- Open Access
Monocyte-derived dendritic cells loaded with a mixture of apoptotic/necrotic melanoma cells efficiently cross-present gp100 and MART-1 antigens to specific CD8+ T lymphocytes
© von Euw et al; licensee BioMed Central Ltd. 2007
- Received: 25 January 2007
- Accepted: 20 April 2007
- Published: 20 April 2007
In the present study, we demonstrate, in rigorous fashion, that human monocyte-derived immature dendritic cells (DCs) can efficiently cross-present tumor-associated antigens when co-cultured with a mixture of human melanoma cells rendered apoptotic/necrotic by γ irradiation (Apo-Nec cells).
We evaluated the phagocytosis of Apo-Nec cells by FACS after PKH26 and PKH67 staining of DCs and Apo-Nec cells at different times of coculture. The kinetics of the process was also followed by electron microscopy. DCs maturation was also studied monitoring the expression of specific markers, migration towards specific chemokines and the ability to cross-present in vitro the native melanoma-associated Ags MelanA/MART-1 and gp100.
Apo-Nec cells were efficiently phagocytosed by immature DCs (iDC) (55 ± 10.5%) at 12 hs of coculture. By 12–24 hs we observed digested Apo-Nec cells inside DCs and large empty vacuoles as part of the cellular processing. Loading with Apo-Nec cells induced DCs maturation to levels achieved using LPS treatment, as measured by: i) the decrease in FITC – Dextran uptake (iDC: 81 ± 5%; DC/Apo-Nec 33 ± 12%); ii) the cell surface up-regulation of CD80, CD86, CD83, CCR7, CD40, HLA-I and HLA-II and iii) an increased in vitro migration towards MIP-3β. DC/Apo-Nec isolated from HLA-A*0201 donors were able to induce >600 pg/ml IFN-γ secretion of CTL clones specific for MelanA/MART-1 and gp100 Ags after 6 hs and up to 48 hs of coculture, demonstrating efficient cross-presentation of the native Ags. Intracellular IL-12 was detected in DC/Apo-Nec 24 hs post-coculture while IL-10 did not change.
We conclude that the use of a mixture of four apoptotic/necrotic melanoma cell lines is a suitable source of native melanoma Ags that provides maturation signals for DCs, increases migration to MIP-3β and allows Ag cross-presentation. This strategy could be exploited for vaccination of melanoma patients.
- Melanoma Cell
- Melanoma Cell Line
- Gp100 Peptide
- Specific CTLs
Dendritic cells (DCs) are able to efficiently capture antigens (Ags) at their immature stage (iDC), process them and initiate immune responses upon interaction with lymphocytes . The specialized functions of mature DCs (mDCs) are essential to start T cell mediated immunity since they can prime naïve T cells. As DCs take up and process Ags and in response to various stimuli [2–6] they undergo a process of maturation and express large amounts of human leukocyte Ags (HLA) – peptide complexes at their surface. To achieve their function, DCs must arrive into the lymph nodes in response to several chemoattracting signals that bind specific cell surface receptors expressed by DCs during the maturation process, such as CCR7 (CC chemokine receptor) . Once DCs have arrived into secondary lymphoid organs, they can stimulate naïve T cells .
A common strategy used for vaccine preparation is to load DCs with exogenous peptides from tumor associated Ags (TAAs) on empty HLA class I molecules [9–11]. This approach, however, has the limitations of peptide restriction to a given haplotype and the induction of responses to only one or few defined Ags. In order to use a broader spectrum of known and yet unknown Ags for DCs loading, the approach of whole tumor cells is preferred. We and others have demonstrated that when murine DCs that had phagocytosed apoptotic B16 melanoma cells were used as vaccines, they were able to induce an effective, long-term protection against challenge with live B16 cells [12, 13].
Since the induction of CD8+ cytotoxic T lymphocytes (CTLs) appears to play a central role in the process of protective immunity, only cross-presentation of tumor Ags acquired from whole tumor cells would confer effective antitumor immunity. Several authors have demonstrated in murine models [14, 15] and in humans . that when DCs engulf apoptotic cells, Ags can be cross-presented for the generation of HLA class I/peptide-complexes, allowing the induction of specific CTLs. However, some conflicting findings have been reported in the human, such as the lack of DCs maturation upon phagocytosis of apoptotic cells [17–19], so the fate and immunogenic potential of DCs that have internalized melanoma apoptotic cells or their debris remain an open issue. Several studies have used tumor cells virally transduced with TAAs or tumor cells apoptotized after infection with recombinant viruses encoding melanoma associated Ags [20–23] but few of these have evaluated the specific cross-presentation of native-melanoma Ags present in apoptotic tumor cells.
While we were writing this manuscript, Palucka et al  published the results of a phase I clinical trial of a vaccine composed of DCs loaded with killed allogeneic melanoma cells which induced objective clinical responses and elicited MART-1 specific CD8+T cells in stage IV patients. Thus, the use of a mixture of apoptotic/necrotic allogeneic melanoma cell lines as a complex source of melanoma Ags for DCs cross-presentation could be further explored to validate and complement these findings in the clinical setting.
The induction of apoptosis by different methods may produce a mixture of apoptotic/late apoptotic and/or necrotic tumor cells that could provide different signals necessary for DCs maturation as well as for CTL priming [4, 25]. To investigate these points we produced and characterized human monocyte-derived DCs and co-incubated them with a mixture of four melanoma cell lines, rendered apoptotic/necrotic by γ radiation. Upon phagocytosis, we analyzed DCs maturation through the expression of surface markers and the decrease of the endocytic capacity, in vitro migration in response to chemokines and, most importantly, demonstrated their ability to cross-present native tumor Ags to specific CTLs in vitro.
Cell lines and clones
Four human melanoma cell lines were used in this study. Mel-XY1, Mel-XY2, Mel-XY3 and Mel-XX4 were cultured in melanoma medium (Dulbecco's Modified Eagle Medium: nutrient Mixture F12 (1:1) supplemented with 2 mM glutamine, 20 nM sodium selenite, 100 μM ascorbic acid, 0.3 mg/ml galactose, 0.15 mg/ml sodium pyruvate and 5 μg/ml insulin, 100 IU/ml penicillin, 10 μg/ml streptomycin) plus 10% fetal bovine serum (FBS) (Natocor, Córdoba, Argentina) in a GMP core facility at the Centro de Investigaciones Oncológicas-FUCA.
CTL clones (HLA A*0201 restricted) specific for MelanA/MART-1 (M27: AAGIGILTV) and gp100 (G154: KTWGQYWQV) Ags were expanded in RPMI medium in 14-day cycles by using 30 ng/ml anti-CD3 antibody (OKT-3, BD Biosciences) and serial 300 UI/ml IL-2 (Chiron BV, Amsterdam, Netherlands) every 3 days plus 10% heat-inactivated AB human serum and antibiotics . Cell lines were periodically tested to be mycoplasma-free.
Preparation of tumor apoptotic/necrotic cells
Apoptotic/necrotic tumor cells (Apo-Nec cells) were prepared as a batch of four cell lines from master cell banks after safety testing for mycoplasma, viruses and bacteria. The complete HLA haplotype of the four melanoma cell lines that compose Apo-Nec cells is: MEL-XY1: A*0201, A23; B18, B37; DR7, DR11, DR52, DR53; MEL XY2: A30, A33; B18, B65; DR1, DR1; MEL-XY3: A*0201, A23; B18, B18; DR11, DR13, DR52 and MEL-XX4: A24 (9), A33 (19); B18, B65 (14); DR1, DR11. All cell lines express Tyrosinase, MAGE 3, MelanA/MART-1 (except MEL-XX4 cell line), TRP-2, gp100, GD2, GD3 and NY-ESO-1 by RT-PCR and/or immunocytochemistry (unpublished results). After gamma irradiation at 70 Gy (Siemens Lineal Accelerator, Instituto Alexander Fleming, Buenos Aires, Argentina), the cells were frozen (50% DMEM, 40% human albumin and 10% DMSO) in liquid nitrogen until use. Cells were then thawed and plated in melanoma medium plus 10% FBS to complete the apoptotic process. After 72 hs the cells were detached from the flasks, washed, counted and resuspended in fresh serum-free AIM-V™ Medium (Therapeutic grade, GIBCO, Invitrogen Corporation, Grand Island, N.Y). Apoptosis and necrosis were assessed by Annexin-V and Propidium iodide (PI) binding (Annexin-V apoptosis detection kit, BD Biosciences, San José, CA) and Flow Cytometric (FACS) analysis (FACSCalibur, BD Biosciences, San José, CA). A soft agar clonogenic assay performed in sextuplicate (1.5 × 104 cells/well) was used to test that irradiated cells have lost their proliferation ability compared to non-irradiated control cells .
Generation of DCs from monocytes
DCs were generated from buffy-coats or leukapheresis products obtained from healthy donors at the Hemotherapy Department of the Instituto Alexander Fleming. Peripheral blood mononuclear cells (PBMCs) were purified by Fycoll-Hypaque density centrifugation. PBMCs were resuspended in AIM-V medium and allowed to adhere to 0.22 μm filter-capped culture flasks (TPP, Germany). After 2 hs at 37°C, the non-adherent cells were removed, and adherent monocytes were subsequently cultured for 5 days in AIM-V supplemented with 800 U/ml rhuGM-CSF (kindly provided by Dr. Esteban Corley, PC-Gen, Buenos Aires, Argentina) and 50 ng/ml IL-4 (Peprotech, Mexico). Phenotypic changes were monitored by light microscopy and FACS. To induce control DCs maturation, 2 μg/ml LPS (Lipopolysaccharide from E. coli J5, Sigma, St. Louis, CA) was added and the cells were further cultured for 48 hs.
Characterization of DCs phenotype was performed at the immature state (iDCs) and after Apo-Nec phagocytosis by staining 5 × 105 cells with fluorochrome-labeled antibodies (Abs) against CD14, CD11c, CD1a, HLA-DR/DP/DQ (HLA-II), CD80, CD86, CD83, CD40, HLA-ABC (HLA-I) and CCR7 (BD Biosciences, San Jose, CA), and FACS analysis was performed with CELLQuest software (Becton Dickinson, San Jose, CA). The corresponding isotype-matched controls used were FITC IgG1, FITC IgG2a and PE Rat IgG2a, (BD Biosciences, San Jose, CA.) Also, to analyze CD83 expression in double-positive PKH26/PKH67 DCs, we used an unlabeled anti-CD83 mAb (IgG1) and the corresponding isotype matched control, revealed with an anti-mouse IgG1-PerCP (BD Biosciences, San José, CA).
DCs endocytosis was evaluated by incubating 1 × 106 cells with 1 mg/ml FITC-dextran (FITC-Dx) (Sigma, St Louis, CA) for 30 min at 37°C. After washing with Phosphate Buffered Saline, cells were analyzed by FACS. Controls included tubes incubated with FITC-Dx at 4°C to inhibit the endocytic process and a basal uptake performed at 0 time point. Uptake was quantified by FACS analysis (10,000 cells per point).
DCs phagocytosis of apoptotic/necrotic tumor cells
Apo-Nec cells (72 hs after irradiation plus culture) were co-cultured with iDCs (five days culture) at different ratios (1:1, 2:1 and 3:1) in fresh AIM-V medium for different time points. In some experiments DCs were dyed red with PKH26 and Apo-Nec cells were dyed green with PKH67-GL (Sigma, St. Louis, CA). After co-culture, FACS analysis was performed and DCs phagocytosis of Apo-Nec cells was defined by the percentage of double-positive cells. Appropriate controls were performed to set the cytometer for each color. A control for non-phagocytic binding of Apo-Nec cells to DCs was set by incubating the cells at 4°C for the same time points.
in vitro DCs migration
DCs migration was assessed in vitro before and after co-culture with Apo-Nec cells, using a 48 wells chemotaxis chamber (AP 48 Neuroprobe Inc., Gaithersburg, MD). In the lower compartment, 10 ng/ml MIP-1α or MIP-3β (Peprotech, Rocky Hill, NJ, USA) were placed diluted in RPMI. Also, random migration was assessed placing RPMI in the lower chamber. DCs were seeded in the upper chamber (3 × 104 DCs/well) in RPMI. Between the upper and lower chamber a 5 μm pore polycarbonate membrane (Neuroprobe, Inc., Bethesda, MD) was placed. After 90 min at 37°C, the cells in the upper face of the membrane were scrapped out and the migrating cells adhered to the lower face of the membrane were stained with Giemsa. Membranes were air-dried, mounted onto a glass slide with Canadax and the cells were counted under the microscope. Five medium-power (400×) fields/well and 3 wells/condition were analyzed. Statistical analysis was performed using Student's t-Test.
The phagocytic process was also studied by electron microscopy. Co-cultured samples were fixed at different time points with 2.5% glutaraldehyde in 0.1 M phosphate buffer pH 7.4, and then post-fixed in 1% OsO4, washed twice with distilled water and contrasted with 5% uranyl acetate for 2 hs. After washing and dehydratation, samples were embedded in Durkupan. Ultrathin slices (70–90 nm) were mounted in copper grids and contrasted with Reynold's lead citrate. Grids were analyzed under a transmition electron microscope Zeiss 109. Alternatively, to obtain whole-cell pictures, ultrathin slides (0.5 μm) were obtained in a ultramicrotome (Reichert-Jung), stained with 0.4% toluidine blue in 0.1 M carbonate buffer pH 7.4, mounted in Durkupan and analyzed under light microscopy (1000× magnification). Pictures were obtained with a Sony Cybershot Digital camera (5 megapixels) and images were further processed with Adobe Photoshop 6.0.
in vitro cross priming assay – IFN-γ secretion
CD14+ monocytes were purified to 98% from a HLA A*0201 donor using anti-CD14 microbeads (Miltenyi Biotec, Germany) and were differentiated to iDCs by 5 days culture as described above. iDCs (6 × 105) were loaded with Apo-Nec cells (2 × 105) for 6, 12, 24 and 48 hs and incubated overnight with MelanA/MART-1 or gp100 specific CTL clones (2 × 105 cells) in 1 ml AIM-V medium. IFN-γ secretion to the supernatant was determined in triplicate by ELISA (OptEIA IFN-γ, Pharmingen BD Biosciences, San Diego, CA) according to the manufacturer's suggestions. A calibration curve was performed for each experiment and the sample concentration was calculated by log-log regression analysis using Cembal 2.2 software. Controls for this experiment included DCs loaded for 3 hs at 37°C with 20 μg/ml MART-1 or gp100 peptides plus 3 μg/ml β2-microglobulin, Ag-expressing live melanoma cell lines HLA A*0201 positive (positive controls) or DCs loaded with the non-specific peptides and Ag-expressing live melanoma cell lines HLA A*0201 negative (negative controls). G154 and M27 clones were also incubated with Apo-Nec cells and Apo-Nec Mel-XY2 (HLA*0201 negative, MelanA/MART-1 positive), and DCs alone.
Measurement of intracytoplasmatic IL-10 and IL-12 cytokines
DCs were cocultured with Apo-Nec cells in a 3:1 ratio after labeling with PKH26 and PKH67 respectively, as described above. At 6, 12, 24 and 48 hs post coculture intracellular cytokines were accumulated by additional 8 hs-treatment with Brefeldin A (Golgi Plug, BD Biosciences, San José CA). After that, the cells were permeabilized with 0.05% saponin and stained with anti-IL10 (rat IgG2a)-APC and anti-IL-12 (p40–p70, mouse, IgG1)-PerCp (BD Biosciences, San José, CA). Isotype matched controls were also included. For FACS analysis the double-stained PKH26/PKH67 population was gated and the cytokines were evaluated in a four-color experiment. DC/Apo-Nec cells were compared to non-co-cultured DCs and Apo-Nec cells at each time point.
Gamma-irradiation induced apoptosis of melanoma cell lines
Generation and characterization of DCs
To avoid deviation of the immune response to FBS-derived Ags, a serum-free culture protocol for in vitro generation of human DCs from PBMCs was developed. We obtained 5 × 106 DCs from 1 × 108 freshly plated PBMCs, after depletion of lymphocytes, which did not adhere to plastic culture flasks, in the presence of rhGM-CSF and IL-4. When cryopreserved PBMCs or leukapheresis products were plated under the same conditions, DCs yield was lower (3 × 106 DCs from 1 × 108 MN cells).
On day 5, aggregates of cells with the typical morphological features of DCs were harvested and phenotypically characterized. Control mDCs were obtained by adding 2 μg/ml LPS on day 5 and further incubating for 48 hs. Morphological changes during DCs maturation were observed by light microscopy and confirmed by FACS.
iDCs efficiently phagocyte Gamma irradiation induced apoptotic/necrotic melanoma cells
Phagocytosis of apoptotic/necrotic melanoma cells induces DC maturation
DCs maturation was also evaluated by the decrease of DCs endocytosis after Apo-Nec cells phagocytosis (Figure 4C). FITC-Dx endocytosis was maximal in iDCS, 99 ± 5% uptake (Mean: 224), decreased to 31 ± 3% (Mean: 19) in LPS-maturated DCs and to 33 ± 5% (Mean: 11) in DC/Apo-Nec cells.
DCs loaded with melanoma apoptotic/necrotic cells express CCR7 and migrate towards MIP-3 beta in vitro
DCs loaded with apoptotic/necrotic melanoma cells cross-present MelanA/MART-1 and gp100 Ags to specific T CD8+ cells
Evaluation of Intracytoplasmatic IL-10 and IL-12
There is now considerable experimental evidence that dying cells are capable of transferring Ags to the immune system for the induction of T cell immunity [19, 28, 29]. Albert et al first demonstrated in the murine model that apoptotic material could be processed and cross-presented by DCs to stimulate specific HLA-restricted CD8+ T cells . In this study, the use of whole apoptotic/necrotic tumor cells to load DCs exploits both the advantage of maturation signals delivered by necrotic cells as proposed by Gallucci et al  and the optimal Ag processing and presentation in HLA class I and class II molecules by DCs  of a vast repertoire of known as well as yet unknown Ags from apoptotic cells for the induction of anti-tumor immune responses. Some controversy has arisen since several authors found that the uptake of pure or early apoptotic tumor cells did not induce proper DCs maturation [17, 18]. However, these studies differ from the current one in several aspects: i) we used a mixture of four apoptotic/necrotic melanoma cell lines expressing several native melanoma-associated Ags instead of the single cell line approach or virally transduced tumor cells expressing exogenous Ags [21, 22], ii) we used serum-free medium instead of FCS or autologous serum to generate monocyte-derived DCs , iii) the method used for tumor Ag preparation consists in gamma irradiation (70 Gy) and 72 hs culture in melanoma medium containing FBS as compared to higher energy radiation and culture in serum free-medium (producing mainly necrotic cells), UVB exposure or apoptosis induced by viral infection or by Fas pathway [17, 33, 34].
Previous reports [17, 18] showed that early apoptotic melanoma cells or pure apobodies failed to induce mDCs, and either TNF-α, Poly I:C or cytokine cocktails were necessary to achieve DCs maturation and Ag presentation. In our case, phagocytosis of the mixture of melanoma cell lines used to load iDCs was enough to generate mDCs without addition of other stimuli. According to the results reported by Sauter et at  we took advantage of DCs maturation induced by necrotic tumor cells present in the mixture of Apo-Nec cell lines, as well as DCs ability of antigen processing and cross-presentation for CTL priming due to the apoptotic cells.
Using this particular melanoma cell lines mixture we wanted to address the question if the uptake of Apo-Nec cells could allow native TAAs to be processed to peptides by iDCs, mDCs and cross-present them to specific CTLs. As we have shown here, high levels of IFN-γ were secreted by M27 and G154 CTL clones after DC/Apo-Nec stimulation and, more importantly, M27 clone was also stimulated after incubation with DCs that have phagocytosed HLA-A*0201 negative MelanA/MART-1+ gp100+ Apo-Nec cells. This results clearly demonstrated that DCs have processed MelanA/MART-1 Ag taken up from the tumor cells and presented it to M27 clone in their own HLA-A*0201 context. As early as 6 hs after DCs loading with Apo-Nec, these cells could efficiently induce IFN-γ release, and we were able to measure CTL cross-presentation even 72 hs post DC/Apo-Nec co-culture. Several authors have identified gp100 as a regression Ag, since the induction of anti-gp100 immunity correlated with the regression of documented metastases in melanoma [25, 35]. Besides, anti-MelanA/MART-1 CD8+T lymphocytes have also been detected in melanoma patients by tetramer staining and ELISPOT, correlating with clinical outcome and regressions . Labarriere et al  reported that the use of purified melanoma apoptotic bodies to load DCs plus maturation with cytokines, efficiently cross-primed CTLs specific for NA-17A Ag but not for MelanA/MART-1 Ag. The authors could not detect MelanA/MART-1 epitopes in apobodies using a MelanA/MART-1 specific mAb. In our case, not only DCs matured after phagocytosis of Apo-Nec cells but the induction of IFN-γ secretion by a CTL clone specific for MelanA/MART-1 peptide was found.
Thus, our results suggest that a vaccine such as DC/Apo-Nec has the potential to initiate an immune response specific for MelanA/MART-1 and gp100 Ags and probably for other Ags expressed by these cells. Recently, Palucka et al . have assayed in a phase I clinical trial a vaccine composed of DCs loaded with killed allogeneic melanoma cells demonstrating objective clinical responses and MART-1 specific CD8+T cell immunity. However, in this study the authors used a single HLA-A*0201 negative allogeneic melanoma cell line killed after a combination of TNF treatment, γ irradiation and culture in serum-free medium, plus the addition of CD40L to activate DCs. Our results further support the use of apoptotic/necrotic allogeneic tumor cells as a complex source of multiple melanoma native Ags to load DCs and show that a good maturation signal could be obtained with this particular mixture of melanoma cells, which also allows the cross-presentation of melanoma Ags to specific CTLs.
As we have demonstrated here, cross-presentation for MelanA/MART-1 and gp-100 Ags was achieved by DCs that have phagocytosed Apo-Nec cells but not by Apo-Nec cells themselves, since Apo-Nec cells (mixture) or HLA-A*0201 positive Apo-Nec cells (MEL-XY3 irradiated cells) were not able to induce INF-γ secretion separately. We have also observed that Apo-Nec cells progressively lost their HLA-A*0201 surface expression after irradiation and that their ability to present MelanA/MART-1 and gp100 peptides to CTLs decreased concomitantly (not shown). These results suggest that Apo-Nec cells are not presenting the peptides to CTLs by themselves but, after their phagocytosis, processed peptides are being cross-presented to CD8+ T cells by DCs. A recent report by Blanchére et al.  suggested in a murine model that apoptotic cells may be critical in processing Ags for cross-presentation, in essence by pre-selection of immunologically important antigenic determinants. In this view, our results in the human setting further support this hypothesis, since tumor dying cells (mainly apoptotic cells) can be used as a source of processed tumor determinants for DCs loading and cross-presentation to CTLs. Furthermore, presentation by DCs of Ags generated in apoptotic melanoma cells has the potential benefit that presentation via HLA class II may generate helper epitopes that support the development of specific CD4+ lymphocytes that might be important for antitumoral immunity. We cannot address if Ag peptides are being processed into Apo-Nec cells and then taken up by DCs and presented in the HLA class I context or if DCs have processed them after Apo-Nec phagocytosis. Besides, tumor-derived exosomes loaded onto DCs have been shown to trigger MART-1 melanoma Ag cross-presentation to specific CTLs  Although we used washed Apo-Nec cells resuspended in fresh AIM-V medium in all experiments and a differential ultracentrifugation of culture supernatants is required to obtain tumor-derived exosomes, we cannot exclude the contribution of exosomes that might be released by Apo-Nec cells during the 48 hs co-culture with DCs. Nevertheless, our main objective has been to assess if this particular mixture of Apo-Nec cells (either entire or their fragments/debris after gamma irradiation) was able to be phagocytosed by iDCs, induce iDCs maturation, migration and cross-presentation of native tumor peptides to specific CD8+ T cells.
We have also evaluated DC/Apo-Nec cells migration to MIP-3β as a measure of their potential homing to lymph nodes. Upon phagocytosis, DCs must reach the lymph nodes in response to chemokine concentration gradients such as MIP-3β in order to prime naïve T cells. It was important to asses if DC/Apo-Nec could respond in vitro to MIP-3β. We found that like fully mature LPS-treated DCs, DC/Apo-Nec cells up-regulated MIP-3β receptor (CCR7) and efficiently migrated in vitro to MIP-3β but not to MIP-1α. Our results are coincident with those reported by Hirao et al, who found specific DCs migration to MIP-3β in vitro and in vivo and CCR7 induction after phagocytosis of UV-treated fibrosarcoma cells .
The production of the pro-inflammatory cytokine IL-12 requires two signals: IFN-γ and a maturation signal provided by CD40 ligation (CD40L) or LPS. Recently, Xu et al [39, 40] have proposed that Toll – like receptor 8 provides a priming signal for high production of IL-12. Production of IL-12 and IL-10 influences DCs maturation and the induction of a potent immune presentation to T cells [40, 41]. Accordingly, we found that upon phagocytosis of Apo-Nec intracellular pro-inflammatory IL-12 transiently increased while IL-10 did not change in DC/Apo-Nec cells.
We believe that our results complement the existing reports about the use of apoptotic and necrotic cells to load iDCs, and demonstrated that they induce DCs maturation, up-regulate CCR7 allowing their migration to MIP-3β, and efficiently process native melanoma Ags and cross-present them to specific CTLs. This particular mixture of apoptotic/necrotic cell lines phagocytosed by iDCs should be tested as a vaccine for melanoma patients since it could provide mature melanoma Ag-loaded DCs for efficient cross-priming to elicit anti-tumor immunity.
We used a mixture of four melanoma apoptotic/necrotic cell lines as a source of native Ags to load human monocyte-derived iDCs. After phagocytosis, we found that Apo-Nec cells induced DCs maturation without addition of further stimuli, increased in vitro migration in response to MIP-3β chemokine and intracellular IL-12 production. Most importantly, we demonstrated the ability of DC/Apo-Nec cells to cross-present native tumor Ags to Ag-specific CTLs in vitro. We suggest that our results with DC/Apo-Nec should be explored as a vaccination strategy in melanoma patients.
This work has been supported by grants from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), Universidad de Buenos Aires, Fundación Sales, Fundación para la Investigación y Prevención del Cáncer (FUCA), Fundación Pedro F. Mosoteguy and Fundación María Calderón de la Barca, Argentina. CY and YQL are Damon Runyon-Lilly clinical investigators supported by: "Rippel Foundation and Burroughs Wellcome Fund". JM and RW are members of CONICET, EMvE is a fellow of Universidad de Buenos Aires and MMB, EML and MB are fellows of Fundación Sales; DF is a fellow of Agencia Nacional de Promoción Científica y Tecnológica. We thank the Hemotherapy Department of the Instituto Alexander Fleming for providing blood from healthy volunteers. We thank Dr Alicia Brusco and Mariana Ravasio from the Instituto de Biología Celular y Neurociencias "Profesor Eduardo de Robertis" LANAIS-MIE, Facultad de Medicina, Universidad de Buenos Aires, for the electron microscopy processing.
- Banchereau J, Steinman R: Dendritic cells and the control of immunity. Nature. 1998, 392: 245-252. 10.1038/32588.View ArticlePubMedGoogle Scholar
- Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, Pulendran B, Palucka K: Immunobiology of dendritic cells. Annu Rev Immunol. 2000, 18: 767-811. 10.1146/annurev.immunol.18.1.767.View ArticlePubMedGoogle Scholar
- De Smedt T, Pajal B, Muraille E, Lespagnard L, Heinen E, De Baetselier P, Leo O, Moser M: Regulation of dendritic cell numbers and maturation by lipopolysaccharide in vivo. J Exp Med. 1996, 184: 1413-1424. 10.1084/jem.184.4.1413.View ArticlePubMedGoogle Scholar
- Sparwasser T, Koch ES, Vabulas RM, Heeg K, Lipford GB, Ellwart JW, Wagner H: Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells. Eur J Immunol. 1998, 28: 2045-2054. 10.1002/(SICI)1521-4141(199806)28:06<2045::AID-IMMU2045>3.0.CO;2-8.View ArticlePubMedGoogle Scholar
- Verdijk RM, Mutis T, Esendam B, Kamp J, Melief CJ, Brand A, Goulmy E: Polyriboinosinic polyribocytidylic acid (poly (I: C)) induces stable maturation of functionally active human dendritic cells. J Immunol. 1999, 163: 57-61.PubMedGoogle Scholar
- Caux C, Massacrier C, Vanbervliet B, Dubois B, Van Kooten C, Durand I, Banchereau J: Activation of human dendritic cells through CD40 cross-linking. J Exp Med. 1994, 180: 1263-1272. 10.1084/jem.180.4.1263.View ArticlePubMedGoogle Scholar
- Penna G, Vulcano M, Sozzani S, Adorini L: Differential migration behavior and chemokine production by myeloid and plasmacytoid dendritic cells. Hum Immunol. 2002, 63: 1164-1171. 10.1016/S0198-8859(02)00755-3.View ArticlePubMedGoogle Scholar
- Mellman I, Steinman RM: Dendritic cells: specialized and regulated antigen processing machines. Cell. 2001, 106: 255-258. 10.1016/S0092-8674(01)00449-4.View ArticlePubMedGoogle Scholar
- Nestle FO, Alijagic S, Gilliet M, Sun Y, Grabbe S, Dummer R, Burg G, Schadendorf D: Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat Med. 1998, 4: 328-332. 10.1038/nm0398-328.View ArticlePubMedGoogle Scholar
- Mayordomo JI, Zorina T, Storkus WJ, Zitvogel L, Celluzzi C, Falo LD, Melief CJ, Ildstad ST, Kast WM, Deleo AB: Bone marrow-derived dendritic cells pulsed with synthetic tumour peptides elicit protective and therapeutic antitumour immunity. Nat Med. 1995, 1: 1297-1302. 10.1038/nm1295-1297.View ArticlePubMedGoogle Scholar
- Celluzzi CM, Mayordomo JI, Storkus WJ, Lotze MT, Falo LD: Peptide-pulsed dendritic cells induce antigen-specific CTL-mediated protective tumor immunity. J Exp Med. 1996, 183: 283-287. 10.1084/jem.183.1.283.View ArticlePubMedGoogle Scholar
- Goldszmid RS, Idoyaga J, Bravo AI, Steinman R, Mordoh J, Wainstok R: Dendritic cells charged with apoptotic tumor cells induce long-lived protective CD4+ and CD8+ T cell immunity against B16 melanoma. J Immunol. 2003, 171: 5940-5947.View ArticlePubMedGoogle Scholar
- Scheffer SR, Nave H, Korangy F, Schlote K, Pabst R, Jaffee EM, Manns MP, Greten TF: Apoptotic, but not necrotic, tumor cell vaccines induce a potent immune response in vivo. Int J Cancer. 2003, 103: 205-211. 10.1002/ijc.10777.View ArticlePubMedGoogle Scholar
- Albert ML, Pearce SF, Francisco LM, Sauter B, Roy P, Silverstein RL, Bhardwaj N: Immature dendritic cells phagocytose apoptotic cells via alphavbeta5 and CD36, and cross-present antigens to cytotoxic T lymphocytes. J Exp Med. 1998, 188: 1359-1368. 10.1084/jem.188.7.1359.PubMed CentralView ArticlePubMedGoogle Scholar
- Chen Z, Moyana T, Saxena A, Warrington R, Jia Z, Xiang J: Efficient antitumor immunity derived from maturation of dendritic cells that had phagocytosed apoptotic/necrotic tumor cells. Int J Cancer. 2001, 93: 539-548. 10.1002/ijc.1365.View ArticlePubMedGoogle Scholar
- Shaif-Muthana M, McIntyre C, Sisley K, Rennie I, Murray A: Dead or alive: immunogenicity of human melanoma cells when presented by dendritic cells. Cancer Res. 2000, 60: 6441-6447.PubMedGoogle Scholar
- Pietra G, Mortarini R, Parmiani G, Anichini A: Phases of apoptosis of melanoma cells, but not of normal melanocytes, differently affect maturation of myeloid dendritic cells. Cancer Res. 2001, 61: 8218-8226.PubMedGoogle Scholar
- Labarriere N, Bretaudeau L, Gervois N, Bodinier M, Bougras G, Diez E, Lang F, Gregoire M, Jotereau F: Apoptotic body-loaded dendritic cells efficiently cross-prime cytotoxic T lymphocytes specific for NA17-A but not for Melan A/Mart-1 antigen. Int J Cancer. 2002, 101: 280-286. 10.1002/ijc.10605.View ArticlePubMedGoogle Scholar
- Demaria S, Santori FR, Ng B, Liebes L, Formenti SC, Vukmanovic S: Select forms of tumor cell apoptosis induce dendritic cell maturation. J Leukoc Biol. 2005, 77: 361-368. 10.1189/jlb.0804478.View ArticlePubMedGoogle Scholar
- Moehler MH, Zeidler M, Wilsberg V, Cornelis JJ, Woelfel T, Rommelaere J, Galle PR, Heike M: Parvovirus H-1-induced tumor cell death enhances human immune response in vitro via increased phagocytosis, maturation, and cross-presentation by dendritic cells. Hum Gene Ther. 2005, 16: 996-1005. 10.1089/hum.2005.16.996.View ArticlePubMedGoogle Scholar
- Russo V, Tanzarella S, Dalerba P, Rigatti D, Rovere P, Villa A, Bordignon C, Traversari C: Dendritic cells acquire the MAGE-3 human tumor antigen from apoptotic cells and induce a class I-restricted T cell response. Proc Natl Acad Sci USA. 2000, 97: 2185-2190. 10.1073/pnas.040540197.PubMed CentralView ArticlePubMedGoogle Scholar
- Motta I, Andre F, Lim A, Tartaglia J, Cox WI, Zitvogel L, Angevin E, Kourilsky P: Cross-presentation by dendritic cells of tumor antigen expressed in apoptotic recombinant canarypox virus-infected dendritic cells. J Immunol. 2001, 167: 1795-1802.View ArticlePubMedGoogle Scholar
- Gaipl US, Beyer TD, Baumann I, Voll RE, Stach CM, Heyder P, Kalden JR, Manfredi A, Herrmann M: Exposure of anionic phospholipids serves as anti-inflammatory and immunosuppressive signal – implications for antiphospholipid syndrome and systemic lupus erythematosus. Immunobiology. 2003, 207: 73-81. 10.1078/0171-2985-00217.View ArticlePubMedGoogle Scholar
- Palucka AK, Ueno H, Connolly J, Kerneis-Norvell F, Blanck JP, Johnston DA, Fay J, Banchereau J: Dendritic cells loaded with killed allogeneic melanoma cells can induce objective clinical responses and MART-1 specific CD8+ T-cell immunity. J Immunother. 2006, 29: 545-557. 10.1097/01.cji.0000211309.90621.8b.View ArticlePubMedGoogle Scholar
- Rohn TA, Schadendorf D, Sun Y, Nguyen XD, Roeder D, Langen H, Vogt AB, Kropshofer H: Melanoma cell necrosis facilitates transfer of specific sets of antigens onto MHC class II molecules of dendritic cells. Eur J Immunol. 2005, 35: 2826-2839. 10.1002/eji.200526299.View ArticlePubMedGoogle Scholar
- Yee C, Thompson JA, Byrd D, Riddell SR, Roche P, Celis E, Greenberg PD: Adoptive T cell therapy using antigen-specific CD8+ T cell clones for the treatment of patients with metastatic melanoma: in vivo persistence, migration, and antitumor effect of transferred T cells. Proc Natl Acad Sci USA. 2002, 99: 16168-16173. 10.1073/pnas.242600099.PubMed CentralView ArticlePubMedGoogle Scholar
- Hamburger AW: The human tumor clonogenic assay as a model system in cell biology. Inter J Cell Cloning. 1987, 5: 89-107.View ArticleGoogle Scholar
- Berard F, Blanco P, Davoust J, Neidhart-Berard EM, Nouri-Shirazi M, Taquet N, Rimoldi D, Cerottini JC, Banchereau J, Palucka AK: Cross-priming of naive CD8 T cells against melanoma antigens using dendritic cells loaded with killed allogeneic melanoma cells. J Exp Med. 2000, 192: 1535-1544. 10.1084/jem.192.11.1535.PubMed CentralView ArticlePubMedGoogle Scholar
- Ferlazzo G, Semino C, Spaggiari GM, Meta M, Mingari MC, Melioli G: Dendritic cells efficiently cross-prime HLA class I-restricted cytolytic T lymphocytes when pulsed with both apoptotic and necrotic cells but not with soluble cell-derived lysates. Int Immunol. 2000, 12: 1741-1747. 10.1093/intimm/12.12.1741.View ArticlePubMedGoogle Scholar
- Albert ML, Sauter B, Bhardwaj N: Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs. Nature. 1998, 392: 86-89. 10.1038/32183.View ArticlePubMedGoogle Scholar
- Gallucci S, Lolkema M, Matzinger P: Natural adjuvants: endogenous activators of dendritic cells. Nature Medicine. 1999, 5: 1249-1255. 10.1038/15200.View ArticlePubMedGoogle Scholar
- Sauter B, Albert ML, Francisco L, Larsson M, Somersan S, Bardwaj N: Consequences of cell death: exposure to necrotic tumor cells, but not Primary Tissue cells or apoptotic cells, induces the maturation of Immunostimulatory Dendritic cells. J Exp Med. 2000, 191: 423-433. 10.1084/jem.191.3.423.PubMed CentralView ArticlePubMedGoogle Scholar
- Jarnjak-Jankovic S, Pettersen RD, Saeboe-Larssen S, Wesenberg F, Gaudernack G: Evaluation of dendritic cells loaded with apoptotic cancer cells or expressing tumour mRNA as potential cancer vaccines against leukemia. BMC Cancer. 2005, 5-20.Google Scholar
- Di Nicola M, Napoli S, Anichini A, Mortarini R, Romagnoli L, Magni M, Matteucci P, Baldassarri P, Bersani I, Carlo-Stella C, Gianni AM: Dendritic cell viability is decreased after phagocytosis of apoptotic tumor cells induced by staurosporine or vaccinia virus infection. Haematologica. 2003, 88: 1396-1404.PubMedGoogle Scholar
- Linette GP, Zhang D, Hodi FS, Jonasch EP, Longerich S, Stowell CP, Webb IJ, Daley H, Soiffer RJ, Cheung AM, Eapen SG, Fee SV, Rubin KM, Sober AJ, Haluska FG: Immunization using autologous dendritic cells pulsed with the melanoma-associated antigen gp100-derived G280-9V peptide elicits CD8+ immunity. Clin Cancer Res. 2005, 11: 7692-7699. 10.1158/1078-0432.CCR-05-1198.View ArticlePubMedGoogle Scholar
- Andersen MH, Keikavoussi P, Broker EB, Schuler-Thurner B, Jonassen M, Sondergaard I, Straten PT, Becker JC, Kampgen E: Induction of CTL responses in melanoma patients by dfendritic cell vaccination: cessation of CTL responses is associated with disease progression. Int J Cancer. 2001, 94: 820-10.1002/ijc.1536.View ArticlePubMedGoogle Scholar
- Blachere NE, Darnell RB, Albert ML: Apoptotic cells deliver processed antigen to dendritic cells for cross-presentation. PLoS Biol. 2005, 3: e185-10.1371/journal.pbio.0030185.PubMed CentralView ArticlePubMedGoogle Scholar
- Wolfers J, Lozier A, Raposo G, Regnault A, Thery C, Masurier C, Flament C, Pouzieux S, Faure F, Tursz T, Angevin E, Amigorena S, Zitvogel L: Tumor-derived exososmes are a source of shared tumor rejection antigens for CTL cross-priming. Nature Medicine. 2001, 7: 297-303. 10.1038/85438.View ArticlePubMedGoogle Scholar
- Hirao M, Onai N, Hiroishi K, Watkins SC, Matsushima K, Robbins PD, Lotze MT, Tahara H: CC chemokine receptor-7 on dendritic cells is induced after interaction with apoptotic tumor cells: critical role in migration from the tumor site to draining lymph nodes. Cancer Res. 2000, 60: 2209-2217.PubMedGoogle Scholar
- Xu S, Koldovsky U, Xu M, Wang D, Fitzpatrick E, Son G, Koski G, Czerniecki : High-avidity antitumor T-cell generation by toll receptor 8-primed, myeloid- derived dendritic cells is mediated by IL- the induction of IL-12 by TLR8 12 production. BJ Surgery. 2006, 140: 170-178. 10.1016/j.surg.2006.03.006.View ArticleGoogle Scholar
- Caetano Reis e Sousa: Dendritic cells in a mature age. Nature Rev Immunol. 2006, 6: 476-483. 10.1038/nri1845.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.