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Immunology |
1 Unit of Immunotherapy of Human Tumors, Istituto Nazionale Tumori; 2 Fondazione D'Amico, Milan, Italy; 3 Microscopy and Image Analysis Consortium; 4 Dipartimento di Neuroscienze e Tecnologie Biomediche, Università Milano-Bicocca, Monza, Italy; and 5 Department of Drug Research and Evaluation, Section of Pharmacogenetic, Drug Resistance and Experimental Therapeutics, Istituto Superiore di Sanità, Rome, Italy
Requests for reprints: Licia Rivoltini, Unit of Immunotherapy of Human Tumors, Istituto Nazionale Tumori, Via Venezian 1, 20133 Milan, Italy. Phone: 39-2-2390-3245; Fax: 39-2-2390-2154; E-mail: rivoltini{at}istitutotumori.mi.it.
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, and transforming growth factor-ß (TGF-ß), and a dose-dependent suppressive activity on activated T-cell–proliferation and cytolytic functions, which could be reversed by anti-TGF-ß–neutralizing antibodies. Microvesicles isolated from plasma of advanced melanoma patients, but not from healthy donors, mediated comparable effects on CD14+ monocytes, skewing their differentiation toward CD14+HLA-DR–/low cells with TGF-ß–mediated suppressive activity on T-cell–functions. Interestingly, a subset of TGF-ß–secreting CD14+HLA-DR– cells mediating suppressive activity on T lymphocytes was found to be significantly expanded in peripheral blood of melanoma patients compared with healthy donors. These data suggest the development in cancer patients of an immunosuppressive circuit by which tumors promote the generation of suppressive myeloid cells through the release of circulating microvesicles and without the need for cell-to-cell contact. Therapeutic interventions on the crucial steps of this pathway may contribute to restore tumor/immune system interactions favoring T-cell–mediated control of tumor growth in cancer patients. (Cancer Res 2006; 66(18): 9290-8) |
Introduction |
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Although the extracellular release of membrane vesicles has been characterized under specific physiologic conditions in different normal cell types (including blood, endothelial, and epithelial cells), the rate of shedding seems to be constitutive and markedly increased in most neoplastic cells (2, 7), thus suggesting a role of this phenomenon in malignant transformation and/or progression (8).
Growing interest has been focused on tumor-released microvesicles since the profusion of these phospholipid particles of 50 to 200 nm size in body fluids of cancer patients with advanced disease was shown (2, 3, 9, 10). In particular, due to microvesicle ability to transport a wide array of biologically active molecules involved in plasma membrane discharge, cell signaling, receptor transfer, and apoptosis, microvesicle shedding could be exploited by neoplastic cells not only to maintain their own growth and survival at the tumor site but also to deliver detrimental signals to the host's immune defenses independently from cell-to-cell contact.
We and others have shown that human cancer cells (including melanoma, colon carcinoma, head and neck, and ovary cancers) produce large amounts of microvesicles bearing proapoptotic molecules, such as Fas ligand (FasL) and tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL; refs. 1–3, 10). These membranous organelles, purified from tumor cell culture supernatants or from cancer patient sera, can induce apoptosis of activated tumor-specific T cells, thus impairing the ability of effector lymphocytes to exert their cytolytic activity on tumor targets. Cancer cells could hence use proapoptotic microvesicles to promote the establishment in the host of an immunoprivileged environment, as it occurs in different physiologic conditions requiring maintenance of immunotolerance, such as pregnancy, or during the downsizing of immune responses after antigen clearance (11–13). However, being T lymphocytes variably susceptible to apoptosis depending on their activation state (2), it would be more advantageous for cancer cells to interfere upstream with the development of antitumor immune responses through the direct impairment of T-cell–priming by dendritic cells. In fact, this cell population plays a key role in the modulation of adaptive immunity, switching on and off T-cell–responses depending on their functional state (14). Patients with different chronic diseases, including cancer, have been reported to display defects in dendritic cell activity (15) together with more generalized dysfunctions in the homeostasis of the myeloid compartment, leading to the in vivo accumulation of cells with suppressive effects on T lymphocytes (the so-called "myeloid suppressor cells"; ref. 16).
Based on these evidences, we chose to investigate the effects of microvesicles released by human cancer cells on the process of monocyte differentiation into dendritic cells. Here, we report that microvesicles produced by human melanoma and colorectal carcinoma cells, both in vitro and in vivo, skew monocyte differentiation toward myeloid cells with altered phenotypic features and functions. These cells, which are expanded in peripheral blood of melanoma patients, exert suppressive activity on T cells mostly mediated by the microvesicle-induced release of transforming growth factor-ß (TGF-ß).
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This study was approved by the Ethics Committee of the Istituto Nazionale Tumori of Milan and informed consent was obtained from each patient and healthy volunteer.
Monocyte isolation and differentiation into dendritic cells. Monocytes were immunosorted from healthy donor PBMC using human CD14 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). The resulting cell population was represented by >95% CD14+ monocytes as assessed by flow cytometry. Immature dendritic cells were obtained by in vitro culture of CD14+ cells (106 per well in 48-well plates) for 3 to 6 days in X-VIVO 15 (BioWhittaker, Walkersville, MD) supplemented with 50 ng/mL granulocyte macrophage colony-stimulating factor (GM-CSF; Peprotech, Rocky Hill, NJ) and 20 ng/mL interleukin (IL)-4 (Peprotech) in the presence or absence of amounts of microvesicles corresponding to 
30 µg protein/106 target cells. Control experiments were done with 30 µg bovine serum albumin (BSA; Sigma-Aldrich Co., St. Louis, MO) or 0.02 µm latex beads (aldehyde/sulfate latex, Interfacial Dynamics Corp., Portland, OR) in a 1:8 monocyte/bead ratio. To purify CD14+HLA-DR+ and CD14+HLA-DR– cells from the PBMC of melanoma patients, monocytes were first isolated by negative selection with Monocyte Negative Isolation kit (Dynal Biotech ASA, Oslo, Norway) and then positively sorted for HLA-DR expression on incubation with 5 µg/106 cells phycoerythrin (PE)–conjugated anti-human HLA-DR monoclonal antibody (mAb; BD PharMingen, San Jose, CA) and anti-PE microbeads (Miltenyi Biotec) according to the manufacturer's instruction. The purity of the immunosorted CD14+HLA-DR+ and CD14+HLA-DR– cell populations was assessed by flow cytometry.
Tumor cell lines and microvesicle purification. The melanoma cell lines 501mel and 624.38mel were described previously (18, 19). The colorectal carcinoma cell line SW403 was purchased from the American Type Culture Collection (Manassas, VA), whereas the 1869col colorectal carcinoma line was kindly provided by Dr. Cristina Maccalli (Istituto Superiore di Sanità, Rome, Italy; ref. 20). Tumor lines were negative for Mycoplasma contamination as routinely tested by enzymatic assay (MycoAlert Mycoplasma Detection kit, Cambrex, Vervier, Belgium). Microvesicles were isolated by serial centrifugations (2) from supernatants of 72-hour confluent tumor cells cultured with 10% fetal bovine serum (FBS; Cambrex) deprived previously of bovine microvesicles by ultracentrifugation (90 minutes at 100,000 x g). The same protocol was used to purify microvesicles released by healthy donor macrophages (differentiated in vitro from 107 CD14+ monocytes cultured for 1 week in RPMI 1640 supplemented with 30% FBS). Microvesicle purification from healthy donors (n = 10) and melanoma patients (n = 10) was obtained after 1:2 plasma dilution in PBS by sequential centrifugations (30 minutes at 2,000 x g, 45 minutes at 12,000 x g, and 2 hours at 110,000 x g). Microvesicle pellets were resuspended in PBS, filtered through a 0.22 µm filter (Stericup, Millipore Corp., Billerica, MA), and further centrifuged for 1 hour at 110,000 x g. After a final 1-hour wash at 100,000 x g, microvesicle pellets were recovered and resuspended in appropriate medium and proteins were quantified by Bradford assay (Bio-Rad Laboratories, München, Germany).
Analysis of apoptosis and phagocytic activity. Immunosorted CD14+ cells and human Jurkat T cells (positive control) were stained with Annexin V/propidium iodide (PI; Bender MedSystems, Vienna, Austria) after a 24-hour incubation (106/mL) at 37°C with melanoma-derived and colorectal carcinoma–derived microvesicles (30 µg) or bioactive concentrations of recombinant FasL (SuperFasLigand, Alexis, Lausen, Switzerland) and TRAIL (R&D Systems, Minneapolis, MN) proteins. The percentage of apoptotic cells was then analyzed by FACSCalibur and the CellQuest software (Becton Dickinson, San Jose, CA). Phagocytic activity of monocyte-derived dendritic cells differentiated for 6 days in the presence or absence of melanoma-derived and colorectal carcinoma–derived microvesicles was assessed in terms of FITC-dextran internalization (1 mg/mL; Sigma-Aldrich) at 37°C or 4°C as specificity control. After a 30-minute incubation, cells were analyzed for green fluorescence emission using FACSCalibur and the CellQuest software.
Analysis of monocyte-microvesicle interaction. CD14+ monocytes and melanoma-derived or colorectal carcinoma–derived microvesicles were stained with the membrane PKH26green and PKH26red fluorescent dyes, respectively (Sigma-Aldrich) and then coincubated at 37°C with microvesicles (30 µg protein/106 monocytes). Samples collected at different time points were analyzed by confocal microscopy in terms of transfer of the microvesicle-derived red fluorescence to the green fluorescent monocytes. Control experiments were done at 4°C on pretreatment of monocytes with 1 µg/mL cytochalasin D (Sigma-Aldrich) or in the presence of the Ca2+-chelating agent EDTA (5 mmol/L). Images were obtained using a Radiance 2100 laser scanning confocal microscope (Bio-Rad Laboratories, Hercules, CA) equipped with a krypton/argon laser. Noise reduction was achieved by "Kalman filtering" during acquisition.
Flow cytometry and confocal microscopy. Differentiating monocytes were stained with the following mouse anti-human IgG mAbs (BD PharMingen): PE anti-CD14, PE anti-HLA-DR, PE anti-CD80, PE anti-CD86, FITC anti-CD40, FITC anti-HLA-DR, and APC-anti-CD14. In each experiment, IgG-matched isotypic controls were used. Events were gated according to light scatter properties, selecting monocyte cell population and excluding cell debris. To standardize the mean channel number values, instrument detector setup for FL-1 channel was set so that the FITC-Calibrite (Becton Dickinson) bead pick gave 560 to 600 linear units in log-scale amplification. Data were acquired and analyzed by a FACSCalibur and the CellQuest software. The same samples were analyzed for surface and intracellular HLA-DR expression by confocal microscopy after culture in Permanox four-well chamber slides (Nunc, Rochester, NY), fixation in 4% buffered paraformaldehyde, and overnight incubation at 4°C with primary mouse anti-human anti-HLA-DR mAb (L243) in a 30% goat serum, 0.3% Triton X-100, 0.45 mol/L NaCl, and 0.02 mol/L phosphate buffer. After suitable washings, slides were incubated for 1 hour at room temperature with a secondary rhodamine-conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch Europe Ltd., Soham, United Kingdom). Images were obtained using a laser scanning confocal microscope (Radiance 2100) equipped with a krypton/argon laser. Noise reduction was achieved by "Kalman filtering" during acquisition.
Evaluation of suppressive activity of microvesicle-treated monocytes and CD14+HLA-DR– cell subset on lymphocytes. Cytokine-treated monocytes differentiated in the presence or absence of microvesicles of different origin were irradiated and added to 105 allogeneic PBMC at different responder/stimulator ratios in round-bottomed 96-well plates (Costar-Corning, New York, NY) in X-VIVO 15. Suppressive activity was analyzed on autologous PBMC proliferation in response to 1 µg/mL OKT3 (anti-CD3 Orthoclone OKT3 agonist antibody, Ortho Biotech, Bridgewater, NJ) coated previously for 2 hours at 37°C in 96-well plates. For blocking experiments, anti-IL-6 (1 µg/mL; Abcam, Cambridge, United Kingdom), anti-TNF- (2 µg/mL; Abcam), or anti-TGF-ß–neutralizing antibody (1 µg/mL; R&D Systems) was added at the beginning of cocultures. The suppressive activity of CD14+HLA-DR– cells isolated from melanoma patients (20 x 103 per well) was evaluated on CD14+-depleted PBMC (105 per well) stimulated for 3 days with 50 ng/mL phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) and 1 µg/mL phytohemagglutinin (PHA; Sigma-Aldrich). T-cell–proliferation was assessed on day 4 by measuring [3H]thymidine incorporation (1 µCi/well; Amersham Biosciences) after 18-hour pulse using a liquid scintillation ß-counter. Each assay was done in quadruplicates.
Cytokine quantification by cytokine bead array and ELISA. Supernatants from monocytes (as bulk or sorted subsets) were collected after 6-hour incubation at 37°C. Samples were tested for the presence of TNF-, IL1ß, IL-6, IL-10 and IL-12 by multiple cytokine analysis with cytokine bead array (CBA; human inflammation kit, Becton Dickinson), and, on acidification, for TGF-ß1 by ELISA (Amersham Biosciences).
Analysis of cytolytic molecules and IFN-
intracellular expression by in vitro activated T cells. PBMC were cocultured for 24 hours at 37°C with autologous monocytes differentiated in the presence or absence of melanoma-derived and colorectal carcinoma–derived microvesicles (4:1, PBMC/monocyte ratio) in round-bottomed 96-well plates coated previously with anti-CD3 OKT3 agonist antibody. Blocking experiments were done using 1 µg/mL anti-TGF-ß1-neutralizing antibody. Nonadherent cells were then collected and surface stained with PerCP-conjugated anti-CD8 antibody (BD PharMingen). Cells were then fixed and permeabilized with Cytofix/Cytoperm kit (BD PharMingen), stained with FITC anti–granzyme B, PE anti-perforin, and APC anti-IFN- antibody (BD PharMingen), and evaluated by fluorescence-activated cell sorting (FACS) analysis.
Microvesicle scanning electron microscopy and Western blot analysis. Microvesicles isolated from plasma of melanoma patients were resuspended in ethanol 100%, laid on cover glasses, and then sputter coated with gold by Edwards S150A (BOC Edwards, Crawley, United Kingdom) for scanning electron microscopy. Samples were examined by Philips XL-40 scanning electron microscope (FEI, Eindhoven, the Netherlands) with a cold cathode field emission gun at an accelerating voltage of 30 kV. All images were recorded using the secondary electron detector. For Western blot analysis, equal amounts of proteins obtained from the plasma-derived microvesicle lysates of healthy donors or stage IV melanoma patients were separated by gel electrophoresis and blotted onto a polyvinylidene difluoride membrane (Amersham Biosciences). Membranes were incubated with mAb specific for gp100 (DAKOCytomation, Glostrup, Denmark) and LAMP-2 (BD PharMingen) molecules. Antibody binding was detected by enhanced chemiluminescence (SuperSignal, Pierce, Rockford, IL).
Statistical analysis. Statistical analysis was done by t test for paired samples, with exception of data reported in Fig. 6A, which were analyzed by Dunnett's test. P < 0.05 was considered as statistically significant.
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CD14+ monocytes were then treated with IL-4 and GM-CSF in the presence or absence of melanoma-derived and colorectal carcinoma–derived microvesicles, and their differentiation into immature dendritic cells was monitored as phagocytic ability and immunophenotypical modifications. The presence of tumor-released microvesicles impaired the ability of differentiating monocytes to acquire phagocytic activity as expected in immature dendritic cells. In fact, whereas the majority of cells were able to internalize FITC-dextran in microvesicle-untreated samples, monocytes differentiated in the presence of either melanoma-derived or colorectal carcinoma–derived microvesicles were unable to uptake this substrate (Fig. 1C). In addition, in microvesicle-treated monocytes, the down-regulation of CD14 surface expression, which physiologically occurs when differentiation into immature dendritic cells is complete, was inhibited in a consistent percentage of cells (Fig. 2A, left ). This seemed to be a persisting effect, as it could be detected even 6 days after microvesicle treatment (Fig. 2A, middle). The dependence of this effect on the tumor origin of microvesicles was shown by the evidence that CD14+ cells treated under the same conditions with microvesicles purified from supernatants of human macrophages underwent physiologic CD14 down-modulation (Fig. 2A, right). Further control experiments, in which CD14+ cells were treated with comparable amounts of BSA or with latex beads, showed a normal loss of CD14 expression (data not shown), thus excluding that the inhibitory effect of tumor-microvesicle on dendritic cell differentiation could be due to endocytic machinery engulfment on microvesicle treatment.
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Together with the down-modulation of HLA-DR molecules, microvesicle-treated monocytes displayed a significant decrease in the expression of costimulatory (CD80 and CD86) and activatory (CD40) molecules (Fig. 2D).
Monocytes differentiated in the presence of tumor-released microvesicles exert TGF-ß–mediated suppressive activity on T-cell–function. Based on the phenotypic alterations mentioned above, we evaluated whether monocytes differentiated in the presence of tumor-derived microvesicles exerted a reduced stimulatory capacity on allogeneic HLA-mismatched PBMC proliferation. Indeed, monocytes differentiated in the presence of melanoma or colorectal carcinoma–released microvesicles exerted no stimulatory effects on allogeneic PBMC proliferation compared with untreated monocytes (Fig. 3A ). In addition, microvesicle-treated monocytes acquired a strong and dose-dependent suppressive activity on autologous T-cell–proliferation in response to T-cell–receptor (TCR) triggering, which was instead potentiated by the presence of microvesicle-untreated cells (Fig. 3B).
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, IL-6, and TGF-ß secretion was detected in microvesicle-treated monocytes compared with untreated cells (Fig. 3C).
The potential role of these cytokines in the suppressive activity of microvesicle-treated monocytes was investigated through the use of neutralizing mAbs. Interestingly, lymphocyte proliferation on TCR triggering was completely restored by TGF-ß neutralization, whereas no boost was induced targeting TNF- and IL-6 (Fig. 4A
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cytolytic molecules by CD8+ T cells on TCR stimulation. Microvesicle-treated monocytes impaired the cytolytic potential of activated CD8+ lymphocytes by significantly reducing the intracellular accumulation of granzyme B, perforin, and IFN- compared with CD8+ T cells cultured with untreated monocytes (Fig. 4B). This effect was mainly mediated by TGF-ß released by microvesicle-treated monocytes, as the expression of these molecules was completely restored by the anti-TGF-ß–neutralizing antibody (Fig. 4B). Microvesicle-treated monocytes exerted their suppressive activity also on the cytotoxic function of tumor-specific T cells, impairing MelanA/MART-1-specific T-cell–ability to mobilize CD107a molecules on degranulation in response to MART-1+ melanoma cells (22) in a TGF-ß–dependent manner (data not shown). No up-regulated arginase activity, a hallmark of myeloid suppressor cells (MSC) in tumor-bearing mice (23, 24), was detected in microvesicle-treated monocytes compared with untreated cells, and no overcome of their suppressive activity on T-cell–proliferation was observed in the presence of arginase inhibitors (see Supplementary Data).
Microvesicles isolated from plasma of melanoma patients skew monocyte differentiation toward TGF-ß–secreting CD14+HLA-DR–/low myeloid cells with suppressive activity. In line with previous studies showing the presence of exosome-like microvesicles in different biological fluids from tumor patients (2, 3, 9, 10) and from healthy donors (25), we isolated microvesicles from plasma of stage IV melanoma patients, where scanning electron microscopy evidenced purified vesicular structures dimensionally compatible with exosomes (diameter, 
200 nm; Fig. 5A
). Microvesicle samples from both melanoma patients and healthy donors expressed LAMP-2, a lysosomal-associated membrane protein usually found in microvesicles and exosomes (25), as shown by Western blotting (Fig. 5B). Additionally, microvesicles from melanoma patients, but not from healthy volunteers, expressed the melanoma antigen gp100, thus confirming the presence of tumor-derived microvesicles in the blood of cancer patients (Fig. 5B).
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Consistent with monocytes differentiated using tumor cell-derived microvesicles, CD14+ cells treated with microvesicles from melanoma patients significantly impaired T-cell–proliferation in a TGF-ß–dependent manner (Fig. 5D). On the contrary, a pronounced stimulatory effect was observed with monocytes treated with healthy donor microvesicles, most likely attributable to their origin from immune cells, such as dendritic cells and B lymphocytes (26).
TGF-ß–secreting CD14+HLA-DR– cells with immunosuppressive activity are expanded in peripheral blood of melanoma patients. To evaluate the in vivo relevance of our findings, we searched for CD14+HLA-DR– cells in PBMC from stage IV melanoma patients (n = 16) and healthy donors (n = 10). A statistically significant (P < 0.005) expansion of this cell subset was detected in patient PBMC (8.4 ± 8.2%) compared with healthy volunteers (0.8 ± 1.0%; Fig. 6A ). Thus, CD14+HLA-DR– cells were immunosorted from patient PBMC (Fig. 6B) and evaluated for spontaneous release of TGF-ß and suppressive activity. CD14+HLA-DR– cells spontaneously secreted TGF-ß ex vivo, although this cytokine was barely detectable in supernatants from the CD14+HLA-DR+ cell subset (Fig. 6C). Moreover, the addition of CD14+HLA-DR– cells to CD14+-depleted PBMC resulted in a significant inhibition of lymphocyte proliferation in response to mitogenic stimuli (Fig. 6D), hence confirming the immunosuppressive potential of this cell subset in melanoma patients.
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release. This effect was consistent with the rapid production by microvesicle-treated monocytes of bioactive TGF-ß, in turn responsible for the suppressive effects on T cells. Comparable phenotypic and functional effects on monocyte differentiation were achieved with microvesicles purified from the plasma of melanoma patients but not from healthy donors. In addition, TGF-ß–secreting CD14+HLA-DR– cells with suppressive activity on autologous T-cell–proliferation were significantly expanded in the same set of patients but barely detectable in healthy volunteers. Hence, these data suggest that the release of vesicular structures may represent a novel pathway enabling tumor cells to modulate host environment for surviving and progressing in vivo. The results here described contribute to highlight the intricate tumor-host interactions, which, in spite of the expression of tumor-associated antigens and the existence of tumor-specific T cells, may render immune responses ineffective in tumor growth control even after stimulation with cancer vaccines. Indeed, although evidences support a potential role of the immune system in containing tumor development (and thus promoting a more favorable prognosis) in the initial phases of neoplastic disease (27), it is undoubted that, thanks to mechanisms not completely defined, tumors can progressively remodel the host environment, shutting off potential interferences with their own survival in vivo. This process could occur even in those tumors (such as melanoma) in which recall responses against certain tumor antigens are apparently detectable ex vivo (28) but most likely as mere indicators of tumor burden rather than effective mediators of antitumor immunity (29). In the context of tumor immune escape strategies, many studies have focused on the ability of cancer cells to eliminate, damage, or anergize T-cell–functions (30). However, the mechanisms exploited by growing tumors to dampen the initial phases of the immune response involving the APC compartment are still poorly defined. Nonetheless, several reports showed the existence in cancer patients of marked systemic defects involving APC, including dendritic cells, as a general consequence of abnormal myeloid differentiation and activation (15). In particular, tumor cells are known to promote the expansion of a myeloid cell population with negative regulatory effect on T-cell–functions, known as MSC (16). These cells, although extensively characterized in tumor-bearing mice, where they display a conserved Gr1+CD11b+ phenotype and common suppressive mechanisms, still need to be defined in the human setting (16). Nevertheless, several findings support the occurrence of aberrant myeloid differentiation in cancer patients, with defects involving decreased frequencies of mature immunocompetent dendritic cells, accumulation of HLA class IIlow immature dendritic cells and expansion of immature myeloid cells (15). In particular, the accumulation of CD14+ monocytes expressing reduced levels of HLA-DR and costimulatory molecules was described by Schadendorf et al. in peripheral blood monocytes from melanoma patients compared with healthy controls in association with disease progression (31).
Thus, we could speculate that the CD14+HLA-DR–/low cell subset we identified as the result of monocyte interaction with tumor-released microvesicles may represent a component of the MSC population. Although the contribution of arginase activity, a hallmark of MSC in tumor-bearing animals (23) and selected human tumor histologies (such as renal carcinoma; ref. 16), could not be detected, we identified TGF-ß as the soluble factor responsible for the immunosuppressive activity of microvesicle-treated monocytes. This cytokine, exerting a well-defined role in immunosuppression through mechanisms ranging from direct effects on T-cell–proliferative and cytolytic potential (21) to the activation of regulatory T cells (32), has been recently identified as the key player of MSC function in defined tumor murine models (33).
The multiple defects in myeloid differentiation and function detected systemically in cancer patients support the hypothesis that circulating factors, able to reach these cells in different tissue compartments, might be involved. Herein, we show that tumor-released microvesicles may play a role in this process. Microvesicles are membrane organelles shed from the cell surface under different physiologic and pathologic conditions by a large array of cells (26). Thanks to their ability to circulate and transport a broad protein spectrum from the membrane and cytoplasmic repertoire of the cell (34), microvesicles may act as important mediators for intercellular cross-talk and molecule delivery. Indeed, since their discovery (35), growing evidence has been collected about the presence of microvesicular organelles in different biological fluids of both cancer patients and healthy individuals (2, 3, 9, 10). The association between microvesicle release and tumor progression, suggested by microvesicle abundance in the body fluids of patients with advanced neoplastic disease, has been recently hypothesized in several studies showing the microvesicle ability to transport bioactive molecules potentially involved in immunosuppressive circuits, such as FasL and TRAIL (1–3, 10). Evidence of the possible detrimental effect of tumor-shed microvesicles on the APC compartment was collected in murine models by Taylor et al., reporting the inhibitory activity of membrane-derived vesicles released by melanoma metastatic variants on MHC class II surface expression in macrophages (36). However, the potential interference of tumor-released microvesicles with APC function has been thus far poorly investigated, being recent studies mainly focused on the use of tumor-derived microvesicles as vehicles for tumor antigen cross-presentation by APC (34). Although the possibility to induce effective immunoprotection in tumor-bearing mice vaccinated with tumor-derived exosomes has been reported (37), these results have not been thus far confirmed in human setting, where the clinical application of microvesicles as cancer vaccines has been mainly focused on dendritic cells instead of tumor-derived exosomes (38).
Secretion of microvesicles capable of skewing monocyte differentiation toward a suppressive phenotype seemed to be a tumor-related phenomenon, as microvesicles released by macrophages, used as normal counterpart, allowed the physiologic differentiation of monocytes into immature dendritic cells. Although it would be interesting to analyze the immunomodulatory effects of microvesicles released by melanocytes, representing melanoma nontransformed histologic counterpart, the lack of FasL expression in these normal cells (39) suggests that production of immunosuppressive microvesicles might be a selective feature of transformed tissues. Tumor specificity of this phenomenon was further evaluated using exosome-like membrane vesicles purified from plasma of both healthy donors and melanoma patients, where the presence of circulating tumor-released microvesicles was proven in terms of expression of the melanoma antigen gp100 (see Fig. 5B). Although tumor cells may not be the only source of microvesicles collected from patients' plasma, the detrimental effect on differentiating monocytes was exclusively induced by microvesicle preparations from melanoma patients and not from healthy donors, thus suggesting that the presence of a growing tumor is linked to the microvesicle-induced suppressive effect.
The mechanism by which microvesicle-treated monocytes may deliver their suppressive signals to antitumor T-cell–effectors without creating systemic immune suppression in the host is presently unknown, being still undefined the pathway used by MSC to expressly target tumor-specific immune responses (16). However, it could be hypothesized that CD14+HLA-DR– cells may cross-present antigenic determinants contained in tumor-microvesicle (37) to specific CD8+ T cells through their conserved expression of HLA class I molecules and then selectively deliver TGF-ß–mediated suppressive signals.
In conclusion, our data suggest the possible development in cancer patients of a circuit primed by tumor cells to create an immunotolerant environment, through the active release of membrane vesicles, able to redirect normal differentiation of circulating monocytes into a CD14+ immature myeloid population with a TGF-ß–mediated suppressive activity on T-cell–proliferation and function. Although further studies are needed to clearly depict the molecular mechanisms involved in the dysregulation of monocyte differentiation by tumor-derived microvesicles, our observations may offer new tools for prognostic and therapeutic strategies in cancer patients.
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Acknowledgments |
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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 Dr. Flavio Arienti (Transfusion Medicine Bank, Istituto Nazionale Tumori) and Dr. Vincenzo Russo (Cancer Immunotherapy and Gene Therapy Program, H.S. Raffaele Scientific Institute) for providing healthy donor and patient blood samples, Dr. Manuela Iero for helpful discussion, Agata Cova for the skillful technical help, Grazia Barp for editorial assistance, and Dr. Goffredo Alfieri and Bracco Imaging S.p.A. (Milan, Italy) for scanning electron microscopy analysis.
Received 5/18/06. Revised 7/12/06. Accepted 7/19/06.
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in metastatic melanoma patients. Cancer Immunol Immunother 2005;8:1–11.[Medline]This article has been cited by other articles:
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C. Eken, O. Gasser, G. Zenhaeusern, I. Oehri, C. Hess, and J. A. Schifferli Polymorphonuclear Neutrophil-Derived Ectosomes Interfere with the Maturation of Monocyte-Derived Dendritic Cells J. Immunol., January 15, 2008; 180(2): 817 - 824. [Abstract] [Full Text] [PDF]
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 G. Ferretti, A. Felici, F. Cognetti, and M. Mandala Transforming Growth Factor-{beta} Signaling and Regulatory T Cells J. Clin. Oncol., October 10, 2007; 25(29): 4695 - 4696. [Full Text] [PDF]
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P. Filipazzi, C. Castelli, V. Roberta, V. Huber, M. Iero, L. Pilla, L. Rivoltini, and G. Parmiani In Reply J. Clin. Oncol., October 10, 2007; 25(29): 4696 - 4697. [Full Text] [PDF]
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P. Filipazzi, R. Valenti, V. Huber, L. Pilla, P. Canese, M. Iero, C. Castelli, L. Mariani, G. Parmiani, and L. Rivoltini Identification of a New Subset of Myeloid Suppressor Cells in Peripheral Blood of Melanoma Patients With Modulation by a Granulocyte-Macrophage Colony-Stimulation Factor-Based Antitumor Vaccine J. Clin. Oncol., June 20, 2007; 25(18): 2546 - 2553. [Abstract] [Full Text] [PDF]
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 C. Liu, S. Yu, J. Kappes, J. Wang, W. E. Grizzle, K. R. Zinn, and H.-G. Zhang Expansion of spleen myeloid suppressor cells represses NK cell cytotoxicity in tumor-bearing host Blood, May 15, 2007; 109(10): 4336 - 4342. [Abstract] [Full Text] [PDF]
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R. Valenti, V. Huber, M. Iero, P. Filipazzi, G. Parmiani, and L. Rivoltini Tumor-Released Microvesicles as Vehicles of Immunosuppression Cancer Res., April 1, 2007; 67(7): 2912 - 2915. [Abstract] [Full Text] [PDF]
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), melanoma-derived microvesicle-treated (
), and colorectal carcinoma–derived microvesicle-treated (
) monocytes at the third day of differentiation with cytokines were incubated for 3 days with allogeneic HLA-mismatched PBMC at the indicated stimulator/target ratios. B, tumor microvesicle-treated monocytes exert suppressive effects on T-cell–proliferation in response to TCR triggering. Untreated (
