Mechanisms of tumor escape from the immune system: Adenosine-producing Treg, exosomes and tumor-associated TLRs

Electronic journal of oncology

Volume 98 • N◦ 2 • février 2011 John Libbey Eurotext

©

Mechanisms of tumor escape from the immune system: Adenosine-producing Treg, exosomes and tumor-associated TLRs Article received on October 20, 2010, accepted on November 16, 2010 Reprint: T.L. Whiteside

Theresa L. Whiteside, Magis Mandapathil, Miroslaw Szczepanski, Marta Szajnik University of Pittsburgh, Cancer Institute, Suite 1.27, 5117, Centre Avenue, Pittsburgh, PA 15213, USA

doi : 10.1684/bdc.2010.1294

To cite this article : Whiteside TL, Mandapathil M, Szczepanski M, Szajnik M. Mechanisms of tumor escape from the immune system: Adenosine-producing Treg, exosomes and tumor-associated TLRs. Bull Cancer 2011 ; 98 : E25-E31. doi : 10.1684/bdc.2010.1294.

Abstract. Human tumors utilize many different mechanisms of immunosuppression to prevent immune cells from exercising their antitumor activities. These mechanisms, which enable the tumor to escape from the host immune system and to progress, are being intensively investigated in hope of finding therapeutically safe and effective inhibitors able to counteract tumor-induced immunosuppression. Three of more recently discovered tumor-related suppression mechanisms, i.e. accumulations of adenosine-producing regulatory T-cells (Treg) in the tumor microenvironment, release by tumors of suppressive microvesicles (TMV) and expression of toll-like receptors (TLR) on the tumor cell surface, are described in this review. All contribute in a varying degree to creating a milieu favo-

rable for the tumor and unfavorable for immune effector cells. Tumor escape has been a major problem in cancer immunotherapy and it has been held responsible for the failure of many immune interventions in cancer. For this reason, it is important to study and understand the various suppressive pathways human tumors utilize. Future antitumor immunotherapies are likely to include inhibitors of tumor-induced suppression with the goal of restoring antitumor immune responses in patients with cancer. 

Introduction

responses, that it is mediated by various mechanisms orchestrated by the tumor and that it gets more pronounced in metastatic than in primary disease. Further, while tumor-induced immune dysfunction is strongest in the tumor microenvironment, it is not local but systemic. However, it has not been clear how tumormediated suppression translates into systemic immune dysfunction demonstrable by immune cells in the peripheral circulation of patients with cancer. We have recently identified three novel mechanisms that human tumors can use to escape from the host immune system. These are:

Human solid tumors have developed numerous ways of avoiding the host immune system (reviewed in [1]). This phenomenon referred to as “tumor escape” has been recently acknowledged to be a major problem responsible for the tumor resistance to immune therapies and for the general lack of success in inducing clinical responses to therapeutic vaccines in patients with cancer [2, 3]. The magnitude of the problem created by the tumor escape has been controversial and has been debated for many years, largely because of differences encountered between tumor types in the ability to induce immune dysfunction, and because it has not been possible to reliably correlate the presence of tumor-induced immune dysfunction with the disease progression. Nevertheless, it is clear today that immune dysfunction accompanies tumor progression, that it is not generalized but confined to tumor-specific immune Bull Cancer vol. 98 • N◦ 2 • février 2011

Key words: tumor escape, regulatory T-cells (Treg), tumorderived microvesicles (TMV), TLR4 on tumor cells

– accumulations of regulatory T-cells (Treg) expressing ectonucleotidases CD39 and CD73 in tumors and peripheral blood of patients with cancer; – the presence in patient’s sera of tumor-derived microvesicles (TMV) or exosomes which induce expansion and augment suppressor functions of Treg;

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– expression on tumor cells of toll-like receptors (TLR) which signal to promote tumor growth and its resistance to immune cells or anticancer drugs.

man. This has been difficult, because Treg are but a minor subset of CD4+ T-cells and because FOXP3, the transcription factor expressed by Treg, is not a selective Treg marker, being transiently expressed in other human cells as well [6]. With the discovery that functional Treg are CD25high , we and others have been able to use flow cytometry to assess the frequency of Treg in the blood of cancer patients [7-11]. As shown in figure 1, a significant enrichment in the percentage of CD4+ CD25high Treg is evident in patients with cancer relative to healthy controls. Further, Treg accumulate in human tumors, and tumor-infiltrating lymphocytes (TIL) contain numerous CD4+ CD25high Treg with elevated suppressor activity (figure 2) compared to that in the patients’ peripheral circulation. The frequency and function of Treg are increased in advanced malignancies [10]. The mechanisms used by Treg for imposing suppression are many, including soluble factors such as IL-10 and TGF-␤ or cell contactdependent mechanisms depending on the involvement of perforin/granzyme B or death receptors in Tregmediated killing of responder cells [8, 12-14]. More recently, we and others have reported the presence of ectonucleotidases, CD39 and CD73, on the surface of natural (n) Treg as well as adaptive (Tr1) Treg [15, 16]. This finding suggested that Treg have the capability to hydrolyze ATP to AMP and AMP to immunosuppressive adenosine. Our in vitro data confirm that human Treg produce adenosine and can

These mechanisms and their involvement in executing tumor escape are reviewed here, with the intent to illustrate the diversity of pathways used by human tumors to promote their own progression and to disarm or block antitumor immunity. It is important to note that the three means of escape involve pathways that are present and functional in normal cells. However, in cancer these pathways are subverted to mediate immune dysfunction and benefit the tumor. A better and more complete understanding of various forms of tumor-induced immune dysfunction is necessary for the development of new, more effective strategies for preventing tumor escape and thus for optimizing antitumor therapies in the future.

Treg in patients with cancer The role of Treg is to maintain immune system in balance, preventing excessive activation and expansion of immune cells. The frequency and suppressive activity of Treg is, therefore, important for downregulating immune responses that are excessive or overly extended. The loss of Treg-mediated control, whether due to the Treg deficiency or excess, occurs in human diseases [4, 5] and for this reason, it has been important to assess the Treg frequency and function in

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Figure 1. An increased frequency of CD4+ CD25high regulatory T-cells (Treg) in the circulation of patients with cancer.

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Figure 2. Regulatory T-cells (Treg) in tumor infiltrating lymphocytes obtained from patients with HNSCC mediate stronger suppression of autologous responder cell (RC) proliferation than Treg in the peripheral circulation of the same patients or of normal controls (NC). The data were obtained in experiments with CFSE-labeled RC and Treg co-cultured at two different S:RC ratios for five days.

utilize the adenosinergic pathway to mediate suppression [15, 17]. We have shown that metabolic inhibitors of this pathway, such as ARL67156 or ZM241385 block suppression of responder cell proliferation [15, 17]. As indicated in figure 3, adenosine binds to adenosine 2A (A2A ) receptors liberally expressed on effector T-cells (Teff) and suppresses their antitumor functions

via the up-regulation of intracytoplasmic cAMP levels [15]. Importantly, CD39, a surface associated molecule with enzymatic activity, is a useful marker for the isolation of Treg and for their characterization [18]. But since CD39 is also expressed on a subset of CD4+ Teff, to increase purity of the Treg fraction, it is necessary to sort for CD39+ CD25+ Treg, which co-express FOXP3 and mediate suppression utilizing adenosine they produce11 . Another useful albeit negative marker for Treg is CD26, a surface glycoprotein coupled to adenosine deaminase (ADA), whose absence on Treg and presence on Teff cells serves to distinguish these T-cell subsets [15]. The absence of ADA, which coverts adenosine to inosine, on the surface of Treg suggests that accumulations of pericellular adenosine that Treg produce but do not degrade is available to them for mediating immune suppression [15]. In addition to adenosine, Treg can also produce PGE2 , and our recent data indicate that the Treg subset producing adenosine and PGE2 might be distinct from that producing IL10 and TGF-␤ and might be especially prominent in patients with COX-2+ tumors and in advanced disease [18]. In aggregate, these data indicate that human Treg comprise several T-cell subsets mediating immune suppression by several distinct mechanisms, which might be orchestrated by the tumor microenvironment to which Treg are recruited.

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Figure 3. A schema illustrating interactions of endonuclease-expressing regulatory T-cells (Treg) with T effector cells expressing adenosine 2A receptors. Bull Cancer vol. 98 • N◦ 2 • février 2011

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(AML) sera were positive for leukemia blast-associated markers CD33, CD34, CD117 and for TGF-␤1 as well as proteins usually associated with exosomes such as LAMP-1. These TMV decreased cytotoxic activity of NK cells from normal controls (NC) (P < 0.002), induced SMAD phosphorylation and down-regulated NKG2D receptor expression. Sera of AML patients contained elevated TGF-␤1 levels (mean: 2.6 ng/mL) and antiTGF-␤1 Abs abrogated TMV-mediated inhibition of NK activity [23].

Tumor-derived microvesicles (TMV) or exosomes Sera of patients with cancer contain tumor-derived biologically active microvesicles or exosomes, which originate from endosomal compartments of various cells, including tumor cells. The microvesicles derived from tumor cells (TMV) are immunosuppressive [1, 19, 20]. At the same time, TMV are also immunogenic, as they carry tumor antigens, which can be used by dendritic, cells for cross-presentation, following the uptake and processing of these TMV [21]. In this way, TMV can also induce T-cell activation and tumor rejection [22]. Thus, effects of TMV on the host immune cells have been a topic of considerable controversy due to the dual capability of TMV to suppress and/or activate antitumor responses. TMV can be isolated from supernatants of tumor cells grown in culture and examined in Western blots for their protein contents. It turns out that TMV with similar protein content are present and can be isolated from plasma of patients with cancer. For example, TMV isolated from sera of patients with acute myeloid leukemia

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We observed that sera of patients with cancer contained significantly higher concentrations of microvesicles than sera of NC [24]. At the same time, these patients had an increased frequency of Treg in the peripheral blood. Further, our in vitro studies showed that TMV isolated from tumor cell supernatants and captured on latex beads contained immunosuppressive cytokines (IL-10, TGF-␤1) and death receptor ligands (e.g., FasL), as shown in figure 4, which are involved in Treg differentiation and activities. We, therefore, hypothesized that TMV induce, expand and up-regulate biologic activities of human Treg. When CD3+ CD4+ T-cells

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Figure 4. A phenotypic profile of CD4+ CD25high T-cells present in seven-day cultures of CD4+ CD25+ T-cells ± TMV or DC-derived MV. T-cells were stained with various mAbs and evaluated by multicolor flow cytometry. The gate is set on CD4+ CD25high T-cells and the data are means ± S.D. from three independent cultures. The flow cytometry data below show MFI for FasL, IL-10, TGF-␤1, granzyme B and perforin expression in CD4+ CD25high T-cells cultured as described above.

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were labeled with CFSE, stimulated with OKT3/antiCD28 Ab-coated beads and cultured in the presence of TMV or DC-derived MV as controls, the frequency of CD4+ CD25+ FOXP3+ Treg was significantly increased only in co-cultures with TMV [25, 26]. Thus, TMV but not DC-derived MV promoted proliferation of Treg in culture. Further, TMV converted CD4+ CD25neg T-cells to CD25high FOXP3+ Treg with enhanced expression of CTLA-4, FasL, CCR7, TGF-␤1, granzyme B and perforin, suggesting that these Treg were highly suppressive. In fact, using flow cytometry-based cytotoxicity assays (FLOCA), we showed that Treg generated in the presence of TMV had a significantly increased ability to inhibit proliferation and survival of autologous responder cells [26]. The co-incubation with TMV also increased levels of secreted cytokine, especially of inhibitory cytokines, TGF-␤1 and IL-10, in Treg. Finally, we demonstrated that Treg generated in the presence of TMV, but not DC-derived MV, were strongly positive for TGF-␤1 and IL-10 and had up-regulated expression of phosphorylated SMAD2/3 and phospholorylated STAT3 relative to controls not exposed to TMV [26]. These data suggested that TMV concomitantly up-regulated phosphorylation of relevant transcription factors in Treg and of immunosuppressive cytokines. Further, pre-incubation of TMV with neutralizing antiTGF-␤1 or anti-IL-10 Abs resulted in a significant reduction in the frequency of Treg generated in cocultures. In aggregate, our in vitro data indicated that Treg induction by TMV is largely mediated by TGF-␤1 and IL-10, which are prominent membrane components in TMV [26]. The capability of TMV to promote expansion and suppressor activity of Treg represents yet another way human tumors manage to interfere with antitumor responses in order to escape from the host immune system.

The TLR4 presence and signaling in human tumors TLRs are expressed on inflammatory cells and play a key role in defense against pathogens benefiting the host [27]. Recent immunohistochemistry (IHC), flow cytometry and RT-PCR data show that TLRs are also expressed on tumor cells [28-30]. As the consequences of TLR signaling in tumor cells have not been clear, we embarked on a series of experiments to determine the Bull Cancer vol. 98 • N◦ 2 • février 2011

role TLR4 plays in tumor progression. Working with HNSCC cell lines, we initially showed that ligands binding to TLR4 (e.g., LPS) enhanced tumor cell proliferation, activated the PI3K/Akt pathway, up-regulated IRAK-4 expression, induced NF-␬B translocation and increased production of several cytokines involved in promoting tumor growth [30]. To confirm the tumor-promoting role of TLR4 expression in other human tumors, we next considered ovarian carcinoma (OvCa). By IHC, we first showed that TLR4 was expressed in all OvCa, tumor cell lines and normal epithelium [31]. Next, working with five OvCa cell lines, we studied binding of two TLR4 ligands, LPS and paclitaxel (PTX) to the receptor these lines express. It appeared that the two ligands of TLR4 had differential effects on proliferation and survival of OvCa cells: LPS induced proliferation in MyD88+ SKOV3 cells but not in MyD88neg A2780 cells; while PTX induced death in A2780 cells, SKOV3 cells were resistant to PTX (figure 5). In SKOV3 cells PPS or PTX binding to TLR4 induced IRAK4 activation and cJun phosphorylation, activated the NF-␬B pathway (figure 6) promoted secretion of survival cytokines and resistance to drug-induced apoptosis. Silencing of TLR4 in SCOV3 cells with small interference RNA resulted in down-regulation of cJun phoshorylation and a loss of PTX resistance. On the other hand in MyD88neg A2780 cells, which were PTX sensitive, TLR4 stimulation up-regulated TRIF, and TLR4 silencing eliminated this effect [31]. The data suggest that TLR4/MyD88 signaling in OvCa cells supports tumor progression and increases its resistance to drug or immune cellmediated death thus promoting its escape from the host immune system.

Conclusions The three examples cited above represent but a small part of a vast repertoire of tricks human tumors have evolved to escape from the host immune system [1]. To be able to succeed with cancer immunotherapy, it will be necessary to consider these various ways of escape and devise strategies for their silencing or elimination [32]. As each individual tumor creates its own microenvironment and develops unique interactions with the host, immune profiling of the tumor might be necessary to be able to select the optimal strategy for inhibiting tumor-derived inhibitors. With increasing

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Figure 6. Western blots of tumor cells that were either untreated or treated with LPS or paclitaxel (PTX) for 30 or 60 minutes. Strong up-regulation of phosphorylated cJun was observed only in SKOV3 cells treated with LPS or PTX but not in A2780 cells, where only the TRIF pathway was activated in response to TLR4 signaling induced by LPS or PTX. Below, Western blots performed after siRNA-mediated silencing of TLR4.

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recognition of molecular pathways involved in tumor escape, such strategies are now being translated to clinical trials for patients with cancer. 

15. Mandapathil M, Hilldorfer B, Szczepanski MJ, et al. Generation and accumulation of immunosuppressive adenosine by human CD4+ CD25high FOXP3+ regulatory T cells (Treg). J Biol Chem 2010 ; 285 : 7176-86.

Conflict of interest : none.

16. Deaglio S, Dwyer KM, Gao W, et al. Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression. J Exp Med 2007 ; 204 : 1257-65.

Acknowledgements. This work was supported in part by the NIH grants PO1-CA109688 and RO-1DE13918 to TLW.

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