A synthetic NOD2 agonist, muramyl dipeptide (MDP)-Lys (L18) and IFN-bsynergistically induce dendritic cell maturation with augmented IL-12production and suppress melanoma growthTaku Fujimura*, Kenshi Yamasaki, Takanori Hidaka, Yumiko Ito, Setsuya AibaDepartment of Dermatology, Tohoku University Graduate School of Medicine, Seiryo-machi 1-1, Aoba-ku, Sendai 980-8574, Japan1. IntroductionInterferon beta (IFN-b) affects growth and development ofmalignant cells 13. IFN-bshows growth inhibitory effects onmelanoma cells and also induces cross-priming of CD8+T cells bydendritic cells (DC) in vitro 4,7, and has been clinically used forthe therapy of malignant melanoma for more than 20 years 5,6.Peritumoral injection of IFN-brecruits the effecter cells includingCD8 and CD4-positive T cells in microenvironment of melanoma8. Although there are supportive evidences for the use of type Iinterferon as an adjuvant therapy for malignant melanoma, tumorregression was observed in only 1520% of the patients treatedwith IFN-b, and significant survival prolongation was not attainedby IFN-btreatment 9.An intracellular molecule nucleotide-binding oligomerizationdomain 2 (NOD2), which was revealed to be a receptor formuramyldipeptide (MDP), plays roles in innate and adaptiveimmunity 10. MDP is the smallest structural unit responsible forthe immunoadjuvant activity of the peptidoglycan of bacterial wall.To date, a number of its analogues and derivatives have beensynthesized. Among them, N2-acetylmuramyl-L-alanyl-D-isoguluta-minyl-N6-stearoyl-L-lysine (MDP-Lys (L18), a stearoyl-MDP deriva-tive,hasbeenreportedtobeanadjuvantthatexhibitsmorebiologicalactivity and less pyrogenenicity than other MDP derivatives 11,12.Several authors reported that MDP-Lys (L18) elicit the antitumorimmunity against the tumor in vivo 1315. MDP-Lys (L18) alsosynergistically induce IL-12 and IFN-gproduction in combinationwith TLR3, TLR4, and TLR9 agonists in dendritic cells (DCs), which inturn induce Th1-lineage immune responses 16.In this study, we addressed if the MDP-Lys(L18) in combinationwith IFN-bcan augment the DC activation and enhance theantitumor effect for melanoma. Our data here showed that MDP-Lys (L18) augmented IFN-b-dependent DCs maturation enhancingTh1-lineage, and the combination of MDP-Lys (L18) and IFN-bsuppressed melanoma growth on mice more efficiently than IFN-balone.Journal of Dermatological Science 62 (2011) 107115A R T I C L EI N F OArticle history:Received 19 October 2010Received in revised form 5 February 2011Accepted 7 February 2011Keywords:MelanomaImmunotherapyMDP-Lys (L18)IFN-bA B S T R A C TBackground:A synthetic NOD2 agonist, muramyl dipeptide (MDP)-Lys (L18), mimics the bacterialpeptidoglycan moiety and acts as a powerful adjuvant that induces cell-mediated immunity.Objective: To investigate the induction of antitumor immune response for malignant melanoma by IFN-bin combination with MDP-Lys (L18) (IFNMDP-Lys (L18).Methods: Human monocyte-derived DCs (MoDCs) are stimulated with IFNMDP-Lys (L18) in vitro. Weassess the expression of costimulatory molecules on MoDCs by FACS. Moreover, we investigate theinduction of Th1 cytokines by real time PCR and ELISA. Further to confirm the anti tumor immuneresponse of IFNMDP-Lys (L18) therapy, we examine the growth of B16F10 melanoma in vivo.Results: The stimulation of human MoDCs with IFNMDP-Lys (L18) significantly augmented theproduction of IL-12p70, TNF-a, and IL-6 compared to that with MDP or that with IFN-balone. IFNMDP-Lys (L18) increased the expression of IL-12p35, IL-12p40, IL-10, TNF-a, IL-6 and IL-1bmRNA by MoDCusing real-time PCR. The expression of CD83 and costimulatory molecules CD40, CD80, and CD86 wasalso augmented in MoDC treated with IFNMDP-Lys (L18), which resulted in their augmented allogeneicT cell stimulation. In vivo, the administration of IFNMDP-Lys (L18) significantly suppressed the growthof B16F10 melanoma, while the monotherapy of IFN-bor MDP-Lys (L18) did not significantly affect thetumor growth.Conclusion: These findings suggest that IFNMDP-Lys (L18) can be a promising adjuvant therapy formalignant melanoma.? 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rightsreserved.* Corresponding author. Tel.: +81 22 717 7271; fax: +81 22 717 7361.E-mail address: (T. Fujimura).Contents lists available at ScienceDirectJournal of Dermatological Sciencejournal homepage: ? 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.jdermsci.2011.02.0022. Materials and methods2.1. Media and reagentsMDP-Lys (L18), recombinant human IFN-band recombinantmurine IFN-bwere provided from Daiichi Pharmaceutical Co., Ltd.,Tokyo, Japan. Recombinant human IFN-awas provided fromTakeda Pharmaceutical Co., Ltd., Osaka, Japan. We used thefollowing antibodies (Abs) for flow cytometry: PE-conjugatedanti-CD86, PE-conjugated anti-CD83, PE-conjugated anti-CD80,PE-conjugated anti-CD40, PE-conj