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*Pancreatic Cancer
The Journal of Immunology, 2004, 172: 7341-7349.
Copyright © 2004 by The American Association of Immunologists

Tumor-Derived MUC1 Mucins Interact with Differentiating Monocytes and Induce IL-10highIL-12low Regulatory Dendritic Cell1

Paolo Monti*, Biagio Eugenio Leone{ddagger}, Alessandro Zerbi*, Gianpaolo Balzano*, Silvia Cainarca{dagger}, Valeria Sordi{dagger}, Marina Pontillo*, Alessia Mercalli{dagger}, Valerio Di Carlo*, Paola Allavena§ and Lorenzo Piemonti2,*,{dagger}

* Laboratory of Experimental Surgery, Surgical Department, and {dagger} Telethon-Juvenile Diabetes Research Foundation Center for {beta} Cell Replacement, San Raffaele Scientific Institute, {ddagger} University of Milan Bicocca, and § Department of Immunology and Cell Biology, "Mario Negri" Institute, Milan, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DC) initiate immunity by the activation of naive T cells and control immunity through their ability to induce unresponsiveness of lymphocytes by mechanisms that include deletion and induction of regulatory cells. An inadequate presentation to T cells by tumor-induced "regulatory" DC, among several mechanisms, can explain tolerance to tumor-associated Ags. In this study, we show that tumor-derived mucin profoundly affects the cytokine repertoire of monocyte-derived DC and switch them into IL-10highIL-12low regulatory APCs with a limited capacity to trigger protective Th1 responses. In fact, DC cocultured with pancreatic tumor cell lines in a Transwell system did not reach full maturation, had low immunostimulatory functions, did not produce IL-12, and released high levels of IL-10. The involvement of known tumor-derived immune-suppressive factors (e.g., vascular endothelial growth factor, TGF-{beta}, IL-6, and IL-10) was considered and excluded. We provide evidence that tumor-derived MUC1 mucins are responsible for the impaired DC maturation and function. DC obtained in the presence of tumor microenvironment preferentially polarized IL-4+ response. Moreover, T cells primed by these regulatory DC became anergic and behaved as suppressor/regulatory cells. These findings identify mucin secretion as a novel mechanism of tumor escape from immune surveillance and provide the basis for the generation of potentially tolerogenic DC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The immune system is potentially able to recognize Ags expressed on human and experimental tumors and to mount a protective immune response (1, 2, 3, 4). However, malignant tumors are tolerated, progress, and ultimately kill their host. Among several mechanisms, tolerance can be explained by an inadequate presentation of tumor-associated Ags to T cells.

Ag presentation is professionally performed by dendritic cells (DC)3 (5, 6, 7, 8) that themselves need to be activated to prime T cell responses. Consistent with DC involvement in the immune unresponsiveness found in tumors, tumor-associated DC usually express an immature phenotype and have defective Ag-presenting ability (9, 10, 11, 12, 13). Accordingly, T cell responses are also impaired in cancer patients. In particular, reports have suggested that Th1-type immunity is impaired and that Th2 responses predominate in some neoplastic diseases (14, 15, 16, 17, 18, 19). The tumor microenvironment may influence the differentiation of DC as well as their capacity to prime T cells in a variety of ways. Tumors have been reported to produce factors including IL-10, vascular endothelial growth factor (VEGF), TGF-{beta}, IL-6, and M-CSF, which inhibit or suppress the differentiation and activation of DC (20, 21, 22, 23, 24, 25, 26, 27, 28).

Mucins are large (>200 kDa) glycoproteins with a high carbohydrate content (50–90% by weight). They are expressed by a variety of normal and malignant epithelial cells (29, 30). MUC1 mucins are giant, complex glycoproteins, comprising a polypeptide core with multiple oligosaccharide side chains in O-linkage to serine or threonine residues. Although the mature molecule is anchored within the cell surface by a characteristic transmembrane domain, most of the mucin is expressed extracellularly in an elongated form extending far beyond most other cell surface-expressed macromolecules. Although the exact function(s) of MUC1 mucins in the normal cell are still a matter for debate, they have had considerable impact as markers of many human carcinomas. In malignant cells, the expression of MUC1 is elevated and its orientation within the tissue is no longer polarized to apical surfaces. Moreover, in many human cancer cells, MUC1 mucins display an altered glycosylation, resulting in tumor-specific exposure of immature carbohydrate structures and peptide epitopes within the tandem repeat (31, 32). In particular, the elongation of the O-linked saccharide chain does not occur, thus leading to the generation of antigenic determinants such as Tn (GalNAc-O-Ser/Thr), sialyl-Tn (NeuAc-GalNAc-O-Ser/Thr), and T (Gal-GalNAc-O-Ser/Thr) (33).

These tumor-specific forms of MUC1 are cleaved off the tumor cells, drain to regional lymph nodes (LNs), and enter the peripheral circulation, where they could potentially modulate immune responses, and indeed, an immunoregulatory role for tumor-derived mucins has been recently suggested (34).

In this study, we investigated the development of DC differentiated in a tumor microenvironment, mimicked by coculturing human monocytes and tumor cells in a Transwell system (Costar, New York, NY). We demonstrate that inhibition of DC differentiation and function is associated with an augmented production of IL-10 by DC cocultured with MUC1-expressing tumor cells. We propose that tumor-derived MUC1 mucins convert them into IL-10-producing IL-12-deficient APCs with a limited capacity to trigger protective Th1 responses and the ability to promote T cell anergy and regulatory activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and reagents

Human pancreatic carcinoma cell lines ASPC-1, Capan-1, MiaPaca-2, Panc-1, HS766T (American Type Culture Collection, Manassas, VA), PT45, PC13, HPAF, CFPAC, T3M4, and PaCa 44 (kindly provided by Prof. A. Scarpa, Department of Pathology, University of Verona, Verona, Italy) were grown in RPMI (Biochrom, Berlin, Germany) 10% FCS (HyClone Laboratories, Logan, UT). Human recombinant granulocyte macrophage CSF (GM-CSF; specific activity, 1.1 x 104 U/mg) was obtained from Novartis Pharmaceuticals (Basel, Switzerland). Human rIL-4 (IL-4; specific activity >2 x 106 U/mg) and IL-12 were obtained from PeproTech EC (London, U.K.). Macrophage-inflammatory protein (MIP)-3{beta}/CC chemokine ligand (CCL)19 was purchased from PeproTech (Rocky Hill, NJ). Neutralizing Abs were used at 10 µg/ml and included anti-IL-10, anti-TGF-{beta}, and anti-VEGF (R&D Systems, Minneapolis, MN). All of the neutralizing mAbs were added from day 0 during all of the differentiation periods. Anti-mouse IgG was from Sigma-Aldrich (St. Louis, MO). Extractive MUC1 (CA15-3) was from Fujirebio Diagnostics (Malvern, PA).

In vitro model of DC differentiation and maturation in a pancreatic cancer microenvironment

Peripheral blood monocytes were obtained by density gradients, as described in Refs. 35 and 36 . To evaluate the effects of the tumor environment on DC, monocytes (106/ml) were seeded with GM-CSF (50 ng/ml) and IL-4 (10 ng/ml) in the lower chamber of 6-well Transwell plates with permeable membrane pores (0.2 µm; Costar). Tumor cell lines or tumor cells from primary cultures (1 x 105 cells) were seeded in the upper chamber. At day 6 of culture, DC were induced to maturation by addition of a transfected CD40 ligand (CD40L) cell line (J558LmCD40L) or with LPS (10 ng/ml). At day 8, DC were harvested and used in functional assays.

Phenotype analysis. Phenotype analysis was performed by flow cytometry with the following mouse mAbs: L243 (IgG2a, anti-MHC class II (MHC II)), W6/32 (IgG2a, anti-MHC class I (MHC I)) from Sigma-Aldrich; SK9 (IgG2b, anti-CD1a) from BD Biosciences (San Jose, CA); PAM-1 (IgG1, anti-mannose receptor (MR) (37)); and 19-2 (IgG1, anti-MR) from BD PharMingen (San Diego, CA); BB1 (IgM, anti-CD80), BU63 (IgG1, anti-CD86), EA-5 (IgG1, anti-CD40) from Ancell (Bayport, MN); HB15a (IgG2b, anti-CD83); SM1/1 (IgG2a, anti-CD95) from Alexis Italia (Vinci, Italy); D12 (IgG2a, anti-CD11b), SHCL-3 (IgG2b, anti-CD11c) from BD PharMingen; and 2D7 (anti-CCR5) and 2H4.1 (anti-CCR7) from BD PharMingen. FITC-conjugated, affinity-purified, isotype-specific, goat anti-mouse Abs (Ancell) were also used. Results are expressed as percentage of positive cells or as mean fluorescence intensity (MFI).

Endocytosis. MR-mediated endocytosis was measured as FITC-dextran uptake as previously described (35, 36) and quantified by flow cytometry.

Chemotaxis. Cell migration was evaluated in modified Boyden chambers for 1.5 h, as previously described (38).

Mixed leukocyte reaction. Priming of allogenic T cells was assessed by [3H]thymidine uptake as previously described (35, 36). Results are presented as cpm.

Polarization of naive T lymphocytes. Cord blood from normal end-stage deliveries was obtained from the Department of Obstetrics and Gynecology (University Vita Salute San Raffaele, Milan, Italy) under protocols approved by the board of the local ethical committee. Cord blood naive T cells were cultured with differently treated DC (10:1) for 10 days. At the end of the incubation, cells were collected and stimulated with PMA and ionomicin (Sigma-Aldrich) for 6 h in the presence of 10 µg/ml brefeldin A for the last 2 h. Cytokine detection was assessed by intrastaining after cell permeabilization with the following mAbs: MP4-25D2 (rat IgG1, anti-IL-4) from Serotec (Oxford, U.K.) and G7-4 (mouse IgG, anti-IFN-{gamma}) from Bender MedSystems (Vienna, Austria).

T cell anergy assay. Allogenic T cells were first primed with either control (Ctrl)-DC or ASPC-1-DC (10:1 ratio). Ten days later, T cells were collected, depleted of residual DC by anti-CD32 or anti-MR-coated beads, and rested for 5 days in medium alone. T cells were then cultured again with fully mature DC from the same donor as the first stimulation or DC from an unrelated donor. [3H]Thymidine incorporation was measured after 5 days of culture.

T cell suppressor assay. Allogenic CD4 T cells were purified and cocultured (1 x 106/ml) for 7 days with irradiated Ctrl-DC or ASPC-1-DC at a 10:1 ratio. T cells were then mixed with freshly isolated T cells at ratios ranging from 1:5 to 1:100 (suppressor/responder ratio), and with Ctrl-DC (10:1). [3H]Thymidine incorporation was measured after 5 days of culture.

Preparation of tumor supernatants (TSNs)

TSNs were prepared by seeding 1 x 106 tumor cells in 1 ml of complete medium, and collected after 24 h. The effects of TSNs on DC were examined by replacing 33% of the culture medium with TSN at the initiation of culture. Ctrl-DC received the same amount of boiled TSNs. Depletion of MUC1 from TSN was obtained by repeated panning on anti-MUC1-mAbs-coated dishes. Anti-MUC1 Abs included the following: tumor-associated glycoprotein (TAG) 72/CA72-4 Ab-1 (clone B72.3, anti-mucin-carried sialylated-Tn epitope) mouse IgG1/k from NeoMarkers (Fremont, CA); CBL 204 (clone SM-3, anti-mucin core protein) mouse IgG1 from Cymbus Biotechnology; and mAbs anti-CA15-3 (DF3 and 115D8 mouse mAbs) from Fujirebio Diagnostics. To test the effect of recovered mucin on DC differentiation and maturation, culture plastic coated with a mixture of anti-MUC1 (CA15-3) mAbs (DF3 and 115D8 mouse mAbs), anti-TAG 72 mAb (that recognize the mucin-carried sialylated-Tn epitope), a Ctrl mAb (anti-CD20), or culture plastic without Abs coating was exposed to TSN from ASPC-1 and Capan-1. After 1 h, TSN was removed and the plastic was washed. Monocytes were subsequently cultured on this culture plastic in the presence of GM-CSF and IL-4.

Expression of MUC1 in Capan-1

The epitope-tagged (Flag) MUC1 (intact MUC1 mucin with 32 tandem repeats) was described previously (39, 40). Briefly, a double-stranded synthetic oligonucleotide was designed to encode the amino acids sequence DYKDDDDQILDMVA, where DYKDDDD is the Flag epitope recognized by the mAb M2. The remainder of the inserted amino acid sequence (QILDMVA) is a result of the addition of two unique restriction enzyme sites. This oligonucleotide was ligated into a Bsm I site at position 232 of the MUC1 cDNA. The MUC1/Flag was inserted between Hind III and Eco RI in pCR3.1 vector (Invitrogen, Carlsbad, CA) by G. Hansson (Department of Medical Biochemistry, Goteborg University, Sweden). The constuct was introduced into Capan-1 pancreatic cancer line by Fugene (Roche, Basel, Switzerland) transfection according to the manufacturer’s instructions. The expression of MUC1 was evaluated by anti-Flag mAb M2 (Sigma-Aldrich), anti-TAG 72 mAb that recognizes the mucin-carried sialylated-Tn epitope, and anti-mucin core protein mAb (SM-3).

Cytokine measurement by ELISA

Cytokine production by Ctrl-DC, tumor-DC, or tumor cells was measured at the indicated times and quantified by ELISA with the following commercial kits: IL-10, IL-12 p70, IL-1{beta}, IL-6, VEGF (Endogen, Boston, MA); and TGF-{beta}1 (R&D Systems).

Statistical analysis

Data are expressed as mean ± SD and compared by Student’s t test or Wilcoxon rank sign test. For all analyses, a two-tailed p value of 0.05 was considered significant. Statistical analyses were performed using the Statistical Package for Social Science (SPSS 11.0; SPSS, Chicago, IL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DC differentiation and maturation in a tumor microenvironment

To evaluate the effects of a tumor environment on DC, monocytes were cultured with GM-CSF and IL-4 in the presence or absence of 11 different pancreatic cancer cell lines in a Transwell system with 0.2-µm porous membrane. After 6 days, DC were induced to maturation by CD40 ligation for 48 h. Fig. 1A shows that 9 of 11 pancreatic cancer cell lines inhibited DC maturation, evaluated as decreased expression of MHC II and CD40 after CD40L exposure. Two cancer cell lines were selected for further studies: ASPC-1 cells as inhibitory tumor cell line and Capan-1 cells as noninhibitory cell line. Both cell lines allowed recovery of >90% DC compared with DC cultured without tumor cells (Ctrl-DC). A complete phenotype analysis of DC after maturation with CD40L was performed. Fig. 1B shows a representative experiment and Table I shows results from 13 independent experiments. Despite similar morphology, DC cocultured with the pancreatic cancer cell line ASPC-1 (ASPC-1-DC) showed lower expression of CD83, as well as other molecules involved in Ag presentation (MHC class I, MHC II, CD80, CD86, CD40), compared with Ctrl-DC. In contrast, DC cocultured with the pancreatic cancer cell line Capan-1 (Capan-1-DC) were not significantly affected. A similar inhibition of DC maturation by ASPC-1 was observed when LPS was used instead of CD40L as the maturation stimulus (data not shown). Interestingly, the only maturation marker that was not down-regulated by ASPC-1-DC was the chemokine receptor CCR7. In maturing DC, CCR7 up-regulation is instrumental in guiding Ag-carrying DC to LNs where primary immune responses take place (41, 42). In chemotaxis assay, ASPC-1-DC migrated in response to CCL19 (the CCR7 ligand) (Fig. 2B).



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  		FIGURE 1. Phenotype analysis of DC differentiated in the presence or absence of pancreatic cancer line. A, DC were differentiated from monocytes with GM-CSF and IL-4 alone (Ctrl-DC) or in the presence of eleven different pancreatic cancer cell lines ("tumor"-DC). Monocytes were plated in the lower compartment and tumor cells were plated in the upper compartment of a Transwell plate (0.2-µm pores). At day 6, a CD40L-transfected cell line was added to induce DC maturation. Shown is the expression of MHC II and CD40 by DC. Data are expressed as mean ± SD and compared by Student’s t test. *, p < 0.05 vs Ctrl-DC. B, DC were differentiated from monocytes with GM-CSF and IL-4 in the absence (Ctrl-DC) or presence of pancreatic cancer lines ASPC-1 (ASPC-1-DC) and Capan-1 (Capan-1-DC) for 7 days. Maturation was induced by culture with a CD40L-transfected cell line for 48 h. FACS profiles are from 1 experiment of 13 performed.

 

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  		Table I. Phenotype analysis of DC differentiated in the presence or absence of pancreatic cancer cell linesa

 


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  		FIGURE 2. Functional activity of DC cocultured with pancreatic cancer cell lines. DC were differentiated and treated as described in Fig. 1. Data are from representative experiments of five performed. A, Endocytic activity of differently treated DC before maturation. Ctrl-DC ({square}) and Capan-1-DC () have high endocytosis of FITC-dextran. In contrast, ASPC-1-DC ({blacksquare}) shows very low levels of endocytosis. B, Chemotactic activity of differently treated DC to CCL19/MIP-3{beta}. Ctrl-DC ({square}), Capan-1-DC (), and ASPC-1-DC ({blacksquare}) migrated to CCL19/MIP-3{beta} (100 ng/ml) after CD40L-maturation. Shown are net numbers of migrated cells. Cells migrating to medium alone were <10 cells. C, Stimulatory activity of DC in MLR. ASPC-1-DC (filled) were poor stimulators of allogenic T cell proliferation compared with Ctrl-DC (open) and Capan-1-DC (gray).

 
Functional activity and cytokine production of DC differentiated in the presence of pancreatic cancer cell lines

Immature DC display potent endocytic activity, which decreases upon maturation. To study the endocytic capacity of ASPC-1-DC, FITC-dextran uptake, a marker of MR-mediated endocytosis, and lucifer yellow uptake, a nonspecific marker of macropinocytosis, were examined (Fig. 2A). Immature ASPC-1-DC showed very low levels of FITC-dextran uptake (MFI at 120 min: 30 ± 12; n = 5) compared with immature Ctrl-DC (MFI at 120 min: 125 ± 18; p = 0.02 vs ASPC-1-DC; Student’s t test) and Capan-1-DC (MFI at 120 min: 140 ± 30; p = 0.03 vs ASPC-1-DC; Student’s t test). Lucifer yellow uptake was also decreased, although to a lesser extent (data not shown). Surface expression of MR was tested in immature ASPC-1-DC and, in line with defective endocytosis, was reduced compared with Ctrl-DC and Capan-1-DC (Ctrl-DC = 84 ± 12 MFI; Capan-1-DC = 88 ± 10 MFI; ASPC-1-DC = 54 ± 6 MFI; p = 0.004 ASPC-1 DC vs Ctrl-DC; n = 13).

We next tested whether ASPC-1-DC were able to stimulate allogenic T lymphocytes in MLR. The immunostimulatory capacity of ASPC-1-DC in MLR was very low compared with that of Ctrl-DC and Capan-1-DC and was not increased by LPS or CD40L stimulation, confirming an impaired capacity to reach maturation (Fig. 2C).

The immunostimulatory capacity of mature DC is associated with their relative amounts of IL-10 and IL-12 secretion (43). Consistent with their reduced immunostimulatory capacity, mature ASPC-1-DC produced on average 6.6 ± 0.7-fold higher levels of IL-10 and 6.7 ± 2.5-fold less IL-12 than Ctrl-DC (IL-10: p = 0.001; IL-12: p = 0.001; n = 5; Student’s t test) (Fig. 3). IL-10 and IL-12 production of Capan-1-DC was not significantly different from that of Ctrl-DC.



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  		FIGURE 3. ASPC-1-DC produce high IL-10 and low IL-12. Left panel, IL-10 production by Ctrl-DC, ASPC-1-DC, and Capan-1-DC at different times of culture. Right panel, IL-12p70 production by Ctrl-DC, ASPC-1-DC, and Capan-1-DC at different times of culture. Data are expressed as mean ± SD and compared by Student’s t test. *, p < 0.05 vs Ctrl.

 
Addition of a blocking anti-IL-10 mAb (10 µg/ml) partially restored the expression of CD40, CD83, CD86, and IL-12 release (data not shown), indicating that the autocrine production of IL-10 negatively affected IL-12 synthesis. Both ASPC-1 and Capan-1 cell lines did not produce detectable IL-10 even after 7-day culture and exposure to GM-CSF/IL-4 or monocytes/DC, confirming that DC were the source of IL-10 in the coculture.

Th polarization of naive T cells and generation of anergic/regulatory T cell by ASPC-1-DC

We investigated the type of Th cell response generated by DC differentiated in the presence of the noninhibitory tumor cell line Capan-1 and with two tumor cell lines inhibiting DC maturation (ASPC-1 and MiaPaca-2). DC differentiated in the tumor microenvironment and Ctrl-DC were cocultured with allogenic naive T cells, and T cells were tested for their capacity to produce IL-4 or IFN-{gamma}. Ctrl-DC and Capan-1-DC stimulated a vigorous IFN-{gamma} response whereas ASPC-1-DC and MiaPaca-2-DC induced a predominant IL-4 response (Fig. 4A). The proportion of IL-4-producing cells was 9 ± 9% for Ctrl-DC and 10 ± 4% for Capan-1-DC, compared with 31 ± 9% for ASPC-1-DC (p < 0.02 vs Ctrl-DC) and 34 ± 9% for MiaPaca-2-DC (p < 0.01 vs Ctrl-DC; n = 6 for each condition).



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  		FIGURE 4. T cell polarization by DC cocultured with cancer cells and generation of anergic regulatory T cell. A, Intrastaining cytokine production by naive T cells cocultured for 6 days with DC differentiated in the presence of ASPC-1, Capan-1, and MiaPaca-2 cancer cell lines. A representative experiment of six performed is shown with anti-IL-4 PE-mAb and anti-IFN-{gamma} FITC-mAb. Numbers are percentages of positive cells. B, Induction of T cell anergy by ASPC-1-DC. Allogenic T cells were first primed with CD40L-stimulated ASPC-1-DC (•) or Ctrl-DC ({circ}). T cell cultures from both groups were restimulated 1 wk later with CD40L-Ctrl-DC generated from the same donor (Auto) or a different unrelated donor (Allo). Shown is T cell proliferation after the second MLR as proliferation index. Data are from a representative experiment of two performed. C, Suppressor cell activity of T cells originally primed by ASPC-1-DC or Ctrl-DC. Allogenic CD4 T cells were purified and cocultured (1 x 106/ml) for 7 days with irradiated Ctrl-DC or ASPC-1-DC at a 10:1 ratio. T cells were then mixed with freshly isolated T cells at ratios ranging from 1:5 to 1:100 (suppressor/responder ratio) and with Ctrl-DC (10:1). [3H]Thymidine incorporation was measured after 5 days of culture. Shown is a representative experiment of two performed.

 
DC with an IL-10highIL-12low phenotype are reported to promote T regulatory cells after stimulation of allogenic T cell in MLR. To determine whether the tumor-matured DC could have a similar property, allogenic T cells were cocultured with Ctrl-DC or ASPC-1-DC for 10 days, and then restimulated in a second coculture with mature Ctrl-DC from the same donor or from an unrelated donor. T cells first primed with Ctrl-DC responded vigorously to restimulation, whereas T cells first primed with ASPC-1-DC were completely unresponsive both when related or unrelated DC were used for restimulation (Fig. 4B). To determine whether these T cells contained cells with a regulatory phenotype, T cells that were primed with immature Ctrl-DC or ASPC-1-DC were isolated and cocultured with fresh T cells and allogenic DC. As expected, T cells stimulated by Ctrl-DC did not suppress the MLR. In contrast, T cells stimulated with ASPC-1-DC suppressed the MLR by 70 ± 10% when added at 1:1 suppressor/responder ratio and by 30 ± 10% at a 1:10 ratio (p = 0.02 and p = 0.05 respectively, Student’s t test).

Identification of tumor-derived factor affecting DC differentiation and function

A number of tumor-derived factors, such as VEGF, TGF-{beta}, IL-10, and IL-6, that have the capacity to alter differentiation or maturation of DC have been described (20, 21, 22, 23, 24, 25, 26, 27, 28). TGF-{beta}, IL-6, and IL-10 were not produced or were produced at very low levels by the 11 tumor cell lines examined, and were undetectable in ASPC-1 cells (data not shown). Low amounts of VEGF were secreted by both ASPC-1 cells and by the noninhibitory cell line Capan-1, suggesting that VEGF did not mediate the inhibitory capacity of ASPC-1. Moreover, the inhibitory effect of ASPC-1 TSN on DC was not reversed by the addition of neutralizing Abs against VEGF and TGF-{beta} (data not shown). Because the inhibitory capacity of ASPC-1 could not be ascribed to reported mediators of DC function, other factors were examined.

Engagement of C-type lectin receptors on DC were described to be able to modify DC function in a tolerogenic fashion (44). Recently we have described that MR (a C-type 1 lectin receptor) engagement on myeloid DC, with selected ligands or Abs, activates an alternative maturation program characterized by a profile of anti-inflammatory and tolerogenic cytokines that would prevent the generation of Th1-polarized responses (45). Because DC stimulated through the MR up-regulate IL-10 production and down-regulate IL-12 (45), we examined whether factor(s) released by tumor cell lines were able to bind MR on DC. Pretreatment of MR-expressing DC (immature DC) with TSN from the ASPC-1 cell line dose-dependently inhibited the binding of anti-MR mAbs, while TSN from the noninhibitory cell line Capan-1 did not (Fig. 5A). Binding of a different mAb (anti-CD40) to DC was not affected by TSN pretreatment (data not shown), ruling out a nonspecific effect. Recent evidence shows that MUC1 mucins, expressed and secreted at a high level in pancreatic cancer, are able to engage MR (46). A soluble form of MUC1 mucins (also named CA15-3) is found in several tumors. Interestingly, the inhibitory cell line ASPC-1 released significantly higher amounts of CA15-3 compared with the noninhibitory Capan-1 cell line (Fig. 5B). Moreover, an association between the ability to influence DC phenotype, the expression of MUC1 mucins carrying the sialylated-Tn epitope, and the ability to inhibit anti-MR mAb binding (but not anti-DC-specific ICAM-3-grabbing nonintegrin (SIGN) mAb binding) was present in all the other cell lines tested (Fig. 5C).



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  		FIGURE 5. MUC1 mucins secretion and MR binding. A, Immature Ctrl-DC (day 6 of culture) were preincubated at 4°C with TSN from ASPC-1 and Capan-1 cancer cell lines. Binding to MR was evaluated as competition of binding of a specific anti-MR (PAM-1). ASPC-1 TSN dose-dependently inhibited anti-MR binding, while Capan-1 TSN did not. Results are from one experiment of four performed. B, Kinetics of MUC1 (CA15-3) release in culture medium during DC/tumor coculture with ASPC-1 and Capan-1 cancer cell lines (ELISA). ASPC-1 and Capan-1 lines analysis for MUC1 expression (TAG 72 epitope) in flow cytometry. Results are from one experiment of two performed. C, upper panel, MUC1 mucins expression on pancreatic cancer cell lines was detected by flow cytometry and expressed as MFI; n = 3. Sialyl Tn, TAG 72 mAb; core protein, CBL 204 mAb. C, lower two panels, Immature Ctrl-DC (day 6 of culture) were preincubated at 4°C with TSN from pancreatic cancer cell lines. Bindings to MR and DC-SIGN were evaluated as competition of binding of a specific anti-MR and anti-DC-SIGN mAb. Results are expressed as change in percentage of MFI vs Ctrl (DC incubated with medium alone); n = 2.

 
To determine whether MUC1 mucins could inhibit DC maturation and function, DC were incubated in the presence of mucins extracted from TSNs. The MUC1 mucins were extracted by incubating TSN from ASPC-1 or Capan-1 cells on plates that were precoated with anti-CA15-3 mAbs (DF3 and 115D8 mouse mAbs), anti-TAG 72 mAb (which recognizes the MUC1-carrying sialylated-Tn epitope), an indifferent mAb (anti-CD20), or medium alone (Ctrl). DC generated on plates where ASPC-1 TSN was extracted with anti-CA15-3 or anti-TAG 72 had lower expression of CD40 and CD86 (Fig. 6A). They also showed decreased IL-12 and increased IL-10 production (p = 0.03 and p = 0.01 vs Ctrl, Student’s t test) (Fig. 7B), as observed in the coculture experiments. Moreover, addition of exogenous MUC1 mucins (CA15-3) during DC differentiation dose-dependently increased IL-10 secretion in CD40L-DC (Fig. 6B). Consistent with these findings, the ASPC-1 TSN, which had been depleted of MUC1 mucins by repeated panning, no longer affected DC maturation (Fig. 6C). These findings indicated that the inhibition of DC maturation and the release of a tolerogenic cytokine profile (low IL-12, high IL-10) induced by ASPC-1 cell line could be mediated by MUC1 mucins. To investigate this further, the Capan-1 line that expressed no or negligible amounts of MUC1 was transfected with vectors containing MUC1 cDNA. After transfection, Capan-1 line expressed the glycosylated form of MUC1 carrying the sialylated-Tn epitope that is recognized by the anti-TAG 72 mAb (Fig. 7A). When monocytes were cocultured in the Transwell system, MUC1 transfected Capan-1 cells, but not Capan-1 cells transfected with Ctrl vector, inhibited DC maturation (lower level of MHC II, CD40, and CD83) (Fig. 7B), and suppressed IL-12 and increased IL-10 production by DC (Fig. 7C). These results unequivocally demonstrated that the inhibition of DC maturation and the release of a tolerogenic cytokine profile (low IL-12, high IL-10) by pancreatic cancer cell lines can be mediated by MUC1 mucins.



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  		FIGURE 6. Effect of extracted mucins on differentiating DC. A, DC were differentiated in the presence or absence of mucins extracted by panning TSN of ASPC-1 or Capan-1 cell lines on dishes coated with mAb anti-MUC1 (anti-TAG 72, anti-CA15-3), irrelevant mAb (anti-CD20), or uncoated plastic (Ctrl). DC were cultured for 7 days and matured with CD40L. Phenotype analysis of CD40 and CD86 expression of mature DC differentiated in the presence of recovered mucins. Results are from one experiment of two performed. B, MUC1 addition induces IL-10 production by DC. IL-10 and IL-12 production by DC differentiated in the presence of mucins: DC were differentiated in the presence or absence of mucins extracted by panning TSN of ASPC-1 or Capan-1 cell lines, and dishes coated with mAb anti-MUC1 (anti-TAG 72). Alternatively DC were differentiated in the presence of exogenous CA15-3. DC were cultured for 7 days and matured with CD40L. Results are expressed as mean ± SD; n = 4; *, p < 0.05 vs Ctrl, Student’s t test. C, Depletion of mucins abrogates the inhibitory effect of ASPC-1 TSN. IL-12 and IL-10 production by DC exposed to ASPC-1 TSN (33% v/v) for 7 days and matured with CD40L. The same ASPC-1 TSN was depleted of MUC1 by repeated panning on anti-MUC1-coated TAG 72 plastic (Depl). Depletion of MUC1 decreased IL-10 production by DC and restored IL-12. Results are expressed as mean ± SD; n = 4; *, p < 0.05 vs Ctrl, Student’s t test.

 


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  		FIGURE 7. MUC1 expression by Capan-1 cell line inhibited maturation, suppressed IL-12 production, and increased the release of IL-10 by DC. A, The MUC1/Flag was inserted between HindIII and EcoRI in pCR3.1 vector and the construct was introduced into Capan-1 cell line by Fugene transfection. MUC1 expression was evaluated by anti-Flag mAb M2, anti-core protein mAb, and anti-TAG 72 mAb that recognizes the mucin-carrying sialylated-Tn epitope. B, Effect of transfected MUC1 on differentiating DC. DC were differentiated from monocytes with GM-CSF and IL-4 in the presence of untransfected Capan-1, pCR3.1 transfected Capan-1, or MUC1pCR3.1 transfected Capan-1. Monocytes were plated in the lower compartment and tumor cells in the upper compartment of a Transwell plate. At day 6, a CD40L-transfected cell line was added to induce DC maturation. Shown is the expression of CD83, CD40, and MHC II by DC. Representative results are shown for one of three experiments performed. C, IL-10 and IL-12 production by DC differentiated in the presence of untransfected Capan-1, pCR3.1 transfected Capan-1, or MUC1/pCR3.1 transfected Capan-1. Results are expressed as mean ± SD; n = 4; *, p < 0.05 vs Ctrl, Student’s t test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we show that a microenvironment characterized by the presence of tumor mucins affects the differentiation and maturation of monocyte DC, and results in APCs with a tolerogenic/regulatory cytokine profile. DC cocultured with some pancreatic tumor cell lines retain an immature phenotype after LPS or CD40L maturation, and behave differently compared with Ctrl-DC, in that they have low accessory cell activity, are unable to produce IL-12, secrete large amounts of IL-10, retain the ability to migrate to CCL19, and preferentially polarize IL-4+/regulatory effectors. In contrast, they retain the ability to up-regulate CCR7 and migrate to CCL19. The cytokine secretion profile (IL-12p70lowIL-10high), the potential responsiveness to CCL19 (ability to migrate to LNs), and the prominent polarization of T cell with an IL-4+/regulatory phenotype propose the DC obtained in a tumor microenvironment as regulatory DC (43, 47).

It is well established that tumor cells produce several factors that may negatively modulate the differentiation and maturation of DC (9, 10, 11, 12, 13). The cell lines used in this study did not produce relevant levels of IL-6, IL-10, and TGF-{beta}. Low levels of VEGF were produced by both ASPC-1 and Capan-1 tumor cells, but addition of blocking anti-VEGF Abs in the assays had no significant effect. Therefore, none of the above factors proved to be responsible for the tumor-associated inhibitory effect. In this study, we have identified MUC1 mucins released by tumor cells, in particular, mucins carrying sialyl-Tn glycoform, as responsible for the observed effect. This conclusion is supported by several pieces of evidence. The inhibitory tumor cell line ASPC-1 released higher levels of CA15-3-reacting MUC1 compared with the "control" Capan-1 cell line. MUC1 mucins depletion from ASPC-1 supernatants abrogated the augmented IL-10 production in DC and almost totally restored their ability to secrete IL-12. In addition, extracted MUC1 mucins produced by cancer cell lines, as well as CA15-3, dose-dependently increased IL-10 production in DC. To confirm the role of MUC1 mucins in inducing DC modification, we transfected the tumor cell line Capan-1 with MUC1. The transfection of Capan-1 with full-length MUC1 (mucin with 32 tandem repeats) resulted in the expression of a sialyl-Tn glycoform of this mucin, as demonstrated by TAG 72 positivity. MUC1 mucins expression by Capan-1 inhibited maturation, suppressed IL-12 production, and increased the release of IL-10 by DC. This experiment unambiguously identified MUC1 mucins as a factor responsible for the observed effects on DC.

The finding that some tumor-derived mucins deliver a negative signal on DC may be a novel strategy by which tumor cells escape immune surveillance. DC with an IL-12p70lowIL-10high phenotype do not promote a protective Th1-type anti-tumor immune response. Rather, as shown in this and other studies, DC producing high amounts of IL-10 induce T cell anergy (48, 49, 50). The fact that tumor-glycosylated mucins may play a relevant role in modulating DC function in cancer was already supported by indirect data. Anti-MUC1 immune responses in cancer patients are characterized by a low frequency of MHC-unrestricted CTL and a lower titer of IgM Abs. Tumor-derived MUC1 mucins glycosylated with short linear carbohydrates, isolated from patients’ sera or ascites, failed to prime Th cells. Moreover, in vitro priming of class I restricted CTL by DC loaded with tumor-derived MUC1 mucins was much less efficient than priming with DC loaded with the synthetic MUC1 peptide (51, 52). In mice, immunization with human MUC1 elicits a protective immune response only when an unglycosylated protein is used (53, 54, 55).

A role of MUC1 mucins as immunosuppressor was also reported in vivo in cancer patients. Expression of elevated MUC1 mucins is associated with poor prognosis and cancer progression in a variety of cancer types (56). Elevated levels of serum MUC1 mucins in patients with metastatic breast, colorectal, and ovarian cancer following immunotherapy are associated with poor survival and a lower anti-tumor immune response (34, 56). Cumulatively, all these results are consistent with an immunoregulatory role for MUC1 mucins.

MUC1 mucins can exist in many different glycoforms. In fact, the pool of O-glycan structures produced by a single cell is the product of complex biosynthetic processes, which are not template-guided and require the ordered action of multiple glycosyltransferases. Accordingly, O-glycan patterns are often cell and tissue specific and may differ substantially from one cell type to another. Cancer cells express aberrant forms of MUC1 mucins. In fact, the expression of distinct oligosaccharide structures, together with differential glycosylation of mucin core proteins, confers on tumor cells an enormous range of potential mucin forms. In our study, a clear characterization of the glycan structure of MUC1 mucins able to modulate DC function was not performed. In fact, we decided to use MUC1-transfected cells as a source of MUC1 mucins, because only "naked" recombinant or some glycoforms of a tandem repeat have been described (57, 58). Our data suggest that probably the critical interaction occurs between MUC1 mucin carbohydrates and C-type lectin receptors on DC, and, therefore, we considered the tumoral form of MUC1 with its unique glycans as the best source. This obviously did not permit us to identify the active glycan structure of MUC1 mucins, but the result obtained using anti-TAG 72 mAb suggested that MUC1 carrying sialylated-Tn epitope is the relevant one. A recombinant glycoform (with sialylated core 1 glycans occupying 90% of potential O-glycosylation sites) was actually tested in our system, and showed similar suppressive activity on DC (data not shown).

We recently reported that DC stimulated through the MR up-regulate IL-10 production and down-regulate IL-12 (45). The glycosylated mucin of tumor cells was a likely candidate as ligand of MR. MUC1 mucins have been shown to recognize MR (46). Mucins released by ASPC-1 cell line and other DC-modulating lines, but not those from the Ctrl cell lines Capan-1 and H766T, inhibited the binding of a specific mAb to MR. The same competition for MR-binding was observed when the extractive MUC1 mucins were used. A nonspecific hindrance is unlikely, as these mucins did not affect the recognition of a nonrelated molecule CD40 by a specific anti-CD40 mAb. Myeloid DC express several C-type lectin receptors specialized in the binding of carbohydrate-rich molecules (59). Therefore, we cannot exclude the possibility that tumor-derived mucins bind to receptors expressed on the surface of DC other than MR. However, the finding that the inhibitory activity of tumor cell lines is strongly associated with a MR-binding activity, together with the evidence that engagement of MR had similar effect on cytokine production (45), suggest that the inhibitory effect of tumor-derived mucins is at least in part mediated via interaction with the MR. Interestingly, competition experiments excluded an involvement of the C-type lectin receptor DC-SIGN.

In this study, DC cocultured with inhibitory tumor cell lines showed a decreased ability to polarize naive T cells into IFN-{gamma}-producing effectors, while IL-4-producing T cells were higher than with Ctrl-DC. It is commonly recognized that an IL-12-induced IFN-{gamma}-dominant immunity is crucial for the induction of a protective anti-tumor response (60, 61). A deviation from a type 1 to a type 2 cytokine response has been described in mouse tumor models (62, 63) and in some human neoplastic diseases (14, 15, 16, 17, 18, 19). We propose that DC in the tumor microenvironment, deficient in IL-12 and major producers of IL-10, are responsible for this altered Th1/Th2 balance. It should also be remembered that evidence has recently accumulated indicating that the carcinoma-associated Tn and sialyl-Tn Ags are also expressed by helminth and protozoan parasites (64). In rodents and in humans, parasite infections are associated with Th2 cytokine responses, eosinophilia, mastocytosis, and high levels of serum IgE. We can speculate that also in these conditions the mechanism described in our study could be responsible for the Th2 response.

In conclusion, we have shown that tumor-derived mucins, probably interacting with MR or other C-type lectin receptors on DC, affect the differentiation and maturation of monocyte DC and result in APCs with a tolerogenic/regulatory cytokine profile. These results shed new light on a novel mechanism of immune escape and open the way to new approaches aimed at restoring a protective anti-tumor response. Moreover, the possibility to obtain DC able to induce the development of T regulatory cells with suppressive activity may open potential novel applications in the field of transplant immunology.


   Acknowledgments
 
We thank Alberto Mantovani ("Mario Negri" Institute, Milan, Italy) and Ezio Bonifacio (San Raffaele Scientific Institute, Milan, Italy) for discussion and suggestions, and Giancarlo Bianchi ("Mario Negri" Institute) for chemotaxis experiments.


   Footnotes
 
1 This work was supported by grants from Ministry of Universities and Research (Cofin. 2002), Italian Association for Cancer Research, Telethon Italy (JT01), and the Juvenile Diabetes Research Foundation (JT01). Back

2 Address correspondence and reprint requests to Dr. Lorenzo Piemonti, Telethon-Juvenile Diabetes Research Foundation Center for {beta} Cell Replacement, San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milan, Italy. E-mail address: piemonti.lorenzo{at}hsr.it Back

3 Abbreviations used in this paper: DC, dendritic cell; CD40L, CD40 ligand; CCL, CC chemokine ligand; Ctrl, control; LN, lymph node; MFI, mean fluorescence intensity; MHC I, MHC class I; MHC II, MHC class II; MIP, macrophage-inflammatory protein; MR, mannose receptor; SIGN, specific ICAM-3-grabbing nonintegrin; TSN, tumor supernatant; VEGF, vascular endothelial growth factor; TAG, tumor-associated glycoprotein. Back

Received for publication February 17, 2004. Accepted for publication April 9, 2004.


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