Treg Cells in Cancer - Science Translational Medicine

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Treg Cells in Cancer: A Case of Multiple Personality Disorder Paula D. Bos1* and Alexander Y. Rudensky1,2* Foxp3+ RORγt–expressing T cells expand in colorectal cancer and contribute to pathogenesis in a mouse model of polyposis.

Regulatory T cells (Treg cells), a CD4 T cell lineage characterized by the expression of the forkhead transcription factor Foxp3, ensure that a protective infammatory immune response is achieved with minimal damage to host tissues. Hence, Treg cell–mediated immune suppression becomes an obvious mechanism that tumors, which are considered “self ” tissues, can co-opt in order to limit infammation and evade immunosurveilance. As evidenced by analyses of FOXP3 expression in tumor-infltrating lymphocytes, Treg cells accumulate and correlate with poor prognosis in many cancer types, including breast, lung, melanoma, and ovarian carcinomas. In addition, lower quantities of Treg cells compared with efector CD8 cells have been observed in patients that respond to chemotherapy. Terapeutically targeting the Treg cell population promotes antitumor immunity and tumor rejection in mouse models of cancer in a variety of experimental settings. Tese observations indicate a prominent tumor-promoting role of Treg cells in cancer (1). However, in colorectal, head and neck, or bladder cancer, FOXP3 expression has been indicative of better prognosis (2). Heterogeneity of Treg cell populations and of infammationdependent phenotypic changes in diferent immune cell types ofers potential explanations for these conficting observations. In this issue of Science Translational Medicine, Blatner and colleagues provide one example of such heterogeneity by describing a polyp growth–promoting subset of CD4 T cells that coexpress FOXP3 and RORγt and accumulate in biopsies of colon cancer patients (3). Tis work highlights the complexity of intratumoral T cell popula1

Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA. 2Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA. *Corresponding authors. E-mail: [email protected] (A.Y.R.); [email protected] (P.D.B.)

tions and their efects on tumor progression. It further emphasizes the importance of investigating the origins and functions of Treg cells in cancer settings in order to improve cancer therapy. TREG CELL HETEROGENEITY Foxp3 is required for Treg cell lineage differentiation, maintenance, and suppressive function. Tis transcription factor likely serves as a node for the integration of extracellular cues that inform the appropriate suppression function required for a specifc immune response. For example, the proinfammatory T helper 17 (TH17) response in the intestine is conditional upon signal transducer and activator of transcription 3 (STAT3) signaling in activated T cells, whereas in Treg cells cooperation between Foxp3 and STAT3 enables efcient suppression of the TH17 response. By sensing the anti-infammatory cytokine interleukin-10 (IL-10), Treg cells can control this TH17 response, preventing excessive infammation that would result in colitis and increased cancer risk (4). In addition to variations imparted by the environmental regulation in diferent tissues and infammatory states, an extra level of heterogeneity of the Treg cell population comes from the fact that Foxp3 can be induced in the thymus (tTreg) and in the periphery (pTreg). Extrathymic generation of pTreg cells requires Foxp3 induction by means of suboptimal antigen stimulation of CD4 cells in the presence of transforming growth factor–β (TGF-β) (Fig. 1). A prevalent site of pTreg cell generation is the gut, an interface rich in food and intestinal commensal community-derived antigens and metabolic products. Although tTreg cells emerging from the thymus feature stable Foxp3 expression due to epigenetic modifcation of the Foxp3 locus, Foxp3 expression during early pTreg cell generation is transient. Furthermore, Foxp3 is transiently upregulated during TH17 cell diferentiation,

and Foxp3+IL-17+ cells have been reported in the gut. Intratumoral Treg cells are thought to arise through a variety of means. Among the possibilities are the preferential recruitment of CCR4-, CCR10-, CCR5-, or CXCR4-expressing Treg cells via tumorderived chemokine secretion, expansion, or preferential survival of tTreg cells over efector T cells in an environment rich in reactive oxygen species, or de novo generation of pTreg facilitated by the tumor. Although there is some evidence supporting each of these possibilities, it is plausible that their relative contribution depends on the organ in which the tumor is developing. Te use of genetic models with conditional ablation of chemokine receptors, or genetic manipulation of cis-regulatory elements controlling extrathymic generation or stability of Foxp3 upon expansion, will enable the evaluation of the role of these mechanisms. In humans, analysis of Treg cells during an immune response is further complicated by the fact that the transient expression of FOXP3 at a relatively low level is observed in activated T cells. Tis fnding necessitates the use of additional markers in combination with high levels of FOXP3 in order to identify human Treg cells in cancer and infection. A variety of activation markers including cytotoxic T lymphocyte–associated antigen 4 (CTLA-4), inducible T cell costimulator (ICOS), and CD25 (IL-2 receptor α-chain), are up-regulated in intratumoral Treg cells. Te latter is most frequently used for Treg cell isolation and functional characterization. Blatner et al. describe the functional contribution of a subset of intratumoral T cells that coexpress FOXP3 and RORγt (the lineage specifcation factor for TH17 cells) to colon cancer pathogenesis (3). Tese cells share features of both Treg and TH17 cells, accumulate in a stage-dependent manner in colon cancer in humans, and promote polyposis in mice (Fig. 1). T cells with similar features have been reported before in normal and infammatory conditions, such as Crohn’s disease (5, 6). Tey are proposed to arise at mucosal sites or at other infamed sites where TGF-β, in combination with proinfammatory cytokines such as IL-6 and IL-1β, can facilitate high levels of RORγt expression in newly generated Treg cells (7). High levels of RORγt and STAT3 activation promote production of IL-17 cytokines and expression of chemokine receptor CCR6 without marked impairment in suppressor function (5). Whether these cells represent a

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Fig. 1. RORγt+ Foxp3+ Treg pathogenic role in intestinal polyposis. Antigen presentation to naïve CD4 T cells by dendritic cells (DCs) in the presence of TGF-β drives cells into becoming Foxp3+ Treg cells. IL-6 and other inflammatory cytokines drive differentiation into TH17 lineage. Recently generated Treg cells sense IL-6, IL-1β, IL-23, or other proinflammatory cytokines and acquire RORγt expression in the polyp microenvironment. Blatner et al. found that RORγt-expressing Treg cells remain T cell–suppressive but lack IL-10 expression and stimulate polyp formation in mice, partly through loss of mast cell regulation. Targeting this population through RORγt antagonists might provide a therapeutic opportunity to prevent early adenoma development.

CREDIT: K. SUTLIFF/SCIENCE TRANSLATIONAL MEDICINE

transitional state between the Treg and TH17 cells or a relatively stable cell population with dual properties remains unknown. RORγt-EXPRESSING TREG CELLS IN MOUSE INTESTINAL POLYPOSIS Similar to human colorectal cancer, expansion of Foxp3+ RORγt+ CD4+ T cells occurs in the APCΔ468 mouse model of hereditary polyposis. Although this population comprises up to 25% of Treg cells isolated from the mouse polyps, Blatner and colleagues showed that gene expression analysis of the whole Foxp3+ CD4+ population displayed a pronounced TH17-expression profle, highlighting the key role of a proinfammatory environment in the emergence of these cells. Consistent with a positive correlation between a TH17 tumor infltrate and poor prognosis in colorectal cancer (8), RORγt defciency in APCΔ468 polyp-prone mice prevented polyp development, signifcantly prolonging survival of these mice (3).

Importantly, Blatner et al. showed that polyposis was signifcantly compromised in chimeric APCΔ468 mice harboring RORγtdefcient Treg cells from Foxp3Cre Rorcfox/fox donors. Tis observation suggests that expression of RORγt in the Foxp3+ Treg population is partially responsible for polyp development in this model. Furthermore, just like adoptive transfer of wild-type Treg cells isolated from naïve mice into APCΔ468 hosts, Treg cells isolated from RORγt-defcient polyp-bearing mice deterred polyposis, suggesting that a small number of RORγtdefcient Treg cells is able to exert some control over the infammatory process governing polyposis, at least for a brief period of time. Te prominent presence of this Treg subset in human colorectal cancer samples suggests that they might have a similar role in human disease. In order to discover a possible mechanism by which this “pathogenic” Treg cell subset contributes to adenomatous polypo-

sis in the mice, the authors revisited their previous observation that Treg cells isolated from polyp-containing mice lack IL-10 production and are thus unable to suppress mast cells, which are essential for the development of the intestinal polyps (9). Ablation of the RORγt gene in Foxp3+ cells in polypprone APCΔ468 mice resulted in a decrease in mast cell density. Additionally, RORγt defciency restored the ability of polyp-isolated Treg cells to suppress mast cell degranulation. In contrast, IL-17A defciency was unable to restore mast cell suppression. Tese correlative observations suggest that pathogenic RORγt-expressing Treg cells might promote polyposis owing to a diminished ability to suppress mast cells. In addition to decreased mast cell numbers, reduced polyposis in RORγt-defcient mice was associated with improved antitumor immunity and reduced protumorigenic features (3). Blatner et al. observed a signifcant increase in the number of granzyme B and perforin double-positive cells in the polyps and a concomitant reduction in macrophages and myeloid suppressor cells infltrating the intestinal tissues. Moreover, these mice exhibited an increase in serum levels of type I cytokines and antiangiogenic factors and in interferon-γ response in T cells stimulated with polyp-pulsed dendritic cells. RORγt expression in other cell types results in developmental defects that include a markedly skewed T cell receptor repertoire, lymphopenia, and altered gut microbial community (7). Tus, it is unclear to what extent the efects observed by Blatner and colleagues are due to the loss of RORγt in Foxp3+ Treg cells present in the polyps. A more detailed analysis of mice with conditional ablation of RORγt in Foxp3+ Treg cells and its impact on the balance of the efector T cell populations will provide further insights into the function of the RORγt+ Foxp3+ CD4 cells. GROWING PROMISE FOR IMMUNOTHERAPY Treg cells can modulate cancer progression in multiple ways. First, by acting as bona fde suppressors they could provide protection from the antitumor immune response. Furthermore, intratumoral Treg cells may facilitate tumor progression through nonimmune means, including production of soluble mediators—such as vascular endothelial growth factor (VEGF), receptor activator of nuclear factor κB ligand (RANKL), or prostaglandins—that act directly on tumor or stromal


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FOCUS cells. Te study by Blatner and coauthors implies that in addition to aforementioned modes of cancer-promoting activities, Treg cells may partake in protumorigenic efector immune responses, depending on the microenvironment of the developing tumor. Te growth in understanding Treg cell function and the infuence that infammatory environments exert over closely related T cell lineages will enable the identifcation of additional intratumoral Treg cell subsets with distinct functional features that provide explanations for their divergent roles in cancer. Te current knowledge of the biology of Treg cells is being leveraged to develop strategies to broadly target Treg cells in cancer and reawaken antitumor immunity. Preclinical studies of a wholesale Treg cell ablation ofer promise for the feld of cancer immunotherapy. However, the autoimmune side efects of this approach will likely equal if not exceed those observed upon CTLA-4 blockade (therapy aimed at enhancing the immune response to cancer) (10). Te work by Blatner et al. (3) provides the framework for testing the e%cacy of the use of RORγt antagonists in patients with precancerous colonic hyperplasia and early-stage colorec-

tal cancer. Te specifcity of this approach is expected to minimize potential side efects. Te identifcation of additional intratumoral Treg cell subsets and characterization of their mechanism of action and homeostatic requirements will provide new molecular targets for a tailored immunotherapy based on Treg cell manipulation.

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REFERENCES AND NOTES 1. A. M. Gallimore, A. K. Simon, Positive and negative influences of regulatory T cells on tumour immunity. Oncogene 27, 5886–5893 (2008). 2. S. Ladoire, F. Martin, F. Ghiringhelli, Prognostic role of FOXP3+ regulatory T cells infiltrating human carcinomas: The paradox of colorectal cancer. Cancer Immunol. Immunother. 60, 909–918 (2011). 3. N. R. Blatner, M. F. Mulcahy, K. L. Dennis, D. Scholtens, D. J. Bentrem, J. D. Phillips, S. Ham, B. P. Sandall, M. W. Khan, D. M. Mahvi, A. L. Halverson, S. J. Stryker, A.-M. Boller, A. Singal, R. K. Sneed, B. Sarraj, M. J. Ansari, M. Oft, Y. Iwakura, L. Zhou, A. Bonertz, P. Beckhove, F. Gounari, K. Khazaie, Expression of RORγt marks a pathogenic regulatory T cell subset in human colon cancer. Sci. Transl. Med. 4, 164ra159 (2012). 4. S. Z. Josefowicz, L. F. Lu, A. Y. Rudensky, Regulatory T cells: Mechanisms of differentiation and function. Annu. Rev. Immunol. 30, 531–564 (2012). 5. K. S. Voo, Y. H. Wang, F. R. Santori, C. Boggiano, Y. H. Wang, K. Arima, L. Bover, S. Hanabuchi, J. Khalili, E. Marinova, B. Zheng, D. R. Littman, Y. J. Liu, Identification of IL-17-

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producing FOXP3+ regulatory T cells in humans. Proc. Natl. Acad. Sci. U.S.A. 106, 4793–4798 (2009). Z. Hovhannisyan, J. Treatman, D. R. Littman, L. Mayer, Characterization of interleukin-17-producing regulatory T cells in inflamed intestinal mucosa from patients with inflammatory bowel diseases. Gastroenterology 140, 957–965 (2011). D. R. Littman, A. Y. Rudensky, Th17 and regulatory T cells in mediating and restraining inflammation. Cell 140, 845–858 (2010). M. Tosolini, A. Kirilovsky, B. Mlecnik, T. Fredriksen, S. Mauger, G. Bindea, A. Berger, P. Bruneval, W. H. Fridman, F. Pagès, J. Galon, Clinical impact of different classes of infiltrating T cytotoxic and helper cells (Th1, th2, treg, th17) in patients with colorectal cancer. Cancer Res. 71, 1263–1271 (2011). E. Gounaris, N. R. Blatner, K. Dennis, F. Magnusson, M. F. Gurish, T. B. Strom, P. Beckhove, F. Gounari, K. Khazaie, Tregulatory cells shift from a protective anti-inflammatory to a cancer-promoting proinflammatory phenotype in polyposis. Cancer Res. 69, 5490–5497 (2009). N. Ohkura, M. Hamaguchi, S. Sakaguchi, FOXP3+ regulatory T cells: Control of FOXP3 expression by pharmacological agents. Trends Pharmacol. Sci. 32, 158–166 (2011).

Acknowledgments: P.D.B is an American Cancer Society Fellow. Competing interests: The authors declare that they have no competing interests.

10.1126/scitranslmed.3005283 Citation: P. D. Bos, A. Y. Rudensky, Treg cells in cancer: A case of multiple personality disorder. Sci. Transl. Med. 4, 164fs44 (2012).


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