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R Provides Efficient and Versatile Vaccination Against Tumors by Dendritic Cells 1
* Department of Experimental Immunology and Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, and
Department of Respiratory Oncology and Molecular Medicine, Institute of Development, Aging, and Cancer, Tohoku University, Sendai, Japan; and
Department of Dermatology, Tohoku University School of Medicine, Sendai, Japan
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Abstract |
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Rs) on DCs. It was found that when compared with ATCs, ATC-ICs were efficiently internalized by DCs via FcRs, and this process induced maturation of DCs, which was more efficient than that of ATCs. Importantly, ATC-IC loading was shown to be more efficient than ATCs alone in its capacity for inducing antitumor immunity in vivo, in terms of cytotoxic T cell induction and tumor rejection. These results show that using ATC-ICs may overcome the limitations and may enhance the immune response of current ATC-based DC vaccination therapy. |
Introduction |
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IgG-complexed Ags (immune complexes (ICs)) internalized through FcR on DCs are efficiently presented to CTLs through the MHC class-I pathway (21, 22, 23, 24, 25). Previous reports suggest that Ag presentation by DCs through FcR-mediated uptake can be increased 100-fold over pinocytosis of soluble Ags, and it has also been shown that ICs themselves can effectively induce DC maturation (21). As in soluble forms, Ags entrapped in liposome can also enhance MHC class I-restricted Ag presentation when targeted to FcR on DCs (26). These results suggest that targeting tumor-derived Ags to FcRs on DCs may enhance antitumor immunity in vivo.
This study was able to demonstrate that DCs can take up ICs and enhance both MHC class I and class II-restricted Ag presentation in vivo. By applying this in vivo positive function of FcR-mediated Ag presentation to tumor immunotherapy, DCs were found to be able to internalize IgG-complexed ATCs (ATC-ICs) and generate a greater amount of tumor-specific CTLs than those internalizing ATCs alone. Furthermore, DCs that internalized ATC-ICs induced protection against inoculated tumors more effectively than those induced by ATCs. These results show that DCs pulsed with ATC-ICs may become a novel and versatile approach for improving the antitumor response induced by recent ATC-mediated DC vaccination therapy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All of the experiments were performed on 6 to 10-wk-old male and female mice. FcR (27) and FcRIIB (28) double deficient (FcRnull) mice were generously provided by Dr. J. V. Ravetch (Rockefeller University, New York, NY). FcRIIB-/- (28) or FcRIII-/- (34) mice were generated by breeding the F2 offspring of crosses between chimeras and C57BL/6 mice (B6, H-2b), and the wild-type (129/B6 hybrid background, H-2b) mice generated by the same breeding protocol were used as controls for FcRIIB-/- and FcRIII-/- animals. FcR-deficient (FcR-/-) mice were generated as described previously (27) and backcrossed to B6 background over eight generations. The mice were housed and bred in the Animal Unit of the Institute of Development, Aging, and Cancer (Tohoku University, Sendai, Japan), an environmentally controlled and specific pathogen-free facility. C57BL/6 mice were obtained from Charles River Breeding Laboratories (Tokyo, Japan).
Abs and flow cytometric analysis
Rabbit anti-OVA IgG was obtained from Biodesign International (Saco, ME). Mouse anti-trinitrophenol (TNP) IgG1 was purified from the ascites of hybridomas by ion exchange chromatography on DEAE-cellulose (Merck, West Point, PA) and by affinity isolation with a protein G column as previously described (29). Purified mouse IgG1 (BD PharMingen, San Diego, CA) was used for control-irrelevant Ab. For immunostaining, the following mAbs were used: FITC-, PE-, or biotin-conjugated anti-CD11c (HL3), anti-CD86 (GL1), and anti-I-Ab (M5/114.15.2). All were from BD PharMingen. Tricolor-conjugated streptavidin (Caltag Laboratories, Burlingame, CA) and Cy3-conjugated streptavidin (Jackson ImmunoResearch Laboratories, West Grove, PA) were used for staining biotin-Abs. FITC-conjugated anti-mouse IgG was obtained from Zymed Laboratories (San Francisco, CA). Cell surface staining was performed according to standard techniques, and flow cytometric analysis was performed with a FACSCalibur using CellQuest software (BD Labware, Mountain View, CA).
Induction of apoptotic cell death and opsonization with IgG
The murine EL4 thymoma cell line, genetically modified to express OVA gene (E.G7 cells, American Type Culture Collection, Manassas, VA), was used to induce apoptotic tumor cell death. Cells were irradiated with x-rays (100 Gy) followed by incubation at 37°C, 5% CO2 in a serum-free medium for 27 h. After apoptotic cell induction, the surface of the cells was bound with TNP. For the preparation of ATC-ICs, the cells were further opsonized with mouse anti-TNP IgG1 (1 µg/105 cells). The opsonization of ATCs was confirmed by staining ATC-ICs with FITC-conjugated anti-mouse IgG. Cell death was confirmed using an apoptosis detection kit (Clontech Laboratories, Palo Alto, CA). OVA-ICs were prepared by incubating OVA with rabbit anti-OVA IgG at 1:10 weight ratio at 37°C for 1 h.
Generation and loading of bone marrow-derived DCs
Bone marrow cells were obtained from mice as previously described (30). After depleting red blood cells and lymphocytes, bone marrow cells were cultured at 1 x 106 cells/ml in the presence of 20 ng/ml murine recombinant GM-CSF (PeproTech, Rocky Hill, NJ). The medium was replaced by a GM-CSF-containing medium on day 4 of culture. For loading OVA/OVA-ICs, cells on day 6 of culture were pulsed with a medium containing 10 µg/ml OVA/OVA-ICs. After 48 h they were collected and characterized by flow cytometry or used for animal vaccination. For loading ATCs/ATC-ICs, cells on day 6 of culture were cocultured with either ATCs or ATC-ICs at a ratio of 5:1. After 3 h of coculture, the cells were harvested and CD11c+ DCs were purified by MACS sorting (Miltenyi Biotech, Auburn, CA) and further cultured for an additional 24 h. DCs were then collected for cytometric analysis or animal vaccination.
Phagocytosis and DC maturation
For OVA uptake, DCs were incubated with 10 µg/ml FITC-conjugated OVA (Molecular Probes, Eugene, OR) or OVA-ICs for 48 h. The cells were then washed in EDTA to separate surface-bound OVA and stained with anti-CD11c-biotin/tricolor. Double positives for tricolor and FITC were enumerated in flow cytometric analysis. For ATC internalization, ATCs or ATC-ICs were dyed green with PKH2 (Zynaxis Cell Science, Malvern, PA) and cocultured with DCs at a DC:tumor cell ratio of 5:1. After 3 h, the cells were harvested and stained with anti-CD11c biotin/tricolor. The uptake of ATC/ATC-ICs by DCs was confirmed by flow cytometric analysis as cells both positive for PKH2 and tricolor. The same experiments were performed at 4°C or on FcRnull DCs. For evaluating DC maturation by OVA/OVA-ICs, DCs were pulsed with OVA/OVA-ICs for 48 h and stained for CD86 and MHC class II (I-Ab). In addition, DCs were pulsed with various concentrations of Ag to evaluate the dose response effect on DC maturation. DC maturation was analyzed by flow cytometry. For evaluating maturation of DCs that had internalized ATC or ATC-ICs, DCs were pulsed with either ATC with or without mouse IgG1 or ATC-ICs for 3 h, and CD11c+ cells were sorted by MACS. After an additional 24 h of coculture, the cells positive for both CD11c and PKH2 were stained with CD86 and MHC class II (I-Ab) and analyzed by flow cytometry. For confocal microscopic analysis, DCs that had internalized PKH2-labeled ATCs were stained with CD11c-biotin/Cy3 and mounted on a slide. The uptake was confirmed using confocal laser scanning microscopy (Leica, Deerfield, IL).
Generation of OVA-specific IL-4 producing Th2 type CD4+ T cells
IL-4 producing OVA-specific Th2 type CD4+ T cells (OVA-T cells) that respond to OVA-derived peptides and produce IL-4 in a MHC class II-restricted manner were established. As previously described (31), female B6 mice were immunized with 100 µg of OVA emulsified in complete Freund’s adjuvant via the footpads. Ten days later, draining lymph nodes were removed and single cell suspensions enriched for T cells were made using nylon wool columns. A total of 4 x 106 cells were then cultured with mitomycin C-treated 1 x 106 syngeneic spleen cells in the presence of 50 µg/ml OVA in a medium containing RPMI 1640 supplemented with 10% heat-inactivated FBS, 10 µM 2-ME, 0.1 U/ml penicillin, and 0.1 µg/ml streptomycin. On day 4 half of the medium was removed and replaced with fresh medium. After 7 days, cells were harvested and cultured with 5 x 106 mitomycin C-treated syngeneic spleen cells with 50 µg/ml OVA. OVA-reactive T cells had been long cultured by 4-day OVA stimulation followed by a 7-day resting culture. More than 98% of the cells were confirmed to be CD4+ cells and produced IL-4 in response to OVA (A. Yada and T. Takai, unpublished observation).
In vitro Ag presenting assay
OVA-T cells were cultured at 1 x 105 cells/well in 96-well flat-bottom culture plates with 5 x 103 irradiated (15 Gy) DCs. Graded doses of OVA or OVA-ICs were added to the cultures, and after 24 h supernatants were collected and measured for IL-4 concentrations by ELISA (BD PharMingen).
Serum anti-OVA antibody detection
Mice were immunized i.v. with 1 x 106 DCs that had internalized either OVA or OVA-ICs. After immunization, sera were collected until day 20, and the IgG (IgG1, IgG2a, and IgG2b) level against OVA was determined by ELISA by measuring absorbance at 450 nm.
Immunohistochemistry analysis
Frozen spleen sections from mice were prepared after day 20 of DC immunization and incubated at 4°C with FITC-conjugated GL7 for 18 h. After washing, slides were mounted and examined with fluorescent microscopy (Olympus, Melville, NY).
In vivo analysis of OVA-specific CD4 T cells
Mice were immunized i.v. through the tail vein with 1 x 106 DCs that had internalized either OVA or OVA-ICs. Seven days after immunization the mice were sacrificed, and CD4+ T cells were purified from the spleen by MACS. CD4 T cells (1 x 105) were then cocultured with irradiated DCs (5 x 103) and various concentrations of OVA in 96-well U-bottom culture plates. The supernatants were harvested from the well after a 24-h incubation, and cytokine concentrations were determined using either IFN- or an IL-4 ELISA kit (BD PharMingen).
Cytotoxicity assays
Groups of three mice were injected i.v. through the tail vein with DCs (1 x 106/200 µl PBS/mouse) loaded with OVA/OVA-ICs or ATCs/ATC-ICs. One to two weeks postvaccination, the mice were sacrificed, and T-enriched cells were prepared from the spleen. Splenocytes were incubated in vitro for 5 days with irradiated (100 Gy) E.G7 cells at an effector-stimulator ratio of 10:1. After restimulation, the target E.G7 cells were labeled with 100 µl Ci/ml Na251CrO4 for 1 h and incubated with the stimulated splenocytes for 4 h at 37°C. CTL activity was measured using an auto well system (Aloka, Tokyo, Japan).
In Vivo antitumor protection effect
Groups of 12 mice were injected s.c. into the left rear leg with either ATC or ATC-IC-pulsed DCs (5 x 105/100 µl PBS/mouse) or PBS alone, and the same immunization was repeated once more a week later. Seven days after the last vaccination the mice were injected s.c. with 1 x 105 E.G7 cells in 100 µl of PBS into the same portion. A tumor with a diameter <5 mm was determined as a tumor-free size and recorded for >80 days.
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Results |
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To analyze the efficiency of DCs in internalizing ICs, DCs were cocultured with FITC-conjugated OVA or OVA-ICs for a period of time. After 48 h of coincubation, uptake of OVA by DCs was measured using flow cytometry. Fig. 1A shows that by 48 h, as much as 84% of DCs had internalized OVA-ICs, while only 69% of DCs had taken up OVA alone, indicating an efficient uptake of OVA-ICs by DCs. Next, the phenotype of DCs that had taken up OVA-ICs was analyzed. DCs were cocultured with either OVA or OVA-ICs for 48 h, and surface molecules that are known to up-regulate during DC maturation were evaluated by flow cytometry. The expression levels of surface molecules such as CD86 and I-Ab were higher in DCs loaded with OVA-ICs than those from OVA, indicating a more efficient maturation by OVA-ICs than by OVA alone (Fig. 1B). The dose-response curve also showed that DC maturation was parallel to increased OVA-IC loading (Fig. 1C). When FcRnull DCs were used, there was no significant difference in the expression levels of these molecules between OVA and OVA-ICs (A. Yada and T. Takai, unpublished observations). Together these results indicate that through FcR on DCs, OVA-ICs can be efficiently internalized by DCs and induce DC maturation more efficiently than OVA alone.
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Following this, the efficiency of DCs that had taken up OVA-ICs in presenting Ag in a MHC class II-restricted manner was also analyzed. In the in vitro assay, OVA-ICs were found to be much more effective than OVA alone in activating OVA-specific Th2 type CD4+ T cell lines (Fig. 2A). IL-4 production by these cells in response to OVA-ICs mixed with DCs was >100-fold compared with those of OVA alone, indicating that OVA-ICs can induce efficient MHC class-II-restricted Ag presentation by DCs, at least in vitro (Fig. 2A, filled bar). The augmented Ag-presenting effect was verified to be dependent on FcR because DCs from FcR-deficient (FcRnull) mice did not significantly induce IL-4 production even in the presence of OVA-ICs (Fig. 2A, dotted bar). To assess this efficiency of OVA-ICs in vivo, we immunized mice with DCs pulsed with either OVA (OVA/DC) or OVA-ICs (OVA-IC/DC). Seven days after the immunization, CD4+ T cells were purified from the spleen of immunized mice, and the cells were cocultured with irradiated DCs and OVA for 24 h. As shown in Fig. 2D, IFN- concentrations of the supernatant were significantly higher in the OVA-IC/DC group than those in the OVA/DC group. Like the results shown in vitro, these results shown in vivo indicate that CD4 T cells derived from the OVA-IC/DC group were much more primed in vivo than the CD4 T cells derived from the OVA/DC group.
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Rnull mice yielded any detectable IgG responses.
In contrast, B cells are activated in germinal centers of lymphoid tissues and thereby become plasma cells and produce Abs. To investigate whether immunization of DCs loaded with OVA-ICs actually results in activation of B cells in germinal centers, DC-immunized spleen sections were stained with FITC-conjugated GL7. It has been shown that GL7 is highly expressed on activated B cells (32). As shown in Fig. 2C, the size of GL7-positive germinal centers in mice immunized with OVA-IC-loaded DCs were found to be much larger than those immunized with OVA-loaded DCs alone. FcRnull DCs did not induce large germinal centers even after OVA-IC loading. Together, these results suggest that, consistent with higher uptake and maturation molecule expression, DCs internalizing OVA-ICs are significantly more efficient than OVA in Ag presentation via the MHC class II pathway. In addition, OVA-ICs can efficiently activate B cells in germinal centers and induce production of OVA-specific Abs that are much higher than those induced by OVA.
Immune complex internalization results in efficient MHC class-I-restricted Ag presentation in vivo
Previous studies have shown in vitro that internalization of ICs elicits efficient MHC class I-restricted Ag presentation by DCs (21, 22, 23, 24, 25). To determine whether this also occurs in vivo, DCs loaded with either OVA or OVA-ICs were injected i.v. into wild-type mice. Splenocytes were harvested 7 days after immunization, and CTL induction was measured using 51Cr-release assay. As shown in Fig. 3A, only mice vaccinated with DCs loaded with OVA-ICs elicited a strong CTL response, while those loaded with OVA were unable to induce CTLs with a high killing rate. These results indicate that only OVA-ICs internalized through FcRs on DCs can induce Ag-specific CTLs in vivo. This response ceased when mice were immunized with FcRnull DCs. Furthermore, as shown in Fig. 3E, DCs pulsed with OVA and irrelevant mouse IgG1 Ab did not enhance this response. Together, these results indicate that the efficient induction by OVA-ICs is FcR-dependent, and the FcR binding without Ag uptake does not induce efficient CTLs. Next, we examined whether Ag presenting capacity of DCs would differ among the strains of other FcR gene targeted mice. As shown in Fig. 3, B–D, each deletion of FcR (FcR, FcRIIB, and FcRIII) lowered the efficiency of OVA-specific killing activity, indicating the presence of an FcR-mediated augmenting pathway for Ag presentation by DCs.
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The next step was to analyze whether the efficiency of FcRs in terms of Ag presentation could also be applied to cellular Ags such as ATCs. First, the efficiency of DCs in phagocytosing ATCs complexed with IgGs (ATC-ICs) was determined. Thymoma E.G7 cells were used as a source of ATCs. Cell death was induced in 
90% of the cells (as confirmed by annexin V binding and propidium iodide staining) after x-ray irradiation (Fig. 4A). The E.G7 cells were opsonized by binding TNP onto the cell surface followed by adding anti-TNP mouse IgG1. Opsonization of apoptotic E.G7 cells was confirmed by flow cytometry (Fig. 4B). According to previous observations, DCs are responsible for the efficient uptake of apoptotic cells (16). To test whether opsonization was able to enhance the uptake of apoptotic cells by DCs, DCs were cocultured with either ATCs or ATC-ICs. To visualize the uptake, a phagocytosis assay was performed by flow cytometric analysis. DCs and ATCs were independently labeled with either red CD11c-biotin/tricolor or green PKH2, respectively, and the uptake of green ATCs by red DCs was defined by the percentage of double-positive cells. As early as 3 h into coculture, DCs were confirmed to be efficiently engulfed ATCs (34% double positives). Furthermore, by 3 h into coculture, 75% of DCs had taken up ATC-ICs, more than twice as much as those that had engulfed unopsonized cells, indicating the more efficient uptake of ATC-ICs than ATCs (Fig. 4C, 37°C). As a control, to distinguish engulfed cells from surface-conjugated cells, cocultures were conducted at 4°C where the uptake of DCs was inhibited (Fig. 4C, 4°C). To further confirm the uptake shown by the flow cytometric analysis, confocal microscopic analysis was performed (Fig. 4E). Immune complexes of IgG1 isotype are taken up by DCs via FcRs. By using DCs from FcRnull mice, the mechanism by which DCs efficiently internalize ATC-ICs was analyzed. The number of cells engulfed by DCs derived from FcRnull mice was comparable between ATCs and ATC-ICs (Fig. 4D), indicating that the efficient uptake of ATC-ICs by DCs is FcR dependent. The effect of ATCs on the expression of surface molecules, which up-regulate upon DC maturation, was then studied. As shown in Fig. 4F, ATC-ICs were more effective than ATCs in inducing DC maturation. To determine whether DCs taking up the apoptotic cells are in fact the same cells that are maturing, we examined the expression of surface molecules only on cells that are positive for both CD11c and PKH2 (Fig. 4G). Furthermore, to exclude FcR binding-induced maturation, irrelevant Ab (mouse IgG1) was added to the ATC group. The results in Fig. 4G show that the expression of surface molecules on DCs that actually internalized apoptotic cells was relatively higher in the ATC-IC group than in the ATC plus irrelevant Ab group.
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To test whether DCs that had taken up ATC-ICs via FcR can efficiently prime tumor-specific CTLs in vivo, B6 mice were immunized i.v. with DCs loaded with either ATCs or ATC-ICs. Spleen cells were harvested after immunization, and tumor-specific CTL generation was assessed using a standard 51Cr-release assay with E.G7 as targets. Spleen cells from mice immunized with DCs that had been pulsed with ATCs showed the killing of E.G7 cells to some extent (Fig. 5A, 
). However, when mice were immunized with DCs that had been pulsed with ATC-ICs, E.G7 cells were killed more effectively compared with those pulsed with ATCs (Fig. 5A, •). Cytolytic activity was significantly higher than that of ATCs, demonstrating an efficient priming of tumor-specific CTLs in vivo by ATC-ICs compared with ATCs. Efficient cytolytic activity depended on FcR on DCs, as confirmed by the results from FcRnull DCs (Fig. 5B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rs on DCs can enhance Ag presentation to T cells in vivo. It also demonstrates that by using this positive role of FcR-mediated Ag presentation, DCs pulsed with ATC-ICs can enhance in vivo generation of tumor-specific CTLs and tumor rejection, as compared with DCs pulsed with ATCs alone.
Our results demonstrate that when DCs capture ICs through FcRs, they can become potent activators for both CD4+ and CD8+ T cells in vivo. Consistent with previous studies, when OVA-ICs are engaged to FcRs on DCs, they can be effectively taken up by DCs and enhance DC maturation (21). Nearly 85% of OVA-ICs were internalized by DCs, while only 69% of OVA were taken up during the 48-h period of incubation (Fig. 1A). In addition, the expression of costimulatory molecules such as CD86 and I-Ab on DCs was relatively higher in OVA-ICs than in OVA alone (Fig. 1, B and C). In vitro Ag presentation assays reveal that only OVA-ICs induced CD4+ T cell activation at OVA concentrations of 1 µg/ml (Fig. 2A). These results demonstrate that FcRs can enhance Ag uptake and DC maturation, which leads to CD4+ T cell activation in vitro. Efficient uptake, enhanced DC maturation, and CD4+ T cell activation in vitro suggest possibilities for effective T cell priming by OVA-ICs in vivo. The results of higher anti-OVA Ab response and of activated B cells in splenic germinal centers, both induced by only OVA-ICs, demonstrate that FcRs can also enhance in vivo activation of MHC class-II-restricted Ag presentation (Fig. 2, B and C). High IFN- concentrations, detected in the supernatant of the OVA-IC/DC group, also confirm that DCs loaded with OVA-ICs can efficiently prime OVA-specific CD4 T cells in vivo (Fig. 2D). Efficient induction of CTLs by DCs via FcR-mediated Ag presentation has not previously been fully characterized in vivo (22). Our results demonstrate that OVA-ICs, but not OVA, induced in vivo Ag-specific CTLs (Fig. 3). A recent report indicates that mouse splenic CD8+ DCs can constitutively cross-present exogenous Ags, whereas CD8- DCs require additional signals such as ligation of FcRs (35). We have confirmed that DCs used in our experiments are CD8- DCs (data not shown), and although it is bone marrow-derived DCs, this may explain the absence of CTL induction in the OVA group. We could also observe the efficient elicitation of killing activity by adoptive transfer of OVA-IC-pulsed DCs either from FcR-/-, FcRIIB-/-, or FcRIII-/- mice, indicating that each FcR on DC has an enhancing effect on Ag presentation for OVA-ICs (Fig. 3, B–D). In contrast, adoptive transfer of FcRnull DCs did not induce Ag-specific CTLs (Fig. 3A). We are aware of the fact that another group had conflicting results on the activity of FcRIIB in CTL induction (36). Although FcRIIB is an immunoreceptor tyrosine-based inhibitory motif -bearing inhibitory receptor for intracellular signaling, our experiments show that FcRIIB on DCs can contribute positively to CTL induction in vivo (Fig. 3, B and C). We are aware that the ratio of Ag to Ab is different between our experimental condition and others. In our experiments ICs were made of OVA and anti-OVA IgG at an Ab excess 1:10 weight ratio, whereas the other group prepared their ICs with a 2:1 weight ratio, an Ag-excess condition. In an Ag-excess condition ICs become too large, which will induce an inhibitory effect by FcRIIB upon internalization. We used an Ab-excess condition that will make proper size of the immune complex for internalization. We have also confirmed that FcR chain-deficient DCs, which only possess FcRIIB, can internalize ICs more efficiently than OVAs alone (A. Yada and T. Takai, unpublished data). Previous reports have indicated that the amount of Ag is one of the major factors determining the outcome of immunity, and cross-priming will only occur when the level of Ag expression is sufficiently high (33). We believe that the enhanced CTL activity of ICs in FcR chain-deficient mice can be explained by the enhanced uptake of ICs, as compared with OVAs. When DCs were pulsed with OVA and irrelevant mouse IgG1 Ab, DCs could not elicit efficient CTLs, suggesting that the uptake via FcR, not the binding, is necessary for inducing effective CTLs (Fig. 3E).
Taken together, these results indicate the positive role of FcRs in enhancing both MHC class I- and class II-restricted Ag presentation in vitro and in vivo.
Recently there has been great progress in tumor immunity. Increasing numbers of TAAs have been identified from various types of tumor cell and used as targets for cancer immunotherapy (5, 6, 7). Although TAAs have been widely used for loading DCs in clinical trials for human cancers, their disadvantages are that they have to be molecularly defined before loading and are prone to develop CTLs that escape other undefined tumor Ags (2). To overcome these drawbacks and to use the DC vaccination approach to tumors that express undefined TAAs, the use of whole tumor Ags, such as ATCs, has recently been developed for loading DCs. Using this method, DCs would be able to present as many tumor Ags as possible to T cells and minimize the occurrence of immune escape variants. In animal experiments, DCs pulsed with ATCs have been shown to produce potent antitumor immunity in vitro and in vivo, indicating the versatility of this approach for eradicating tumors with uncharacterized TAAs (10, 11, 12, 13, 14, 15, 16). In contrast, recent data has demonstrated that ATCs are somewhat insufficient for activating the immune system, possibly because of their inability to induce DC maturation (17, 18). However, DCs regain their capacity for T cell activation when they are stimulated with proinflammatory cytokines such as TNF-
and IL-1
, indicating the need for external activating signals after ATC internalization in ATC-mediated DC vaccination (19).
Exploiting the positive role of FcRs in DC maturation and CTL induction, we tried to augment the ATC-induced antitumor response by loading DCs with ATC-ICs. To test whether FcR-mediated ATC-IC internalization could augment the antitumor response of ATCs, the cytolytic activity of DCs pulsed with ATC-ICs or ATCs alone was compared. About 90% of E.G7 cells were found to have undergone apoptosis by x-ray treatment (Fig. 4A), and as in OVA-ICs, ATC-ICs were taken up efficiently by DCs via FcRs. By 3 h of co-incubation, nearly 75% of DCs had engulfed ATC-ICs, while only 34% of DCs were able to engulf ATCs alone (Fig. 4C). This enhanced uptake by ATC-ICs was FcR-dependent, as confirmed by experiments on FcRnull DCs (Fig. 4D). By 24 h of coculture, ATC-ICs induced relatively higher levels of costimulatory molecules on DCs than ATCs alone (Fig. 4F). The maturation of DCs was also confirmed on DCs that actually engulfed apoptotic cells (Fig. 4G). As described above, there is still conflicting evidence as to whether DCs can actually mature efficiently and present Ags derived from ATCs without further exogenous maturation signals. In our results, although ATCs induced maturation molecules on DCs to a certain level, ATC-ICs were more effective than ATCs in inducing costimulatory molecules on DCs. This result shows that additional activation through the uptake of ATCs via FcRs can enhance maturational signal of ATCs. Although the exact mechanism is yet to be defined, DC maturation induced by ATC-IC uptake may be the result of FcR-mediated uptake, because ATC-ICs pulsed to FcRnull DCs were not effective in inducing these maturational molecules (A. Yada and T. Takai, unpublished data). In addition, not only the binding but the uptake of Ag through FcR may be inevitable in this maturation signaling because the binding of the irrelevant Abs, which was independent from uptake of ATCs, did not induce full maturation (Fig. 4G). Finally, DCs pulsed with ATC-ICs were identified as capable of priming a substantial tumor-specific CTL response in vivo that allowed a strong reaction against E.G7 cells. Compared with ATCs, ATC-ICs induced CTLs with a higher killing activity, and E.G7 cells were also efficiently rejected by prior ATC-IC vaccination (Fig. 5, A and B). The efficient antitumor response induced by ATC-IC vaccination may be mainly caused by higher uptake of Ags by ATC-ICs, because DCs are able to present much higher amounts of Ag-derived peptides than ATC-pulsed DCs. Previous reports have indicated that the outcome of cross-presentation, whether it leads to cross-priming or cross-tolerance, depends on the level of Ag expressed by cells taken up by DCs (33). In ATC-ICs the dose of Ag internalized by DCs may exceed the threshold for the induction of efficient CTLs. The enhanced DC maturation by ATC-ICs may also support the better in vivo generation of CTLs. To further enhance the antitumor efficiency induced by DCs pulsed with ATC-ICs, we doubled the immunization of DCs before tumor inoculation. Although the antitumor efficiency was slightly enhanced, mice vaccinated four times with DCs did not show significant rejection of tumors as compared with mice vaccinated two times with the same DCs (data not shown). The result indicates that additional experiments should be made to establish a more optimal model for DC vaccination.
In conclusion, our study demonstrates that ATC-ICs enhanced in vivo antitumor response. Furthermore, tumor cells were more efficiently rejected by ATC-ICs than ATCs alone. The recent in vitro experiments on human DCs also indicate the efficiency of FcRs in the induction of antitumor CTLs (23). These results suggest that ATC-ICs may be more useful than ATCs in inducing antitumor response against tumors with uncharacterized tumor Ags. Thus, ATC-ICs may become an efficient and versatile source for DC-based antitumor vaccination.
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Acknowledgments |
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Rnull mice. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Toshiyuki Takai, Department of Experimental Immunology, Institute of Development, Aging, and Cancer, Tohoku University, 4-1 Seiryo, Sendai 980-8575, Japan. E-mail: tostakai{at}idac.tohoku.ac.jp
3 Abbreviations used in this paper: TAA, tumor-associated Ag; ATC, apoptotic tumor cell; DC, dendritic cell; IC, immune complex; TNP, trinitrophenol.
Received for publication February 11, 2002. Accepted for publication November 27, 2002.
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Y. Inoue, T. Kaifu, A. Sugahara-Tobinai, A. Nakamura, J.-I. Miyazaki, and T. Takai Activating Fc{gamma} Receptors Participate in the Development of Autoimmune Diabetes in NOD Mice J. Immunol., July 15, 2007; 179(2): 764 - 774. [Abstract] [Full Text] [PDF]
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D. Thomas-Rudolph, T. W. Du Clos, C. M. Snapper, and C. Mold C-Reactive Protein Enhances Immunity to Streptococcus pneumoniae by Targeting Uptake to Fc{gamma}R on Dendritic Cells J. Immunol., June 1, 2007; 178(11): 7283 - 7291. [Abstract] [Full Text] [PDF]
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D. H. Schuurhuis, N. van Montfoort, A. Ioan-Facsinay, R. Jiawan, M. Camps, J. Nouta, C. J. M. Melief, J. S. Verbeek, and F. Ossendorp Immune complex-loaded dendritic cells are superior to soluble immune complexes as antitumor vaccine. J. Immunol., April 15, 2006; 176(8): 4573 - 4580. [Abstract] [Full Text] [PDF]
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T. Dao, M. Gomez-Nunez, C. Antczak, B. Kappel, J. Singh Jaggi, T. Korontsvit, V. Zakhaleva, and D. A. Scheinberg Natural Killer Cells License Dendritic Cell Cross-Presentation of B Lymphoma Cell-Associated Antigens Clin. Cancer Res., December 15, 2005; 11(24): 8763 - 8772. [Abstract] [Full Text] [PDF]
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J. Bayry, S. Lacroix-Desmazes, M. D. Kazatchkine, O. Hermine, D. F. Tough, and S. V. Kaveri Modulation of Dendritic Cell Maturation and Function by B Lymphocytes J. Immunol., July 1, 2005; 175(1): 15 - 20. [Abstract] [Full Text] [PDF]
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K. M. Dhodapkar, J. L. Kaufman, M. Ehlers, D. K. Banerjee, E. Bonvini, S. Koenig, R. M. Steinman, J. V. Ravetch, and M. V. Dhodapkar Selective blockade of inhibitory Fc{gamma} receptor enables human dendritic cell maturation with IL-12p70 production and immunity to antibody-coated tumor cells PNAS, February 22, 2005; 102(8): 2910 - 2915. [Abstract] [Full Text] [PDF]
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L. J. Brady Antibody-Mediated Immunomodulation: a Strategy To Improve Host Responses against Microbial Antigens Infect. Immun., February 1, 2005; 73(2): 671 - 678. [Full Text] [PDF]
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A. Getahun, J. Dahlstrom, S. Wernersson, and B. Heyman IgG2a-Mediated Enhancement of Antibody and T Cell Responses and Its Relation to Inhibitory and Activating Fc{gamma} Receptors J. Immunol., May 1, 2004; 172(9): 5269 - 5276. [Abstract] [Full Text] [PDF]
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) or Fc
). C, The germinal centers of spleen in DC-immunized mice were stained with B cell activation marker, GL7. One representative experiment of at least three is shown. Data are shown as the mean of triplicate samples ± SD. D, CD4 T cells (1 x 105) purified from the spleen of immunized mice were cocultured with irradiated DCs (5 x 103) in the absence or presence (20 µg/ml) of OVA. Supernatants were collected after 24 h of incubation, and cytokine concentrations of IFN-
