Plasmacytoid dendritic cells (pDC) are key regulators of antiviral immunity. In previous studies, we reported that pDC-infiltrating human primary breast tumors represent an independent prognostic factor associated with poor outcome. To understand this negative impact of tumor-associated pDC (TApDC), we developed an orthotopic murine mammary tumor model that closely mimics the human pathology, including pDC and regulatory T cell (Treg) infiltration. We showed that TApDC are mostly immature and maintain their ability to internalize antigens in vivo and to activate CD4+ T cells. Most importantly, TApDC were specifically altered for cytokine production in response to Toll-like receptor (TLR)–9 ligands in vitro while preserving unaltered response to TLR7 ligands (TLR7L). In vivo pDC depletion delayed tumor growth, showing that TApDC provide an immune-subversive environment, most likely through Treg activation, thus favoring tumor progression. However, in vivo intratumoral administration of TLR7L led to TApDC activation and displayed a potent curative effect. Depletion of pDC and type I IFN neutralization prevented TLR7L antitumoral effect. Our results establish a direct contribution of TApDC to primary breast tumor progression and rationalize the application of TLR7 ligands to restore TApDC activation in breast cancer. Cancer Res; 73(15); 4629–40. ©2013 AACR.

Despite active immunosurveillance, some tumors still progress and escape through immunosubversion processes (1, 2). The understanding of the paradoxical role of the immune system during cancer development is a major challenge for new immunotherapy strategies.

Dendritic cells, the most powerful antigen-presenting cells (APC), play a key role in orchestrating adaptive immune responses. Most cancers, including breast tumors, are highly infiltrated by dendritic cell. Two main populations of dendritic cell, namely myeloid dendritic cell (mDC) and plasmacytoid dendritic cell (pDC), are found in mouse and human tissues. Functional alteration of mDC by the tumor microenvironment has been described as a mechanism to escape immunosurveillance. However, there is also increasing evidence implicating pDC in tumor immunity (3, 4).

pDC are key regulators of antiviral immunity at the interface of innate and adaptive immunity (5). pDC secrete rapidly large amounts of type I IFNs, inflammatory cytokines, and chemokines in response to microbial and self-RNA or DNA recognized by endosomal Toll-like receptor (TLR)–7 and TLR9, respectively (6–8). After their encounter with viruses, pDC differentiate into mature dendritic cell and present viral antigens directing T-cell responses with considerable flexibility (9). Uncontrolled production of type I IFN by chronically activated pDC contributes to autoimmune diseases (10). In contrast to immune activation, pDC were also shown to suppress or limit inflammatory responses to allo-Ag, allergens, or oral Ag (11–14). In human breast cancer, we previously reported that pDC infiltrating the primary tumor represent an independent prognostic factor associated with poor outcome (15). pDC infiltrate other solid tumors with various consequences on immune response (16–18). We and others reported that tumor-associated (TA)pDC are altered for IFN-α production (17, 19, 20) and favor regulatory T cell (Treg) expansion via ICOS–ICOSL interaction (20) that may contribute to tumor progression and explain their negative impact on patient survival (15). In contrast, TApDC were shown to become efficient therapeutic targets after recruitment and activation by TLR7L in skin cancers (3, 21).

Depending on the context, TApDC could thus have negative or positive impact on antitumor immune responses. This study in orthotopic mouse mammary tumor model was designed to understand whether TApDC directly contribute to breast tumor progression and whether they could be mobilized to favor tumor regression. We showed that NEU15 mammary tumor cell line implanted in immunocompetent mice are highly infiltrated by both TApDC and TATreg. Importantly, TApDC are functionally altered in their response to TLR9L and their in vivo depletion delays tumor growth. However, TApDC can be activated in vivo via TLR7L to induce tumor regression through a type I IFN-mediated mechanism. This study shows a direct contribution of TApDC to breast tumor progression and identifies TLR7 ligands as new therapeutic strategies in breast cancer.

Mice

Wild-type FVB/N and C57BL/6 (Charles River Laboratory), homozygous or heterozygous (named MMTV-Neu F1) FVB/N-MMTVneu-202Mul transgenic female mice (Jackson Laboratory; ref. 22) were used at 6 to 8 weeks of age. Mice were maintained in pathogen-free animal facility “AniCan” at the Cancer Research Center of Lyon. Experiments were conducted in accordance with the European and French laws and were validated by the local animal ethical evaluation committee (Comite d'Equique Commun CLB Animal transit ENS PBES P4).

Tumor cell line and reagents

The NEU15 cell line was established from a spontaneous mammary tumor harvested from an MMTVneu transgenic female mouse. NEU15 cells were grown in vitro with 5% CO2 in DMEM (Life Technologies) supplemented with 10% heat inactivated fetal calf serum (PAA Laboratories), 100 U/mL penicillin, 100 μg/mL streptomycin, and 1% l-glutamine (Sigma-Aldrich).

TLR7 ligands were formaldehyde-inactivated influenza virus (A/Wisconsin/67/05; 1,000 HAU/mL; gift from Sanofi Pasteur), CL075 (3 μg/mL) and R848 (5 μg/mL; Cayla SAS), and SM360320 (5 μg/mL, Janssen Infectious Diseases-Diagnostics BVBA; ref. 23). TLR9 ligands were CpG-A/ODN-2336 and CpG-B/ODN-1826 (5 μg/mL; Cayla SAS).

In vivo transplanted orthotopic mammary tumor models

Wild-type (WT) FVB or MMTV-neu F1 mice were injected with 5 × 106 NEU15 cells into the fourth mammary fat pad. Tumor volume was calculated by using the ellipsoidal formula, π/6 × length × width2. Tumor-bearing mice were euthanized at the experimental endpoint (volume > 2,000 mm3). TLR7L administration was conducted as intratumoral (i.t.) or subcutaneous contralateral injection (50 μL) as indicated. In pDC depletion experiment, mice were injected intraperitoneally with ascite-derived mab927 (200 μL half diluted ascite; ref. 24) or purified 120G8 antibody (150 μg/injection; BioXcell; ref. 25). Purified total IgG (Sigma-Aldrich) or rat IgG1 (BioXcell) were used as control. Anti-mouse IFNAR1 and rat IgG1 (50 μg) were from eBioscience.

Enzymatic digestion and dendritic cell purification

After enzymatic digestion [30 minutes at 37°C, type IA collagenase (1 mg/mL) and DNase (0.1 mg/mL)], red blood cells were lysed with Pharmlyse Buffer (BD Biosciences). For cell sorting, dendritic cell were enriched by anti-CD11c microbeads (Miltenyi Biotec), then stained with anti-CD11c-PE-Cy5, B220-PE, and CD11b-FITC antibodies in presence of Fc Block. pDC were sorted as CD11c+CD11bB220+ cells among live cells on a FACS Vantage sorter (BD Biosciences). Purity was routinely above 98%. For infiltrate analysis, CD45+ cells were first enriched from tumor single-cell suspension by magnetic selection (Miltenyi Biotec) as detailed in Supplementary Materials and Methods.

Cytokine secretion assays

pDC were cultured at 0.25 × 106 cells/mL with TLR7 and TLR9 ligands in RPMI medium supplemented with 10% fetal calf serum, penicillin/streptomycin, l-glutamine, nonessential amino acids, and 20 ng/mL of human recombinant Flt3-ligand (PeproTech). Supernatants were collected after 40 hours and assessed for cytokine production. CCL5 (R&D Systems) and IFN-α (PBL) levels were measured by specific ELISA. Interleukin (IL)-6 and CCL3 were measured by Luminex multiplex bead cytokine assay (MILLIPLEX Mouse Cytokine/Chemokine kit; Millipore).

Mixed lymphocyte reaction

Allogeneic CD4+ T cells were purified using anti-CD4–coated microbeads (Miltenyi Biotec) from naive spleen of C57BL/6 mice depleted of Gr1, MHC II, CD11b, and CD8-expressing cells. CD4+ T cells (5 × 104 per well) were cultured in triplicate for 5 days with pDC using indicated TLR ligands. At day 4, half of the medium was collected for IFN-γ determination by ELISA (R&D Systems). T-cell proliferation was assessed by [3H]-thymidine incorporation during the last 18 hours (0.5 μCi/well; GE Healthcare). [3H]-Thymidine incorporation was measured as counts per minute (cpm) by liquid scintillation counting (MicroBeta; Perkin-Elmer).

Statistical analysis

Statistical analyses were conducted using Prism 5 software. Differences between groups were analyzed using the Mann–Whitney test for nonparametric and unpaired samples. Gehan Breslow Wilcoxon test was used to compare survival curves. P values less than 0.05 were considered significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Transplantable HER2/neu-expressing tumors escape immune response in WT mice while preserving HER2/neu expression

To characterize the potential role of pDC in primary breast tumor progression, we selected a clinically relevant murine mammary tumor model. HER-2/neu FVB/N transgenic mice express the rat proto-oncogene her2/neu under the control of the mammary-specific MMTV promoter. Those mice develop focal, poorly differentiated ER/neu+ spontaneous mammary carcinomas (Supplementary Fig. S1A and S1B). Because of long and variable latency of tumor appearance, a stable HER-2/neu-expressing cell line (referred to as NEU15) was derived in vitro from a spontaneous tumor. NEU15 was then transplanted by orthotopic injection into syngeneic mice. Transgenic mice tolerated rat HER2/neu as a self-protein as evidenced by the absence of specific immune responses due to central tolerance mechanisms and the aggressive tumorigenicity of NEU15 tumor in MMTV-neu F1 mice (named NEU15F1; Fig. 1A). In contrast, rat HER2/neu was perceived as a foreign antigen by the immune system of WT mice, as evidenced by the induction of high levels of anti-HER2/neu antibody of IgG1 isotype that resulted in delayed NEU15 tumor (named NEU15WT) growth (Fig. 1A and B). Accordingly, NEU15WT tumor-bearing mice displayed (data not shown). Despite immune pressure, NEU15WT tumors maintained similar HER2/neu expression to that of NEU15F1 tumors (Fig. 1C). The resistance of NEU15WT tumor cells to immunoediting implies that sustained HER2/neu expression is essential for NEU15 tumor cell survival and suggests an immunosubversion by the tumor microenvironment.

Figure 1.

NEU15WT tumors escape immunosurveillance without immunoediting. A, NEU15 tumor cells derived from an MMTVneu spontaneous tumor were injected into WT or MMTVneu F1 female mice. Data represent the mean ± SEM of the calculated tumor volume for 5 individual mice. B, concentrations of anti-Neu antibodies in sera from MMTVneu mice bearing NEU15WT or NEU15F1 tumors were measured. C, representative immunohistochemical staining on paraffin-embedded sections of NEU15WT and NEU15F1 tumors using HPS stain or anti-HER2/Neu antibody as indicated. Magnification, ×200. TUM, tumor bed. ***, P < 0.001.

Figure 1.

NEU15WT tumors escape immunosurveillance without immunoediting. A, NEU15 tumor cells derived from an MMTVneu spontaneous tumor were injected into WT or MMTVneu F1 female mice. Data represent the mean ± SEM of the calculated tumor volume for 5 individual mice. B, concentrations of anti-Neu antibodies in sera from MMTVneu mice bearing NEU15WT or NEU15F1 tumors were measured. C, representative immunohistochemical staining on paraffin-embedded sections of NEU15WT and NEU15F1 tumors using HPS stain or anti-HER2/Neu antibody as indicated. Magnification, ×200. TUM, tumor bed. ***, P < 0.001.

Close modal

NEU15WT tumors are highly infiltrated by pDC and Treg

Although leukocytes are found in all tumors, NEU15WT tumors seemed more infiltrated by CD45+ cells (22.9 ± 7.3%) than NEU15F1 tumors (8.9 ± 5.3%; Supplementary Fig. S2A).

A thorough analysis of immune cell infiltrate was conducted by multiparametric flow cytometry on NEU15WT tumor single cell suspension (Fig. 2A). T cells (CD3+) and NK cells (NKp46+) represented about 10% to 15% of leukocytes, whereas B (CD19+) represented a minor part of the infiltrate (Fig. 2B). Macrophages (CD11b+MHCIIintLy6G/C), monocytes (CD11b+MHCIIintLy6C+) and neutrophils (CD11b+CD11cintLy6G+) infiltration (also described as MDSC) represented a moderate part of the immune infiltrate, in contrast to more aggressive mammary tumor models (4T1 and TS/A). Interestingly, SiglecF+ myeloid cells (most likely eosinophils or SiglecF+ macrophages) represented almost 20% of the infiltrate. Remarkably, dendritic cell represented the most important infiltrating population in all tumors thus identifying this model as particularly relevant for TADC functional characterization. CD11b+ dendritic cell (CD11c+CD11b+MHCIIhi) represented the major part (about 20% of leukocytes) when compared to CD8α+ dendritic cell (CD11c+CD8α+SiglecH) and pDC (CD11c+ SiglecH+; Fig. 2B).

Figure 2.

Increased TApDC and regulatory T-cell infiltration in NEU15WT tumors escaping immunosurveillance. A and C, NEU15WT tumors were analyzed for immune infiltration according to the gating strategy presented in A. B, percentage of immune cells were evaluated among leukocytes from NEU15WT tumor single cell suspensions. C and D, percentages of tumor-infiltrating pDC and Treg cells (gating strategy detailed in Supplementary Fig. S3A and S3B) were evaluated in NEU15WT and NEU15F1 tumors. ***, P < 0.001. E, representative immunohistochemical staining on paraffin-embedded sections of NEU15WT tumors using anti-BST2 or anti-Foxp3 antibodies. Magnification, ×200. SiglecF+ m.c., myeloid cells; Neutro., neutrophil; Mφ, macrophage; Mono., monocyte.

Figure 2.

Increased TApDC and regulatory T-cell infiltration in NEU15WT tumors escaping immunosurveillance. A and C, NEU15WT tumors were analyzed for immune infiltration according to the gating strategy presented in A. B, percentage of immune cells were evaluated among leukocytes from NEU15WT tumor single cell suspensions. C and D, percentages of tumor-infiltrating pDC and Treg cells (gating strategy detailed in Supplementary Fig. S3A and S3B) were evaluated in NEU15WT and NEU15F1 tumors. ***, P < 0.001. E, representative immunohistochemical staining on paraffin-embedded sections of NEU15WT tumors using anti-BST2 or anti-Foxp3 antibodies. Magnification, ×200. SiglecF+ m.c., myeloid cells; Neutro., neutrophil; Mφ, macrophage; Mono., monocyte.

Close modal

Frequency of most immune cells was similar in both tumor types (data not shown) except for pDC and Treg that were more abundant in NEU15WT than NEU15F1 tumors (Fig. 2C and D and Supplementary Fig. S2B). Finally, histological analyses confirmed that both TApDC and TATreg are found within the tumor mass, with pDC mostly localized in the tumor bed and Treg in both tumor bed and immune infiltrate areas (Fig. 2E).

Taken together, these results show that NEU15WT tumor represents an interesting immunosubversion model closely mimicking our observations in human breast cancer with increased pDC and Treg recruitment possibly contributing to escape to immunosurveillance (1520, 26).

TApDC are functionally immature and can mediate CD4+ T-cell activation

TApDC were gated based on their high expression of CD11c and Siglec-H (Fig. 2A). They expressed high levels of BST2, B220, and Ly6C while lacking Ly6G and CD11b thus confirming their identity (Fig. 3A). Moreover, they showed heterogeneous CD8α expression. TApDC were immature with no surface expression of CD40, CD80, and CD86, and intermediate levels of MHC-II (Fig. 3A). This phenotype resembled to the one of pDC found in naive spleen (data not shown). Furthermore, in vivo phagocytic activity was weak but similar to spleen-derived pDC (Supplementary Fig. S3) suggesting that immature TApDC may uptake tumor Ags.

Figure 3.

NEU15WT TApDC are phenotypically and functionally immature. Freshly isolated TApDC were characterized in NEU15WT tumors. A, representative expression profile of surface molecules on gated TApDC are shown as overlay of specific (gray) and control (white) stainings. B and C, resting and activated TApDC or naive WT spleen pDC were incubated with allogeneic CD4+ T cells. T-cell proliferation (B) and IFN-γ production (C) were determined. Data are expressed as mean ± SEM of 7 of 8 experiments. Statistical analyses of TLRL-treated versus medium [(−)] condition are shown for spleen and tumors. *, P < 0.05; **, P < 0.01.

Figure 3.

NEU15WT TApDC are phenotypically and functionally immature. Freshly isolated TApDC were characterized in NEU15WT tumors. A, representative expression profile of surface molecules on gated TApDC are shown as overlay of specific (gray) and control (white) stainings. B and C, resting and activated TApDC or naive WT spleen pDC were incubated with allogeneic CD4+ T cells. T-cell proliferation (B) and IFN-γ production (C) were determined. Data are expressed as mean ± SEM of 7 of 8 experiments. Statistical analyses of TLRL-treated versus medium [(−)] condition are shown for spleen and tumors. *, P < 0.05; **, P < 0.01.

Close modal

pDC were sorted from tumor or spleen and cultured with allogeneic naive CD4+ T cells in the presence or not of various TLR ligands. Regardless of their tissue of origin, freshly isolated pDC did not activate CD4+ T cells (Fig. 3B and C). However, pDC maturation through TLR7 ligands, and to less extent TLR9 ligands, induced effector T-cell proliferation (Fig. 3B) as well as IFN-γ production [mean IFN-γ (pg/mL) for spleen vs. tumor pDC, respectively: Ctrl, 30 vs. 6; TLR7 ligands, 764–2312 vs. 415–2275; TLR9 ligands, 84–278 pg/mL vs. 55–88 pg/mL; Fig. 3C]. In conclusion, TApDC are phenotypically and functionally immature and may acquire abilities to activate antitumor effector T cells upon TLRL activation, as spleen-derived pDC.

TApDC exhibit an abrogated cytokine response to TLR9 but not TLR7 ligands

As the capacity of APC-derived cytokines is crucial to shape the immune response, we measured the ability of TApDC to secrete cytokines after in vitro TLR stimulation. Interestingly, IFN-α production by TApDC in response to CpG-A was strongly inhibited, with 30-fold less IFN-α than in naive spleen pDC (294.4 ± 295.9 vs. 10.65 ± 27.1; Fig. 4A). This alteration was confirmed using CpG-B/ODN-1826 with a 5-fold decrease in IFN-α production (Fig. 4A). In contrast, TApDC were as potent as naive spleen pDC to produce IFN-α in response to Flu. Other TLR7 ligands, such as CL075 or SM360320 (23), 2 synthetic TLR7 ligands, did not trigger significant IFN-α even in spleen pDC.

Figure 4.

NEU15WT TApDC exhibit an abrogated cytokine response to TLR9 but not TLR7 ligands. TApDC and naive WT spleen pDC were incubated with indicated TLRL. Supernatants were collected and analyzed for IFN-α (A) and MIP-1α and IL-6 (B) production. Data represent the mean ± SD from 3 to 12 independent samples. *, P < 0.05.

Figure 4.

NEU15WT TApDC exhibit an abrogated cytokine response to TLR9 but not TLR7 ligands. TApDC and naive WT spleen pDC were incubated with indicated TLRL. Supernatants were collected and analyzed for IFN-α (A) and MIP-1α and IL-6 (B) production. Data represent the mean ± SD from 3 to 12 independent samples. *, P < 0.05.

Close modal

Similarly the production of inflammatory cytokines by TApDC was specifically altered in response to CpG-A/B whereas responses to Flu, CL075, and SM360320 remained mostly unchanged (Fig. 4B). In particular, production of MIP-1α (323.7 ± 261.8 vs. 83.4 ± 144.1 for CpG-A) and IL-6 (67.8 ± 60.9 vs. 11.9 ± 17.5 for CpG-A; 1,999.8 ± 292.7 vs. 292.7± 344.6 for CpG-B) by TApDC in response to CpG-A and/or CpG-B were significantly reduced when compared to naive spleen pDC.

We then assessed a role for TLR9 downregulation in this alteration. Both TLR9 and TLR7 mRNA expression were slightly but similarly reduced in tumor versus spleen-derived pDC (Supplementary Fig. S4). Furthermore, production of cytokine such as TNF-α was not altered in tumor versus spleen-derived pDC in response to TLR9L (data not shown). Altogether, this shows that TLR9 receptor downregulation cannot merely explain the specific alteration of cytokine production in response to TLR9L when compared to TLR7L.

pDC depletion delays tumor growth in vivo

To determine whether such TApDC contributes to tumor growth, pDC were depleted in vivo using anti-BST2 depleting mAbs (24, 25). WT mice were treated every other day by intraperitoneally injection from the day before tumor implantation until the experimental endpoint. A significant decrease of the tumor volume was observed upon pDC depletion from day 14 postimplantation (Fig. 5A). Tumor growth was followed over time and mice were euthanized when tumor size reached the endpoint. Survival curve analysis showed an increase in median survival times from 35 to 43 days (Fig. 5B). Specific and effective pDC depletion in the tumor upward of 80% was validated by flow cytometry (Fig. 5D) and functional IFN-α response to TLR7-L intratumoral injection at the endpoint (Fig. 5C). Those data show that effective pDC depletion in the tumor microenvironment delays tumor growth and increases mice survival. These results are in concordance with our observation in human breast cancer showing that recruitment of pDC within the tumor directly contributes to poor clinical outcome (15, 27).

Figure 5.

pDC depletion delays tumor growth in vivo. A–C, WT mice (n = 20 per group) were treated with purified pDC-depleting 120G8 or rat IgG1 control antibody from 1 day before tumor implantation and every other day until tumors reached the experimental endpoint. A, data represent the mean tumor volume mean ± SEM for 20 mice. B, Kaplan–Meier survival plot of mice stratified in those with pDC depletion versus none. Data are from 1 representative out of 3 independent experiments. C and D, in vivo depletion of pDC was analyzed functionally (C) and phenotypically (D) by flow cytometry in tumors. C, TLR7L (10 μg) was administered by i.t. injection at experimental endpoint and serum was harvested 3 hours after for IFN-α level measurement (•, rat IgG1 control; ○, pDC-depleted). D, dot plot representation of CD11b/SiglecF staining gated on CD45+CD11c+lin cells after dead cells and doublet exclusion for one representative mouse of each group. **, P < 0.01, ***, P < 0.001.

Figure 5.

pDC depletion delays tumor growth in vivo. A–C, WT mice (n = 20 per group) were treated with purified pDC-depleting 120G8 or rat IgG1 control antibody from 1 day before tumor implantation and every other day until tumors reached the experimental endpoint. A, data represent the mean tumor volume mean ± SEM for 20 mice. B, Kaplan–Meier survival plot of mice stratified in those with pDC depletion versus none. Data are from 1 representative out of 3 independent experiments. C and D, in vivo depletion of pDC was analyzed functionally (C) and phenotypically (D) by flow cytometry in tumors. C, TLR7L (10 μg) was administered by i.t. injection at experimental endpoint and serum was harvested 3 hours after for IFN-α level measurement (•, rat IgG1 control; ○, pDC-depleted). D, dot plot representation of CD11b/SiglecF staining gated on CD45+CD11c+lin cells after dead cells and doublet exclusion for one representative mouse of each group. **, P < 0.01, ***, P < 0.001.

Close modal

TLR7 triggering induces tumor regression in vivo

As breast TApDC respond to TLR7L in vitro, we next assessed the possibility to activate TApDC via SM360320, a TLR7 agonist shown to be in vivo a robust IFN-α inducer and a potent adjuvant (23), to revert their tumor-promoting ability. Intratumoral injections of TLR7-L (50 μg) lead to potent tumor regression when compared to vehicle-treated mice (Fig. 6A). TLR7L treatment induced a strong increase in complete response with 90% in TLR7L-treated group versus 30% of spontaneous regression in the vehicle-treated group (P = 0.0198, data not shown). Importantly, 100% of cured mice were protected against a subsequent orthotopic contralateral challenge of NEU15 cells (3 months later; data not shown). In contrast to intratumoral injection, contralateral subcutaneous injection of TLR7L did not induce significant tumor regression (Fig. 6B). Both intratumoral and contralateral subcutaneous injections led however to similar range of plasmatic IFN-α levels suggesting that intratumoral route is necessary to mobilize antitumor activity of TApDC (Fig. 6C).

Figure 6.

Intratumoral TLR7L injection induces tumor regression. Mice were treated with 50 μg SM360320 TLR7L at day 13 and 20 post-NEU15 tumor implantation by intratumoral (A–E) or subcutaneous contralateral (B, C) injections. Controls were injected with vehicle only. A and B, data represent the mean tumor volume ± SEM for 10 mice. **, P < 0.01. C, serum was harvested 3 hours after TLR7L injection for IFN-α level measurement. D, intratumoral dendritic cell activation was assessed 24 hours postintratumoral TLRL injection by flow cytometry. Representative expression profiles of costimulatory molecules on gated TApDC, CD8α+, or CD11b+ dendritic cell are shown. E, fold changes in gene expression upon intratumoral TLR7L injection. TLDA mouse immune array was conducted on tumor samples from control and TLR7L treated mice. Data were calculated as fold changes over nontreated control condition and presented as Heat Map. Genes with fold changes over 10 are listed here.

Figure 6.

Intratumoral TLR7L injection induces tumor regression. Mice were treated with 50 μg SM360320 TLR7L at day 13 and 20 post-NEU15 tumor implantation by intratumoral (A–E) or subcutaneous contralateral (B, C) injections. Controls were injected with vehicle only. A and B, data represent the mean tumor volume ± SEM for 10 mice. **, P < 0.01. C, serum was harvested 3 hours after TLR7L injection for IFN-α level measurement. D, intratumoral dendritic cell activation was assessed 24 hours postintratumoral TLRL injection by flow cytometry. Representative expression profiles of costimulatory molecules on gated TApDC, CD8α+, or CD11b+ dendritic cell are shown. E, fold changes in gene expression upon intratumoral TLR7L injection. TLDA mouse immune array was conducted on tumor samples from control and TLR7L treated mice. Data were calculated as fold changes over nontreated control condition and presented as Heat Map. Genes with fold changes over 10 are listed here.

Close modal

Analyses of intratumoral immune infiltrates exhibited an increase in leukocyte frequency as soon as 24 hours post-TLR7L injection (Supplementary Fig. S6A). Although no major changes in the frequency of most immune cells could be noticed (data not shown), a significant and specific increase in monocyte infiltration was observed (Supplementary Fig. S6B). As pDC are the most likely target of such TLR7L in vivo, we explored whether intratumoral TLR7L injection affected pDC frequency and function in vivo. Although pDC frequency remained unchanged (data not shown), TLR7L induced strong increase in MHC-II expression and costimulatory molecules at their surface (Fig. 6D). In contrast, CpG did not activate TApDC thus confirming our in vitro data. Specific activation of TApDC was confirmed, as neither TA-CD8α+ dendritic cell nor TA-CD11b+ dendritic cell displayed increase in MHC-II (nor CD80, CD86, data not shown) expression upon TLR7 triggering (Fig. 6D).

Finally, changes in gene expression were analyzed in NEU15WT tumors 8 hours post-intratumoral injection of TLR7L by TaqMan low-density array (TLDA) mouse immune assay (Supplementary Materials and Methods). Data are presented as fold changed over the nontreated conditions and displayed as a heat map (Fig. 6E) and detailed in Supplementary Fig. S5. Genes that displayed fold changes higher than 10 were selected to highlight the most significant changes in gene expression upon TLR7 intratumoral injection. These data depicted an increased infiltration in immune cells such as myeloid cells (H2-Ea-ps, Nos2) and in particular cytotoxic T cells (Cd3e, Cd8, Tbx21), via chemokine-mediated recruitment (Cxcl11, 2,413 ± 3,409 fold changes; Ccr7, 58 ± 53 fold changes). The increase in perforin and granzyme genes (125 ± 147 and 185 ± 209 fold changes, respectively) also indicated a potent cytotoxic response. A Th1-type T-cell response was seen via the sharp increase in genes related to APC maturation (Cd28, Cd40, IL12b), IFNγ (123 ± 151 fold changes) and IFN-γ–induced genes (Cxcl10, Ccl5, Stat1) production. Interestingly, the induction of this genes, with the exception of IL10, was type I IFN-dependent as gene increase was no longer seen when anti-IFNAR1 antibody was coinjected with TLR7L (Supplementary Fig. S5A). In parallel, an increase in type I IFN genes (IFNa and Mx1) was seen in samples treated by TLR7L by quantitative PCR (Supplementary Fig. S5B).

Altogether these data showed the induction of type I IFN response as well as Th1 cytotoxic T cells response leading to tumor regression.

In contrast to CpG, in vivo TLR7 antitumoral activity requires type I IFN production by pDC

To explore whether TLR7L antitumoral activity was mediated by pDC, pDC were depleted the 2 days before TLRL injection. As previously observed with a dose of 50 μg, 10 μg of TLR7L also lead to a significant decrease of tumor volume (day 35; Fig. 7A). pDC depletion in such short-term schedule led to a delay in tumor growth as previously observed (Fig. 7A). Finally, pDC depletion completely abrogated TLR7L-mediated antitumoral effect (1,046 ± 221.58 mm3 for w/o pDC vs. 1,266.5 ± 335.67 mm3 for TLR7L w/o pDC), showing that pDC mediate the antitumor effect of TLR7L in vivo (Fig. 7C). The efficiency of pDC depletion was confirmed phenotypically (data not shown) and functionally at the type I IFN systemic plasmatic level (Fig. 7B).

Figure 7.

Therapeutic activity of TLR7L depends on TApDC and type I IFN production. NEU15WT-bearing mice were treated with 10 μg TLR7L (A–C) or CpG-B (D–E) at day 13 and 20 (n = 10–15/group). A and D, pDC were depleted from WT mice the 2 days before TLRL injection. B, serum were harvested 3 hours after TLR7L injection for IFN-α level measurement. C and E, mouse IFNAR1 or rat IgG1 control antibodies were coinjected with TLRL. A and C–E, data represent the mean tumor volume ± SEM. *, P < 0.05; **, P < 0.01.

Figure 7.

Therapeutic activity of TLR7L depends on TApDC and type I IFN production. NEU15WT-bearing mice were treated with 10 μg TLR7L (A–C) or CpG-B (D–E) at day 13 and 20 (n = 10–15/group). A and D, pDC were depleted from WT mice the 2 days before TLRL injection. B, serum were harvested 3 hours after TLR7L injection for IFN-α level measurement. C and E, mouse IFNAR1 or rat IgG1 control antibodies were coinjected with TLRL. A and C–E, data represent the mean tumor volume ± SEM. *, P < 0.05; **, P < 0.01.

Close modal

We then assessed whether inhibition of type IFN signaling affected TLR7L activity. For that matter, anti-IFNAR1 or control antibodies were coadministered intratumoral with TLR7L (10 μg). IFNAR inhibition resulted in a significant decrease of TLR7L antitumoral activity showing the requirement for type I IFN in TLR7L activity (752.1 ± 135.3 mm3 for TLR7L vs. 1,392.1 ± 181.8 mm3 for TLR7L w/o IFNAR). The efficacy of anti-IFNAR antibodies was confirmed by qPCR on tumor extracts with a decrease in IFN-stimulated gene expression upon TLR7L treatment in the context of type I IFN blockade (data not shown).

As TApDC were shown to lack both in vivo and in vitro the ability to respond to CpG, the effect of this TLR9L on tumor growth was assessed. Unexpectedly, intratumoral administration of CpG-B induced tumor regression (771.7 ± 295.3 mm3 for CpG vs. 1,826.6 ± 196.8 mm3 for control). Interestingly, inhibition of tumor growth by CpG was delayed in comparison with TLR7L, which started to decrease the tumor volume as soon as after the first injection. However, the mechanism seems different as intratumoral CpG injection did not lead to any detectable IFN-α production in contrast with TLR7L (data not shown). Surprisingly, pDC depletion altered CpG antitumoral activity (Fig. 7D), however inhibition of type I IFN signaling did not (Fig. 7E). These results suggest that pDC indirectly affect CpG activity through a distinct type I IFN-independent mechanism.

Altogether, those data show that in contrast to CpG, TLR7L antitumoral activity is mediated by pDC and their ability to secrete large amount of type I IFN.

In line with the negative prognostic value of pDC infiltration in human breast tumors (15), we show for the first time in vivo that mouse mammary TApDC favor primary tumor growth but can be activated via TLR7 triggering to mediate antitumoral response and subsequent tumor regression.

We developed a clinically relevant tumor model in which the HER2/neu+ NEU15 cell line developed in WT hosts escapes from immunosurveillance through pDC and Treg-mediated immunosubversion, thus closely mimicking our observations in human breast cancer (15, 20, 26).

First, we showed in vitro and in vivo that TApDC from NEU15WT tumors were specifically altered in their ability to respond to TLR9 but not TLR7 ligands. These data are consistent with the specific alteration of TLR9 response previously reported by us (19, 20) and others on both human tumor and immune cells (17, 28). Although pDC hyporesponsiveness to TLR9 ligands might be explained by receptor downregulation (17, 28), we showed that it is rather due to a specific interference of the tumor microenvironment, for instance via IRF7 downregulation (29). Tumor-induced TLR9 loss of function might represent an immunosubversion mechanism to avoid immune alert by putative endogenous ligands for TLR9 within the tumor microenvironment, such as described in autoimmune disorders (30–32).

Second, we showed for the first time in primary breast tumors that TApDC favored breast tumor progression. In vivo depletion of pDC indeed significantly slowed down tumor growth. Although antibody-mediated pDC depletion is quite efficient, variation in its efficacy might account for discrepancies in the beneficial effect of pDC depletion in short-term depletion settings (Fig. 7B and D). The use of a pDC-deficient mouse model (33) will formerly show the extent of pDC role in tumor progression. To our knowledge this is a unique proof of a direct role of pDC in favoring primary tumor growth in solid tumors. pDC were previously shown to regulate growth of multiple myeloma cells (34) and more recently to favor bone metastasis of breast cancer cells (35).

Accumulating evidences have shown a specialized role of pDC in the induction of peripheral tolerance (11–14, 36) through Ag-specific T-cell deletion (12) or differentiation/expansion of suppressive T cells (11, 14, 31, 37–44). NEU15WT tumors were indeed more infiltrated by Treg. For instance, we observed in preliminary experiments a decrease in TATreg infiltration after pDC depletion. This role of pDC would be consistent with our recent reports in human breast cancer, showing that IFN-α–deficient TApDC from human breast tumors favor Treg expansion via ICOS–ICOSL interaction (20, 27) that may contribute to tumor progression and explain their negative impact on patient survival (15). Whether tumor pDC are able to modulate Treg function/differentiation remains to be addressed.

Despite their negative impact on tumor progression, TApDC could be activated in vivo via intratumoral injection of TLR7L, thus mediating a Th1 signature, as evidenced by TLDA analysis, and subsequent tumor regression. TLR7L induced long-term protective memory response as 100% of cured mice were protected against subsequent tumor challenge.

In vivo depletion of pDC abrogated the therapeutic activity of TLR7L, showing the central role for TApDC in TLR7L-mediated antitumor response. Importantly, we showed that this therapeutic activity is mediated by locally induced and not systemic type I IFN. This points toward the importance of intratumoral TApDC activation leading to type I IFN production and subsequent additional activities (cross-presentation, Treg neutralization). We indeed observed that type I IFN neutralization led to the inhibition of the intratumoral Th1 signature. The antitumor activity of Imiquimod, another TLR7L, is dependent upon CD8α+ pDC that harbor direct tumor killing activity mediated by granzyme B and/or TRAIL leading to subsequent capture and antigen cross-presentation (3, 21). However, we did not observe any increase in TRAIL expression on our TApDC upon TLR7L treatment (data not shown) suggesting a different mechanism of action. However, considering our results in human models that type I IFN led to the inhibition of TATreg proliferation (20), the potential role of Treg inhibition in this TLR7L antitumoral activity will be carefully assessed.

Although we showed both in vitro and in vivo that CpG could not activate TApDC, this TLR9L unexpectedly induced tumor regression. Although is antitumoral activity was reduced upon pDC depletion, IFNAR inhibition did not affect their therapeutic action showing a different mechanism of action than TLR7L. We hypothesize that pDC depletion somehow impact on key effectors of CpG-mediated antitumoral response. We indeed observed an increase in macrophage infiltration upon CpG treatment (Supplementary Fig. S6B) that was reduced upon pDC depletion (data not shown). Although tumor-associated macrophages (TAM) are commonly associated with tumor development and progression, antitumor activity can be achieved by targeting TAM recruitment and polarization toward M1 phenotype, a process in which CpG was shown to participate (45, 46). CpG-mediated antitumoral mechanisms will need to be further clarified.

Based on promising results of the literature, ongoing phase I/II clinical trials are currently evaluating the therapeutic potential of TLR agonists for the treatment of various type of cancer (47). In the light of our observation that TApDC remain responsive to TLR7L while lacking response to CpG ODN, the therapeutic potential of TLR7 agonists in human breast tumors shall be considered.

No potential conflicts of interest were disclosed.

Conception and design: I. Le Mercier, J. Vlach, J.-Y. Blay, N. Bendriss-Vermare, I. Puisieux, N. Goutagny

Development of methodology: I. Le Mercier, D. Poujol, V. Sisirak, M. Gobert, B. Dubois, J. Marvel, J. Vlach, J.-Y. Blay, I. Puisieux, N. Goutagny

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): I. Le Mercier, D. Poujol, A. Sanlaville, M. Gobert, I. Treilleux, B. Dubois, J.-Y. Blay, I. Puisieux, N. Goutagny

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): I. Le Mercier, D. Poujol, J.-Y. Blay, N. Bendriss-Vermare, C. Caux, I. Puisieux, N. Goutagny

Writing, review, and/or revision of the manuscript: I. Le Mercier, J.-Y. Blay, N. Bendriss-Vermare, C. Caux, I. Puisieux, N. Goutagny

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D. Poujol, A. Sanlaville, V. Sisirak, J. Vlach, I. Puisieux, N. Goutagny

Study supervision: J.-Y. Blay, C. Caux, I. Puisieux, N. Goutagny

The authors thank S. Goddard-Léon and M. Pratviel for technical support, M. Paturel and A. Besse for precious advice on statistical analysis, and Janssen Infectious Diseases-Diagnostics BVBA for providing SM360320.

This work is supported by The BCRF, Ligue Contre le Cancer, Institut National du Cancer (INCa ACI-63-04, METESCAPE, ANR-10-LABX-0061, ARC 3364, EML 2009, LYRIC, INCa_466), and Lyon Biopole.

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.
de Visser
KE
,
Eichten
A
,
Coussens
LM
. 
Paradoxical roles of the immune system during cancer development
.
Nat Rev Cancer
2006
;
6
:
24
37
.
2.
Fricke
I
,
Gabrilovich
DI
. 
Dendritic cells and tumor microenvironment: a dangerous liaison
.
Immunol Invest
2006
;
35
:
459
83
.
3.
Drobits
B
,
Holcmann
M
,
Amberg
N
,
Swiecki
M
,
Grundtner
R
,
Hammer
M
, et al
Imiquimod clears tumors in mice independent of adaptive immunity by converting pDCs into tumor-killing effector cells
.
J Clin Invest
2012
;
122
:
575
85
.
4.
Liu
C
,
Lou
Y
,
Lizee
G
,
Qin
H
,
Liu
S
,
Rabinovich
B
, et al
Plasmacytoid dendritic cells induce NK cell-dependent, tumor antigen-specific T cell cross-priming and tumor regression in mice
.
J Clin Invest
2008
;
118
:
1165
75
.
5.
Kadowaki
N
,
Antonenko
S
,
Lau
JY
,
Liu
YJ
. 
Natural interferon alpha/beta-producing cells link innate and adaptive immunity
.
J Exp Med
2000
;
192
:
219
26
.
6.
Colonna
M
,
Trinchieri
G
,
Liu
YJ
. 
Plasmacytoid dendritic cells in immunity
.
Nat Immunol
2004
;
5
:
1219
26
.
7.
Gilliet
M
,
Cao
W
,
Liu
YJ
. 
Plasmacytoid dendritic cells: sensing nucleic acids in viral infection and autoimmune diseases
.
Nat Rev Immunol
2008
;
8
:
594
606
.
8.
Kawai
T
,
Sato
S
,
Ishii
KJ
,
Coban
C
,
Hemmi
H
,
Yamamoto
M
, et al
Interferon-alpha induction through Toll-like receptors involves a direct interaction of IRF7 with MyD88 and TRAF6
.
Nat Immunol
2004
;
5
:
1061
8
.
9.
Fonteneau
JF
,
Gilliet
M
,
Larsson
M
,
Dasilva
I
,
Munz
C
,
Liu
YJ
, et al
Activation of influenza virus-specific CD4+ and CD8+ T cells: a new role for plasmacytoid dendritic cells in adaptive immunity
.
Blood
2003
;
101
:
3520
6
.
10.
Blanco
P
,
Palucka
AK
,
Gill
M
,
Pascual
V
,
Banchereau
J
. 
Induction of dendritic cell differentiation by IFN-alpha in systemic lupus erythematosus
.
Science
2001
;
294
:
1540
3
.
11.
de Heer
HJ
,
Hammad
H
,
Soullie
T
,
Hijdra
D
,
Vos
N
,
Willart
MA
, et al
Essential role of lung plasmacytoid dendritic cells in preventing asthmatic reactions to harmless inhaled antigen
.
J Exp Med
2004
;
200
:
89
98
.
12.
Goubier
A
,
Dubois
B
,
Gheit
H
,
Joubert
G
,
Villard-Truc
F
,
Asselin-Paturel
C
, et al
Plasmacytoid dendritic cells mediate oral tolerance
.
Immunity
2008
;
29
:
464
75
.
13.
Fugier-Vivier
IJ
,
Rezzoug
F
,
Huang
Y
,
Graul-Layman
AJ
,
Schanie
CL
,
Xu
H
, et al
Plasmacytoid precursor dendritic cells facilitate allogeneic hematopoietic stem cell engraftment
.
J Exp Med
2005
;
201
:
373
83
.
14.
Wei
S
,
Kryczek
I
,
Zou
L
,
Daniel
B
,
Cheng
P
,
Mottram
P
, et al
Plasmacytoid dendritic cells induce CD8+ regulatory T cells in human ovarian carcinoma
.
Cancer Res
2005
;
65
:
5020
6
.
15.
Treilleux
I
,
Blay
JY
,
Bendriss-Vermare
N
,
Ray-Coquard
I
,
Bachelot
T
,
Guastalla
JP
, et al
Dendritic cell infiltration and prognosis of early stage breast cancer
.
Clin Cancer Res
2004
;
10
:
7466
74
.
16.
Vermi
W
,
Bonecchi
R
,
Facchetti
F
,
Bianchi
D
,
Sozzani
S
,
Festa
S
, et al
Recruitment of immature plasmacytoid dendritic cells (plasmacytoid monocytes) and myeloid dendritic cells in primary cutaneous melanomas
.
J Pathol
2003
;
200
:
255
68
.
17.
Hartmann
E
,
Wollenberg
B
,
Rothenfusser
S
,
Wagner
M
,
Wellisch
D
,
Mack
B
, et al
Identification and functional analysis of tumor-infiltrating plasmacytoid dendritic cells in head and neck cancer
.
Cancer Res
2003
;
63
:
6478
87
.
18.
Zou
W
,
Machelon
V
,
Coulomb-L'Hermin
A
,
Borvak
J
,
Nome
F
,
Isaeva
T
, et al
Stromal-derived factor-1 in human tumors recruits and alters the function of plasmacytoid precursor dendritic cells
.
Nat Med
2001
;
7
:
1339
46
.
19.
Labidi-Galy
SI
,
Sisirak
V
,
Meeus
P
,
Gobert
M
,
Treilleux
I
,
Bajard
A
, et al
Quantitative and functional alterations of plasmacytoid dendritic cells contribute to immune tolerance in ovarian cancer
.
Cancer Res
2011
;
71
:
5423
34
.
20.
Sisirak
V
,
Faget
J
,
Gobert
M
,
Goutagny
N
,
Vey
N
,
Treilleux
I
, et al
Impaired IFN-alpha production by plasmacytoid dendritic cells favors regulatory T-cell expansion that may contribute to breast cancer progression
.
Cancer Res
2012
;
72
:
5188
97
.
21.
Stary
G
,
Bangert
C
,
Tauber
M
,
Strohal
R
,
Kopp
T
,
Stingl
G
. 
Tumoricidal activity of TLR7/8-activated inflammatory dendritic cells
.
J Exp Med
2007
;
204
:
1441
51
.
22.
Guy
CT
,
Webster
MA
,
Schaller
M
,
Parsons
TJ
,
Cardiff
RD
,
Muller
WJ
. 
Expression of the neu protooncogene in the mammary epithelium of transgenic mice induces metastatic disease
.
Proc Natl Acad Sci U S A
1992
;
89
:
10578
82
.
23.
Dharmapuri
S
,
Aurisicchio
L
,
Neuner
P
,
Verdirame
M
,
Ciliberto
G
,
La
MN
. 
An oral TLR7 agonist is a potent adjuvant of DNA vaccination in transgenic mouse tumor models
.
Cancer Gene Ther
2009
;
16
:
462
72
.
24.
Blasius
AL
,
Giurisato
E
,
Cella
M
,
Schreiber
RD
,
Shaw
AS
,
Colonna
M
. 
Bone marrow stromal cell antigen 2 is a specific marker of type I IFN-producing cells in the naive mouse, but a promiscuous cell surface antigen following IFN stimulation
.
J Immunol
2006
;
177
:
3260
5
.
25.
Asselin-Paturel
C
,
Brizard
G
,
Pin
JJ
,
Briere
F
,
Trinchieri
G
. 
Mouse strain differences in plasmacytoid dendritic cell frequency and function revealed by a novel monoclonal antibody
.
J Immunol
2003
;
171
:
6466
77
.
26.
Gobert
M
,
Treilleux
I
,
Bendriss-Vermare
N
,
Bachelot
T
,
Goddard-Leon
S
,
Arfi
V
, et al
Regulatory T cells recruited through CCL22/CCR4 are selectively activated in lymphoid infiltrates surrounding primary breast tumors and lead to an adverse clinical outcome
.
Cancer Res
2009
;
69
:
2000
9
.
27.
Faget
J
,
Bendriss-Vermare
N
,
Gobert
M
,
Durand
I
,
Olive
D
,
Biota
C
, et al
ICOS-ligand expression on plasmacytoid dendritic cells supports breast cancer progression by promoting the accumulation of immunosuppressive CD4+ T cells
.
Cancer Res
2012
;
72
:
6130
41
.
28.
Hirsch
I
,
Caux
C
,
Hasan
U
,
Bendriss-Vermare
N
,
Olive
D
. 
Impaired Toll-like receptor 7 and 9 signaling: from chronic viral infections to cancer
.
Trends Immunol
2010
;
31
:
391
7
.
29.
Sisirak
V
,
Vey
N
,
Goutagny
N
,
Renaudineau
S
,
Malfroy
M
,
Thys
S
, et al
Breast cancer-derived TGF-beta and TNF-alpha compromise IFN-alpha production by tumor-associated plasmacytoid dendritic cells
.
Int J Cancer
2013
;
133
:
771
8
.
30.
Barrat
FJ
,
Meeker
T
,
Gregorio
J
,
Chan
JH
,
Uematsu
S
,
Akira
S
, et al
Nucleic acids of mammalian origin can act as endogenous ligands for Toll-like receptors and may promote systemic lupus erythematosus
.
J Exp Med
2005
;
202
:
1131
9
.
31.
Vollmer
J
,
Tluk
S
,
Schmitz
C
,
Hamm
S
,
Jurk
M
,
Forsbach
A
, et al
Immune stimulation mediated by autoantigen binding sites within small nuclear RNAs involves Toll-like receptors 7 and 8
.
J Exp Med
2005
;
202
:
1575
85
.
32.
Ganguly
D
,
Chamilos
G
,
Lande
R
,
Gregorio
J
,
Meller
S
,
Facchinetti
V
, et al
Self-RNA-antimicrobial peptide complexes activate human dendritic cells through TLR7 and TLR8
.
J Exp Med
2009
;
206
:
1983
94
.
33.
Cisse
B
,
Caton
ML
,
Lehner
M
,
Maeda
T
,
Scheu
S
,
Locksley
R
, et al
Transcription factor E2-2 is an essential and specific regulator of plasmacytoid dendritic cell development
.
Cell
2008
;
135
:
37
48
.
34.
Chauhan
D
,
Singh
AV
,
Brahmandam
M
,
Carrasco
R
,
Bandi
M
,
Hideshima
T
, et al
Functional interaction of plasmacytoid dendritic cells with multiple myeloma cells: a therapeutic target
.
Cancer Cell
2009
;
16
:
309
23
.
35.
Sawant
A
,
Hensel
JA
,
Chanda
D
,
Harris
BA
,
Siegal
GP
,
Maheshwari
A
, et al
Depletion of plasmacytoid dendritic cells inhibits tumor growth and prevents bone metastasis of breast cancer cells
.
J Immunol
2012
;
189
:
4258
65
.
36.
Wang
RF
. 
Regulatory T cells and Toll-like receptors in cancer therapy
.
Cancer Res
2006
;
66
:
4987
90
.
37.
Kuwana
M
. 
Induction of anergic and regulatory T cells by plasmacytoid dendritic cells and other dendritic cell subsets
.
Hum Immunol
2002
;
63
:
1156
63
.
38.
Bilsborough
J
,
George
TC
,
Norment
A
,
Viney
JL
. 
Mucosal CD8alpha+ DC, with a plasmacytoid phenotype, induce differentiation and support function of T cells with regulatory properties
.
Immunology
2003
;
108
:
481
92
.
39.
Ito
T
,
Yang
M
,
Wang
YH
,
Lande
R
,
Gregorio
J
,
Perng
OA
, et al
Plasmacytoid dendritic cells prime IL-10-producing T regulatory cells by inducible costimulator ligand
.
J Exp Med
2007
;
204
:
105
15
.
40.
Gilliet
M
,
Liu
YJ
. 
Human plasmacytoid-derived dendritic cells and the induction of T-regulatory cells
.
Hum Immunol
2002
;
63
:
1149
55
.
41.
Moseman
EA
,
Liang
X
,
Dawson
AJ
,
Panoskaltsis-Mortari
A
,
Krieg
AM
,
Liu
YJ
, et al
Human plasmacytoid dendritic cells activated by CpG oligodeoxynucleotides induce the generation of CD4+CD25+ regulatory T cells
.
J Immunol
2004
;
173
:
4433
42
.
42.
Kang
HK
,
Liu
M
,
Datta
SK
. 
Low-dose peptide tolerance therapy of lupus generates plasmacytoid dendritic cells that cause expansion of autoantigen-specific regulatory T cells and contraction of inflammatory Th17 cells
.
J Immunol
2007
;
178
:
7849
58
.
43.
Ouabed
A
,
Hubert
FX
,
Chabannes
D
,
Gautreau
L
,
Heslan
M
,
Josien
R
. 
Differential control of T regulatory cell proliferation and suppressive activity by mature plasmacytoid versus conventional spleen dendritic cells
.
J Immunol
2008
;
180
:
5862
70
.
44.
Sharma
MD
,
Baban
B
,
Chandler
P
,
Hou
DY
,
Singh
N
,
Yagita
H
, et al
Plasmacytoid dendritic cells from mouse tumor-draining lymph nodes directly activate mature Tregs via indoleamine 2,3-dioxygenase
.
J Clin Invest
2007
;
117
:
2570
82
.
45.
Guiducci
C
,
Vicari
AP
,
Sangaletti
S
,
Trinchieri
G
,
Colombo
MP
. 
Redirecting in vivo elicited tumor infiltrating macrophages and dendritic cells towards tumor rejection
.
Cancer Res
2005
;
65
:
3437
46
.
46.
Colombo
MP
,
Mantovani
A
. 
Targeting myelomonocytic cells to revert inflammation-dependent cancer promotion
.
Cancer Res
2005
;
65
:
9113
6
.
47.
Goutagny
N
,
Estornes
Y
,
Hasan
U
,
Lebecque
S
,
Caux
C
. 
Targeting pattern recognition receptors in cancer immunotherapy
.
Target Oncol
2012
;
7
:
29
54
.