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The Journal of Immunology, 2006, 176: 4573-4580.
Copyright © 2006 by The American Association of Immunologists

Immune Complex-Loaded Dendritic Cells Are Superior to Soluble Immune Complexes as Antitumor Vaccine1

Danita H. Schuurhuis2,*, Nadine van Montfoort3,*, Andreea Ioan-Facsinay3,4,{dagger}, Reshma Jiawan*, Marcel Camps*, Jan Nouta*, Cornelis J. M. Melief*, J. Sjef Verbeek{dagger} and Ferry Ossendorp5,*

* Department of Immunohematology and Blood Transfusion and {dagger} Department of Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cells (DCs) play an important role in the induction of T cell responses. Fc{gamma}Rs, expressed on DCs, facilitate the uptake of complexed Ag, resulting in efficient MHC class I and MHC class II Ag presentation and DC maturation. In the present study, we show that prophylactic immunization with DCs loaded with Ag-IgG immune complexes (ICs) leads to efficient induction of tumor protection in mice. Therapeutic vaccinations strongly delay tumor growth or even prevent tumors from growing out. By depleting CD4+ and CD8+ cell populations before tumor challenge, we identify CD8+ cells as the main effector cells involved in tumor eradication. Importantly, we show that DCs that are preloaded in vitro with ICs are at least 1000-fold more potent than ICs injected directly into mice or DCs loaded with the same amount of noncomplexed protein. The contribution of individual Fc{gamma}Rs to Ag presentation, T cell response induction, and induction of tumor protection was assessed. We show that Fc{gamma}RI and Fc{gamma}RIII are capable of enhancing MHC class I-restricted Ag presentation to CD8+ T cells in vitro and that these activating Fc{gamma}Rs on DCs are required for efficient priming of Ag-specific CD8+ cells in vivo and induction of tumor protection. These findings show that targeting ICs via the activating Fc{gamma}Rs to DCs in vitro is superior to direct IC vaccination to induce protective tumor immunity in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Because T cells are critical mediators in the protection against tumors, induction of a strong T cell response against tumor cells is a main goal in cancer immunotherapy. Dendritic cells (DCs)6 are crucial in the initiation of primary CD4+ and CD8+ T cell responses (1). Immature DCs are very efficient in capturing Ag, but they lack the costimulatory signals for efficient T cell activation. DC maturation can be induced by different microbial and inflammatory products as well as CD4+ Th-dependent CD40 ligation (1). Ag uptake capacity decreases upon DC maturation, whereas the expression of costimulatory molecules and MHC molecules increases, and concomitantly T cell stimulatory capacity is enhanced. Furthermore, mature DCs produce IL-12, a key cytokine in the induction of Th1 and CTL responses (2, 3). Maturation of DCs is required for the induction of efficient CTL responses in vivo (4).

DCs are professional APCs specialized in processing and presentation of antigenic peptides derived from external sources. Exogenous Ag, taken up by fluid-phase pinocytosis, receptor-mediated endocytosis, or phagocytosis, is processed in the endosomal/lysosomal pathway and presented on MHC class II molecules (5). However, parts of the internalized Ags enter the cytosolic (endogenous) pathway, by which intracellular Ags are usually processed and presented, and are presented on MHC class I molecules, a process called cross-presentation (6, 7). Cross-presentation of Ag internalized by fluid phase pinocytosis is inefficient and requires high Ag concentrations. Uptake can be improved by delivering the Ag in bacteria (8) or apoptotic cells (9) or by complexing it to beads (10) or Ab (11, 12). Ab-mediated uptake of Ag is generally dependent on FcRs, as shown for several cell types, including B cells (13, 14), macrophages, (15) and DCs.

Loading DCs with Ag-IgG immune complexes (ICs) leads to efficient Ag uptake, maturation of the DCs, and efficient MHC class I- and class II-restricted Ag presentation in vitro (11, 12, 16, 17, 18) and T cell priming in vivo (12, 19, 20, 21). These processes are Fc{gamma}R-mediated. Incubation of DCs with ICs bypasses the "license to kill" signal (22, 23, 24) and enables DCs to directly prime specific CD8+ CTL responses in vivo (12).

The in vivo contribution of Fc{gamma}R-facilitated Ag presentation to activation of CD4+ and CD8+ T cells (11, 12) and to the induction of Ab responses (25) has been shown. Mice deficient for the FcR {gamma}-chain, required for surface expression and signaling of the activating Fc{gamma}Rs (Fc{gamma}RI and Fc{gamma}RIII) and Fc{epsilon}RI, exhibit impaired delayed-type hypersensitivity and CD4+ Th responses to model Ag, compared with wild-type (wt) controls (26). As a consequence of impaired activation of Th cells, mice deficient for FcR {gamma}-chain displayed impaired Ag-specific Ab responses. We recently showed that DCs loaded in vitro with ICs gain the ability to induce efficient CD8+ Ag-specific responses in vivo (12). Together these findings support the requirement of Fc{gamma}R-mediated enhanced Ag presentation for efficient induction of CD4+ and CD8+ T cell responses and Ab responses in vivo. Recent studies showed that IC-mediated Ag presentation induces tumor immunity. These studies are contradictory regarding the role of the inhibitory Fc{gamma}R (Fc{gamma}RIIB) on IC-mediated Ag presentation and T cell priming by wt DCs. By immunizing with IC-loaded DCs lacking the inhibitory Fc{gamma}R, or by blocking this Fc{gamma}R, it was shown that selective engagement of activating Fc{gamma}Rs by ICs promotes DC maturation, T cell priming, and induction of effective antitumor immunity (20, 27, 28).

However, another study showed that protective antitumor immunity was induced with IC-loaded wt DCs, without the need for blocking the inhibitory Fc{gamma}R (21).

DC-based vaccination strategies used for tumor immunotherapy include loading of DCs with tumor-associated Ag (29, 30), viral vectors (31), tumor mRNA (32), or whole tumor cell contents (apoptotic tumor cells (33, 34, 35, 36) and fusion of tumor cells with DCs (37, 38). The use of Ag-IgG complexes for loading of DCs may be useful, because no additional maturation-inducing stimulus is required and the loading of both MHC class I and MHC class II molecules with IC-derived epitopes is very efficient. Furthermore, loading of DCs with ICs may be advantageous because it does not require prior knowledge of the immunodominant epitopes present in the tumor Ag or knowledge of the HLA of the patient.

In the present study we show that IC-loaded DCs (both freshly isolated bone marrow-derived DCs and D1 cell line DCs) induce protective immunity against tumors expressing the Ag present in the IC. IC-loaded DCs are much more efficient in inducing protective immunity than are DCs loaded with soluble protein. Fc{gamma}RI and Fc{gamma}RIII are important for cross-presentation and cross-priming of ICs by DCs. Furthermore, we show that expression of Fc{gamma}Rs on DCs is required for the induction of tumor protection by IC-pulsed DCs. Importantly, we show that vaccination with IC-loaded DCs is superior to vaccination with soluble ICs in inducing protection against highly aggressive tumor growth.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6 (B6; H-2b) mice were purchased from IFFA Credo. Both Fc{gamma}RI–/– mice (39) and Fc{gamma}RIII–/– mice (40) were generated in our laboratory. The Fc{gamma}RII knockout was provided by T. Takai (Tokoku University, Sendai, Japan) (41). The knockout mice were backcrossed on the B6 inbred strain, Fc{gamma}RIII–/–, Fc{gamma}RI–/–, and Fc{gamma}RII–/– for 12, 5, and 3 generations, respectively. Double knockout mice were generated by intercrossing the single knockout strains. For all knockout lines, wt control mice were used with the same genetic background. Homozygous MHC H-2b mice were selected and used for experiments. All animals were maintained under specific pathogen-free conditions in the Animal Facility of the Leiden University Medical Center and were used at 6–10 wk of age in accordance with national legislation and under supervision of the Animal Experimental Committee of the University of Leiden.

Cells and reagents

B6 mouse embryo cells transformed with murine CD80 (B7.1), H-2Kb, and a construct expressing an endoplasmic reticulum-targeting signal sequence, followed by the OVA257–264 CTL epitope SIINFEKL (SigSIINFEKL), were generated as described (42). B3Z is a T cell hybridoma, specific for SIINFEKL in H-2Kb, which carries a beta-galactosidase construct driven by NF-AT elements from the IL-2 promoter (43). Cell lines were cultured in IMDM (BioWhittaker) supplemented with 8% heat-inactivated FCS (Greiner), 100 IU/ml penicillin, 2 mM L-glutamine, and 20 µM 2-ME (complete medium). The MO-5 tumor cell line, a variant of the melanoma B16F10 tumor line that expresses full-length OVA as neo-Ag (44), was a gift from K. L. Rock (University of Massachusetts Medical Center, Worcester, MA) and was cultured as described. D1 cell line, a long-term growth factor-dependent immature splenic DC line derived from B6 mice, was kindly provided by P. Ricciardi-Castagnoli (University of Milano-Bicocca, Milan, Italy) and cultured as described (45). Bone marrow-derived primary DC cultures (BM DCs) were generated as described (46) and consisted of >95% CD11c+ cells. Both nonadherent and adherent (detached using 2 mM EDTA) DCs were collected and used. LPS of Escherichia coli (serotype 026:B6) was purchased from Sigma-Aldrich. Synthetic peptide used was OVA257–264 (SIINFEKL).

Ab and cell surface immunofluorescence

The following Abs were purchased from BD Pharmingen: PE-coupled anti-CD8beta Ab (Ly-3.2) and allophycocyanin conjugated OVA257–264-loaded H-2Kb tetramers, which were prepared as described (47, 48, 49). Staining for CD8+, OVA257–264-specific T cells was performed as described (12). Stained cells were analyzed using a FACScan flow cytometer equipped with CellQuest software (BD Biosciences).

MHC class I-restricted Ag presentation

OVA-ICs were preformed by incubating different concentrations of soluble OVA (grade V; Sigma-Aldrich) with 25 µg/ml purified polyclonal rabbit IgG anti-OVA (rIgG{alpha}OVA; ICN Biomedicals) for 30 min at 37°C in 96-well flat-bottom plates. Increasing concentrations of SIINFEKL peptide, or soluble OVA alone or preincubated with 25 µg/ml control purified rabbit IgG, were used as controls. Concentrations shown in the figure are the final concentrations after addition of the DCs. ICs were preformed in 3-fold higher concentrations in 50 µl. After 30-min preincubation, 100 µl containing 5 x 104 BM DCs were added and incubated for 24 h at 37°C. After incubation, 5 x 104 B3Z T cells were added to each well and incubated for another 24 h at 37°C. Presentation of SIINFEKL in H-2Kb was measured by the activation of B3Z cells, measured by a colorimetric assay using chlorophenol red-beta-D-galactopyranoside as substrate to detect lacZ activity in B3Z lysates.

Induction of CTL responses in vivo

D1 cell line DCs or BM DC cultures generated from wt or Fc{gamma}RI/III–/– mice were incubated with OVA-ICs (1 µg/ml OVA, 25 µg/ml rIgG{alpha}OVA) for 48 h. As a control, BM DCs were incubated with 10 µg/ml LPS for 48 h and exogenously loaded with 1 µg/ml OVA257–264 (SIINFEKL) peptide for 2 h at 37°C. After extensive washing, 106 BM DCs or D1 cells were injected i.v. in B6 mice in PBS with 0.5% BSA. Soluble OVA-ICs were preformed in PBS (200 µg/ml OVA, 5000 µg/ml rIgG{alpha}OVA) and injected i.v. into mice in different concentrations as described in the legend of Fig. 7. As a control, soluble OVA in PBS was injected i.v. Mice were depleted for CD4+ cells by i.p. injection of 100 µg of purified anti-CD4 Ab GK1.5 in PBS at days 5, 3, and 1 before and at days 1 and 7 after injection of DCs. The levels of CD4+ cells in peripheral blood of depleted mice were checked by flow cytometry. More than 95% of CD4+ cells were depleted. After 10 days, spleen cells (5 x 106 cells/well) were cocultured in vitro with irradiated (10,000 rad) B7.1-, H-2Kb-, and SigSIINFEKL-transfected mouse embryo cells (5 x 105 cells/well) to restimulate OVA257–264-specific responses in 1.5-ml cultures in 24-well plates in the absence of additional cytokines. After 7 days, the percentage of OVA257–264-specific CD8+ T cells was assessed by staining with tetrameric complexes.


Figure 7
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  		FIGURE 7. DCs loaded with ICs are a more potent vaccine than ICs. OVA-ICs were preformed by incubating OVA and rIgG{alpha}OVA. D1 cells loaded with OVA-ICs by 48 h incubation or pure OVA-ICs were injected i.v. in B6 mice, followed by s.c. injection of OVA-expressing MO-5 tumor cells 14 days later (A) or by in vitro restimulation of splenocytes (B). Amounts of OVA in ICs used to load 1 x 106 D1 cells or injected directly into mice are indicated. A, The percentage survival at day 70 after tumor challenge is presented; p values were calculated using the log-rank test, based on survival curves of ICs either directly injected or loaded on DCs (Expt. 1, 10 µg of ICs vs 10 µg of D1-IC, p = 0.0049; 3 µg of ICs vs 3 µg of D1-IC, p = 0.0185; Expt. 2, 10 µg of ICs vs 10 µg of D1-IC, p = 0.1805; 1 µg of ICs vs 1 µg of D1-IC, p = 0.0043; 0.1 µg of ICs vs 0.1 µg of D1-IC, p = 0.0007). Day 70 was used as endpoint of survival analysis because mice will develop tumors at ultimately day 65, as can be seen in Fig. 3B. Each group consisted of six mice. B, Restimulated splenocytes from immunized and naive mice were analyzed for CD8+ cells capable of binding H-2Kb-SIINFEKL tetrameric complexes. Data indicated are the amount of specific CD8+ cells as a percentage of the total amount of CD8+ cells. Data shown are the means of five mice ± SEM.

 
Immunization with Ag-loaded DCs or ICs followed by tumor challenge

OVA-ICs were preformed by incubating 20 µg/ml OVA and 0.5 mg/ml rIgG{alpha}OVA for 30 min at 37°C. D1 cells or B6 BM DCs were loaded with preformed OVA-ICs, soluble OVA, rIgG{alpha}OVA, or 10 µg/ml LPS together with soluble OVA for 48 h. A total of 1 µg/ml OVA (uncomplexed or in ICs) or 3-fold or 10-fold dilutions (as described in Results) was used to load the DCs. After washing, 106 cells were injected i.v. in B6 mice. Alternatively, preformed OVA-ICs in PBS were injected directly into mice. At the indicated time points before or after immunization, mice were challenged s.c. with 5 x 104 OVA-expressing MO-5 tumor cells in PBS with 0.5% BSA. When indicated, CD4+ and/or CD8+ cell populations were depleted by i.p. injection of 100 µg of purified anti-CD4 Ab GK1.5 and/or anti-CD8 Ab 2.43 in PBS at day 1 before tumor challenge and days 2, 4, 6, and 13 after tumor challenge. More than 95% of CD4+ cells and 90% of CD8+ cells were depleted. Mice were checked two times each week for the presence of tumors and were sacrificed when tumors reached a volume of 1000 mm3. For statistical analyses, mean survival was compared using the log-rank test. Differences were considered significant when p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Both Fc{gamma}RI and Fc{gamma}RIII on DCs mediate MHC class I-restricted Ag presentation of OVA-ICs

We and others (11, 12, 39) have previously shown that incubation of DCs with Ag-IgG ICs results in more efficient uptake of Ag and MHC class I-restricted presentation of IC-derived peptides than does incubation with soluble Ag. Furthermore, ICs induce maturation of the DCs, improving their costimulatory capacity. The enhanced Ag presentation of ICs is mediated by the activating Fc{gamma}RI and Fc{gamma}RIII, as the absence of either of these receptors leads to partial reduction of Ag presentation. Deficiency of the common FcR {gamma}-chain (resulting in absence of surface expression of all activating Fc{gamma}R) results in the lack of Ag presentation (11, 39). In contrast with previous experiments in which BM DCs, OVA-ICs, and T cells were coincubated (39), we preincubated BM DCs with OVA-ICs or soluble OVA and control rabbit IgG for 24 h, before the addition of the OVA257–264 (SIINFEKL)-specific, H-2Kb-restricted, costimulation-independent T cell hybridoma B3Z. OVA-ICs contained titrated amounts of OVA protein and a fixed concentration of rIgG{alpha}OVA (complexes were preformed by incubating OVA protein and rIgG{alpha}OVA). Optimal OVA-IC formation was achieved at ~1 µg/ml OVA at a fixed Ab concentration of 25 µg/ml (12). We studied the role of the individual Fc{gamma}Rs on Ag presentation in MHC class I by generating BM DC cultures from Fc{gamma}R-deficient mice and their interbreeds. As shown in the upper panel of Fig. 1, BM DCs deficient for either Fc{gamma}RI or Fc{gamma}RIII ({square}) presented SIINFEKL epitope processed from OVA-ICs as efficiently as wt control BM DCs ({blacksquare}) to B3Z hybridoma cells. In contrast, Fc{gamma}RI/III–/– double knockout and Fc{gamma}RI/II/III–/– triple knockout BM DCs lacked Fc{gamma}R-mediated enhancement of Ag presentation (Fig. 1, lower panel). Soluble OVA and SIINFEKL synthetic peptide in increasing concentrations were presented by all BM DCs with similar efficiency. These data show that the presence of either Fc{gamma}RI or Fc{gamma}RIII is sufficient to mediate IC uptake and MHC class I-restricted presentation of antigenic peptides.


Figure 1
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  		FIGURE 1. Both Fc{gamma}RI and Fc{gamma}RIII mediate MHC class I-restricted Ag presentation of OVA-ICs. BM DC cultures generated from mice deficient for Fc{gamma}RI, Fc{gamma}RIII, Fc{gamma}RI/III, or Fc{gamma}RI/II/III (open symbols) and their respective wt controls (filled symbols) were incubated with the indicated concentrations of OVA and preincubated with 25 µg/ml control rabbit IgG (ova + ctrl rIgG) or rIgG{alpha}OVA (OVA-IC). As a control, SIINFEKL peptide was used (SIINFEKL). After 24 h, B3Z cells were added for 24 h to measure T cell activation (see Materials and Methods). Data indicated are the means of triplicates ± SD. One representative experiment of two performed is presented. Every individual figure consists of two figures; the left part of each individual figure represents titrated amounts of SIINFEKL peptide presented by the BM DCs (triangles), and the right part of each individual figure represents presentation of titrated amounts of OVA protein preincubated either with control Ab (circles) or with rIgG{alpha}OVA (squares).

 
Activating Fc{gamma}R I and III on DCs are required for efficient in vivo priming of CD8+ CTL by OVA-IC-treated DCs

We previously showed that OVA-IC-treated, but not OVA-treated, DCs have the capacity to prime SIINFEKL-specific CD8+ CTL in vivo, in the absence of CD4+ T cell help (12). To establish the contribution of activating Fc{gamma}Rs in this process, we stimulated wt and Fc{gamma}RI/III–/– double knockout BM DCs with OVA-ICs in vitro and injected them i.v. into wt B6 mice. As a control, LPS-activated BM DCs were loaded with synthetic OVA257–264 SIINFEKL and injected into wt B6 mice. Ten days later, spleens were removed and splenocytes restimulated with SIINFEKL-transfected cells. After 7 days, the amount of SIINFEKL-specific CD8+ cells as a percentage of the total amount of CD8+ cells was assessed in the restimulated splenocytes by staining with tetrameric complexes. Only wt BM DCs efficiently primed SIINFEKL-specific CD8+ T cell responses in vivo (Fig. 2A), showing that the activating Fc{gamma}RI and Fc{gamma}RIII are required for this process. The inability of the Fc{gamma}RI/III–/– BM DCs to induce CD8+ T cell priming was not due to an intrinsic defect of these cells, as LPS-activated Fc{gamma}RI/III–/– BM DCs loaded with the OVA257–264-synthetic peptide primed SIINFEKL-specific CD8+ T cells as efficiently as did their wt counterparts (Fig. 2B). Thus, Fc{gamma}RI and Fc{gamma}RIII are critical for the ability of IC-loaded DCs to cross-prime CD8+ T cells in vivo to the protein Ag present in the ICs.


Figure 2
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  		FIGURE 2. BM DCs lacking Fc{gamma}RI/III do not prime SIINFEKL-specific CD8+ CTL responses in vivo after incubation with OVA-ICs in vitro. OVA-IC-treated (A) or LPS-activated SIINFEKL peptide (OVA257–264)-loaded (B) BM DCs derived from B6 wt and Fc{gamma}RI/III–/– double knockout mice were injected i.v. in wt B6 recipients. In vitro restimulated splenocytes from immunized and naive mice were analyzed for CD8+ cells capable of binding H-2Kb-SIINFEKL tetrameric complexes. Data indicated are the amount of specific CD8+ cells as percentage of the total amount of CD8+ cells. Data shown are the means of five mice ± SEM. Upper panels show the flow cytometric data from one representative mouse of each of the three groups.

 
OVA-IC-loaded DCs induce efficient tumor immunity

We have shown before that in addition to BM DCs, D1 DC line loaded with OVA-ICs efficiently primes CD8+ CTL responses in vivo (12). We have used this well-characterized myeloid-type DC line to show the physiological significance of IC-mediated enhanced T cell priming in the induction of protective immunity in vivo. The ability of OVA-IC-loaded DCs to induce prophylactic or therapeutic immunity against an OVA-expressing tumor was evaluated. D1 cells were treated with equimolar amounts of soluble OVA or preformed OVA-ICs for 48 h before i.v. injection into B6 mice. Because OVA-ICs induce DC activation as well as enhanced Ag uptake, D1 cells treated with an equimolar amount of soluble OVA combined with LPS to induce DC maturation were used to study the effect of maturation as such. Two weeks later, mice were challenged with OVA-expressing MO-5 tumor cells (B16F10 melanoma cells stably transfected with OVA cDNA) (44) and were monitored for the development of palpable tumors. All mice immunized with OVA-IC-loaded D1 cells developed protective immunity to the tumor challenge (zero of nine mice developed a tumor). In contrast, all naive mice (eight of eight) and mice immunized with D1 cells treated with soluble OVA (seven of seven), rIgG{alpha}OVA (four of four), or soluble OVA and LPS (four of four) developed large tumors (Fig. 3, A and B). These data show that OVA-IC-loaded D1 cells induce protection against OVA-expressing tumors. We have shown before that activation of the DCs by itself is not sufficient to enhance MHC class I-restricted presentation of peptides derived from exogenous Ag (12). Fig. 3B shows that maturation of the DCs by itself is not sufficient to induce tumor immunity, because DCs matured with LPS and loaded with soluble OVA do not induce protective immune responses. The antitumor response was OVA-specific, as mice immunized with OVA-IC-treated D1 cells and challenged with the parental B16 tumor cells were not protected (Fig. 3C). Next to the D1 cell line, we tested the capacity to induce tumor immunity of freshly isolated B6 BM DCs. OVA-IC-treated B6 BM DCs induced similar protective immune responses against B16-OVA tumor challenge (one of seven mice developed a tumor compared with seven of eight in the untreated group) (Fig. 3D). These data show that IC-loaded DCs induce significant antitumor responses in vivo.


Figure 3
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  		FIGURE 3. IC-treated DCs induce prophylactic responses against tumors expressing the Ag present in the ICs. B6 recipients were injected i.v. with D1 DCs treated with equimolar amounts of OVA provided as preformed OVA-ICs (D1 + OVA-IC; n = 9), soluble OVA (D1 + OVA; n = 7), soluble OVA combined with LPS (D1 + OVA + LPS; n = 4) or with rIgG{alpha}OVA (D1 + r{alpha}OVA; n = 4) (A and B), with D1 cells treated with preformed OVA-IC (n = 8) (C), or with freshly isolated B6 BM DCs treated with OVA-ICs (n = 7) (D). Two weeks later, immunized and naive (n = 8) mice were challenged s.c. with MO-5 OVA-expressing tumor cells (A, B, and D) or with the parental B16F10 tumor cells (C) and were monitored for development of palpable tumors. Data shown are tumor growth curves of individual mice (A) and survival curves of treatment groups (B–D). D1-IC-treated mice are significantly different from all of the other treatment groups and the naive mice (p < 0.0001). These other treatment groups are not significantly different from the naive mice (p = 0.65, p = 0.92, and p = 0.43 for D1-OVA-, D1-OVA + LPS-, and D1-rIgG{alpha}OVA-treated mice compared with naive mice, respectively) (A). Statistical analysis was performed using the log-rank test.

 
Fc{gamma}Rs are required for the induction of tumor protection by IC-loaded DCs

To establish whether Fc{gamma}Rs are required for the induction by DCs of protective immunity against tumors, BM DCs obtained from B6 wt mice or from Fc{gamma}RI/II/III–/– mice were treated with OVA-ICs and injected i.v. into B6 wt mice. Two weeks after immunization, mice were challenged with MO-5 OVA-expressing tumor cells and were monitored for the development of palpable tumors. Mice immunized with OVA-IC-treated wt BM DCs developed protective immunity to the tumor challenge (Fig. 4). However, mice immunized with OVA-IC-treated BM DCs lacking Fc{gamma}Rs developed fatal tumors comparable to those of nonimmunized mice. Thus, the in vivo protective immunity induced by the injection of DCs loaded in vitro with IC requires expression of Fc{gamma}R on the DCs.


Figure 4
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  		FIGURE 4. Fc{gamma}Rs are required for the induction of tumor protection in vivo. B6 mice were injected i.v. with OVA-IC-treated BM DCs derived from B6 wt and Fc{gamma}RI/II/III–/– triple knockout mice. Two weeks later, immunized and naive mice were challenged s.c. with MO-5 OVA-expressing tumor cells and were monitored for development of palpable tumors. Data shown are survival curves of treatment groups. Each group consisted of eight mice. B6-IC-treated mice vs naive mice, p = 0.02; Fc{gamma}RI/II/III–/– triple knockout DC-IC-treated mice vs naive mice, p = 0.47. Statistical analysis was performed using the log-rank test.

 
IC-loaded DCs have the capacity to induce therapeutic immunity

To measure the capacity of IC-loaded DCs to induce therapeutic immunity, mice were first inoculated with MO-5 tumor cells and subsequently treated with OVA-IC-loaded D1 DCs (Fig. 5A) or freshly isolated B6 BM DCs (Fig. 5B) at different time points after tumor inoculation. Mice were monitored for the development of palpable tumors. Therapeutic treatment was compared with optimally protected mice immunized 14 days before tumor challenge with OVA-IC-loaded DCs. A statistically significant increase in median survival time compared with untreated mice was observed for all therapeutically treated mice, even when treatment was started 10 days after tumor inoculation (Fig. 5). More than 50% of the mice treated at day 0 or 3 after tumor inoculation were still protected at day 70. Thus, IC-loaded DCs induce significant antitumor responses in vivo, also in a therapeutic setting.


Figure 5
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  		FIGURE 5. Therapeutic vaccination with IC-loaded DCs. B6 mice were injected with either D1 DCs (A) or B6-derived BM DCs (B) incubated with preformed OVA-ICs for 48 h. Immunization was performed at different time points (as indicated in the figure) before and after s.c. inoculation of MO-5 tumor cells. Day 0 is the day of tumor inoculation. Shown are survival curves of treatment groups. Each treatment group consisted of eight mice. All treatment groups were statistically different from naive mice. A, p < 0.0001, p = 0.0004, p = 0.0002, and p = 0.0004 for days –14, 0, 3, and 10 vs naive, respectively. B, p < 0.0001, p = 0.0001, and p = 0.0384 for days –14, 0, and 6 vs naive, respectively. Statistical analysis was performed using the log-rank test.

 
CD8+ cells are the main effector cell population involved in tumor protection induced by OVA-IC-loaded DCs

Fc{gamma}R-mediated uptake of ICs by DCs leads to efficient presentation of Ag-derived peptides in both MHC class I and class II molecules and to activation of peptide-specific CD8+ and CD4+ T cells in vitro (11, 12, 16, 17, 18) and in vivo (12, 20, 21). To assess the role of CD8+ and CD4+ cells in the effector phase of OVA-IC-treated DC-induced tumor protection, we depleted these effector cell populations in mice immunized with either freshly isolated BM DCs (Fig. 6B) or D1 cell line DCs (Fig. 6A), both loaded with OVA-ICs, before tumor challenge. As shown in Fig. 6, mice depleted for CD8+ cells developed large tumors and had a short survival time compared with nondepleted mice, indicating an important role for CD8+ cells in tumor eradication. Additional depletion of CD4+ cells (Fig. 6A) further decreased the survival and resulted in accelerated tumor growth, suggesting that CD4+ cells were also involved in tumor protection. Depletion of CD4+ cells alone did not have a significant effect on tumor development. These results show that CD8+ cells are the main effector cells in tumor protection induced by IC-loaded DCs.


Figure 6
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  		FIGURE 6. CD8+ cells are the main effector cell population involved in tumor protection induced by IC-treated DCs. D1 DCs (A) or B6 BM DCs (B) incubated with OVA-ICs were injected i.v. in B6 mice, followed by s.c. injection of OVA-expressing MO-5 tumor cells 14 days later. Naive mice were challenged and used as a control. When indicated, CD4+ and/or CD8+ cell populations were depleted by i.p. injections of 100 µg of purified anti-CD4 GK1.5 Ab and/or anti-CD8 2.43 Ab on day 1 before tumor challenge and days 2, 4, 6, and 13 after tumor challenge. The percentage survival upon tumor challenge for the different treatment groups is presented. Each treatment group consisted of eight mice. Two representative experiments of four performed are shown. Statistical analysis was performed using the log-rank test. DC-IC-treated mice and CD8-depleted DC-IC-treated mice are statistically different. A, D1-IC vs naïve, p < 0.0001; D1-IC CD4– vs D1-IC, p = 0.96; D1-IC CD8– vs D1-IC, p = 0.049; D1-IC CD4–CD8– vs D1-IC, p = 0.007. B, DC-IC vs naïve, p < 0.0001; DC-IC CD4– vs DC-IC, p = 0.3173; DC-IC CD8– vs DC-IC, p = 0.0016.

 
OVA-IC-loaded DCs are much more efficient in inducing protective immunity than are OVA-ICs

Prophylactic and therapeutic immunity against tumors are induced by IC-loaded DCs (Fig. 3 and Refs.4 and 21). We next compared whether direct administration of ICs into mice was as efficient in inducing tumor protection as ICs preloaded in vitro on DCs. Fourteen days before tumor challenge mice were immunized with either titrated amounts of preformed OVA-ICs or with D1 cells loaded in vitro with equal amounts of preformed OVA-ICs. ICs were formed by incubating OVA and anti-OVA Abs for 30 min at 37°C before injection or loading on DCs. DCs were incubated with 3-fold (Expt. 1) or 10-fold (Expt. 2) dilutions of these ICs, starting with 10 µg of OVA in ICs per 106 DCs injected into an individual mouse. After challenge with MO-5 tumor cells, mice were monitored for the development of palpable tumors. As shown in Fig. 7A, mice immunized with 10 µg down to as little as 0.001 µg of OVA in ICs loaded on D1 cells were protected from the development of tumors (Expt. 1: 3 of 5 mice at 10 µg were protected, 6 of 6 mice at all other concentrations tested were protected; Expt. 2: 5 of 6 mice at 10 µg were protected, 6 of 6 mice at 1 and 0.1 µg, 3 of 6 mice at 0.01 µg, 2 of 6 mice at 0.001 µg; 0 of 6 naive mice were protected). Importantly, even mice injected with 0.001 µg of ICs loaded on DCs showed a delay in median survival time of at least 38 days compared with naive mice (data not shown). In contrast, the same amount of OVA in ICs injected directly into mice was less efficient in inducing tumor protection (Expt. 1: 2 of 6 mice injected with 30 µg of ICs, 0 of 6 mice injected with 10 and 3 µg of ICs were protected; Expt. 2: 3 of 6 mice injected with 10 µg of ICs, 1 of 6 mice injected with 1 µg of ICs, and 0 of 6 mice injected with 0.1 µg of ICs were protected). Taking into account that the IC-pulsed DCs are also washed before injection, these data show that ICs loaded on DCs are at least 1000-fold more efficient in inducing tumor protection compared with ICs injected directly into mice, and they induce tumor protection over a broad range of concentrations. Furthermore, ICs loaded on DCs are much more efficient in inducing effective CTL responses than are ICs injected directly into mice. As shown in Fig. 7B, even high amounts (40 µg) of OVA in ICs injected directly into mice could not induce strong CTL responses against the immunodominant epitope (SIINFEKL) of the OVA protein. In contrast, efficient CTL responses were induced when mice were immunized with DCs loaded in vitro with relatively low amounts (3 µg) of OVA in ICs.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This study shows that ICs loaded on DCs (both freshly isolated BM DCs and the in vitro established splenic DC cell line D1) are highly efficient in inducing protective immune responses in vivo. OVA-IC-loaded DCs induced both prophylactic and therapeutic immunity against OVA-expressing tumors. Tumor immunity is mainly CD8+ T cell-mediated and the expression of Fc{gamma}RI and III on the DCs is required for efficient cross-presentation, cross-priming, and induction of efficient antitumor responses. In addition, we show that IC-loaded DCs are much more efficient than direct IC injection in the induction of tumor protection in vivo.

Very low concentrations of ICs are needed to load DCs to induce tumor protection, whereas only high amounts of ICs alone induce significant protection. The difference in efficiency is most probably due to the fact that the injected ICs are taken up and cleared by various types of cells expressing Fc{gamma}Rs other than professional APCs, in particular macrophages. Indeed, ICs have been shown to be quickly cleared after i.v. injection by both Kupffer cells and liver endothelial cells (50, 51). Another possibility is that ICs are targeted to cells that express relatively higher levels of the inhibitory receptor Fc{gamma}RII rather than the activating Fc{gamma}Rs, which does not lead to efficient induction of T cell immunity. Even different DC subsets may express different levels of the Fc{gamma}Rs, leading to differences in MHC class I-restricted Ag presentation and differences in response induction. When preloaded in vitro on DCs, ICs will be presented only by this professional APC. Because IC-loading induces maturation of DCs (11, 12), injection of these DCs induces efficient T cell responses in vivo (4, 12). Indeed we show that direct injection of ICs is inefficient in priming CTL responses in contrast to preloaded DCs (Fig. 7B). Another crucial issue is that direct injection of ICs is difficult to control in vivo. High amounts of injected ICs (above 40 µg/mouse) frequently cause acute shock syndrome in the mice. This has never been observed by injection of DCs preloaded with ICs.

DC-based antitumor therapies are emerging as a potential means of inducing protective tumor-specific responses (52), because DCs are capable of efficiently priming naive T cells (1) and cross-presenting exogenous Ag (e.g., tumor Ag) on MHC class I molecules, thereby priming Ag-specific CD8+ T cell responses (7). In the present study, we formally demonstrate the in vivo CTL priming by IC-treated DCs to be dependent on activating Fc{gamma}R I and III. It was previously shown that internalization of Ag/IgG immune complexes by the activating Fc{gamma}Rs on DCs facilitates MHC class I- and class II-restricted presentation of Ag in vitro (11) and that IC-induced enhanced presentation enables DCs to prime Ag-specific CD4+, CD8+, and humoral responses and to induce tumor protection in vivo (12, 20, 21). Furthermore, IC-induced maturation of DCs bypasses the requirement for CD4+ T cell help for priming of Ag-specific CD8+ responses in vivo (12). Together, these data indicate that Fc{gamma}R targeting on DCs can initiate protective antitumor responses. In contrast with recent studies (20, 27, 28) where the absence of Fc{gamma}RIIB was required for the efficient induction of tumor protection, in our study and in the study of Rafiq et al. (21), IC-loaded wt DCs are very efficient in inducing tumor protection in contrast to DCs lacking Fc{gamma}Rs. Different experimental set-ups might explain these discrepancies; these are the composition of the ICs used to load the DCs, different incubation times of the DCs with ICs before injection, the route of administration of IC-loaded DCs, and differences in the phenotype of the DCs. We and others have shown that the ratio of Ag and Ab used to preform the ICs influences the MHC class I-restricted Ag presentation and the IL-12 production, both being highest upon incubation of DCs with "optimal" ICs (11, 12). Furthermore the culture conditions may influence the expression levels of the different Fc{gamma}Rs on the DCs. It has been shown that D1 cells and BM DCs cultured as described (45, 46) express at least the three Fc{gamma}Rs (Fc{gamma}RI, Fc{gamma}RII, and Fc{gamma}RIII) (11, 12). In these DCs maturation is induced by ICs. In contrast, the DC cultures used by Kalergis and Ravetch (20) express mainly Fc{gamma}RII, and efficient maturation by ICs is only induced when this receptor is not present on the DCs. The relative levels on the DCs of activating and inhibitory Fc{gamma}Rs will determine the outcome of IC treatment of the cells. In the present study, we show that the absence of cell surface expression of Fc{gamma}RI and III is sufficient to abrogate efficient Ag presentation by MHC class I (Fig. 1), CTL priming (Fig. 2), and tumor protection (Fig. 4). Therefore, a role for the newly identified Fc{gamma}RIV (53) is likely to be limited in these processes.

In the above-mentioned studies, ICs were composed of OVA and rabbit IgG{alpha}OVA. Next to the rabbit IgG ICs we tested ICs composed of OVA and mouse IgG anti-OVA. These mouse IgG ICs induced DC maturation and enhanced MHC class I-restricted Ag presentation in vitro as efficiently as did rabbit IgG ICs. Importantly, mouse IgG-ICs also induce highly efficient protective immunity to MO-5 OVA-expressing tumors (data not shown).

Depletion of CD8+ cells leads to impaired tumor protection in immunized mice, indicating a substantial contribution of CD8+ cell responses to tumor protection. This is the first study showing the importance of CD8+ cells in the effector phase of tumor protection after immunization with IC-loaded DCs. Additional depletion of CD4+ cells further decreases survival of immunized and challenged mice, suggesting that besides CD8+ cells, CD4+ cell populations are also involved in protection against the tumor. However, depletion of CD4+ cells alone did not significantly alter protection, indicating that CD4+ responses are not required for an efficient antitumor response. It was previously shown that the protective effect of CD4+ Th cells can be either direct, by inhibiting tumor growth, or indirect, by augmenting protective CD8+ responses or attracting and activating macrophages or NK cells (22, 54, 55). Our data suggest that efficient priming of Ag-specific CD8+ responses by IC-treated DCs does not require further CD4+-dependent help for tumor eradication. In the absence of CD8+ cells, however, CD4+-dependent inhibition of tumor growth may play a role in tumor protection. The data of Rafiq et al. (21) show no tumor protection when mice are immunized with IC-loaded MHC class I–/– or MHC class II–/– DCs, suggesting a requirement for both CD8+ and CD4+ cells for tumor protection. In the present study, we specifically study the role of CD4+ and CD8+ cells in the effector phase of the tumor protection. CD4+ cells are dispensable during the effector phase in the presence of CD8+ cells, but they are important in the absence of CD8+ cells. Notably, mice depleted for both CD4+ and CD8+ cell populations are still partially protected against the tumor, suggesting the existence of other protective mechanisms that need to be investigated further. Candidates for additional effector cells are macrophages and NK cells, which can be activated by DCs (56, 57).

In conclusion, immunization with ex vivo IC-loaded DCs induces efficient tumor-specific CD8+ and CD4+ responses that are important for tumor protection in vivo. IC-loaded DCs are much more effective than are direct IC injection or soluble protein loaded on DCs. IC loading of DCs in vitro appears to be a very efficient vaccination strategy against cancer or infectious diseases. Advantages of this approach include the lack of a requirement for knowledge of the CTL and Th epitopes in the protein Ag and of the patient’s HLA type. The DCs are loaded with relatively low amounts of Ag that are likely to cause sustained Ag presentation; very efficient Fc{gamma}R-mediated Ag uptake and MHC loading is achieved and Fc{gamma}R-mediated DC maturation is induced, licensing the DCs to induce strong tumoricidal CD8+ CTL responses. Therefore, selective targeting of Fc{gamma}Rs on the relevant autologous APCs in vitro is highly advantageous for optimal effective induction of immunity against cancer.


   Acknowledgments
 
We thank P. Ricciardi-Castagnoli for the kind gift of the D1 cell line, M. Mulders and H. Krohn for animal care, and Drs. D. Roelen and R. Toes for critical reading of this manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from Anosys (to D.H.S.), the Netherlands Organization for Scientific Research (Grant 901-12-242 to A.I.-F.), Netherlands Cancer Foundation (Grant UL 2004-3008 to N.v.M.), the European Union Network of Excellence "DC Thera" Project (Grant FP6-512074 to F.O.), European Economic Community Project QLRT-1999-30470, and European Economic Community Project QLRT-1999-00064. Back

2 Current address: Department of Tumor Immunology, NCMLS, Radboud University Nijmegen Center for Molecular Life Sciences, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Back

3 N.v.M. and A.I.-F. contributed equally to this work. Back

4 Current address: Department of Rheumatology, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands Back

5 Address correspondence and reprint requests to Dr. Ferry Ossendorp, Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Albinusdreef 2, P.O. Box 9600, 2300 RC Leiden, The Netherlands. E-mail address: f.a.ossendorp{at}LUMC.nl Back

6 Abbreviations used in this paper: DC, dendritic cell; IC, immune complex; wt, wild type; BM DC, bone marrow-derived primary DC culture; rIgG{alpha}OVA, rabbit IgG anti-OVA. Back

Received for publication July 16, 2004. Accepted for publication January 23, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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N. van Montfoort, J. M. H. de Jong, D. H. Schuurhuis, E. I. H. van der Voort, M. G. M. Camps, T. W. J. Huizinga, C. van Kooten, M. R. Daha, J. S. Verbeek, F. Ossendorp, et al.
A Novel Role of Complement Factor C1q in Augmenting the Presentation of Antigen Captured in Immune Complexes to CD8+ T Lymphocytes
J. Immunol., June 15, 2007; 178(12): 7581 - 7586.
[Abstract] [Full Text] [PDF]

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D. M. Da Silva, S. C. Fausch, J. S. Verbeek, and W. M. Kast
Uptake of Human Papillomavirus Virus-Like Particles by Dendritic Cells Is Mediated by Fc{gamma} Receptors and Contributes to Acquisition of T Cell Immunity
J. Immunol., June 15, 2007; 178(12): 7587 - 7597.
[Abstract] [Full Text] [PDF]

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S. A. Perez, M. V. Karamouzis, D. V. Skarlos, A. Ardavanis, N. N. Sotiriadou, E. G. Iliopoulou, M. L. Salagianni, G. Orphanos, C. N. Baxevanis, G. Rigatos, et al.
CD4+CD25+ Regulatory T-Cell Frequency in HER-2/neu (HER)-Positive and HER-Negative Advanced-Stage Breast Cancer Patients
Clin. Cancer Res., May 1, 2007; 13(9): 2714 - 2721.
[Abstract] [Full Text]