Angiopoietin 2 (ANGPT2) is a proangiogenic cytokine whose expression is often upregulated by endothelial cells in tumors. Expression of its receptor, TIE2, defines a highly proangiogenic subpopulation of myeloid cells in circulation and tumors called TIE2-expressing monocytes/macrophages (TEMs). Genetic depletion of TEMs markedly reduces tumor angiogenesis in various tumor models, emphasizing their essential role in driving tumor progression. Previously, we demonstrated that ANGPT2 augments the expression of various proangiogenic genes, the potent immunosuppressive cytokine, IL-10, and a chemokine for regulatory T cells (Tregs), CCL17 by TEMs in vitro. We now show that TEMs also express higher levels of IL-10 than TIE2− macrophages in tumors and that ANGPT2-stimulated release of IL-10 by TEMs suppresses T cell proliferation, increases the ratio of CD4+ T cells to CD8+ T cells, and promotes the expansion of CD4+CD25highFOXP3+ Tregs. Furthermore, syngeneic murine tumors expressing high levels of ANGPT2 contained not only high numbers of TEMs but also increased numbers of Tregs, whereas genetic depletion of tumor TEMs resulted in a marked reduction in the frequency of Tregs in tumors. Taken together, our data suggest that ANGPT2-stimulated TEMs represent a novel, potent immunosuppressive force in tumors.
TIE2-expressing monocytes/macrophages (TEMs) are a subpopulation of myeloid cells characterized by the expression of monocyte/macrophage markers as well as the angiopoietin receptor TIE2 (1, 2). TEMs are found in circulation and tumors of both human and mice, and conditional TEM deletion in various mouse tumor models has established their profound effect on tumor angiogenesis and progression (2–4). We recently reported that human circulating TEMs are preprogrammed in the bone marrow or peripheral blood for proangiogenic functions before these cells reach the tumor microenvironment (5). We also found that exposure to angiopoietin 2 (ANGPT2)—a cytokine overexpressed by endothelial cells in tumors (6–8)—further augments the ability of TEMs to stimulate angiogenesis through upregulation of such proangiogenic enzymes as thymidine phosphorylase and cathepsin B (5).
In addition, we demonstrated that ANGPT2 skews the phenotype of human TEMs toward an M2-like macrophage polarization in vitro via downregulation of IL-6 and increased expression of mannose receptor (MRC1), CCL17, and IL-10 (5, 9, 10). TEMs display several characteristics of M2-polarized macrophages in murine tumors (11), and our data indicate that ANGPT2 may drive this phenotype (5). Because M2 macrophages are thought to promote tumor progression through angiogenesis as well as suppression of antitumor immune responses (reviewed in Refs. 10 and 12), we hypothesized that ANGPT2 may stimulate the immunosuppressive properties of TEMs.
In this paper, we show that ANGPT2-stimulated human TEMs suppress T cell proliferation and expand CD4+CD25+FOXP3+ regulatory T cells (Tregs) via IL-10–dependent mechanisms. We also demonstrate that murine tumor TEMs express high levels of IL-10 and are markedly immunosuppressive. These data illustrate a novel protumorigenic role for TEMs.
Materials and Methods
Human leukocyte isolation, coculture, and flow cytometry
Monocytes were isolated from human Buffy coats as described previously (3, 5). Autologous CD3+ T cells were isolated by negative selection using StemCell Technologies’ Rosettesep kit. CD3+ 5 monocytes to 5 × 105 CD3+ + cells cultured without monocytes, unlabeled CD3+
Mice and cell lines
Tie1-tTA–driven human ANGPT2 double-transgenic (DT) mice were generated as described previously (5, 13, 14). ANGPT2 DT and wild-type (WT) control mice were depleted of doxycycline 2 wk before implantation of tumor cells. Syngeneic Lewis lung carcinoma cells (2 × 106) were inoculated s.c. and allowed to propagate for 2 wk. Animals were cared for in accordance with German Legislation on the Care and Use of Laboratory Animals.
To generate tumors without TEMs, syngeneic N202.1A mammary tumor cells were injected into FVB mice (Charles River Laboratories) who had received bone marrow transplantation from FVB/Tie2-tk transgenic mice (2). Mice were then given gancyclovir (GCV) to specifically deplete TEMs or PBS as control. Tumors were harvested after 3 wk. All procedures were conducted in accordance with the Animal Care and Use Committee of the Fondazione San Raffaele del Monte Tabor (IACUC 324,335) and communicated to the Ministry of Health in Italy and local authorities in Milan.
MMTV-PyMT mice on the FVB/n-Tg background were a gift from Z. Werb (University of California, San Francisco, San Francisco, CA). Late-stage mammary tumors were excised at 11–12 wk. 4T1 cells were maintained as described previously (15). BALB/c mice (Harlan Laboratories) at 5 wk old were injected s.c. with 106 4T1 mammary carcinoma cells. Animal experiments were conducted in accordance with U.K. Home Office regulations and were approved by the University of Sheffield Ethical Committee.
Immunofluoresence confocal microscopy
Flow cytometry and FACS of murine cells
For FACS, 4T1 single-cell suspensions were stained with anti-F4/80 and anti-TIE2 as described above. F4/80+TIE2− tumor-associated macrophages (TAMs) and F4/80+TIE2+
Western blot analysis
One-way ANOVA, followed by Newman-Keuls post test, was used to determine p values by using GraphPad Prism software. A p value <0.05 was considered statistically significant. All data shown are means ± SEs.
ANGPT2-treated human TEMs suppress T cell proliferation
Because our previous studies showed that ANGPT2 upregulates IL-10 in TEMs (5), we investigated whether ANGPT2 can convert these cells into immunosuppressive cells. Human monocytes and autologous CD3+ lymphocytes were cocultured in the presence of ANGPT2 or medium alone. IFN-γ and IL-4 were used as controls in coculture assays as they induce an M1/immunostimulatory and M2/immunosuppressive phenotype in monocytes, respectively (9, 10, 16). Monocytes in medium alone had some suppressive function corroborating results from other laboratories (17), and this was significantly enhanced by exposure to the type II/Th2 cytokine IL-4 (Fig. 1). Treatment of cocultures with the type I/Th1 cytokine IFN-γ increased T cell proliferation. Strikingly, ANGPT2-treated TEMs not only suppressed T cell proliferation when compared with control but also its effect was significantly greater than that seen with IL-4 (Fig. 1B). Proliferation of CD3+ T lymphocytes cultured without monocytes was not inhibited by the cytokines tested (data not shown). TIE2 receptor expression was never detected on cultured T cells or during coculture with monocytes (data not shown). Moreover, the suppressive effect of ANGPT2-treated TEMs was not due to increased differentiation of the monocytes into macrophages, because CD14 expression on ANGPT2-treated cells was significantly greater than control cells (data not shown).
Addition of an IL-10–neutralizing Ab, but not a control Ab, significantly reversed the suppressive functions of ANGPT2-treated monocytes (Fig. 1B), indicating that ANGPT2-induced IL-10 mediates the suppressive ability of TEMs. However, TEM-T cell contact was also required, because inhibition of ANGPT2-induced IL-10 in coculture experiments using Transwell inserts did not significantly reduce T cell suppression (data not shown).
Treg expansion is induced by human TEMs exposed to ANGPT2
We next investigated individual T cell populations in our lymphocyte/monocyte cocultures to determine whether they were affected by cytokine-treated monocytes. We gated on CD4+ and CD8+ cells and calculated their frequency. The percentage of CD4+ cells significantly increased, and CD8+ cells decreased with ANGPT2 treatment when compared with control (Fig. 2). Interestingly, ANGPT2 induced a prominent CD3+CD4high T cell population that was not observed with IFN-γ, IL-4, or control groups. Moreover, neutralization of ANGPT2-induced IL-10 reversed this ratio back toward control frequencies. These data suggest that ANGPT2 skews the ratio of CD8+ cytotoxic T lymphocytes to CD4+ cells in favor of the latter.
We then gated on CD4+ T cells and analyzed expression of CD25 and FOXP3—two markers of Tregs (17, 18). In the ANGPT2-treated group, CD4+CD25highFOXP3− cells were significantly more abundant than control, indicating an increased activation status of the CD4+ population (Fig. 3A). In addition, ANGPT2-treated TEMs induced significant expansion of CD4+CD25+FOXP3+ cells when compared with control and IL-4–treated TEMs. IFN-γ and IL-4 treatment had no effect when compared with control (Fig. 3A, 3B). Monocytes were required for expansion of CD4+CD25+FOXP3+ cells, because cytokines alone did not affect the T cells (data not shown). The expansion of CD4+CD25+FOXP3+ cells by ANGPT2-treated TEMs was inhibited by neutralization of IL-10 but not by control Abs, underscoring the functional importance of this cytokine in the generation of Tregs.
After coculture with ANGPT2-treated and control monocytes, the nonadherent T cells were added to CFSE-labeled, autologous CD3+ T cells and incubated for another 5 d. The cells induced by ANGPT2-treated TEMs significantly suppressed T cell proliferation when compared with control (Fig. 3C). These data indicate that the Tregs expanded by ANGPT2-treated TEMs exhibit suppressive properties.
Tregs are more abundant in ANGPT2-overexpressing tumors
Given the observations above, we next investigated whether exposure of tumor TEMs to high levels of ANGPT2 in the tumor microenvironment correlates with increased numbers of tumor Tregs. Tumors were propagated in ANGPT2 DT or WT mice as before (5, 14), and then, Tregs were identified as CD3+CD4+FOXP3+ cells. As shown in Fig. 4A and 4B, both the total number of Tregs and the proportion of FOXP3+ cells in the CD3+CD4+ population were significantly increased in ANGPT2 DT tumors when compared with WT tumors (Fig. 4A, 4B). This observation was not due to the general infiltration of T cells, because neither the number of CD3+ T cells nor the number of CD3+CD4+ cells was significantly different between tumor types (Fig. 4B). The proportion of CD3+CD4+ cells was also similar between tumors overexpressing ANGPT2 and WT tumors (Fig. 4B).
Tregs are reduced in murine mammary tumors following genetic depletion of TEMs
We then examined the effect of TEM depletion on the number of Tregs in N202 mammary tumors by using the conditional model described previously (2). In tumors without TEMs (GCV), there was a trend toward greater CD3+ T cell infiltration (Fig. 4D). Both the total number of CD4+ T cells and the percentage of CD4+ cells among the entire CD3+ population were significantly increased, indicative of an antitumor immune response (Fig. 4D). The total number of CD3+CD4+FOXP3+ cells did not significantly differ between tumors with TEMs (PBS) and without TEMs (GCV) (Fig. 4C, 4D). However, the proportion of FOXP3-expressing CD3+CD4+ cells was significantly decreased in TEM-depleted tumors, suggesting that TEMs suppress antitumor immunity by expanding or recruiting the Treg population into tumors.
Tumor TEMs express higher levels of IL-10 than TIE2− TAMs
F4/80+TIE2− TAMs and F4/80+TIE2+ TEMs were analyzed for expression of intracellular IL-10 by flow cytometry in two different mammary tumor models: the spontaneous MMTV-PyMT and syngeneic 4T1 models. CD11b+Gr1+ cells from the spleens were also analyzed. In both tumor models, TEMs were the main source of IL-10 among the total F4/80+ macrophage population, and IL-10 expression by TEMs was comparable to CD11b+Gr1+ cells (Fig. 5A). These data suggest that in mammary tumors—where ANGPT2 is upregulated (6–8)—TEMs are stimulated to express high levels of IL-10 to suppress antitumor immunity and to expand the Treg population.
ANGPT2 expression in tumors upregulates TEM-derived IL-10 and Treg infiltration
We measured ANGPT2 expression in the 4T1 tumor model by Western blot over the course of several weeks after tumor cell injection (day 0). Unexpectedly, we observed a dynamic, not constitutive, pattern of ANGPT2 expression (Fig. 5B). Several isoforms of ANGPT2 were detected including the unglycosylated (55 kDa) and glycosylated forms (70 kDa) (19). Lysates from tumor tissue displayed high levels of ANGPT2 during the early days of tumor growth and then decreased as tumors became larger. Interestingly, IL-10 expression from the same tumor lysates followed a similar dynamic pattern to ANGPT2 expression. IL-10 was also upregulated during the early days of tumor growth (Fig. 5B).
We then quantified intracellular IL-10 in TEMs to determine whether increased levels of ANGPT2 induce TEMs to upregulate IL-10. Tumors from various days posttumor cell injection were enzymatically digested and analyzed by flow cytometry. As expected, F4/80+TIE2+ TEMs displayed increased IL-10 expression during the early time points when ANGPT2 levels were highest (Fig. 5C). We then hypothesized that TEM-derived IL-10 may influence Treg prevalence in tumors. Strikingly, the proportion of tumor CD3+CD4+FOXP3+ Tregs was greatest on day 12 mimicking ANGPT2 expression patterns and upregulation of IL-10 by TEMs (Fig. 5C). On day 12, the frequency of FOXP3-expressing CD3+CD4+ cells increased ∼10-fold when compared with other days. These data provide a direct link between ANGPT2 expression, IL-10 production by TEMs, and Treg abundance in tumors.
Tumor TEMs are immunosuppressive ex vivo
To determine whether TEMs in tumors modulate T cell behavior, we isolated F4/80+TIE2− TAMs and F4/80+TIE2+ TEMs from 4T1 tumors as well as splenic CD11b+Gr1+ cells by FACS (Supplemental Fig. 1). Myeloid subpopulations were then incubated with CFSE-labeled CD3+ T cells ex vivo. TEMs and CD11b+Gr1+ cells exhibited a significantly greater ability to inhibit the proliferation of T cells when compared with TIE2- TAMs (Fig. 6). Blocking IL-10 activity with Abs reduced the suppressive effect of TEMs and CD11b+Gr1+ cells, suggesting that IL-10 mediates the ability of TEMs to prevent T cell proliferation.
To our knowledge, this study shows for the first time that ANGPT2 stimulates human TEMs to markedly suppress T cell proliferation. We previously reported that the immunosuppressive cytokine IL-10 is upregulated by TEMs in response to ANGPT2 stimulation (5), and we demonstrate in this study that TEMs are the predominant source of IL-10 among all F4/80+ macrophages in two different mammary tumor models. Moreover, ANGPT2-induced, TEM-derived IL-10 suppressed T cell proliferation and expanded CD4+CD25highFOXP3+ Tregs in vitro. These data indicate that TEMs promote tumor progression not only by angiogenic stimulation but also through subversion of antitumor immunity and induction of tolerance via expansion of Tregs.
We chose to use mixed cultures of both TIE2− and TIE+ human monocytes in our cocultures, because this represents the monocyte heterogeneity present in peripheral blood and tumors. Because TIE2− monocytes do not respond to ANGPT2 (4, 5), the effects elicited by ANGPT2 in our cocultures represent the responses of TEMs to this cytokine.
The main source of ANGPT2 in tumors is endothelial cells (6–8), so newly recruited TEMs undergoing diapedesis likely encounter high levels of ANGPT2 as they enter the tumor. ANGPT2 may then skew TEM phenotype toward a more M2-like polarization through upregulation of proangiogenic and immunosuppressive genes (3, 5, 11). Our data indicate that ANGPT2 is critical for the suppressive ability of TEMs as well as their expression of IL-10, and this notion is supported by several observations presented in this study. The varied expression levels of ANGPT2 as tumors progressed and expanded surprised us. However, publications by other groups using ANGPT2 knockout mice have shown that ANGPT2 is essential during the early stages of tumorigenesis but dispensable for later stages (20). Our data corroborate their findings and suggest that ANGPT2 helps to establish the initial immunosuppressive environment via upregulation of IL-10 by TEMs and Treg expansion.
In addition to IL-10, other macrophage-derived cytokines and enzymes have been implicated in suppression of T cell proliferation including TGF-β, cyclooxygenase-2–induced PGE2, inducible NO synthase (iNOS)2, and arginase 1 (ARG1) (10, 12, 18, 21–23). Pucci et al. (11) reported that tumor TEMs express higher levels of Arg1 and lower levels of Tgfβ, Cox-2, and Nos2 mRNA than TIE2− TAMs, although differences at the protein level for these molecules have yet to be confirmed. It is possible that ANGPT2 also stimulates the expression of one or more of these immunosuppressive factors by TEMs. Il-10 mRNA expression levels did not differ between TEMs and TAMs (11), whereas we found TEMs expressed significantly more IL-10 protein than TAMs. This is not altogether surprising considering IL-10 is constitutively transcribed in many cell types and highly dependent on posttranscriptional regulation (24–27).
Myeloid-derived suppressor cells (MDSCs) also produce IL-10 and ARG1 (18, 21–23). However, TEMs and the classical CD11b+Gr1+ MDSCs do not appear to be overlapping populations. TEMs do not express Gr1—a commonly used marker to identify MDSCs in secondary lymphoid organs and blood of tumor-bearing mice as well as circulating inflammatory monocytes (18, 22). Rather, TEMs share a related gene signature with circulating Gr1− resident monocytes (1, 2, 11), and splenic CD11b+Gr1+ MDSCs from lymphoma-bearing mice fail to express TIE2 (22). It remains to be seen whether TEMs in tumors and MDSCs in distant, secondary lymphoid organs work in concert to suppress antitumor immune responses. In addition, TEMs can now be classified as MDSCs, because the heterogeneous myeloid cells within this group are defined by their functional ability to inhibit T cell proliferation, rather than their surface marker expression (28, 29).
Although TEMs and classic MDSCs are likely different myeloid cell populations, a recent publication characterizing CD11b+F4/80+ TAMs as MHC-IIhigh and MHC-IIlow subsets indicate that TEMs make up a fraction of the MHC-IIlow cell population (30). In fact, the gene signature, phenotype, and superior proangiogenic abilities of TEMs and MHC-IIlow TAMs are analogous (11, 30). Interestingly, Ag processing by MHC-IIlow cells is less efficient than MHC-IIhigh cells, but both subpopulations inhibit T cell proliferation when compared with splenic dendritic cells. MHC-IIhigh and MHC-IIlow cells are equally capable of suppressing anti–CD3-stimulated T cells with each subset using different mechanisms to accomplish this. MHC-IIhigh cell-mediated suppression is iNOS dependent, whereas the molecule(s) used by MHC-IIlow cells is unknown. These data, together with the data presented in this paper, suggest that TEMs use IL-10, not ARG1 or iNOS, to inhibit T cell proliferation. However, the equivalent immunosuppressive potential of MHC-IIhigh and MHC-IIlow cells contrasts with our own data showing that TEMs possess significantly greater ability to suppress T cell proliferation than TIE2− TAMs. Varying levels of ANGPT2 in different tumor models may explain this discrepancy, because ANGPT2 may drive TEMs’ T cell-inhibitory functions. Moreover, the shaping of different T cell populations (i.e., expansion of Tregs by TEMs) in the tumor microenvironment may be more important to tumor progression than inhibiting T cell proliferation (30).
In summary, we demonstrate a novel tumor-promoting function for TEMs: the ability to suppress T cell proliferation and expand Tregs via ANGPT2-induced IL-10. These data suggest that inhibition of the ANGPT2–TIE2 interaction in tumors could impede angiogenesis and reinitiate antitumor immunity (31).
The authors have no financial conflicts of interest.
We thank Dr. Zena Werb for the MMTV-PyMT mice, Dr. Dan Dumont for the ANGPT2 DT mice, and Susan Newton and Kay Hopkinson (University of Sheffield Flow Cytometry Core Facility).
This work was supported by the Breast Cancer Campaign, U.K. (to C.E.L., C.M., and M.D.P.) and the German Research Foundation (Grant SFB/TR23 [Project C1] to Y.R. and K.H.P.).
The online version of this article contains supplemental material.
Abbreviations used in this article:
- angiopoietin 2
- arginase 1
- inducible NO synthase
- myeloid-derived suppressor cell
- tumor-associated macrophage
- TIE2-expressing monocyte/macrophage
- regulatory T cell.
- Received August 18, 2010.
- Accepted January 27, 2011.
- Copyright © 2011 by The American Association of Immunologists, Inc.