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Human Cancer Biology

TIE-2 and VEGFR Kinase Activities Drive Immunosuppressive Function of TIE-2–Expressing Monocytes in Human Breast Tumors

Mark Ibberson, Sylvian Bron, Nicolas Guex, Eveline Faes-van't Hull, Assia Ifticene-Treboux, Luc Henry, Hans-Anton Lehr, Jean-François Delaloye, George Coukos, Ioannis Xenarios and Marie-Agnès Doucey
Mark Ibberson
1Vital-IT, Swiss Institute of Bioinformatics; 2Ludwig Cancer Research Center; 3Centre du Sein, CHUV, University of Lausanne, Lausanne, Switzerland; and 4Institute of Pathology, Medizin Campus Bodensee, Friedrichshafen, Germany
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Sylvian Bron
1Vital-IT, Swiss Institute of Bioinformatics; 2Ludwig Cancer Research Center; 3Centre du Sein, CHUV, University of Lausanne, Lausanne, Switzerland; and 4Institute of Pathology, Medizin Campus Bodensee, Friedrichshafen, Germany
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Nicolas Guex
1Vital-IT, Swiss Institute of Bioinformatics; 2Ludwig Cancer Research Center; 3Centre du Sein, CHUV, University of Lausanne, Lausanne, Switzerland; and 4Institute of Pathology, Medizin Campus Bodensee, Friedrichshafen, Germany
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Eveline Faes-van't Hull
1Vital-IT, Swiss Institute of Bioinformatics; 2Ludwig Cancer Research Center; 3Centre du Sein, CHUV, University of Lausanne, Lausanne, Switzerland; and 4Institute of Pathology, Medizin Campus Bodensee, Friedrichshafen, Germany
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Assia Ifticene-Treboux
1Vital-IT, Swiss Institute of Bioinformatics; 2Ludwig Cancer Research Center; 3Centre du Sein, CHUV, University of Lausanne, Lausanne, Switzerland; and 4Institute of Pathology, Medizin Campus Bodensee, Friedrichshafen, Germany
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Luc Henry
1Vital-IT, Swiss Institute of Bioinformatics; 2Ludwig Cancer Research Center; 3Centre du Sein, CHUV, University of Lausanne, Lausanne, Switzerland; and 4Institute of Pathology, Medizin Campus Bodensee, Friedrichshafen, Germany
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Hans-Anton Lehr
1Vital-IT, Swiss Institute of Bioinformatics; 2Ludwig Cancer Research Center; 3Centre du Sein, CHUV, University of Lausanne, Lausanne, Switzerland; and 4Institute of Pathology, Medizin Campus Bodensee, Friedrichshafen, Germany
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Jean-François Delaloye
1Vital-IT, Swiss Institute of Bioinformatics; 2Ludwig Cancer Research Center; 3Centre du Sein, CHUV, University of Lausanne, Lausanne, Switzerland; and 4Institute of Pathology, Medizin Campus Bodensee, Friedrichshafen, Germany
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George Coukos
1Vital-IT, Swiss Institute of Bioinformatics; 2Ludwig Cancer Research Center; 3Centre du Sein, CHUV, University of Lausanne, Lausanne, Switzerland; and 4Institute of Pathology, Medizin Campus Bodensee, Friedrichshafen, Germany
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Ioannis Xenarios
1Vital-IT, Swiss Institute of Bioinformatics; 2Ludwig Cancer Research Center; 3Centre du Sein, CHUV, University of Lausanne, Lausanne, Switzerland; and 4Institute of Pathology, Medizin Campus Bodensee, Friedrichshafen, Germany
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Marie-Agnès Doucey
1Vital-IT, Swiss Institute of Bioinformatics; 2Ludwig Cancer Research Center; 3Centre du Sein, CHUV, University of Lausanne, Lausanne, Switzerland; and 4Institute of Pathology, Medizin Campus Bodensee, Friedrichshafen, Germany
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DOI: 10.1158/1078-0432.CCR-12-3181 Published July 2013
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Abstract

Purpose: Tumor-associated TIE-2–expressing monocytes (TEM) are highly proangiogenic cells critical for tumor vascularization. We previously showed that, in human breast cancer, TIE-2 and VEGFR pathways control proangiogenic activity of TEMs. Here, we examine the contribution of these pathways to immunosuppressive activity of TEMs.

Experimental Design: We investigated the changes in immunosuppressive activity of TEMs and gene expression in response to specific kinase inhibitors of TIE-2 and VEGFR. The ability of tumor TEMs to suppress tumor-specific T-cell response mediated by tumor dendritic cells (DC) was measured in vitro. Characterization of TEM and DC phenotype in addition to their interaction with T cells was done using confocal microscopic images analysis of breast carcinomas.

Results: TEMs from breast tumors are able to suppress tumor-specific immune responses. Importantly, proangiogenic and suppressive functions of TEMs are similarly driven by TIE-2 and VEGFR kinase activity. Furthermore, we show that tumor TEMs can function as antigen-presenting cells and elicit a weak proliferation of T cells. Blocking TIE-2 and VEGFR kinase activity induced TEMs to change their phenotype into cells with features of myeloid dendritic cells. We show that immunosuppressive activity of TEMs is associated with high CD86 surface expression and extensive engagement of T regulatory cells in breast tumors. TIE-2 and VEGFR kinase activity was also necessary to maintain high CD86 surface expression levels and to convert T cells into regulatory cells.

Conclusions: These results suggest that TEMs are plastic cells that can be reverted from suppressive, proangiogenic cells into cells that are able to mediate an antitumoral immune response. Clin Cancer Res; 19(13); 3439–49. ©2013 AACR.

Translational Relevance

TIE-2–expressing monocytes (TEM) participate in human breast tumor angiogenesis and cancer development. VEGF, the most potent proangiogenic factor, has been the focus of many preclinical studies in breast cancer. However, these antiangiogenic therapies have, so far, shown limited benefit, and the disease eventually progresses. In addition to their proangiogenic activity, we report that TEMs infiltrating human carcinoma of the breast display a strong immunosuppressive activity. A combined blockade of TIE-2 and VEGF receptor (VEGFR) kinase activities abolished both the proangiogenic and immunosuppressive activities of TEMs isolated from breast carcinoma. Furthermore, this combined treatment reverted TEM from suppressive, proangiogenic cells into cells that are able to mediate an antitumoral immune response. Therefore, a better understanding of TIE-2 and VEGFR signaling in TEMs may enable the design of efficient anti–breast cancer therapies disrupting both tumor angiogenesis and immune suppression.

Introduction

There is increasing evidence highlighting the role of the immune system in the induction of angiogenesis in cancer and vice versa (1, 2). Myeloid cells and, particularly, myeloid-derived suppressor cells (MDSC; ref. 3), tumor-associated macrophages (4), and immature dendritic cells (DC) play prominent roles in the processes of both immunosuppression and angiogenesis (2). Recently, TIE-2–expressing monocytes (TEM) have been characterized as functionally distinct paracrine inducers of angiogenesis, accounting for apparently all angiogenic activity of bone marrow-derived cells in mouse experimental models of cancer (5, 6). Furthermore, human TEMs derived from peripheral blood of healthy subjects and stimulated with ANG-2 were shown to exhibit T-cell–suppressive function in vitro (7).

CD86 and CD80 are ligands for the CD28 and CTLA-4 receptors expressed on the surface of T cells (8) and may preferentially induce Th-1 or Th-2-type cytokines, respectively (9–11). CD86 is constitutively expressed on antigen-presenting cells (APC), whereas CD80 expression is induced upon APC activation. Cancers, including breast cancers (12–15), show an abundance of immature DCs with impaired capacity to stimulate antitumor immunity (16). In cancer, the main mechanisms of immune evasion resulting in impaired antitumor T-cell response are (i) the lack of expression of costimulatory ligands such as CD80 and CD86 on APCs (8) and (ii) the increased number of T regulatory cells (Treg; ref. 17).

In this study, we examined the impact of TEMs on DC-mediated antitumor T-cell response in human breast cancer. We previously reported that placental growth factor (PlGF) produced by breast tumor cells is critically involved in programming the proangiogenic activity of CD11b+ monocytes during their differentiation from hematopoietic progenitor cells (18). We have shown that breast tumor-associated TEMs display a high proangiogenic activity that is shaped by specific signals in the local tumor microenvironment and controlled by the synergistic action of TIE-2 and VEGFR-1 pathways (Xenarios et al., under revision). Thus, in breast cancer, the role of the tumor microenvironment is 2-fold: first, it is involved in the commitment of myeloid precursors to proangiogenic monocytes and, second, it is necessary for the induction of the proangiogenic phenotype of TEMs when they reach the tumor microenvironment. Here, we report that, in human breast cancer, tumor-associated TEMs are able to suppress tumor-specific immune responses. Interestingly, we found that both proangiogenic and suppressive functions of TEMs are driven by activities of TIE-2 and VEGFR kinase. Indeed, blockade of these receptors with specific kinase inhibitors abrogated proangiogenic activity of tumor TEMs and restored tumor-specific immune responses. Furthermore, this treatment reverted TEMs from proangiogenic suppressive monocytes into immunocompetent APCs with features of myeloid DCs. In addition, we show that TEMs infiltrating breast carcinoma expressed high levels of the costimulatory ligand CD86, and that these levels are maintained by TIE-2 and VEGFR kinase activities. High levels of CD86 expression are required for TEMs to convert T cells into Tregs and suppress tumor-specific immune responses. Taken together, our data reveal that tumor-associated TEMs are plastic cells displaying a reversible functional state controlled by the activation state of TIE-2 and VEGFR kinases.

Materials and Methods

A more detailed description is provided in Supplementary Data.

Patient and tissue specimens

This study was approved by the ethics committee of the University Hospital of Lausanne (CHUV, Lausanne, Switzerland). Patient or subject tissue specimens were obtained according to the declaration of Helsinki. A series of primary invasive breast carcinoma (T1–T2 < 3 cm, lymph node status positive for 25% of the patients, grades I–III) were resected from untreated patients with breast cancer at CHUV; Supplementary Table S1). Peripheral blood from patients was collected before surgery and tumor specimens were freshly dissociated with collagenase I.

Confocal image analysis and quantification

The plugin “particle analyzer” from ImageJ was used for image quantification. The fraction of TEMs and CD11c cells in tumor zones was quantified as follows: (nb of pixel CD14)/(nb of pixel CD14+ nb of pixel CD11c) × 100. TIE-2, HLA-DR was quantified in TEMs and CD11c+ cells using a custom algorithm written in C and is available upon request.

Results

TEMS suppress tumor-specific T-cell responses

We examined the impact of TEMs on antitumor T-cell responses in a group of untreated breast cancer patients with primary invasive breast carcinoma (Supplementary Table S1). We first examined the presence of tumor-specific T cells in the peripheral blood of the patients. To this end, DCs were generated in vitro from peripheral blood monocytes of patients, pulsed with autologous tumor lysate, and cocultured with autologous T cells labeled with the cell tracker CFSE. T-cell proliferation was monitored at day 5 by measuring CFSE dilution by flow cytometry. Peripheral CD4 T cells from patients proliferated in response to DCs pulsed with autologous tumor lysate and the specificity of these responses for the tumor was shown by the lower frequency of T cell proliferation when alone or in the presence of unpulsed DCs (Supplementary Fig. S1).

On the basis of these observations, we next measured peripheral blood T-cell response induced by tumor TEMs and DCs. Flow cytometric analysis of freshly dissociated breast tumors revealed that TEMs and CD11c+ DCs together represented 80% of the CD45+ hematopoietic infiltrate and that CD11c+ DCs were 2.6 (±0.3)-fold more abundant than TEMs (22% ± 2.7% of CD45+ cells, n = 10). Moreover, flow cytometry and confocal microscopic analysis revealed that, in breast tumors, the vast majority (>95%) of CD14+ cells coexpressed TIE-2 and VEGFR-1 (Supplementary Fig. S2). In order to measure tumor-specific T-cell responses, we immunomagnetically isolated TEMs and DCs from freshly dissociated breast tumors and cocultured them immediately with autologous T cells isolated from peripheral blood and labeled with CFSE. In this assay, tumor antigens are presented by CD11c+ DCs isolated from the tumor, and T-cell proliferation in response to tumor antigens was monitored by measuring CFSE dilution using flow cytometry. Among the patients examined (n = 6), the frequency of proliferating T cells following exposure to CD11c+ DC ranged between 0.8% and 12.8% and was set at 100% for normalization (Fig. 1A and B and Supplementary Table S2). However, when TEMs and CD11c+ DCs were cocultured with T cells, their proliferation was significantly (P < 0.01) reduced (Fig. 1A and B). The proliferation of CD4 and CD8 T cells was similarly and significantly (P < 0.05) impacted by TEMs (Fig. 1C), suggesting that TEMs suppresses DC-mediated T-cell proliferation. Following 5-day exposure of CD4 T cells to conditioned medium of TEMs, CD11c+ cells, or TEMS/CD11c+ coculture, the T-cell viability was not significantly affected, thus excluding any toxic effect of TEMs on T cells (Fig. 1D).

Figure 1.
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Figure 1.

TEMs suppress CD11c+ DC–mediated tumor-specific T-cell proliferation. CD11c+ DCs and TEMs from breast tumors were exposed to autologous CFSE-labeled T cells and their proliferation was assessed 5 days later by flow cytometry. A, representative dot plots showing the percentage of proliferating CD4 and CD8 T cells. B and C, graphic representation of the changes of proliferating T cells normalized to T-cell proliferation in the presence of CD11c+ DC [(% CFSElow T cells in the presence of DC) − (% CFSElow T cells when alone) = 100%]. D, CD4 T cells negatively selected from peripheral blood were exposed for 5 days to the conditioned medium of TEM, DC, or TEM/DC coculture and their viability was assessed by flow cytometry following DAPI staining. Cumulated data from 6 independent experiments and 6 distinct patients are shown (B–D); *, P < 0.05; **, P < 0.01.

Immunosuppressive and proangiogenic functions of TEMs are driven by TIE-2 and VEGFR kinase activities

In breast tumor, TEMs express high levels of TIE-2 and VEGFR-1 (Supplementary Fig. S2), both of which control their high proangiogenic activity (Xenarios et al., under revision). We assessed whether combined blockade of TIE-2 and VEGFR kinase activities with specific receptor tyrosine kinase inhibitors (RTKI, i.e., TIE-2 kinase inhibitor compound 7 and VEGFR kinase inhibitor PTK787) would reduce the proangiogenic activity of TEMs. To this end, we measured in vitro the ability of tumor TEMs to induce sprouting of human umbilical vein endothelial cells (HUVEC; see Materials and Methods). Tumor TEMs treated with RTKI induced the formation of much shorter HUVEC sprouts relative to untreated TEMs (Fig. 2A), thus confirming that the proangiogenic activity of TEMs is controlled by the activities of TIE-2 and VEGFR kinase. We next examined the impact of these RTKI on the ability of TEMs to suppress CD11c+ DC-mediated T-cell proliferation. TEMs were treated for 6 hours with RTKI, extensively washed, and cocultured for 5 days with tumor DCs and CFSE-labeled autologous T cells. Importantly, we observed that RTKI treatment simultaneously abolished the proangiogenic activity of tumor TEMs (Fig. 2A) and increased significantly (P < 0.05) the fraction of proliferating T cells (T cells + DC = 100%, T cells + DC + TEMS = 44% ± 18, T cells + DC + TEMS/RTKI = 68% ± 27) and the number of cell divisions (Fig. 2B). We, therefore, calculated the T-cell proliferative index, defined as the average number of cell division that the responding cells underwent assessed on the basis of CFSE dilution measurements (see Materials and Methods). The T-cell proliferation index was significantly increased (P < 0.05) when TEMs were treated with RTKI. In addition, CD4 and CD8 tumor-specific T-cell responses were similarly enhanced (Fig. 2C). Furthermore, activities of TIE-2 and VEGFR kinase contribute more to the suppressive function of TEMs than the immunosuppressive cytokines VEGF and IL-10 (Fig. 2C), both of which are released in much larger amounts by TEMs than CD11c+ DCs (Fig. 2D). Thus, RTKI treatment revealed that the proangiogenic and suppressive activities of TEMs operate through common pathways involving activities of TIE-2 and VEGFR kinase.

Figure 2.
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Figure 2.

Proangiogenic and suppressive functions of tumor TEMs are driven by TIE-2 and VEGFR kinase activities. A, TEMs were isolated from breast tumor and treated with RTKI or VEGF function-blocking antibodies and their proangiogenic activity was measured by sprouting of HUVEC cultured on microcarrier beads. The percentage of cumulated sprout length normalized to untreated TEM is shown. B, representative histograms showing proliferating CD4 T cells cocultured for 5 days with DC and untreated TEMs (filled histogram) or TEMs treated for 6 hours with RTKI (black line histogram). The number of cell divisions on the basis of CFSE content is indicated. C, TEMs were treated with RTKI or VEGF and IL-10 function-blocking antibodies and the T-cell proliferation index was calculated as the average number of cell division undergone by the responding cells. Changes in the proliferation index normalized to coculture with untreated TEMs are shown. Control experiments conducted in the absence of treatment or with isotype control antibodies showed the same proliferation index. D, VEGF and IL-10 release measured in a conditioned medium of TEMs and DC 30 hours postisolation from breast tumors. Cumulative data for TEMs from 5 independent experiments and 5 distinct patients are shown in A, B, and D. *, P < 0.05; **, P < 0.01.

Tumor TEMs support a weak T-cell proliferation and expand regulatory T cells

We found that tumor CD11c+ DCs were significantly (P < 0.01) more potent than tumor TEMs at inducing T-cell proliferation (Fig. 1A and B). Indeed, tumor TEMs induced a weak but significant CD4 T-cell proliferation (Fig. 3A, n = 8), which was abolished by neutralizing antibodies to MHC (Fig. 3A and B). These results indicate that tumor TEMs are functional APCs displaying, however, a much weaker aptitude to present antigen to T cells relative to CD11c+ DCs. Interestingly, tumor TEMs, but not CD11c+ DCs, induced a massive (>20%) conversion of CD4 T cells into CD25+ or FOXP3+ CD4 Tregs. Importantly, RTKI treatment inhibited the ability of TEMs to induce FOXP3 expression on CD4 T cells (Fig. 3C and D). These CD4+CD25+FoxP3+ cells endowed a phenotype and functional characteristics of Treg as they efficiently suppressed the proliferation of CD4+ CD45RA− CD25− effector cells in vitro (Fig. 3C, right). These results suggest that activities of Tie2 and VEGFR kinase control both, the ability of TEMs to support T-cell proliferation and the conversion of T cells into Treg.

Figure 3.
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Figure 3.

Tumor TEMs support T-cell proliferation and expand regulatory CD4 T cells. TEMs from breast tumors were exposed to autologous CFSE-labeled CD4 T cells and their proliferation was assessed 5 days later by flow cytometry. A, percentage of proliferating T cells in eight distinct patients. B, changes in CD4 T cell proliferation exposed to TEMs in the presence or absence of MHC I and II neutralizing antibodies; representative dot blots are shown. C, frequency of CD25+FOXP3+ cells among CD4 T cells (left). Following 5-day coculture with TEMs, CD4+CD25+ were isolated by immunomagnetic selection and cocultured for 2.5 days with CD4+CD45RA−CD25− effector cells previously labeled with DDAO and activated with CD3 and CD28 antibodies. Histograms show DDAO dilution in proliferating effector cells (right). D, frequency of CD25+FOXP3+ cells among CD4 T cells in the presence of CD80 or CD86 blocking antibodies normalized to CD4 + TEM (100%). Cumulative data from 4 independent experiments and 4 distinct patients are shown in B and C. *, P < 0.05; **, P < 0.01.

Antiangiogenic treatments alter TEMs phenotype toward myeloid DCs in vitro

We recently reported that, in TEMs from breast tumor or differentiated in vitro from cord blood CD34+ precursors, Tie2 and VEGFR1 pathways synergized with the TNF-α and TGF-β pathways to induce and reduce the proangiogenic activity of TEMs respectively (Xenarios et al., under revision). We compared the impact of RTKI and ANG-2/TGF-β treatments on the proangiogenic activity of TEMs using the HUVEC sprouting assay (see Materials and Methods section). ANG-2/TGF-β (Fig. 4A; Xenarios et al., under revision) or RTKI treatments resulted in a marked reduction in sprout length, confirming that TIE-2 and VEGFR pathways are critical for the proangiogenic activity of both TEMs differentiated in vitro (Fig. 4A) and tumor TEMs (Fig. 2A).

Figure 4.
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Figure 4.

Antiangiogenic treatments shift the gene expression profile and the functional phenotype of TEMs differentiated in vitro toward that of myeloid dendritic cells. A, proangiogenic activity of TEMs differentiated in vitro in response to ANG-2/TGF-β and TIE-2 and VEGFR kinase inhibitors (RTKI) measured by sprouting of HUVEC cultured on microcarrier beads. The percentage of cumulative sprout length normalized to untreated TEMs is shown. B, a total of 355 genes were significantly differentially expressed (adjusted P value ≤ 0.05) between ANG-2/TGF-β and untreated cells. Differentially expressed genes were manually annotated and classified in categories. The genes functionally related to angiogenesis, immune response, cell differentiation, and antigen processing are shown. C, monitoring by flow cytometry of markers of DC differentiation and activation in TEMs treated with ANG-2/TGF-β or TIE-2 and RTKI. Percentage of increase in the expression levels relative to untreated TEMs are shown. D, in vitro differentiated TEMs were cocultured with CFSE-labeled autologous T cells previously stimulated for 5 days with anti-CD3 and CD28 antibodies. In the absence of TEMs, the rate of proliferating T cells, as assessed by CFSE content, was 5% and 30% for stimulated (filled histogram) and unstimulated T cells respectively. TEMs were either untreated or treated with ANG-2/TGF-β or RTKI before coculture with T cells. Mean cumulative data of 5 experiments from 5 patients and 3 distinct cord blood samples are shown in A, C, and B respectively. A representative experiment out of 4 is shown in D. *, P < 0.05; **, P < 0.01.

To gain insight into the impaired proangiogenic phenotype of TEMs, we conducted gene expression profiling of TEMS differentiated in vitro treated with ANG-2/TGF-β (Xenarios et al., under revision; Supplementary Methods). We identified 355 genes that were significantly differentially expressed, with false discovery rate (FDR) 5% or less, upon ANG-2/TGF-β treatment relative to untreated TEMs. Approximately 70% of these genes were downregulated and encoded for functions related to cell cycle, metabolism, apoptosis, and motility (Xenarios et al., under revision). ANG-2/TGF-β treatment downregulated proangiogenic genes, while genes encoding for angiostatic proteins (VASH1: vasohibin 1, UCN: urocortin, the angiostatic cytokine CXCL9; ref. 19) were upregulated (Fig. 4B). This impaired proangiogenic activity was associated with the downregulation of genes whose products are involved in immune suppression (IL-10; HSD11B1, hydroxysteroid 11-β dehydrogenase 1; SLC16A7, solute carrier family 16, member 7) including S100A9 (S100 calcium-binding protein A9), which inhibits DC differentiation and promotes MDSC accumulation in mouse tumors (20). Upregulation of genes enhancing the immune response was observed with increased expression of genes mediating T-cell activation and chemotaxis or associated with the synthesis of proinflammatory cytokines (Fig. 4B). Moreover, ANG-2/TGF-β treatment induced downregulation of genes related to the maturation of monocytes into macrophages and, conversely, upregulation of genes encoding for markers of a promyelocytic stage. In addition, genes encoding for DC markers showed upregulated expression (Fig. 4B). Some of these genes have been previously reported to be upregulated upon in vitro maturation of DCs following exposure to lipopolysaccharide and interferon-γ (21, 22). Consistent with this observation, the expression of genes related to antigen processing and presentation, which are specific functions of APCs, were also upregulated (Fig. 4B). These data indicate that TEMs are plastic cells that shift their gene expression profile toward one resembling myeloid DC in response to treatments that impair their proangiogenic function.

In order to validate this phenotypic reversion, we monitored by flow cytometry the expression of markers of DC differentiation and activation in TEMs treated with ANG-2/TGF-β or RTKI. Relative to untreated TEMs, short and longer exposure to RTKI treatment resulted in significant (P < 0.05) increase in the expression of antigen-presenting molecules HLA-DR, integrin CD61, as well as costimulatory molecules CD80 and CD86. Moreover, CD11c expression was significantly (P < 0.05) induced by RTKI but not ANG-2/TGF-β treatment, whereas CD14 expression was strongly reduced by both treatments (Fig. 4C). These results confirmed the phenotypic change toward a more myeloid-DC-like phenotype that we observed in the gene expression profiling experiment. Furthermore, in order to test whether these treated TEMs could have altered their immunosuppressive function, we cocultured them with CFSE-labeled autologous T cells that had been previously stimulated with anti-CD3, anti-CD28 antibodies and IL-2 for 24 hours and rested 5 days (see Materials and Methods). In vitro differentiated TEMs treated with ANG-2/TGF-β or RTKI significantly increased the frequency of proliferating T cells relative to untreated TEMs (Fig. 4D). A stronger effect was seen with RTKI compared with ANG-2/TGF-β treatment (42% and 57% CFSElow T cells respectively, compared with 31% CFSElow T cells with untreated TEMs; Fig. 4D).

Thus, our data confirm that TEMs differentiated in vitro are plastic cells that can be induced to change from a proangiogenic immunosuppressive phenotype to one of immunocompetent cells resembling myeloid DCs. This phenotypic reversion was induced by modulating pathways driving the proangiogenic function of TEMs, indicating that the angiogenic and immunosuppressive functions of TEMs are probably linked.

In breast tumors, TEMS and CD11c+ DCs express markers of APCs

The reversion of the suppressive phenotype that we observed for TEMs differentiated in vitro in response to antiangiogenic treatments (Fig. 4) prompted us to examine whether TEMs and CD11c+ DCs display overlapping phenotypes in breast tumors. To this end, we characterized TEM and DC populations by confocal microscopy in sections of frozen breast tumor. Our results showed that TEMs were CD14+, CD11c−, TIE-2+, VEGFR-1+ CD31−, whereas CD11c+ DCs were CD14−, CD11c+, TIE-2−, VEGFR-1low CD31−, and CD141− (Supplementary Fig. S2). Both TEMs and DCs expressed APC markers, such as HLA-DR, CD80, CD86, and CD1a (Fig. 5A), consistent with their ability to function as APCs (Figs. 1A, 3A, and 3B). Quantification of confocal microscopy images (see Materials and Methods) indicated that TEMs expressed relatively higher levels of HLA-DR, CD86 [MFI CD86 (TEMs) = 93.3 ± 3.7; MFI CD86 (CD11c+ cells) = 28.4 ± 8.5; P < 0.01] and CD1a [MFI CD1a (TEMs) = 20.5 ± 12.1; MFI CD1a (CD11c+ cells) = 11.2 ± 6.9; P < 0.05] relative to CD11c+ DCs, but comparable [MFI CD80 (TEMs) = 57.2 ± 21.4, MFI CD80 (CD11c+ cells) = 56.6 ± 14.1] expression levels of CD80 (Fig. 5A). Thus in human breast tumor, while TEMs and CD11c+ DC populations show no detectable overlap of CD14 and CD11c expression, they share, to various extents, the APC markers HLA-DR, CD1a, CD80, and CD86 (Fig. 5A). Finally, in contrast to CD11c+ DCs, and consistent with their suppressive function (Fig. 1), we measured in confocal microscopic images that TEMs expressed a 34-fold higher expression level of arginase-1, a marker associated with MDSCs in the mouse, than did CD11c+ DCs (P < 0.05; ref. 3; Fig. 5A). These data suggest that, in breast tumor, CD11c+ DCs show an APC phenotype, whereas TEMs display rather a phenotype of proangiogenic suppressive APCs.

Figure 5.
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Figure 5.

In breast tumors, TEMs and CD11c+ DC express markers of APC, and TEMs alter the function and the maturation of CD11c+ DC. A, sections of frozen breast tumor tissues were examined by confocal microscopy for the expression of HLA-DR, CD80, CD86, CD1a, and arginase-1 and representative images from 6 tumors and 57 images per staining are shown (insert, high magnification). B, DCs display heterogeneous expression of CD11c, with HLA-DR expression significantly lower in CD11clow cells (<40% of maximal CD11c normalized intensity) relative to CD11chigh cells (≥40% of maximal CD11c normalized intensity). C, CD11c expression levels were quantified (A.U. arbitrary units) in tumor zones containing increasing rates of TEM infiltration. Quantification and analysis of confocal microscopic images were carried out as described in Materials and Methods. **, P < 0.01. Scale bar, 25 μm.

TEMS impair CD11c+ DC maturation in breast tumors

Because tumor TEMs suppressed tumor-specific T-cell responses mediated by tumor CD11c+ DCs (Fig. 1), we next examined whether TEMs may directly alter the ability of DCs to present antigen by lowering their HLA-DR expression levels. By quantifying confocal microscopic images of breast tumor sections (see Materials and Methods), we observed that the CD11c+ DC population was heterogeneous and encompassed a subset of CD11clow and CD11chigh cells. The expression levels of CD11c were normalized in each patient to the maximal CD11c expression detected (100%) and CD11c+ DC was classified into subsets of CD11clow (<40% maximal CD11c intensity) and CD11chigh (≥ 40% maximal CD11c intensity) cells. Results show that HLA-DR expression was significantly higher (P < 0.01) in CD11chigh cells relative to CD11clow cells (Fig. 5B). Importantly, CD11c expression levels were strongly dependent on the local rate of TEM infiltration. Using immunofluorescence images, we defined the rate of TEM infiltration relative to CD11c+ cells in a tumor zone as follows: [TEMs surface area/(TEMs + CD11c+ cells) surface area] * 100. We measured a significant reduction (P < 0.01) of CD11c expression with increasing rates of TEMs infiltration (Fig. 5C), indicating that TEMs impaired DC function and maturation by reducing their HLA-DR expression.

In breast tumors, high levels of CD86 expression are required for TEMs to engage Treg cells and to suppress tumor-specific immune responses

In order to understand further how TEMs suppress T-cell responses in tumor, we examined the distribution of TEMs and T cells in frozen sections of breast tumor tissues. By analyzing images of the sections, we could estimate that 60% of infiltrating T cells were in close proximity and potentially engaged by TEMs and CD11c+ cells. Surprisingly, while TEMs were 2.6 (±0.3)-fold less abundant than CD11c+ DCs, TEMs engaged 80% (±6.8%) of T cells compared with 27% (±12%) for CD11c+ DCs (Fig. 6A). Importantly, we observed that T cells were 1.6-fold less abundant in tumor zones containing TEMs, relative to TEMs-depleted zones. Furthermore, the vast majority of T cells (>71%) in TEMs-containing zones expressed the Treg marker CD25 and were preferentially (>92%) engaged by TEMs (Fig. 6B). We observed that, in breast tumor, Treg (CD3+CD25+ FOXP3+ cells) accounted for by 39% (±7%) of infiltrating T cells and were enriched in tumor zones containing TEMs where they massively (92% ±7%) form multiple tight conjugates with TEMs (Fig. 6B) but not with CD11c+ cells (7% ± 4%).

Figure 6.
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Figure 6.

In breast tumors, TEMs express high levels of CD86, thus ensuring engagement of regulatory T cells and their suppressive function. A, sections of frozen breast tumor tissues were examined by confocal microscopy for T-cell–TEM and T-cell–CD11c+ cell conjugates and the percentage of T cells engaged by TEMs or DCs was quantified from 117 images from 8 distinct tumors; scale bar, 25 μm. B, percentage of T cells, Treg (CD3+CD25+ cells) among T cells, and Treg–TEM conjugates among T-cell–TEM conjugates were examined in TEM-depleted (−) and TEM-containing (+) tumor zones. 98 images from 10 distinct tumors were examined; scale bar, 5 μm. C, CD86 to CD80 expression ratio was calculated in 126 confocal microscopic images from 7 distinct tumors (top). Tumor-specific proliferation of T cells (as measured in Fig. 4D) in response to CD86-specific blocking antibodies (bottom). D, changes in the expression levels of CD86 at the surface of tumor TEM measured by flow cytometry following treatment with RTKI. Cumulative data from 5 independent experiments and 5 distinct patients are shown in C, bottom and in D. *, P < 0.05; **, P < 0.01.

The immune response to tumor is often ineffective due to the downmodulated expression of HLA-DR and costimulatory ligands CD80 and CD86 on the surface of the APC, which renders tumor-specific T cells anergic (23). However, we measured by confocal microscopy that CD80 expression was comparable in TEMs and CD11c+ DCs while TEMs display a 3.4 (±1.1, P < 0.01)-fold increase in CD86 expression levels relative to CD11c+ cells. Consequently, the ratio of CD86 to CD80 expression was 3.3 higher in TEMs as compared with CD11c+ DCs (Fig. 6C, top). To test whether excessive CD86 engagement may support the suppressive function of TEMs, we treated TEMS with CD86-blocking antibodies before coculturing them with CD11c+ DCs and CFSE-labeled autologous T cells. We observed that blocking CD86 engagement increased the T-cell proliferation index to the same extent as RTKI treatment (compare Figs. 6C, bottom, and 2C) indicating that TEMs suppressed tumor-specific T-cell proliferation by excessive CD86 engagement. Interestingly, RTKI treatment decreased significantly the expression of CD86 at the surface of tumor TEMs (Fig. 6D). Furthermore, using specific antibodies, we observed that blocking CD86 engagement impaired, to a larger extent, TEMS-induced conversion of T cells into Treg relative to blocking CD80 engagement (Fig. 3D). These data indicate that CD86 contributes largely to this process. Taken together, our results suggest that the basal kinase activity of TIE-2 and VEGFR of tumor TEMs (Fig. 2A) supports the suppressive function of TEMs by maintaining high levels of CD86 expression at their surface (Fig. 6C, top, and 6D), resulting in massive conversion of T cells into Treg (Fig. 3D) and extensive engagement of Treg cells (Fig. 6B).

Discussion

In this study, we show that the suppressive function of TEMs isolated from breast tumor is reversible and, like their proangiogenic activity, is driven by the activities of TIE-2 and VEGFR kinase (Figs. 2A and C, 3C, 4 and 6D). In breast tumor, TEMs extensively engaged Tregs (Fig. 6B) and RTKI treatment disabled TEMs suppressive activity by impairing their capacity to massively convert T cells into Tregs (Fig. 3C). The accumulation of Treg cells in many solid tumors, including breast cancer in humans, has been associated with poor survival (17, 24), tumor angiogenesis (25, 26), hypoxia (27), and increased tumor tolerance (17). Conversely, depletion of CD25+ or CCR10+ cells eliminated Tregs from the tumor microenvironment and significantly reduced angiogenesis (17, 28). Thus, targeting Treg cells currently represents an attractive therapeutic strategy for cancer treatment (28). The clinical efficacy of such a strategy will, however, necessitate targeting the processes converting T cells into Tregs rather than by blocking or depleting Tregs; the aim being to recover sustained and protective antitumor immunity. Our results suggest that, in breast cancer, RTKI may represent an efficient treatment by suppressing the ability of TEMs to convert T cells into Tregs and reverting TEMs into APCs promoting an antitumor T-cell response (Figs. 2C, 3C and 6D). Concomitantly, RTKI treatment fully suppressed the angiogenic activity of TEMs (Fig. 2A) and is, therefore, likely to limit tumor growth and to recover sustained antitumor T-cell responses. Furthermore, our results reveal that the highly angiogenic suppressive functional phenotype of breast tumor TEMs does not reflect a final differentiation state but rather a druggable plastic phenotype. We identify here mechanisms underlying the suppressive function of TEMs such as the alteration of CD11c+ DC function and maturation (Fig. 5), the secretion of VEGF and IL-10 cytokines, which suppress T-cell functions (Fig. 2C and 2D), and the extensive engagement of Tregs (Fig. 6B). In line with our observations, VEGF- or IL-10–impaired DC maturation has been reported in cancer patients (refs. 29–31; Fig. 5B and C). However, while Tregs may differentiate and expand following stimulation by tumor-associated DCs (17, 28), in breast cancer, TEMs, but not CD11c+DCs, appear to control this process (Fig. 3C and D).

We suggest a new mechanism whereby, in TEMs, activities of TIE-2 and VEGFR kinase maintain high levels of CD86 expression, which leads to the conversion of T cells into Tregs (Fig. 3D), extensive engagement of Treg in the tumor (Fig. 6B), and suppression of tumor-specific T response mediated by CD11c+ DCs (Fig. 6C, bottom). Although CD86 expressed on APCs was reported to play a dominant role in inducing and enhancing T–cell-mediated antitumor immunity in some models, this has shown limited therapeutic efficacy (8, 9). Similar heterogeneous observations were made with treatments targeting CTLA-4 with specific antibodies (17, 28). These apparent discrepancies may be due to specific tumor microenvironmental signals enhancing the CD86 expression of TEMs, for example, ligands of TIE-2 and VEGFR. In agreement with this, in ovarian cancer, specific tumor factors upregulated the expression of the PD-L1 on myeloid DCs (31) and B7-H4 on tumor ascite macrophages (32, 33), subsequently leading to impaired T-cell antitumor immunity. Thus, the regulation of the expression of costimulatory molecules on the surface of myeloid cells by the tumor microenvironment might be a more general mechanism promoting both immune suppression and angiogenesis. The well-known heterogeneity of breast cancer may produce variations of the tumor microenvironment likely to explain the variability of the T-cell response and the suppressive activity of TEMs we measured (Figs. 1, 2 and Supplementary Table S2). Nevertheless, combined targeting of the activities of TIE-2 and VEGFR kinase may offer new prospects for breast cancer therapies by leading to disruption of both suppressive and angiogenic activities of TEMs while concomitantly promoting an antitumor immune response.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Authors' Contributions

Conception and design: H-A. Lehr, M-A. Doucey

Development of methodology: N. Guex

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Bron, E. Faes-van't Hull, L. Henry, H-A. Lehr, J-F. Delaloye

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Bron, N. Guex, E. Faes-van't Hull, H-A. Lehr, J. Xenarios, M-A. Doucey

Writing, review, and/or revision of the manuscript: M. Ibberson, L. Henry, H-A. Lehr, J-F. Delaloye, G. Coukos, M-A. Doucey

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Bron, E. Faes-van't Hull, H-A. Lehr

Study supervision: J. Xenarios, M.-A. Doucey

Other: A. Ifticene-treboux

Grant Support

This work was financially supported by grants from the Medic Foundation, Oncosuisse (02069-04-2007; to M.-A. Doucey), the Swiss National Foundation (310030-120473, to M.-A. Doucey; and CR32I3_135073, to J-F. Delaloye), and the ENFIN FP6 Program (I.X. project LSHG-CT-2005-518254, to J. Xenarios).

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.

Acknowledgments

The authors thank Novartis for providing the VEGFR kinase inhibitor PTK787.

Footnotes

  • Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

  • Received October 10, 2012.
  • Revision received April 16, 2013.
  • Accepted April 19, 2013.
  • ©2013 American Association for Cancer Research.

References

  1. 1.↵
    1. Tartour E,
    2. Pere H,
    3. Maillere B,
    4. Terme M,
    5. Merillon N,
    6. Taieb J,
    7. et al.
    Angiogenesis and immunity: a bidirectional link potentially relevant for the monitoring of antiangiogenic therapy and the development of novel therapeutic combination with immunotherapy. Cancer Metastasis Rev 2011;30:83–95.
    OpenUrlCrossRefPubMed
  2. 2.↵
    1. Motz GT,
    2. Coukos G
    . The parallel lives of angiogenesis and immunosuppression: cancer and other tales. Nat Rev Immunol 2011;11:702–11.
    OpenUrlCrossRefPubMed
  3. 3.↵
    1. Gabrilovich DI,
    2. Nagaraj S
    . Myeloid-derived suppressor cells as regulators of the immune system. Nat Rev Immunol 2009;9:162–74.
    OpenUrlCrossRefPubMed
  4. 4.↵
    1. Biswas SK,
    2. Sica A,
    3. Lewis CE
    . Plasticity of macrophage function during tumor progression: regulation by distinct molecular mechanisms. J Immunol 2008;180:2011–7.
    OpenUrlAbstract/FREE Full Text
  5. 5.↵
    1. De Palma M,
    2. Venneri MA,
    3. Galli R,
    4. Sergi Sergi L,
    5. Politi LS,
    6. Sampaolesi M,
    7. et al.
    Tie2 identifies a hematopoietic lineage of proangiogenic monocytes required for tumor vessel formation and a mesenchymal population of pericyte progenitors. Cancer Cell 2005;8:211–26.
    OpenUrlCrossRefPubMed
  6. 6.↵
    1. Venneri MA,
    2. De Palma M,
    3. Ponzoni M,
    4. Pucci F,
    5. Scielzo C,
    6. Zonari E,
    7. et al.
    Identification of proangiogenic TIE2-expressing monocytes (TEMs) in human peripheral blood and cancer. Blood 2007;109:5276–85.
    OpenUrlAbstract/FREE Full Text
  7. 7.↵
    1. Coffelt SB,
    2. Chen YY,
    3. Muthana M,
    4. Welford AF,
    5. Tal AO,
    6. Scholz A,
    7. et al.
    Angiopoietin 2 stimulates TIE2-expressing monocytes to suppress T cell activation and to promote regulatory T cell expansion. J Immunol 2011;186:4183–90.
    OpenUrlAbstract/FREE Full Text
  8. 8.↵
    1. Vesosky B,
    2. Hurwitz AA
    . Modulation of costimulation to enhance tumor immunity. Cancer Immunol Immunother 2003;52:663–9.
    OpenUrlCrossRefPubMed
  9. 9.↵
    1. Yang G,
    2. Hellstrom KE,
    3. Chen L
    . The role of B7-2 (CD86) in tumour immunity. Expert Opin Investig Drugs 1997;6:677–84.
    OpenUrlCrossRefPubMed
  10. 10.↵
    1. Freeman GJ,
    2. Boussiotis VA,
    3. Anumanthan A,
    4. Bernstein GM,
    5. Ke XY,
    6. Rennert PD,
    7. et al.
    B7-1 and B7-2 do not deliver identical costimulatory signals, since B7-2 but not B7-1 preferentially costimulates the initial production of IL-4. Immunity 1995;2:523–32.
    OpenUrlCrossRefPubMed
  11. 11.↵
    1. Kuchroo VK,
    2. Das MP,
    3. Brown JA,
    4. Ranger AM,
    5. Zamvil SS,
    6. Sobel RA,
    7. et al.
    B7-1 and B7-2 costimulatory molecules activate differentially the Th1/Th2 developmental pathways: application to autoimmune disease therapy. Cell 1995;80:707–18.
    OpenUrlCrossRefPubMed
  12. 12.↵
    1. Gervais A,
    2. Leveque J,
    3. Bouet-Toussaint F,
    4. Burtin F,
    5. Lesimple T,
    6. Sulpice L,
    7. et al.
    Dendritic cells are defective in breast cancer patients: a potential role for polyamine in this immunodeficiency. Breast Cancer Res 2005;7:R326–35.
    OpenUrlCrossRefPubMed
  13. 13.↵
    1. Satthaporn S,
    2. Aloysius MM,
    3. Robins RA,
    4. Verma C,
    5. Chuthapisith S,
    6. McKechnie AJ,
    7. et al.
    Ex vivo recovery and activation of dysfunctional, anergic, monocyte-derived dendritic cells from patients with operable breast cancer: critical role of IFN-alpha. BMC Immunol 2008;9:32.
    OpenUrlCrossRefPubMed
  14. 14.↵
    1. Poindexter NJ,
    2. Sahin A,
    3. Hunt KK,
    4. Grimm EA
    . Analysis of dendritic cells in tumor-free and tumor-containing sentinel lymph nodes from patients with breast cancer. Breast Cancer Res 2004;6:R408–15.
    OpenUrlCrossRefPubMed
  15. 15.↵
    1. Bell D,
    2. Chomarat P,
    3. Broyles D,
    4. Netto G,
    5. Harb GM,
    6. Lebecque S,
    7. et al.
    In breast carcinoma tissue, immature dendritic cells reside within the tumor, whereas mature dendritic cells are located in peritumoral areas. J Exp Med 1999;190:1417–26.
    OpenUrlAbstract/FREE Full Text
  16. 16.↵
    1. Fainaru O,
    2. Almog N,
    3. Yung CW,
    4. Nakai K,
    5. Montoya-Zavala M,
    6. Abdollahi A,
    7. et al.
    Tumor growth and angiogenesis are dependent on the presence of immature dendritic cells. FASEB J 2010;24:1411–8.
    OpenUrlAbstract/FREE Full Text
  17. 17.↵
    1. Facciabene A,
    2. Motz GT,
    3. Coukos G
    . T-regulatory cells: key players in tumor immune escape and angiogenesis. Cancer Res 2012;72:2162–71.
    OpenUrlAbstract/FREE Full Text
  18. 18.↵
    1. Laurent J,
    2. Hull EF,
    3. Touvrey C,
    4. Kuonen F,
    5. Lan Q,
    6. Lorusso G,
    7. et al.
    Proangiogenic factor PlGF programs CD11b(+) myelomonocytes in breast cancer during differentiation of their hematopoietic progenitors. Cancer Res 2011;71:3781–91.
    OpenUrlAbstract/FREE Full Text
  19. 19.↵
    1. Arenberg DA,
    2. Kunkel SL,
    3. Polverini PJ,
    4. Morris SB,
    5. Burdick MD,
    6. Glass MC,
    7. et al.
    Interferon-gamma-inducible protein 10 (IP-10) is an angiostatic factor that inhibits human non-small cell lung cancer (NSCLC) tumorigenesis and spontaneous metastases. J Exp Med 1996;184:981–92.
    OpenUrlAbstract/FREE Full Text
  20. 20.↵
    1. Cheng P,
    2. Corzo CA,
    3. Luetteke N,
    4. Yu B,
    5. Nagaraj S,
    6. Bui MM,
    7. et al.
    Inhibition of dendritic cell differentiation and accumulation of myeloid-derived suppressor cells in cancer is regulated by S100A9 protein. J Exp Med 2008;205:2235–49.
    OpenUrlAbstract/FREE Full Text
  21. 21.↵
    1. Autiero M,
    2. Waltenberger J,
    3. Communi D,
    4. Kranz A,
    5. Moons L,
    6. Lambrechts D,
    7. et al.
    Role of PlGF in the intra- and intermolecular cross talk between the VEGF receptors Flt1 and Flk1. Nat Med 2003;9:936–43.
    OpenUrlCrossRefPubMed
  22. 22.↵
    1. Jin P,
    2. Han TH,
    3. Ren J,
    4. Saunders S,
    5. Wang E,
    6. Marincola FM,
    7. et al.
    Molecular signatures of maturing dendritic cells: implications for testing the quality of dendritic cell therapies. J Transl Med 2010;8:4.
    OpenUrlCrossRefPubMed
  23. 23.↵
    1. Pinzon-Charry A,
    2. Maxwell T,
    3. Lopez JA
    . Dendritic cell dysfunction in cancer: a mechanism for immunosuppression. Immunol Cell Biol 2005;83:451–61.
    OpenUrlCrossRefPubMed
  24. 24.↵
    1. Curiel TJ,
    2. Coukos G,
    3. Zou L,
    4. Alvarez X,
    5. Cheng P,
    6. Mottram P,
    7. et al.
    Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med 2004;10:942–9.
    OpenUrlCrossRefPubMed
  25. 25.↵
    1. Giatromanolaki A,
    2. Bates GJ,
    3. Koukourakis MI,
    4. Sivridis E,
    5. Gatter KC,
    6. Harris AL,
    7. et al.
    The presence of tumor-infiltrating FOXP3+ lymphocytes correlates with intratumoral angiogenesis in endometrial cancer. Gynecol Oncol 2008;110:216–21.
    OpenUrlCrossRefPubMed
  26. 26.↵
    1. Gupta S,
    2. Joshi K,
    3. Wig JD,
    4. Arora SK
    . Intratumoral FOXP3 expression in infiltrating breast carcinoma: its association with clinicopathologic parameters and angiogenesis. Acta Oncol 2007;46:792–7.
    OpenUrlCrossRefPubMed
  27. 27.↵
    1. Facciabene A,
    2. Peng X,
    3. Hagemann IS,
    4. Balint K,
    5. Barchetti A,
    6. Wang LP,
    7. et al.
    Tumour hypoxia promotes tolerance and angiogenesis via CCL28 and T(reg) cells. Nature 2011;475:226–30.
    OpenUrlCrossRefPubMed
  28. 28.↵
    1. Zou W
    . Regulatory T cells, tumour immunity and immunotherapy. Nat Rev Immunol 2006;6:295–307.
    OpenUrlCrossRefPubMed
  29. 29.↵
    1. Della Porta M,
    2. Danova M,
    3. Rigolin GM,
    4. Brugnatelli S,
    5. Rovati B,
    6. Tronconi C,
    7. et al.
    Dendritic cells and vascular endothelial growth factor in colorectal cancer: correlations with clinicobiological findings. Oncology 2005;68:276–84.
    OpenUrlCrossRefPubMed
  30. 30.↵
    1. Gabrilovich DI,
    2. Chen HL,
    3. Girgis KR,
    4. Cunningham HT,
    5. Meny GM,
    6. Nadaf S,
    7. et al.
    Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells. Nat Med 1996;2:1096–103.
    OpenUrlCrossRefPubMed
  31. 31.↵
    1. Curiel TJ,
    2. Wei S,
    3. Dong H,
    4. Alvarez X,
    5. Cheng P,
    6. Mottram P,
    7. et al.
    Blockade of B7-H1 improves myeloid dendritic cell-mediated antitumor immunity. Nat Med 2003;9:562–7.
    OpenUrlCrossRefPubMed
  32. 32.↵
    1. Kryczek I,
    2. Zou L,
    3. Rodriguez P,
    4. Zhu G,
    5. Wei S,
    6. Mottram P,
    7. et al.
    B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma. J Exp Med 2006;203:871–81.
    OpenUrlAbstract/FREE Full Text
  33. 33.↵
    1. Kryczek I,
    2. Wei S,
    3. Zou L,
    4. Zhu G,
    5. Mottram P,
    6. Xu H,
    7. et al.
    Cutting edge: induction of B7-H4 on APCs through IL-10: novel suppressive mode for regulatory T cells. J Immunol 2006;177:40–4.
    OpenUrlAbstract/FREE Full Text
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TIE-2 and VEGFR Kinase Activities Drive Immunosuppressive Function of TIE-2–Expressing Monocytes in Human Breast Tumors
Mark Ibberson, Sylvian Bron, Nicolas Guex, Eveline Faes-van't Hull, Assia Ifticene-Treboux, Luc Henry, Hans-Anton Lehr, Jean-François Delaloye, George Coukos, Ioannis Xenarios and Marie-Agnès Doucey
Clin Cancer Res July 1 2013 (19) (13) 3439-3449; DOI: 10.1158/1078-0432.CCR-12-3181

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TIE-2 and VEGFR Kinase Activities Drive Immunosuppressive Function of TIE-2–Expressing Monocytes in Human Breast Tumors
Mark Ibberson, Sylvian Bron, Nicolas Guex, Eveline Faes-van't Hull, Assia Ifticene-Treboux, Luc Henry, Hans-Anton Lehr, Jean-François Delaloye, George Coukos, Ioannis Xenarios and Marie-Agnès Doucey
Clin Cancer Res July 1 2013 (19) (13) 3439-3449; DOI: 10.1158/1078-0432.CCR-12-3181
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