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The Journal of Immunology, 2007, 179, 6009-6015
Copyright © 2007 by The American Association of Immunologists, Inc.
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A Crucial Role for Dendritic Cell (DC) IL-10 in Inhibiting Successful DC-Based Immunotherapy: Superior Antitumor Immunity against Hepatocellular Carcinoma Evoked by DC Devoid of IL-101

Yu-Xiao Chen2,*, Kwan Man{dagger}, Guang Sheng Ling*, Yichen Chen*, Bai-Shun Sun{dagger}, Qiao Cheng{dagger}, On Hong Wong*, Chi-Kin Lo*, Irene Oi-Lin Ng*, Li Chong Chan*, George K. Lau{ddagger}, Chen-Lung Steve Lin, Fanglu Huang§ and Fang-Ping Huang3,*,¶

* Department of Pathology, {dagger} Department of Surgery, and {ddagger} Department of Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China; § Department of Chemistry, University of Cambridge, United Kingdom; and Faculty of Medicine, Imperial College London, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The dendritic cell (DC)-based tumor immunotherapy has been a new promise of cure for cancer patients, but animal studies and clinical trials have thus far only shown limited success, especially in treating established tumors. Certain immunosuppressive mechanisms triggered by tumor cells or the derivatives are believed to be a major obstacle. We studied the role of DC-derived IL-10 and its negative impact on vaccine efficacy in mouse models. Liver tumor cells were injected via the portal vein, giving rise to disseminated intrahepatic tumors, or s.c. to form solid but extrahepatic tumors. Bone marrow-derived DCs were generated from normal or IL-10-deficient mice and used as the vector to deliver tumor Ags. We demonstrate here that DCs devoid of IL-10, a potent immunosuppressive cytokine, are superior over conventional DCs in triggering antitumor immunity. The IL-10–/–DCs were highly immunogenic, expressed enhanced levels of surface MHC class II molecules, and secreted increased amounts of Th1-related cytokines. By inducing tumor-specific killing and through the establishment of immunological memory, the vaccines delivered by IL-10–/–DCs could evoke strong therapeutic and protective immunity against hepatocellular carcinoma in the mouse models. These findings will have great clinical impact once being translated into the treatment of malignant, and potentially infectious, diseases in humans.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The immune system plays important roles in the fight against cancer, and there is evidence indicating the capacity of the normal immune system in rejecting tumors (1). Tumors may however evade the immune system by interacting actively with host immune cells to block their functions (1, 2, 3). Moreover, tumors are clones of mutated cells arisen from the body’s own tissues. Although the mutations may give rise to the so-called tumor-associated Ags (TAA),4 these newly derived or "altered self-"neoantigens are mostly poor immunogens upon exposure to the host immune system (2, 4). Many TAA-specific T and B lymphocytes have been identified in patients, but most of these cells are found in an unresponsive or anergized state (5, 6). These may explain why conventional vaccination approaches often fail to induce effective antitumor immunity (7). It has become a central question in tumor immunology as to how these TAA-specific clones are tolerized or suppressed and whether they can be reactivated to induce effective antitumor immunity (8).

The dendritic cell (DC)-based tumor immunotherapy aims to promote specific immunity to cancer cells within the tumor-bearing host (8, 9, 10, 11, 12, 13). By such an approach, DCs are used not only as a vector to deliver tumor Ags, but also a "natural adjuvant" to boost the vaccine efficiency. Although the main function of DCs is to present Ags to T cells, what make DCs special are their potent immunological adjuvanticity and diversified regulatory capacities (14, 15). Importantly, DCs can provide critical molecules, cytokines, or costimulatory signals to the T cells they interact with for activation. However, DCs are not a homogenous population (16, 17, 18, 19) and the type or functional condition, hence the immunogenic "quality" or nature, of the DC vector employed is essential (13, 17). It has gradually become clear that under certain conditions DCs can even exert tolerogenic or immunosuppressive effects on the immune system (1, 2, 15, 20, 21). DCs in the liver in particular are characterized by their tolerogenic nature, possibly due to the microenvironment within the organ (22), which is one of the major obstacles in the design of immunotherapies against liver cancer. Hepatocellular carcinoma (HCC) is a common liver cancer worldwide, with poor prognosis, and a majority of the patients are presented in advanced stages, which are often inoperable when diagnosed. In this study, we chose HCC as a model to test how DCs functionally conditioned targeting the negative arm of immune regulation can be of therapeutic value for this otherwise incurable disease.

IL-10 (23) is a potent immunosuppressive cytokine secreted by a variety of immune cell types including DCs, which can inhibit T cell activation, while the DC functional activities are in return tightly regulated by this very cytokine (24). Moreover, some tumor cells may produce IL-10 directly to suppress host immunity by blocking the DC functions (25). We observed recently that upon interactions with certain types of tumor cells, HCC and melanoma, for example, DCs could be switched to change their functional phenotype down-regulating their immunogenic capacity. In an attempt to improve the efficacy of DC-based tumor vaccines, we generated DCs from mice with an ablated gene encoding for IL-10 and used them as the vectors to deliver tumor Ags. The IL-10–/– DC vaccines were then tested categorically for their ability to induce therapeutic and protective immunity in mouse HCC models.


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

The IL-10 knockout (strain B6.129P2-Il10tm1Cgn/J, stock no. 002251, generation: N10F33, JAXR Mice Database-002251 B6.129P2-Il10<tm1Cgn>/J) and the wild-type (Wt) control C57BL/6 (stock no. 000664) mouse strains (both H-2b) were obtained from The Jackson Laboratory and set up as breeding pairs in the specific pathogen-free laboratory animal unit, University of Hong Kong Medical School. All experiments involving live animals were conducted strictly according to the protocols under licenses approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR 681-02, 1301-06), the University of Hong Kong.

The mouse HCC cell line (Hepa 1-6)

The Hepa 1-6 mouse liver cancer cell line is a derivative of C57BL/6 mouse hepatoma (H-2b). The tumor cell line was obtained from American Type Culture Collection and maintained in completed DMEM containing 10% heat-inactivated FBS, 1% L-glutamine, 100 µg/ml streptomycin, and 100 U/ml penicillin (Invitrogen Life Technologies). The adherent tumor cells were subcultured (~90% confluence) twice a week, and the cells used for in vivo experiments were of 50–70% confluent.

Establishment of the portal vein (PV) HCC tumor mouse model

Normal C57BL/6 mice (>3 mo of age, male) were used to develop the PV-HCC tumor model. The operations were performed under pentobarbital anesthesia (i.p. injection, 40 mg/kg). A middle incision of the abdomen was made and the PV was exposed. Live tumor cells (Hepa 1-6: 1, 2, 5, and 10 x 106 cells/mouse) in 100 µl of PBS were injected via the PV into the mouse liver. The injection site was covered by a cotton ball for 2–3 min to stop bleeding, before closing and sewing up the abdominal cavity. The mice normally recovered soon after operation. Groups of the tumor-bearing mice were sacrificed at 1, 2, 3, 4, or 5 wk after the PV tumor cell implantation, and the tumor development was recorded and kinetics of tumor development were determined. Subsequently, all experimental mice were terminated at 2 wk, unless indicated otherwise, after the PV tumor cell implantation (2 x 106 tumor cells/mouse).

Establishment of the intraflank HCC tumor mouse model

The intraflank HCC tumor model was developed based on a protocol previously developed by Lee et al. (26). Briefly, under anesthesia (isofluorane inhalation), normal C57BL/6 mice were injected in the left flank s.c. with live tumor cells (Hepa 1-6: 0.1, 0.5, 2.5, 5, or 10 million cells/mouse in 100 µl of PBS). Tumor development was monitored by measuring the size of the tumor (length, width, and depth) every 2–3 days. According to the kinetic data, 10 million cells/mouse were used in all subsequent experiments.

DC generation

DCs were propagated from mouse bone marrow precursors for 7 days in RPMI 1640 culture medium containing mouse GM-CSF (2% culture supernatant of the X63-Ag8 cell line provided by Prof. A N. Barclay, Medical Research Council Cellular Immunology Unit, Sir William Dunn School of Pathology, Oxford, U.K.) with or without IL-4 (4 ng/ml; PeproTech). The mice used for DC generation were generally 6–8 wk of age, and the IL-10–/– mice with signs of chronic enterocolitis (27) were excluded. DC purity was assessed by flow cytometry.

Tumor lysate preparation and pulsing

Tumor lysates were used as the source of TAA to load onto DCs. To prepare the tumor lysates, Hepa 1-6 cells (1 x 106/ml) were subjected to three cycles of freezing-thawing to acquire complete cell lysis. Aliquots of the lysates were stored at –20°C until use. For DC pulsing, the tumor lysate equivalents to 5 x 105 Hepa 1-6 cells were added to 5 x 106 DCs in cultures and incubated overnight to produce the DC vaccines or for various length of time for DC phenotypic analysis and cytokine expression.

Flow cytometry

DC purity and phenotypes were assessed by flow cytometry using Abs to DC surface markers (MHC class II, CD11c, CD80, CD86, CD40; BD Pharmingen). For lymphocyte phenotypic analysis, splenocytes and lymph node cells were stained with Abs to T (CD3, CD4, CD8) and B (CD19) cell markers (BD Pharmingen). Flow cytometry was used to detect and quantify the in vivo tumor-specific cytotoxic killing activity in the vaccinated mice.

Quantitation of cytokine production by DCs

Cytokine production by DCs in vitro, with or without tumor Ag preloading (as described above), spontaneously or in response to LPS stimulation (5 µg/ml) was determined by using commercial ELISA kits for mouse IL-10, IL-12(p70), IFN-{gamma}, TNF-{alpha} (OptEIA; BD Biosciences). Samples (culture supernatants) were collected from replicated cultures at time intervals and measured without dilution.

Protocols for DC therapy and vaccination

To determine the in vivo antitumor effects, DCs generated above from the IL-10–/– and Wt control mice were preloaded in vitro with tumor Ags (Hepa 1-6 cell lysates) overnight. After washing two times in PBS, the cells (106 DCs/mouse) were injected through the tail vein into the mice. For therapeutic effect, the Ag-loaded DCs were injected into the tumor-bearing mice 8 days after the tumor implantation (for both the PV and intraflank models). For vaccination purposes, naive young mice (6–8 wk of age) were given two i.v. injections of the Ag-loaded DCs at biweekly intervals, followed by intraflank tumor implantation 2 wk after the last vaccination. Control groups included mice injected with DC alone (without pulsing of the tumor lysates), untreated, or PBS-treated mice with or without tumor implantation.

Clinical and pathological assessments

At the end point of each experiment, the mice were sacrificed for assessments of tumor development and regression. Different tissues and organs, including liver, spleen, lymph nodes, pancreas, and lungs, were collected. The liver and spleen were also weighed against total body weight to evaluate the extent of tumor development. Some of the tissues were then fixed in 4% buffered formaldehyde, embedded in paraffin, and sections stained with H&E for histological examination.

Mixed Lymphocyte Reaction

Syngenic and allogenic T cell responses to IL-10–/– DCs and WtDCs were determined in the MLR assay. Briefly, day 6 DCs generated from bone marrow precursors of the IL-10 knockout (IL-10–/– DCs) and Wt control (WtDCs) mice (both C57BL/6 background, H-2b), with (killed DCs) or without (live DCs) mitomycin C treatment, were used as the APC. Splenocytes from BALB/c (H-2d, allogenic), C57/BL6 (H-2b, syngenic), and the C57BL/6IL-10–/– mice (H-2b, syngenic) were used as the responder cells and added, respectively, at different effector:responder (DC spleen cells (SPC)) ratios. The effects of exogenous IL-10 were also determined by adding serial concentrations of recombinant mouse IL-10 (1023-ML-010; R&D Systems) in the selected cocultures of IL-10–/– DCs with the splenocytes. Cell proliferation was measured by [3H]thymidine incorporation (0.5 µCi/well added for the last 8 h) at days 1, 2, 3, and 4.

In vivo cytotoxic killing assay

Normal naive C57BL/6 mice were immunized twice as described above for DC vaccination. Two weeks after the last vaccination, the mice were injected with tumor Ag-pulsed and fluorescent-labeled target cells based on and modified from a protocol previously developed by Dercamp et al. (28). Briefly, mouse splenocytes were isolated and equally divided into two portions. One cell population was pulsed with tumor Ags (Hepa 1-6 cell lysates) and cultured overnight at 37°C, while the other population was cultured in medium alone. The two populations of cells were then washed and differentially labeled with the fluorescent dye CFSE at the final CFSE concentrations of 10 and 2 µmol/L for the tumor lysate-pulsed (CFSEhigh) and unpulsed (CFSElow) populations, respectively. After washing, the two cell populations were mixed at 1:1 ratio, and 2 x 107 total cells (per mouse) were injected i.v. into the vaccinated or nonvaccinated mice. At the time intervals indicated, groups of the mice were killed and cells isolated from the spleens and hepatic and mesenteric lymph nodes were analyzed by flow cytometry. The percentage of Hepa 1-6-specific lysis was determined by the relative proportion of the pulsed vs unpulsed target cells, i.e., ratio of the remaining CFSEhigh over CFSElow populations in the corresponding lymphoid organs, and expressed as the percentage of specific tumor killing: [(M2 – M1)/M2] x R x 100), where M2 = percentage of CFSElow (unpulsed target events), M1 = percentage of CFSEhigh (pulsed target events), and R = the mean ratio of CFSEhigh:CFSElow targets from naive recipients.

Statistics

Student’s t test (two-tailed) was used for statistical analysis of the data between groups and a p ≤ 0.05 (* or *) was considered significant and a p ≤ 0.01 (** or *) was considered highly significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
DCs devoid of IL-10 are superior in triggering therapeutic immunity against established tumors in mouse models

To understand the role of DC-derived IL-10 on DC-based tumor immunotherapy, we generated DCs from IL-10-deficient mice and used them as the vectors to deliver tumor Ags. The effectiveness of IL-10–/– DC vaccine was tested repeatedly in two different mouse models of liver cancer. Crucial to the present study, a novel liver cancer model was first developed in normal C57BL/6 mice by direct injection of the tumor cells (Hepa 1-6 mouse HCC cell line, C57BL/6 origin) into the liver through the PV (see Materials and Methods). These mice soon developed progressive hepatomegaly and splenomegaly (Fig. 1A). Histological examination confirmed the development of numerous tumor nodules in the livers (Fig. 1B, micrographs), and splenomegaly as a result of portal hypertension. The PV-HCC mouse model was subsequently used to evaluate the in vivo anti-tumor effects of the DC vaccines. Fig. 1, B and C, shows that the IL-10–/– DCs (group 3) were superior over the WtDCs (group 2) as a cell vector to deliver the therapeutic vaccines. Remarkably, the majority of the mice that had received only one injection of the IL-10–/– DC vaccine (group 3) rejected the tumors effectively within 2 wk, and the livers were found to be grossly and microscopically tumor free (Fig. 1B, micrographs). Their anti-tumor potential was also well reflected by the "liver:body" and "spleen:body" ratios for each of the corresponding treatment groups, respectively (Fig. 1C), with reference to that of the PBS-treated (group 1) and normal (group 4) control mice. The DCs used in the experiment shown in Fig. 1, B and C, were generated in the presence of GM-CSF alone (GM-DCs). Similarly enhanced anti-tumor effects were also observed when the DCs used were generated in the presence of a combination of GM-CSF and IL-4 (G4-DCs) tested in the PV-HCC model (data not shown) and in the intraflank model (Fig. 2B, see below).


Figure 1
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  		FIGURE 1. A superior therapeutic vaccine against HCC in the PV-HCC mouse model. A, Establishment of the PV-HCC liver tumor model in immune competent mice. Normal C57BL/6 mice were injected through the PV with Hepa 1-6 cells (5 million cells/mouse) or PBS (control), and the mice were sacrificed at 2 wk postinjection. Hepatomegaly and splenomegaly were observed in the Hepa 1-6 cell-injected mouse group (right). Histological examination confirmed the development of numerous tumor nodules in the livers of Hepa 1-6 cells (Hepa 1-6, right), but not PBS (control, left), injected mice. TN, Tumor nodule. B and C, Superior antitumor immunity evoked by DC devoid of IL-10. Normal C57BL/6 mice were injected through the PV with live Hepa 1-6 cells (groups 1–3) or PBS only (group 4, normal control). After 1 wk, the mice were treated (i.v.) with either the WtDCs (group 2) or IL-10–/– DCs (group 3) preloaded with tumor Ags (Hepa 1-6 cell lysates) or injected with PBS only (groups 1, 4). The DCs used in B and C were generated from syngenic mouse bone marrow precursor cells in the presence of GM-CSF alone (GM-DCs) as described in Materials and Methods. The mice were sacrificed 2 wk after the PV tumor implantation. Hepatomegaly and splenomegaly were observed grossly in the untreated and the WtDC vaccine-treated, but not the IL-10–/– DC vaccine-treated, mice (B, upper and middle panels). Histological examination of the livers (B, lower panels) confirmed the presence of numerous tumor nodules in the livers of tumor-bearing mice. Tumor development and regression were assessed by calculating the relative liver:body and spleen:body ratios for each of the corresponding treatment and control groups (n = 4). *, p ≤ 0.05; **, p ≤ 0.01 (Student’s t test).

 

Figure 2
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  		FIGURE 2. Therapeutic potential of the IL-10–/– DC vaccines reconfirmed in the intraflank HCC liver tumor model. Normal C57BL/6 mice were injected in the left flank s.c. with 10 million Hepa 1-6 tumor cells at day 0 (solid arrow). By day 7, sizable tumors were measurable (7–10 mm in diameter) and, at day 8 (open arrow), the mice were given i.v. one injection of the tumor Ag-loaded WtDCs or IL-10–/– DCs. Tumor development was measured at daily intervals and expressed as diameters of the tumor mass. Control groups (Control) were tumor-bearing mice treated with PBS only. DCs generated in the presence of GM-CSF alone (GM-DCs, results combined from two repeated experiments, n = 9, A) or GM-CSF plus IL-4 (G4- DCs, n = 5, B), respectively, were compared. *, Significant differences between the IL-10–/– DC and WtDC vaccine-treated groups and *, significant difference between the IL-10–/– DC vaccine-treated group and the PBS-treated tumor control group (Student’s t test, * or *, p ≤ 0.05; ** or **, p ≤ 0.01).

 
To study the kinetics of tumor development, we next adopted another HCC mouse model by injecting the tumor cells s.c. into the left flank of mice, based on a protocol previously developed by Lee et al. (26). This intraflank HCC model allowed close monitoring of the tumor growth and regression and was used to test further and to confirm our observations in the PV-HCC model above. The therapeutic potency and superiority of the IL-10–/– DC vaccine, as compared with that of the WtDCs, in rejecting the extrahepatic tumor cells was again clearly demonstrated (Fig. 2, • vs {blacktriangleup}). Rapid tumor regression was evident in the mice after only one injection of the tumor Ag-loaded IL-10–/– DCs and similar effects were observed whether the DCs used were generated in the presence of GM-CSF alone (Fig. 2A) or a combination of GM-CSF and IL-4 (Fig. 2B).

DCs developed in the absence of IL-10 had a highly immunogenic phenotype

To study the immunological mechanisms, the functional phenotype of IL-10–/– DCs was next characterized. DCs generated in the absence of IL-10 showed markedly enhanced expression of surface MHC class II molecules and moderately increased MHC class I expression, as compared with that of the WtDCs (Fig. 3, A and B). A clear difference in the MHC class IIhigh population (Fig. 3B), particularly for those generated in the presence of GM-CSF alone (GM-DCs), was observed. No or little difference was detected in the expression of CD40 or CD80, but a moderate and consistently observable CD86 up-regulation was noted also on the IL-10–/– DCs compared with the WtDCs (Fig. 3B). Importantly, the absence of IL-10 also resulted in an enhanced production of Th1-type cytokines by the cells upon stimulation. This was indicated by their expression of IL-12(p70) (for both GM-DCs and G4-DCs) and IFN-{gamma} (G4-DCs) (Fig. 3C). Moreover, a spontaneous release of IFN-{gamma} by the IL-10–/– DCs without stimulation was also detected (Fig. 3C, IFN-{gamma} production by G4-DCs, {circ}). In contrast, there was no or little difference observed in the production of TNF-{alpha} between the IL-10–/– DCs and WtDCs. Although a reduction in the LPS-induced IL-12 production was noted following treatment of DCs with tumor Ags (Hepa 1-6 cell lysates), the relative levels were still significantly higher in the IL-10–/– DCs as compared with the corresponding tumor Ag-treated or even untreated WtDC cultures (data not shown). To confirm further the role of DC-derived IL-10 in regulating DC functions and to exclude the possible alloreactivity being a mechanism involved in tumor rejection, syngenic and allogenic T cell responses to the IL-10–/– DCs and WtDCs were determined and compared in the MLR assay. The IL-10–/– DCs also displayed a heightened activity when used as APC for the stimulation of both allogeneic and syngenic T cell responses (Fig. 3D, left panels). The differences could be however largely cancelled out either by mitomycin C treatment of the DCs (killed DCs, Fig. 3D, right panels) or by addition of rIL-10 (Fig. 3E). These results thus demonstrated the essential role of DC-derived IL-10 in modulating DC immunogenicity.


Figure 3
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  		FIGURE 3. Phenotypic and functional analysis of IL-10–/– DCs. A and B, DCs developed in the absence of IL-10 expressed high MHC class II molecules. DCs were generated from mouse bone marrow precursors in the presence of GM-CSF, with (G4-DCs) or without (GM-DCs) IL-4. At day 7, the DC phenotypes were determined by flow cytometry using specific Abs to different DC phenotypic and functional markers. A, MHC class II, CD11c, CD80, CD86, and CD40 expression on IL-10–/– DCs (filled histogram) and WtDCs (gray solid line), which were day 7 DCs generated in the presence of GM-CSF alone. Dotted line, Isotyopic control. B, Frequency of DCs expressing high MHC class II molecules (MHC IIhigh DCs) as determined by gating on CD11c+MHChigh cells (oval gated region) and expressed as the percentage of total CD11c+ cells, and the bars also compare the MHC IIhigh cell frequencies for both the GM-DCs and G4-DCs. C, DCs devoid of IL-10 produced markedly enhanced levels of Th1-type cytokines. DCs were generated as above and, at day 6, the GM-DCs and G4-DCs were stimulated with LPS (1 µg/ml, •, {blacktriangleup}) or cultured without stimulation ({circ}, {triangleup}) for 12, 24, and 48 h. Cytokine levels in the culture supernatants were measured by ELISAs specific for IL-10, IL-12, IFN-{gamma}, and TNF-{alpha}, respectively (see Materials and Methods). Statistical significance of the differences between IL-10–/– DCs and WtDCs is indicated for both the LPS-induced (*) and spontaneous (*) cytokine release, respectively (Student’s t test, * or *, p ≤ 0.05; ** or **, p ≤ 0.01). D and E, Syngenic and allogenic T cell responses to IL-10–/– DCs in the presence or absence of exogenous IL-10. Day 6 DCs generated from bone marrow precursors of the IL-10 knockout (IL-10–/– DCs) and WtDC mice (both C57BL/6 background, H-2b, with (killed DCs) or without (live DCs) mitomycin C treatment, were used as the APCs (effectors). Splenocytes from BALB/c (H-2d, allogenic), C57BL/6 (H-2b, syngenic), and the C57BL/6IL-10–/– mice (H-2b, syngenic) were used as the responder cells and added, respectively, at different effector:responder (DC:SPC) ratios. D, Comparison of allogenic and syngenic T responsiveness to live and killed (mitomycin C-treated) DCs. E, Effects of rIL-10 on the IL-10–/– DC-mediated MLR responses. The effects of exogenous IL-10 were determined by adding a fixed amount (10 ng/ml) or serial concentrations (as indicated in the graph) of recombinant mouse IL-10 (1023-ML-010; R&D Systems) in the selected cocultures of live IL-10–/– DCs and splenocytes from the three different mouse strains, respectively. Cell proliferation was measured by [3H]thymidine incorporation (0.5 µCi/well added for the last 8 h, and data shown at day 2).

 
The IL-10–/– DC vaccine is highly effective in inducing protective antitumor immunity through establishment of specific immunological memory

To understand further the underlying immunological mechanisms, groups of mice were injected with the DC vaccines before tumor implantation. Fig. 4A shows that the vaccine delivered by DCs devoid of IL-10 is also very effective in triggering protective immunity against the tumor. After two immunizations at biweekly intervals with the tumor Ag-loaded IL-10–/– DCs, these mice were effectively protected from tumor development (Fig. 4A, •). Such protection was also observable, although to a much lesser extent, in mice immunized with the WtDC vaccine (Fig. 4A, {blacktriangleup}). These results indicated the establishment of specific immunological memory in the vaccinated mice. An in vivo cytotoxic killing assay was then adopted (28) for assessing the tumor-specific immunity. As shown in Fig. 4, B and C, mice immunized with the IL-10–/– DC vaccine were significantly more efficient, as compared with the WtDC-vaccinated mouse group, in mounting the Hepa 1-6 Ag-specific killing in vivo.


Figure 4
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  		FIGURE 4. The IL-10–/– DC vaccine is highly effective in triggering protective antitumor immunity through establishment of immunological memory. A, Effective induction of protective immunity against liver cancer by immunization with the IL-10–/– DC vaccine. Groups of normal C57BL/6 mice (n = 4) were first given two injections of the DC vaccines at biweekly intervals (open arrows), followed by tumor implantation at day 28 (solid arrow). Tumor development was measured at daily intervals after tumor implantation and expressed as diameters (upper graph) and volume (lower graph) of the tumor mass. B and C, In vivo Hepa 1-6 Ag-specific cytotoxic killing in lymphoid organs of the DC-vaccinated mice. Normal naive C57BL/6 mice were immunized twice as described above in A. Two weeks after the second vaccination, the mice were injected with tumor Ag-pulsed and CFSE-labeled target cells based on and modified from a protocol previously developed by Dercamp et al. (28 ; see Materials and Methods for details). Forty-eight hours after the target cell injection, the mice were killed and different lymphoid organs, including spleens (Sp), hepatic lymph nodes (hLN) and mesenteric lymph nodes (mLN), were removed for analysis by flow cytometry. B, Representative FACS profiles showing relative frequencies of the remaining tumor Ag-pulsed (CFSEhigh) and unpulsed control (CFSElow) target cells detected in the spleen and hepatic lymp nodes of the vaccinated mice. The inserted figure represents the absolute ratio of the tumor Ag-pulsed (CFSEhigh) over the total CFSE+ (CFSEhigh + CFSElow) target cells detected in the corresponding lymphoid organs. The percentage of tumor-specific killing (lysis) in the lymphoid organs of the vaccinated mice was then individually calculated (see Materials and Methods for details) and is shown in C. *, Significant difference between the IL-10–/– DC vaccine and the WtDC vaccine-treated groups, and *, Significant difference between the IL-10–/– DC vaccine-treated group and the PBS-treated control group (* or *, p ≤ 0.05; ** or **, p ≤ 0.01, Student’s t test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we demonstrated a crucial role of DC-derived IL-10 in inhibiting successful immunotherapy against HCC in mouse models. It drew particular attention to the negative regulator of DC functions, critical to the outcome of such a live cell-based anti-tumor approach. We showed that DCs generated in the absence of endogenous IL-10 acquired a highly immunogenic phenotype and could be a desirable vector for delivering tumor vaccines with high efficacy. The novel IL-10–/– DC vaccine was repeatedly tested and confirmed in two HCC mouse models and shown to induce effectively both therapeutic and protected immunity against the tumor.

It is worth pointing out that DCs in the liver are characterized by their tolerogenic nature, possibly due to the microenvironment within the organ (22), which may present as a major obstacle in the design of immunotherapies against the tumor (29). In addition, most of the models of liver cancers currently used for experimental studies are established in immunodeficient animals. To study DCs whose main function is to activate T cells, the key mediators of antitumor immunity, it has been necessary to develop these tumor models in immunocompetent animals. In the present study, the intrahepatic PV-HCC model was successfully established in the normal inbred C57BL/6 strain of mice. The PV-HCC model was useful to determine how the primary liver microenvironment could affect the outcome of DC therapy. We have also adopted the intraflank HCC model (26), which allowed a more detailed monitoring of the kinetics of tumor growth and regression. Although some spontaneous tumor regression was observed in this extrahepatic model, the superiority of IL-10–/– DC vaccine was again unequivocally demonstrated and confirmed. Furthermore, the strong antitumor immunity against liver cancer demonstrated in our study also points to the unlimited potential of such a molecularly programmed and immunologically optimized approach for treating other types of tumors. Findings from our parallel study (our manuscript in preparation) have further shown that the IL-10–/– DC approach could also be highly effective in treating melanoma, a skin cancer of unrelated origin.

The superiority of IL-10–/– DCs could be explained immunologically first by the absence of IL-10, a potent immunosuppressive cytokine (24). This allowed enhanced surface expression of MHC class II, and class I, molecules on the IL-10–/– DCs as compared with control WtDCs, which would result in more efficient Ag presentation (signal 1) to both helper T cells and CTLs, respectively. In particular, we showed that DCs generated in the presence of either GM-CSF alone or GM-CSF plus IL-4, but in the absence of IL-10, all expressed markedly increased levels of the class II molecules. The effects were however not simply due to changes in DC maturation status, hence efficiency of these cells to present Ags, but also to their ability to provide other important costimulatory signals for T cell activation. Although the effects on DC surface expression of CD80, CD86, and CD40 (signal 2) were marginal, the IL-10–/– DCs secreted significantly more IL-12(p70) (GM-DCs and G4- DCs), which was the major polarizing cytokine for Th1-type responses and IFN-{gamma} (G4-DCs only) the very marker and effector mechanism of Th1 immunity. The two essential Th1-type cytokines (signal 3) are known to be crucial for anti-tumor immunity (12). In addition, although different combinations of these DC functional changes appeared to be responsible for the heightened immunogenicity observed, we showed further that DCs generated either in the presence of GM-CSF alone or with IL-4 were both efficient cell vectors for vaccine delivery. They were highly effective in triggering tumor-specific CTL killing and, through establishment of immunological memory, in inducing durable protective anti-tumor immunity.

Taken together, our findings above demonstrate clearly that targeting the negative regulatory regulators of DCs can be an effective way to maximize DC immunogenicity. With many currently available molecular approaches, siRNA, for example, this can be achieved by a selective knock-down transcriptionally or posttranscriptionally (30) of the genes encoding for IL-10 and other known immunosuppressive molecules (31). These approaches will soon prove to be the way forward in the design of novel DC-based immunotherapies, to fight against many different malignant and potentially infectious diseases, and to benefit the mankind.


   Acknowledgments
 
We thank Prof. G. Gordon MacPherson and Prof. Foo Y. Liew for scientific discussions and advice, Dr. Liliane M. Fossati-Jimack for mouse genetics information, and Alan Chan for photographic processing.


   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 Hong Kong Research Grant Council Grants RGC HKU 7246/01M and 7291/02M and grants from the Hong Kong University Research Grant Committee. Y.-X.C. is a Cheng Yu Tong Visiting Scholar from Xiang Ya School of Medicine, Central South University, Hunan, China. Back

2 Current address: Deparment of Immunology, School of Basic Medical Sciences, Xiang Ya School of Medicine, Central South University, Hunan, China. Back

3 Address correspondence and reprint requests to Dr. Fang-Ping Huang, Room 333, 3/F, Block L, Molecular Genetics and Rheumatology Section, Imperial College Faculty of Medicine, Hammersmith Hospital, Du Cane Road, London, W12 ONN U.K. E-mail address: fp.huang{at}imperial.ac.uk Back

4 Abbreviations used in this paper: TAA, tumor-associated Ag; DC, dendritic cell; HCC, hepatocellular carcinoma; IL-10–/–DC, DC generated from IL-10 knockout mice; SPC, spleen cells; WtDC, DC generated from wild-type control mice; PV, portal vein. Back

Received for publication May 14, 2007. Accepted for publication August 27, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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