1 to 1.5 mg/mL.

Monoclonal antibodies and flow cytometry analysis. Anti-human LIGHT antibodies (Peprotech or R&D Systems, Minneapolis, MN) were used followed by appropriate FITC-labeled secondary antibodies. Intracellular staining for LIGHT in melanoma cells was done after permeabilization with 70% methanol at 4°C for 30 minutes. Staining for vimentin was used as a positive control for cell permeabilization as described ( 21). Microvesicles were resuspended in 0.1 μm filtered PBS and then stained as described ( 25) for 1 hour at 4°C with antibodies to LIGHT (Peprotech and R&D Systems), HLA-DR-FITC, LAMP-1 [CD107a-phycoerythrin (PE)], LAMP-2 (CD107b-FITC), CD63-PE (BD PharMingen, San Diego, CA), and gp100 (HMB45, DakoCytomation, Glostrup, Denmark). Irrelevant, isotype-matched antibodies to green fluorescent protein followed by secondary FITC-labeled antibody (BD Biosciences, Franklin Lakes, NJ) or PE-labeled anti-CD3 (BD Biosciences) were used as negative controls for microvesicle immunofluorescence analysis. After staining with unlabeled primary antibodies (anti-LIGHT and anti-gp100), microvesicles were incubated with the secondary antibodies for 30 minutes at 4°C. The samples were then diluted to 300 μL with 0.1 μm filtered PBS and analyzed. Microvesicles were gated by flow cytometry as a population of 40 to 100 nm particles after calibration of log forward scatter versus log side scatter dot plots with 0.05 to 1 μm Fluoresbrite yellow-green microspheres (Polysciences, Inc., Warrington, PA). Flow cytometry analysis of T cells was done with the following mouse anti-human monoclonal antibodies (mAb): APC-anti-CD8 or PerCp-anti-CD8 and PE-anti-CD3 or PerCp-anti-CD3 (BD PharMingen). To detect CCR7, cells were stained with IgM anti-CCR7 (BD PharMingen) followed by biotin-conjugated rat anti-mouse IgM and by Cy-Chrome-conjugated streptavidin (BD PharMingen). PE-labeled tetramers of HLA-A*0201-containing peptides from Melan-A/Mart-126-35 ( 26, 27) or gp100209-217 ( 28) were purchased from ProImmune (Littlemore, United Kingdom). Negative controls for tetramer staining and staining conditions were as described ( 20, 22). To detect apoptotic cells, lymphocytes were stained with PE- or FITC-labeled Annexin V (BD Biosciences). All samples were analyzed by a dual-laser FACSCalibur cytofluorimeter (BD Biosciences) using the CellQuest software (BD Biosciences).

Immunohistochemical analysis. Immunohistochemical analysis was done as described previously ( 5– 20). Cytospin preparations (Shandon, Pittsburgh, PA) of melanoma cell lines were fixed with formalin for 10 minutes at room temperature and then analyzed by immunohistochemistry. The following antibodies were used: anti-CD3 (Novocastra Laboratories, Newcastle upon Tyne, United Kingdom); anti-CD8 (DakoCytomation); anti-CD45RO (DakoCytomation); anti-GMP-17/TIA-1 (Beckman Coulter); anti–granzyme B (Monosan, Uden, the Netherlands); anti-LIGHT, anti-LTβR, anti-HVEM, and anti-6Ckine/CCL21 (Santa Cruz Biotechnology, Santa Cruz, CA); and anti-Ki-67 (MIB-1 antibody, Immunotech, Marseilles, France). Sections were subsequently treated with the appropriate biotinylated secondary antibody and then with streptavidin-horseradish peroxidase (DakoCytomation). Tissue sections subjected to the same treatment but without incubation with primary mAb or incubated with normal IgG serum of the appropriate species were used as negative controls. Apoptosis was detected with the terminal deoxynucleotidyl transferase in situ apoptosis detection kit (Genzyme, Cambridge, MA) as described ( 5). Negative controls for detection of apoptosis in tissue sections were as described ( 5). Analysis of serial sections was done as described ( 5) by acquiring digital images of 10 areas of each tissue section (at ×400) on a Zeiss Axiovert 100 microscope (Carl Zeiss, Oberkochen, Germany).

T-cell cultures and CFSE assay. Lymphocytes were stained with 2 μmol/L CFSE (Molecular Probes, Eugene, OR) as described ( 22). CFSE-stained lymphocytes were then cultured (at 1 × 106 per mL) in 24-well plates precoated or not with anti-CD3 mAb (0.1 or 1 ng/mL) and with anti-HVEM mAb (Abcam, Cambridge, United Kingdom) at 0.2 to 0.8 μg/mL through cross-linking mediated by 10 μg/mL goat anti-mouse IgG (Sigma-Aldrich, St. Louis, MO) as described ( 22). Plates were also fed with or without IL-2 (250 IU/mL). Lymphocyte-melanoma cocultures were carried out at a lymphocyte/tumor ratio of 10:1 with irradiated (200 Gy) melanoma cells and with CFSE-stained lymphocytes from HLA class I and II–mismatched healthy donors in the presence of IL-2 (250 IU/mL). In some experiments, before adding lymphocytes, irradiated melanoma cells expressing LIGHT were preincubated or not with suboptimal doses (50 ng/mL) of anti-HLA class I mAb (w6/32) and/or with neutralizing anti-LIGHT antibody (R&D Systems). Alternatively, 2 × 106 lymphocytes from healthy donors were cocultured with melanoma-derived microvesicle preparations (total protein concentration, 1.5 mg/mL) at 100 and 50 μL/mL in the presence of 250 IU/mL IL-2. Microvesicles were preincubated or not with neutralizing anti-LIGHT antibody (R&D Systems) or with control antibodies directed to HLA-DR or gp100. All T-cell cultures were analyzed by flow cytometry after 4 to 6 days by staining with anti-CD3, anti-CD8, and anti-CCR7 mAbs or with Annexin V-PE.

Results

LIGHT expression in human melanoma cells and melanoma-derived microvesicles. By screening a panel of human primary and metastatic melanoma cell lines with the Human Common Cytokine GEArray cDNA Macroarray, LIGHT was found expressed in several lines along with 36 of 96 genes coding for members of the fibroblast growth factor family, the interleukins, the IFNs, and the TNF family ( Fig. 1A ). Hierarchical cluster analysis of expression profiles revealed two main clusters of tumors ( Fig. 1A, 1P-12M and 3P-11M) characterized by differential expression levels of several growth factor and interleukin genes (such as FGF21, HGF, VEGF-B, VEGF-D, IL-15, IL-16, and IL-19) compared with the average expression detected in a reference tumor set. LIGHT was found expressed at higher levels than in the reference set in some tumors from both clusters ( Fig. 1A). Repeated evaluation with the Human Common Cytokine GEArray confirmed the differences in the expression levels of LIGHT in several tumors of both clusters ( Fig. 1B). By Western blot analysis, LIGHT was identified in lysates of melanoma cell lines with a pattern similar to phytohemagglutinin (PHA)–activated peripheral blood lymphocytes (PBL; Fig. 1C). A LIGHT-specific ELISA indicated release of soluble LIGHT in supernatants of PHA-activated PBLs but not in supernatants from any of 12 LIGHT+ melanoma cell lines (data not shown). To assess the cellular localization of LIGHT in melanoma cells, cell surface or intracellular staining (after permeabilization) with anti-LIGHT antibody was done followed by flow cytometry analysis. PBLs activated or not with PHA were used as positive or negative controls for LIGHT staining ( Fig. 2A ). In all melanoma cell lines investigated (n = 20), LIGHT, if expressed, was found mainly in the intracellular compartment, whereas cell surface expression was either negative or found only on a limited fraction of cells ( Fig. 2B for representative stainings). Similar results were obtained with two different anti-human LIGHT antibodies (data not shown). We next evaluated whether LIGHT was expressed on the surface of tumor-derived microvesicles known to be released by melanoma cells ( 25). LIGHT was expressed on most microvesicles isolated from LIGHT+ melanomas ( Fig. 2C for representative results on microvesicles isolated from a LIGHT+ melanoma). Melanoma-derived microvesicles had a size compatible with that of exosomes (50-100 nm) isolated from different cell types ( 23– 25, 29, 30) and expressed also several markers found previously in exosomes (CD63, CD107a, CD107b, and HLA-DR; Fig. 2C) and the melanocyte-lineage antigen gp100 found previously in melanoma-derived exosomes ( 23– 25, 29, 30).

Figure 1.
Figure 1.

Expression of LIGHT in melanoma cell lines. A, hierarchical cluster analysis of gene expression levels as determined by the Human Common Cytokine GEArray in 23 melanoma cell lines from primary (P) or metastatic (M) lesions. Up-regulated (red) or down-regulated (green) genes in comparison with the average expression found in a reference set of 25 melanoma cell lines. B, normalized expression levels of LIGHT in 10 melanoma cell lines (average of three determinations) compared with the expression levels in the reference set (STD). C, Western blot analysis for LIGHT in PBL activated with PHA for 72 hours (PBL+PHA) and in 9 melanoma cell lines.

Figure 2.
Figure 2.

LIGHT expression in melanoma cells and melanoma-derived microvesicles. A, PBLs were activated or not for 72 hours with PHA and then stained at the cell surface with anti-LIGHT antibody (white histograms) or with secondary FITC-labeled antibody only (gray histograms). B, expression of LIGHT in melanoma cells lines permeabilized (to detect intracellular LIGHT) or not (to detect cell surface expression of LIGHT). White histograms, staining with anti-LIGHT; gray histograms, staining with secondary FITC-labeled antibody only. C, phenotype of melanoma cell-derived microvesicles. Microvesicles were stained with antibodies to the indicated markers (white histograms) and with FITC- or PE-labeled irrelevant antibodies (gray histograms).

In vivo expression of LIGHT and of its receptors in metastatic melanoma lesions. Sections from 20 metastatic lesions from melanoma patients were characterized by immunohistochemistry by staining for LIGHT, LIGHT receptors (LTβR and HVEM), T-cell markers (CD3, CD4, and CD8), and T-cell maturation antigens (CD45RO, TIA-1, and granzyme B). As shown in Table 1 (and Supplementary Fig. S1A for representative LIGHT stainings), expression of LIGHT on variable proportion of neoplastic cells and with weak to strong intensity was found in the tumor cells of 18 of 20 lesions investigated. In the same metastases, LTβR was frequently found expressed in melanoma cells and even on the stromal tissue surrounding the neoplastic cells (see Supplementary Fig. S1B for representative sections showing either LTβR-negative or LTβR-positive stromal cells). By staining with anti-CD3 mAb, the lesions were classified according to extent of T-cell infiltrate of the neoplastic tissue by the “brisk/nonbrisk/absent” pattern ( 31). Interestingly, lesions with a “brisk” infiltrate of T cells in the neoplastic tissue showed a higher extent of expression of LTβR on the stromal cells (in terms of fraction of stromal cells stained with the anti-LTβR antibody) compared with lesions containing a “nonbrisk” or an “absent” pattern of T-cell infiltration (P < 0.001; Table 1). The 20 lesions were also stained with an antibody to the T-cell attractant CCL21 chemokine whose production has been shown recently to be promoted by LIGHT-dependent LTβR triggering on stromal cells present in the tumor environment ( 11). Staining for CCL21 was mainly found on stromal cells in lymph node areas surrounding the neoplastic tissue but only in the seven lesions with a brisk T-cell infiltrate (see Supplementary Fig. 1C, 12-15, for representative results from three patients in the brisk group). In contrast, all lesions with a nonbrisk or absent T-cell infiltrate ( Table 1, lesions from patients 8-20) either were completely negative (as in patient 11 in Supplementary Fig. S1C, 11) or showed CCL21 expression only on rare stromal cells (data not shown). The LIGHT receptor HVEM was often found on intratumoral lymphocytes ( Table 1), with the highest frequency of expression in lesions with a brisk lymphocytic infiltrate (P = 0.043 for the comparison of brisk versus nonbrisk groups). The pattern of expression of LIGHT, LTβR, and HVEM was confirmed by staining of a panel of 35 metastatic lesions (Supplementary Fig. S2A). Even in this extended panel, LIGHT was expressed on neoplastic cells of most lesions, whereas extent of LTβR on stromal component and of HVEM on infiltrating T cells was significantly higher in the subset of lesions with a brisk T-cell infiltrate (P < 0.001 and 0.01 for the brisk versus nonbrisk subset comparison, respectively; Supplementary Fig. S2A). Interestingly, only lesions with high expression of LIGHT on tumor cells and of HVEM on lymphocytes contained intratumoral proliferating T cells (as documented by expression of Ki-67 proliferation marker detected with MIB-1 mAb; Supplementary Fig. S2B and C). Similarly, scattered apoptotic intratumoral lymphocytes (as documented by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling assay) were found only in the lesions with high level of expression of LIGHT on neoplastic cells and of HVEM on infiltrating lymphocytes (see Supplementary Fig. S2B for representative data). The phenotype of intratumoral lymphocytes in the panel of lesions indicated presence of CD3+ T cells, in most instances with predominance of the CD8+ subset ( Table 1). These infiltrating T cells showed a profile consistent with differentiation toward the effector and memory stages as documented by expression of CD45RO and of TIA-1 (a marker of cytolytic granules) and by variable expression of the cytolytic factor granzyme B ( Table 1). In selected patients from this panel (those expressing HLA-A*0201) by ex vivo T-cell staining with HLA-A2 tetramers, we found that the lesions contained a high frequency of MDA-specific T cells directed to Melan-A/Mart-126-35 and/or gp100209-217 (Supplementary Fig. S3A). Furthermore, a large fraction of the tetramer+ T cells expressed a Teffector memory (CCR7−CD45RA−) or Tcentral memory (CCR7+CD45RA−) phenotype (Supplementary Fig. S3B). Taken together, these results indicate that expression of LIGHT is frequent in melanoma cells from metastatic lesions and suggest that the LIGHT-LTβR interaction may play a role in regulating T-cell infiltration of neoplastic tissue, in agreement with results obtained recently in a murine tumor model ( 11, 32). Moreover, the results suggest also that the LIGHT-HVEM interaction may play a role in proliferation/differentiation and apoptosis of T cells at tumor site.

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Table 1.

Expression of LIGHT and its receptors and phenotype of intratumoral lymphocytes in metastatic melanoma lesions

LIGHT expressed on melanoma cells or on melanoma-derived microvesicles costimulates CD3+CD8+ T-cell proliferation. Preliminary experiments were aimed at assessing the effect of HVEM engagement on CD3+CD8+ T-cell proliferation and/or differentiation induced by TCR triggering and by cytokines as IL-2. To this end, we evaluated proliferation and CCR7 phenotype of CD3+CD8+ T cells in response to immobilized anti-CD3 and IL-2 in the presence or absence of immobilized anti-HVEM mAb. HVEM engagement alone did not elicit any T-cell response (Supplementary Fig. S4, 4 versus 1) but costimulated T-cell proliferation (evaluated by CFSE staining) in both CCR7− and CCR7+ fractions in response to a suboptimal dose (1 ng/mL) of immobilized anti-CD3 mAb (Supplementary Fig. S4, 6 versus 3). In the presence of IL-2, engagement of HVEM promoted T-cell proliferation even in response to a 10-fold lower dose (0.1 ng/mL) of anti-CD3 mAb (Supplementary Fig. S4, 11 versus 8). At the highest dose of anti-CD3 (1 ng/mL) and in the presence of IL-2, HVEM engagement further promoted extensive T-cell proliferation in both CCR7+ and CCR7− subsets (Supplementary Fig. S4, 12 versus 9). The effect of HVEM engagement and IL-2 (in the presence of anti-CD3) was synergic as documented by T-cell recovery in such cultures at day 4 compared with cultures stimulated with either IL-2 plus anti-CD3 or anti-HVEM plus anti-CD3 (data not shown). The same CFSE/CCR7 assay was then used to evaluate CD3+CD8+ T-cell responses after lymphocyte coculture with allogeneic, HLA-mismatched LIGHT+/− tumors (to provide TCR engagement by allogeneic HLA molecules expressed on the tumor cells). CD8+ T cells from a healthy donor showed increased proliferation in response to a LIGHT+ tumor compared with a LIGHT− melanoma or to IL-2 alone (Supplementary Fig. S5). To assess the possible role of LIGHT in these responses, PBLs from a HLA-mismatched healthy donor were cocultured with an irradiated LIGHT+ melanoma (showing cell surface LIGHT expression on ∼30% of the cells) in the presence of IL-2. The response of CD3+CD8+ T cells was not inhibited by preincubation of tumor cells with anti-LIGHT antibody alone ( Fig. 3 , 2 versus 1). Similarly, no effect on T-cell response was seen by tumor preincubation with a suboptimal dose of anti-HLA class I mAb used to reduce, without preventing, the extent of TCR engagement by allogeneic HLA molecules on melanoma cells ( Fig. 3, 3 versus 1). However, when tumor cells had been preincubated with both antibodies, the proliferative response of CD3+CD8+ T cells to tumor cells was markedly inhibited in the CCR7+ and CCR7− fractions ( Fig. 3, 4 versus 1). No inhibition of proliferation was observed in the presence of these antibodies on the CD3−CD8+ fraction present in the same cultures ( Fig. 3, 5-8). These results suggest that LIGHT expressed on melanoma cells is functional and may contribute to costimulate CD3+CD8+ T-cell expansion on TCR engagement by HLA class I molecules on melanoma cells.

Figure 3.
Figure 3.

Role of LIGHT expressed on melanoma cells in the proliferative response of CD3+CD8+ T cells. CFSE-stained PBLs from a healthy donor were cocultured for 6 days in the presence of IL-2 (250 IU/mL) with irradiated HLA-mismatched melanoma cells (showing cell surface LIGHT expression on ∼30% of the cells). Melanoma cells were preincubated or not with suboptimal dose (50 ng/mL) of anti-HLA class I (w6/32), with neutralizing anti-LIGHT antibody, or with both antibodies. Results show CCR7 phenotype versus CFSE fluorescence of gated CD3+CD8+ T cells (top) or CD3−CD8+ cells (bottom). Top, the two vertical lines distinguish three CFSE fluorescence sections. Numbers in dot plots, proportion of cells in each section/quadrant.

Because LIGHT was expressed on the microvesicles derived from LIGHT+ melanomas, we then evaluated whether such molecule could play a role on the outcome of T-cell coculture with such microvesicles. To this end, T cells from a healthy donor were cultured in the presence of two doses of microvesicles derived from a HLA-mismatched, LIGHT+ melanoma. T-cell coculture with melanoma-derived microvesicles led to enhanced and dose-dependent proliferation mostly in the CCR7− fraction of CD3+CD8+ T cells compared with the response induced by IL-2 alone ( Fig. 4 , 7 and 4 versus 2). Anti-LIGHT antibody did not affect T-cell response to IL-2 alone ( Fig. 4, 3 versus 2). However, preincubation of microvesicles with anti-LIGHT antibody was effective in inhibiting in a dose-dependent fashion the enhanced proliferative response of the CCR7−CD8+ T-cell fraction to microvesicles ( Fig. 4, 5 and 6 versus 4; 8 versus 7). Control experiments indicated that the costimulatory role of microvesicles on T-cell proliferation was inhibited by anti-LIGHT antibody but not by antibodies directed to different molecules (HLA-DR or gp100) expressed by these microvesicles (see Supplementary Fig. S6 for representative results with anti-gp100 antibody). Taken together, these results suggest that LIGHT on microvesicles has a role in the enhanced proliferative response compared with IL-2 only, induced by coculture of CD3+CD8+ T cells with these microvesicles.

Figure 4.
Figure 4.

LIGHT expressed on melanoma-derived microvesicles costimulates CD3+CD8+ T-cell proliferation. CFSE-stained PBLs from a healthy donor were cultured for 4 days in the presence of IL-2 (250 IU/mL) with microvesicles (total protein concentration, 1.5 mg/mL) at 100 and 50 μL/mL (indicated as microvesicles 1:2) isolated from a HLA-mismatched LIGHT+ melanoma. Microvesicles were preincubated or not with neutralizing anti-LIGHT antibody at two doses (50 or 100 ng/mL). Results show CCR7 phenotype versus CFSE fluorescence of gated CD3+CD8+ T cells. Numbers in dot plots, proportion of cells in each of the four quadrants.

Coculture of CD3+CD8+ T cells with melanoma-derived microvesicles is associated with a LIGHT-dependent apoptotic response. Triggering of TCR and/or culture with IL-2 can lead not only to T-cell proliferation but also to promotion of T-cell apoptosis ( 33). To assess whether HVEM engagement could have a role in costimulation of T-cell apoptosis, we tested the response of CD8+ T cells in the presence of immobilized anti-CD3 mAb ± IL-2 to increasing concentrations of immobilized anti-HVEM mAb. Anti-HVEM used alone at a concentration between 0.2 and 0.8 μg/mL did not elicit any apoptotic response as evaluated by Annexin V binding by CD8+ T cells ( Fig. 5A ). Similarly, no increase in binding of Annexin V could be observed in CD8+ T cells at any anti-HVEM doses in the presence of anti-CD3 but in the absence of IL-2. However, an increasing proportion of CD8+ T cells became Annexin V+ ( Fig. 5A) in the presence of anti-CD3 and IL-2 when immobilized anti-HVEM was used at concentrations (0.4 and 0.8 μg/mL; Fig. 5A) higher than that (0.2 μg/mL) shown previously (Supplementary Fig. S4) to be effective in costimulating T-cell proliferation. These results (together with those shown in Supplementary Fig. S4) indicated that triggering of the LIGHT receptor on T cells can costimulate the proliferative and/or the apoptotic responses, but these two outcomes seem to depend on the extent of HVEM engagement.

Figure 5.
Figure 5.

Role of LIGHT in the apoptotic response of CD3+CD8+ T cells after lymphocyte coculture with melanoma-derived microvesicles. A, PBLs from a healthy donor were cultured for 4 days in the presence of immobilized anti-CD3 mAb (1 ng/mL), IL-2 (250 IU/mL), and increasing doses of immobilized anti-HVEM mAb and then analyzed for Annexin V binding after gating on CD3+CD8+ cells. B, CFSE-stained PBLs from a healthy donor were cultured for 4 days in the presence of IL-2 (250 IU/mL) and with two doses of melanoma-derived microvesicles (as in Fig. 4) or at the highest dose of microvesicles preincubated with neutralizing anti-LIGHT antibody (50 ng/mL) and then analyzed for CFSE fluorescence versus Annexin V-PE binding after gating on CD3+CD8+ T cells. Numbers in dot plots, proportion of cells in each of the four quadrants.

Then, we assessed whether coculture of T cells from healthy donors with melanoma-derived microvesicles in the presence of IL-2 could promote an apoptotic response, and not only proliferation, in a LIGHT-dependent fashion. To this end, we assessed at the same time the extent of T-cell proliferation (by CFSE staining) and the binding of Annexin V-PE by T cells. As shown in Fig. 5B, lymphocyte coculture with two doses of melanoma-derived microvesicles led to enhanced CD8+ T-cell proliferation compared with IL-2 only ( Fig. 5B, upper left quadrants of 2 and 3 versus 1). Moreover, most of the proliferating CD8+ T cells, in response to melanoma-derived microvesicles, became Annexin V+ ( Fig. 5B, 2 and 3 versus 1). However, the proportion of Annexin V+ proliferating T cells was markedly reduced by preincubation of microvesicles with anti-LIGHT antibody ( Fig. 5B, 4 versus 2). The promotion of CD8+ T-cell apoptosis was not observed after coculture of lymphocytes from healthy donors with LIGHT+ melanomas in the presence of IL-2 (data not shown). Taken together, these results suggest that LIGHT expressed on melanoma-derived microvesicles has a role in the apoptotic response of CD3+CD8+ T cells.

Discussion

The results of this study indicated that human melanoma cells can express the costimulatory molecule LIGHT and suggested that this TNF family member through binding to its receptors may play a role in regulating several aspects of the host-tumor interaction. Remarkably, the extent of expression of the LTβR on the stromal cells of invaded nodes correlated with the presence of a brisk infiltrate of CD3+CD8+ T cells in the neoplastic tissue. Furthermore, in lesions with a brisk T-cell infiltrate, stromal cells in lymph node areas surrounding the neoplastic tissue also stained for the T-cell attractant chemokine CCL21, which can be produced on LTβR triggering by LIGHT ( 11). This suggested a role of the LIGHT/LTβR interaction in promoting chemokine-dependent T-cell recruitment in the tumor tissue. The LTβR plays a central role in organization of lymphoid tissues during organogenesis and in the adult ( 34– 36) and regulates expression of homing chemokines that contribute to the compartmentalization of lymphoid tissues and to T-cell and B-cell migration into such tissues ( 37). In fact, triggering of LTβR expressed on radioresistant stromal cells of lymphoid tissues ( 36, 37) leads to production of chemokines as CXCL13, which attracts B cells, and CCL21, which attracts naive CCR7+ T cells. In addition, LTβR engagement can induce expression of adhesion molecules as MAdCAM-1, which contribute to T-cell homing to secondary lymphoid tissues ( 11). Furthermore, a recent study has shown that forced expression of LIGHT in murine fibrosarcoma cells triggers LTβR on stromal component. This in turn results in the production of chemokines, including CCL21, which promote T-cell infiltration of neoplastic tissues ( 11). Our results suggest that a similar pathway could be constitutively active in melanoma metastases. Interestingly, constitutive expression of LTβR in lymphoid tissues is thought to be required for maintenance of normal architecture and organ function ( 38, 39). Thus, the reduced or absent expression of this receptor in the stromal cells of several melanoma metastases remains to be clarified. One possibility is that LTβR expression is linked to the process of tumor stromagenesis ( 40). Alternatively, expression of LTβR on stromal cells at tumor site may be actively suppressed. This in turn could reduce production of chemokines that promote T-cell infiltration of neoplastic tissue, thus providing an efficient tumor escape mechanism from immune recognition. In agreement with the latter hypothesis, recent results have indicated that tumor-invaded lymph nodes from melanoma patients contain reduced mRNA levels for CCL21 compared with tumor-free nodes ( 41).

The phenotype of intratumoral lymphocytes and the high frequency of differentiated (Teffector memory or Tcentral memory) MDA-specific T cells were consistent with T-cell activation at tumor site. Such response could be promoted/amplified by expression of costimulatory molecules, as LIGHT, on melanoma cells. Direct antigen presentation at tumor site by neoplastic cells expressing costimulatory molecules would even contribute to explain the immunogenicity of self-epitopes as Melan-A/Mart-126-35. In fact, current models predict a tolerogenic role for professional APCs by cross-presentation of self-antigens ( 4). However, self-epitopes as Melan-A/Mart-126-35 are poorly generated by the immunoproteasome of mature dendritic cells ( 7) and may not induce tolerance ( 42). Therefore, such self-epitopes may become immunogenic when directly presented by melanoma cells expressing costimulatory molecules. By contrast, other self-determinants may still require breaking of tolerance for achieving T-cell priming at tumor site even in the presence of costimulation. Interestingly, only the T-cell frequency to Melan-A/Mart-126-35 and not to other MDA epitopes (such as gp100 and tyrosinase) was found recently to be significantly higher in tumor-invaded lymph nodes compared with peripheral blood in a large panel of HLA-A*0201+ metastatic melanoma patients ( 5).

In previous studies aimed at assessing the costimulatory role of LIGHT, LIGHT+ cells or immobilized recombinant LIGHT were used to show that T-cell proliferation after TCR ligation can be enhanced by HVEM engagement ( 10, 17). In agreement with these findings, we found that triggering of the LIGHT receptor HVEM on T cells by immobilized anti-HVEM mAb could costimulate proliferation in response to anti-CD3 mAb ± IL-2. By allogeneic lymphocyte-tumor coculture experiments, we found that the proliferative response of CD3+CD8+ T cells could be inhibited by neutralizing antibody to LIGHT in the presence of suboptimal doses of anti-HLA class I antibody. This indicated that LIGHT on tumor cells could contribute to amplify the response elicited by TCR engagement. Similarly, we found that LIGHT+ melanoma-derived microvesicles could enhance CD3+CD8+ T-cell proliferation compared with the response of T cells kept in IL-2 alone, and the response could be inhibited by anti-LIGHT antibody. In addition, T-cell coculture with melanoma-derived microvesicles provided evidence for a role of LIGHT in costimulating the apoptotic response of CD3+CD8+ T cells. Such effect was not observed in mixed lymphocyte-tumor cultures, and based on experiments with immobilized anti-HVEM, it required a level of HVEM engagement higher than needed for costimulating T-cell proliferation. These results suggest that interaction of T cells with LIGHT+ melanoma-derived microvesicles may lead to quite distinct outcomes, such as promotion of T-cell activation versus elimination of antitumor T cells.

The mechanism of LIGHT-mediated induction of apoptosis in CD3+CD8+ T cells remains to be elucidated. On one hand, existing data point to a role of LIGHT in promoting apoptosis of normal or neoplastic cells by a LTβR-dependent mechanism rather than by HVEM engagement ( 43). In these instances, LTβR engagement has been shown to result in TRAF3 recruitment ( 44) and in down-regulation of antiapoptotic Bcl-2 family members ( 45). LIGHT can even costimulate apoptosis of thymocytes bearing TCRs with high affinity for MHC/self-peptides ( 46), although this takes place by an indirect mechanism requiring expression of LTβR not on thymocytes but on thymic APCs ( 46). On the other hand, recent results have indicated that HVEM engagement in B lymphoma cells can up-regulate expression of CD95, thus increasing lymphoma cell susceptibility to Fas-dependent apoptosis ( 47). However, experiments of T-cell response to immobilized HVEM indicated that engagement of this LIGHT receptor on CD3+CD8+ T cells did not costimulate up-regulation of CD95 induced in response to immobilized anti-CD3 and IL-2 (data not shown). Thus, although melanoma-derived microvesicles may express functional CD95-L ( 25, 48), it is possible that the contribution of LIGHT on melanoma microvesicles to T-cell apoptosis may be due to a signaling role of HVEM independent from the extent of expression of CD95 on T cells.

In conclusion, the results of this study suggest that constitutive expression of a costimulatory molecule as LIGHT in human melanoma might contribute to both positive and negative regulation of T-cell-mediated immunity to the tumor.

Acknowledgments

Grant support: Italian Association for Cancer Research (Milan, Italy), Ministry of Health (Rome, Italy), Compagnia di S. Paolo (Turin), Fondazione Hoepli (Milan, Italy), and Ministero dell'Università dell'Istruzione e della Ricerca (Rome, Italy) Fondo per gli Investimenti della Ricerca di Base grant RBNE017B4C.

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.

Footnotes

  • Received September 10, 2004.
  • Revision received December 29, 2004.
  • Accepted February 1, 2005.

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Cancer Research: 65 (8)
April 2005
Volume 65, Issue 8

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Constitutive Expression and Costimulatory Function of LIGHT/TNFSF14 on Human Melanoma Cells and Melanoma-Derived Microvesicles
Roberta Mortarini, Alessia Scarito, Daisuke Nonaka, Marina Zanon, Ilaria Bersani, Elisabetta Montaldi, Elisabetta Pennacchioli, Roberto Patuzzo, Mario Santinami and Andrea Anichini
Cancer Res April 15 2005 (65) (8) 3428-3436; DOI: 10.1158/0008-5472.CAN-04-3239
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