TLR Ligands in the Local Treatment of Established Intracerebral Murine Gliomas 1 Oliver M. Grauer,* † Johan W. Molling, † Erik Bennink, † Liza W. J. Toonen, † Roger P. M. Sutmuller, 2† Stefan Nierkens, † and Gosse J. Adema 3† Local TLR stimulation is an attractive approach to induce antitumor immunity. In this study, we compared various TLR ligands for their ability to affect murine GL261 cells in vitro and to eradicate established intracerebral murine gliomas in vivo. Our data show that GL261 cells express TLR2, TLR3, and TLR4 and respond to the corresponding TLR ligands with increasing MHC class I expression and inducing IL-6 secretion in vitro, while TLR5, TLR7, and TLR9 are essentially absent. Remarkably, CpG- oligonucleotides (CpG-ODN, TLR9) appeared to inhibit GL261 cell proliferation in a cell-type specific, but CpG-motif and TLR9-independent manner. A single intratumoral injection of CpG-ODN most effectively inhibited glioma growth in vivo and cured 80% of glioma-bearing C57BL/6 mice. Intratumoral injection of Pam3Cys-SK4 (TLR1/2) or R848 (TLR7) also produced a significant survival benefit, whereas poly(I:C) (TLR3) or purified LPS (TLR4) stimulation alone was not effective. Additional studies using TLR9 / wild-type and TLR9 / knockout mice revealed that the efficacy of local CpG-ODN treatment in vivo required TLR9 expression on nontumor cells. Additional experiments demonstrated increased frequencies of tumor-infiltrating IFN- producing CD4  and CD8  effector T cells and a marked increase in the ratio of CD4  effector T cells to CD4  FoxP3  regulatory T cells upon CpG-ODN treatment. Surviving CpG-ODN treated mice were also protected from a subsequent tumor challenge without further addition of CpG-ODN. In summary, this study underlines the potency of local TLR treatment in antiglioma therapy and demonstrates that local CpG-ODN treatment most effectively restores antitumor immunity in a thera- peutic murine glioma model. The Journal of Immunology, 2008, 181: 6720 – 6729. M alignant gliomas are highly aggressive brain tumors characterized by intense heterogeneity, high prolifer- ative activity and local invasiveness. These tumors have developed multiple mechanisms to escape from immune sur- veillance (1, 2). We have recently demonstrated that the suppres- sion of antiglioma immune responses is strongly associated with the intratumoral accumulation of CD4 + FoxP3 + regulatory T cells (Treg) 4 (3). Several clinical phase I/II dendritic cell (DC) vacci- nation trials have been conducted with the objective to break im- mune tolerance against high grade gliomas (4 – 6). Although all studies led to the induction of antitumor CTLs and lymphocyte infiltration into tumors in situ, survival benefit remained low. Ob- jective tumor regressions appeared to relate to the absence of a bulky tumor mass secreting TGF-2 and, importantly, on the mat- uration status of DCs inside and around the tumor (7, 8). Thera- peutic strategies combining abrogation of local immunosuppres- sion with potent immune stimulation are therefore of particular interest in the search for efficient treatments of malignant gliomas. The CNS is considered as a unique immunological site. The pres- ence of the blood-brain barrier, graft acceptance, lack of conventional lymphatics, low T cell trafficking, and low but inducible MHC ex- pression levels all point to low immune reactivity to prevent acciden- tal inflammation within the CNS (9). However, in case of a CNS infection strong immune responses against the invading pathogens develop indicating that potent immune responses can occur. The immune system is equipped with a panel of innate receptors that sense a broad spectrum of pathogens and alert cells to respond accordingly. The best characterized receptors are the TLRs that recognize a set of conserved molecular structures, so called patho- gen-associated molecular patterns shared by large groups of mi- croorganisms (10, 11). Currently, 13 TLR have been identified. TLR1–9 are expressed in both mice and humans, TLR10 is present in mice only as a degenerate pseudogene, and TLR11–13 are ex- pressed in mice but lack human orthologs. TLR2 can associate with TLR1 and TLR6, and recognizes bacterial lipoproteins, pep- tidoglycan, and lipoteichoic acid. TLR3 recognizes viral double- stranded RNA and synthetic double-stranded RNAs, such as polyi- nosinic-polycytidylic acid (poly(I:C)). TLR4 binds LPS from Gram-negative bacteria and viral envelope proteins, while TLR5 recognizes flagellin. TLR7 and TLR8 recognize viral single stranded RNA and synthetic molecules like imidazoquinoline or derivates. TLR9 recognizes unmethylated CpG motifs within bac- terial and viral DNA. The specific ligand of TLR10 is currently unknown. The recently discovered TLR11 recognizes uropatho- genic bacteria (12, 13). *Department of Neurology, University of Regensburg, Regensburg, Germany; and † Department of Tumor Immunology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands Received for publication March 31, 2008. Accepted for publication September 5, 2008. 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. 1 This work was supported by a grant from the German Research Foundation (Dr. Oliver M. Grauer, GR2089/1-1) and funds from the Dutch Cancer Society (KWF2003-2893, Prof. Gosse J. Adema), and was performed in part within the frame- work of project D1-101 of Top Institute Pharma. Dr. Roger P. Sutmuller was sup- ported by a VENI grant from the Netherlands Organisation for Scientific Research (NOW grant 91656130). 2 Current address: N.V. Organon, Target Discovery, Oss, The Netherlands. 3 Address correspondence and reprint requests to Prof. G. J. Adema, Tumor Immu- nology Laboratory, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail address: G.Adema@ncmls.ru.nl 4 Abbreviations used in this paper: Treg, regulatory T cell; DC, dendritic cell; poly(I: C), polyinosinic-polycytidylic acid; CpG-ODN, CpG-oligonucleotide; TIL, tumor- infiltrating lymphocyte; Teff, effector T cell; BMDC, bone marrow derived dendritic cell; cLN, cervical lymph node; mLN, mesenterial lymph node. 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