Published OnlineFirst October 2, 2015; DOI: 10.1158/0008-5472.CAN-14-3499 Cancer Microenvironment and Immunology Research Suppression of Intratumoral CCL22 by Type I Interferon Inhibits Migration of Regulatory T Cells and Blocks Cancer Progression David Anz1,2, Moritz Rapp1, Stephan Eiber1,2, Viktor H. Koelzer1, Raffael Thaler1, Sascha Haubner1, Max Knott1, Sarah Nagel1, Michaela Golic1, Gabriela M. Wiedemann1, Franz Bauernfeind3, Cornelia Wurzenberger1, Veit Hornung3,4, Christoph Scholz5, Doris Mayr6, Simon Rothenfusser1, Stefan Endres1, and Carole Bourquin1,7 Abstract The chemokine CCL22 is abundantly expressed in many tion. Mechanistic investigations indicated that Treg blockade types of cancer and is instrumental for intratumoral recruit- was a consequence of reduced intratumoral CCL22 levels ment of regulatory T cells (Treg), an important subset of caused by type I IFN. Notably, stable expression of CCL22 immunosuppressive and tumor-promoting lymphocytes. In abrogated the antitumor effects of treatment with RLR or this study, we offer evidence for a generalized strategy to TLR ligands. Taken together, our findings argue that type I blunt Treg activity that can limit immune escape and promote IFN blocks the Treg-attracting chemokine CCL22 and thus tumor rejection. Activation of innate immunity with Toll-like helps limit the recruitment of Treg to tumors, a finding with receptor (TLR) or RIG-I–like receptor (RLR) ligands prevented implications for cancer immunotherapy. Cancer Res; 75(21); 1– accumulation of Treg in tumors by blocking their immigra- 11. Ó2015 AACR. Introduction effector T-cell function ex vivo (1). Indeed, suppression of anti- cancer immunity is mediated predominantly by intratumoral Treg The chemokine CCL22 is abundantly expressed in the tissue of that suppress CD8 T-cell responses locally at the tumor site (6). many types of human cancer (1, 2). Its cognate receptor is CCR4, a þ High numbers of tumor-infiltrating FoxP3 Treg correlate with transmembrane protein expressed predominantly and constitu- poor prognosis and have been identified as a significant predictor tively by regulatory T cells (Treg), a subpopulation of immuno- of patient death in several types of human cancer (1, 2). suppressive T lymphocytes (3). Several studies show that CCL22 The aim of cancer immunotherapy is to promote antitumor leads to the recruitment of Treg to the cancer tissue (1, 2). immunity and to overcome tumor-induced immunosuppression. Although the role of Treg in cancer remains to be definitely Activation of the innate immune system with ligands for pattern established, many reports on human and murine cancers dem- recognition receptors (PRR) or with cytokines such as IFNa can onstrate a tumor-promoting effect: Treg progressively accumulate suppress tumor growth (7, 8). In mice, CpG oligonucleotides in the blood and lymphoid organs (4) and abundantly infiltrate (CpG) that stimulate the Toll-like receptor 9 (TLR9) improve the the tumor tissue itself (5). These tumor-infiltrating Treg that efficacy of anticancer vaccines and can also be used as single agent express CD25 and the transcription factor FoxP3 strongly suppress to reduce tumor size (9, 10). In humans, imiquimod, a synthetic agonist for TLR7, is used to treat basal cell carcinoma and vulvar intraepithelial neoplasia, and the cytokines IL2 and IFNa are used 1Center of Integrated Protein Science Munich (CIPS-M), Division of for selected patients with melanoma or renal cell cancer (11, 12). € € Clinical Pharmacology, Klinikum der Universitat Munchen, Munich, Furthermore, many TLR activators are currently under investiga- Germany. 2Medizinische Klinik und Poliklinik IV, Klinikum der Uni- versitat€ Munchen,€ Munich, Germany. 3Institute of Molecular Medicine, tion in clinical trials (13). Universitätsklinikum Bonn, Bonn, Germany. 4Gene Center and Depart- The impact of PRR-activating ligands on Treg in the tumor- ment of Biochemistry, Ludwig-Maximlians-Universität, Munich, Ger- bearing host is still unclear. It has been shown that PRR activation many. 5Department of Obstetrics and Gynecology, Ulm University Medical Centre, Ulm, Germany. 6Department of Pathology, Ludwig- can prevent Treg-mediated suppression of T effector cell prolif- Maximilians-Universitat,€ Munich, Germany. 7Department of Medicine, eration in the lymph node (14, 15), but an effect of PRR activation University of Fribourg, Fribourg, Switzerland. on Treg in the tumor tissue has so far not been described. In Note: Supplementary data for this article are available at Cancer Research particular, it is unknown whether trafficking and tumor infiltra- Online (http://cancerres.aacrjournals.org/). tion of Treg can be affected by agonists for TLRs and RIG-I–like D. Anz and M. Rapp contributed equally to this article. helicases. Corresponding Author: Stefan Endres, Division of Clinical Pharmacology, Lindwurmstrasse 2a, 80337 Munich, Germany. Phone: 49-89-440057300; Fax: Materials and Methods 49-89-440057330; E-mail: [email protected] Mice and cell lines doi: 10.1158/0008-5472.CAN-14-3499 Female BALB/c and C57BL/6 mice were from Harlan-Winkel- Ó2015 American Association for Cancer Research. mann. IFNI receptor (IFNAR)-deficient mice on C57BL/6 www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst October 2, 2015; DOI: 10.1158/0008-5472.CAN-14-3499 Anz et al. background were kindly provided by Dr. Z. Waibler (Paul-Ehrlich All patients had been treated surgically between 2004 and 2008 at Institute, Langen, Germany). Mice were 5 to 10 weeks of age at the the same institution (Department of Obstetrics and Gynecology onset of experiments. Animal studies were approved by the local Maistrasse, University of Munich, Munich, Germany). Tissue regulatory agency (Regierung von Oberbayern, Munich, Ger- was stained with the following unconjugated primary antibodies many). The human cell lines Jurkat, A-375, A-431, A-549, and the respective isotype control antibodies: rabbit anti-hCCL22 CAMA-1, MCF-7, MDA-MB-231, MDA-MB-435s, Panc1, SK-BR- (Peprotech), mouse anti-hCD14 (Novocastra), mouse anti- 3, and SW480 and the murine cell lines 4T1, CT26, EL-4 (EG-7), hCD68 (Dako), rat anti-hDC-LAMP (Dendritics), mouse anti- and B16-F1 were obtained from ATCC, where short tandem hDC-SIGN (Abcam), and purified rabbit IgG (Biozol). For detec- repeat (STR) analysis is used for authentication and were used tion, the following secondary antibodies were used: biotinylated within 6 months after resuscitation (ATCC). IMIM-PC1 was goat anti-rabbit (Vector), Cy2 goat anti-rat, and Alexa Fluor 488 kindly provided by Prof. P. Michl (University of Marburg, Mar- donkey anti-mouse (both Jackson ImmunoResearch). For imag- burg, Germany), Panc02 by Prof. C. Bruns, MethA by Prof. ing, a confocal laser scanning microscope (LSM 510, Carl Zeiss) W. Zimmermann, and mGC8 by Dr. J. Nockel€ (all Klinikum der was used and images were processed using Adobe Photoshop for Universit€at Munchen,€ Munich, Germany). Cell lines were authen- adjustment of contrast and size. ticated using STR (LGC Standards) and were cultured in complete DMEM or RPMI medium (PAA Laboratories) and routinely tested Flow cytometry and adoptive T-cell transfer for mycoplasma contamination by MycoAlert Mycoplasma Tumors were mechanically disrupted, incubated with 1 mg/mL Detection Kit (LONZA). The CT26-CCL22dox cell line was gen- collagenase and 0.05 mg/mL DNAse (both Sigma Aldrich), and erated by lentiviral transduction with a construct containing a passed through a cell strainer. Single-cell suspensions were resus- doxycycline (Dox)-inducible CCL22 expression cassette as pended in 44% Percoll (Biochrome) and layered over 67% Percoll described (16). For tumor induction, 0.25 Â 106 (CT26 or prior to centrifugation at 800 Â g for 30 minutes. Lymphocytes CT26-CCL22dox), 1 Â 106 (B16 and Panc02), and 7.5 Â 106 from the interphase were stained with anti-CD3-PerCP, anti-CD4- (EG-7) tumor cells were injected subcutaneously into the flank. APC, anti-CD103-Fitc (all BD Biosciences) and anti-CCR4-Pe Mice with subcutaneous CT26-CCL22dox tumors were fed with a (Biolegend) followed by intracellular detection of FoxP3 using normal or 25 mg/kg doxycycline-containing diet (ssniff Spezial- either anti-FoxP3-PE or anti-FoxP3-Pacific Blue antibody and di€aten GmbH). Tumor size was expressed as the product of the premixed regulatory T-cell staining reagents (Ebioscience). Events perpendicular diameters of individual tumors (mm2). were measured on a FACS Canto II flow cytometer (BD Bio- sciences) and analyzed with FlowJo software (TreeStar). For Application of TLR ligands adoptive transfer, single-cell suspensions from the spleen and 0 The fully PTO-modified CpG oligonucleotide 1826 (5 -TCCAT- lymph nodes of healthy mice were labeled with carboxyfluores- 0 GACGTTCCTGACGTT-3 ) (Coley Pharmaceutical Group) was cein diacetate succinimidyl ester (CFSE; Invitrogen) according injected subcutaneously either peritumorally or in the contralat- to the manufacturer's instructions and injected intravenously eral flank (100 mg CpG in PBS). EG-7 tumor–bearing mice were (3 Â 107 cells per recipient mouse) into tumor-bearing recipients injected with 10 mg CpG complexed with 50 mg of liposomal (mean tumor size, 80 mm2). transfection reagent DOTAP (Roche). Poly(I:C) (Amersham Bioscience) was applied intraperitoneally (250 mg). Lipopoly- Cytokine assays of tissue lysates saccharide (LPS; Sigma-Aldrich) and resiquimod (R848; Alexis Tissue homogenates