عزیز محمودزاده - Dept. of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
علی اکبر پور فتح اله - Dept. of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
محمد حسین کریمی - Transplantation Research Center, Nemazee Hospital, Shiraz University of Medical Sciences, Shiraz, Iran
سید محمد موذنی - Dept. of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

Induction of Tolerogenic Murine Dendritic Cells by Downregulating the Co-stimulatory Molecule of CD۴۰ Using Lentivirus Vector Mahmoodzadeh A۱, Pourfatollah AA۱, Karimi MH۲, Moazzeni SM۱ ۱Dept. of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran ۲Transplantation Research Center, Nemazee Hospital, Shiraz University of Medical Sciences, Shiraz, Iran. Correspond Author: Pourfatollah AA, Dept. of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran E-mail: Pourfa@modares.ac.ir Received: ۱۴ May ۲۰۱۴ Accepted: ۱۳ Jul ۲۰۱۴ Background and Objective: The important role of co-stimulatory molecules in immunological responses has been clearly demonstrated. With the development of new methods in molecular immunology, preventing the co-stimulatory signals in order to induce tolerance has emerged as a novel strategy. RNA interference is a new method that can specifically and effectively reduce target gene expression. Materials and Methods: In this study, we used a lentiviral system to target the expression of CD۴۰ gene in dendritic cells. ShRNA sequence is able to reduce the expression of CD۴۰ gene on murine dendritic cells. Results: Our results indicate ۶۴-fold decreased expression of CD۴۰ mRNA in the group receiving lentiviral shRNACD۴۰ as well as ۳۱% decrease in expression of CD۴۰ protein on the surface of dentridic cells (p<۰.۰۰۰۱). Also, dendritic cells cannot stimulate T cells upon contact with them. Conclusion: This study indicates that a decrease in expression of CD۴۰ molecule induces tolerogenic dendritic cells and prevents Th۱ immunological response. Prevention of Th۱ response would shift the immunological responses to Th۲ response, and this type of response can be effective in autoimmune diseases including rheumatoid arthritis and type I diabetes. References ۱- Groux H, Fournier N, Cottrez F. Role of dendritic cells in the generation of regulatory T cells. Sem in Immunol. ۲۰۰۴ ۱۶: ۹۹-۱۰۶. ۲- Kanako L, Reizis L, Reizis B. Dendritic Cells: Arbiters of Immunity and Immunological Tolerance. Cold Spring Harb Perspect Biol. ۲۰۱۲ ۴: a۰۰۷۴۰۱. Avalable from: www.cshperspectives.org. ۳- Morel PA. Dendritic cell subsets in type۱diabetes :friend or foe? Frontiers in Immunol. ۲۰۱۳ ۴. Avalable from: http://dx.doi.org/ ۱۰.۱۱۵۵/ ۲۰۱۳/۹۷۲۸۶۵. ۴- Machen J, Harnaha JO, Lakomy R, Styche A, Trucco M, Giannoukakis N. Antisense oligonucleotides down-regulating costimulation confer diabetes-preventive properties to nonobese diabetic mouse dendritic cells. The J Immunol. ۲۰۰۴ ۱۷۳: ۴۳۳۱-۴۱. ۵- Maldonado R, Andrian UH. How tolerogenic dendritic cells induce regulatory T cells. Adv Immunol. ۲۰۱۰ ۱۰۸: ۱۱۱-۱۶۵. ۶- Peters AL, Stunz LL, Bishop GA. CD۴۰ and autoimmunity: the dark side of a great activator. Sem Immunol. ۲۰۰۹ ۲۱: ۲۹۳-۳۰۰. ۷- Li L, Wang H, Wang B. Anergic cells generated by blocking CD۲۸ and CD۴۰ costimulatory pathways in vitro ameliorate collagen induced arthritis. Cell Immunol . ۲۰۰۸ ۲۵۴: ۳۹-۴۵. ۸- Zheng X, Suzuki M, Zhang Z, et al. RNAi-mediated CD۴۰-CD۱۵۴ interruption promotes tolerance in autoimmune arthritis. Arth Res & Ther. ۲۰۱۰ ۱۲: R۱۳. ۹- Pletinckx K, Dohler A, Pavlovic V, Lutz M.B. Role of dendritic cells maturity/ costimulation for generation, homeaostasis and suppressive activity of regulatory T cells. Fronties in Immunol. ۲۰۱۱ ۲: ۷-۲۲. ۱۰- Gavrilov K, Saltzman WM. Therapeutic siRNA: Principles, challenges and strategies. Yale J Biol Med. ۲۰۱۲ ۸۵(۲): ۱۸۷-۲۰۰. ۱۱- Manjunath N, Wu H, Subramanya S, Shankar P. Lentiviral delivery of short hairpin RNAs. Adv Drug Delivery Rev. ۲۰۰۹ ۶۱: ۷۳۲-۴۵. ۱۲- Inaba K, Inaba M, Romani M, et al. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with GM-CSF. J Exp Med. ۱۹۹۲ ۱۷۶: ۱۶۹۳-۷۰۲. ۱۳- Virus packaging protocol. Stem Cell Biotechnology Research Center protocol, Iran. ۲۰۱۳. Avalable from: www.stemcellstech. com/index.php/fa/. ۱۴- Muller G, Muller A, Jonuleit H. Fetal calf serum –free generation of functionally active murine dendritic cells suitable for invivo therapeutic approaches. J Inv Dermatol. ۲۰۰۰ ۱۱۴: ۱۴۲-۸. ۱۵- Marin-Gallen S, Clemente-Casares X, Planas R, et al. Dendritic cells pulsed with antigen-specific apoptotic bodies prevent experimental type ۱ diabetes. Clin and Exp Immunol. ۲۰۰۹ ۱۶۰: ۲۰۷-۱۴. ۱۶- Hasse C, Ejrnaes M, Juedes AE, Wolf T. Immunomodulatory dendritic cells require autologous serum to circumvent nonspecific immunosuuressive activity in vivo. Blood. ۲۰۰۵ ۱۰۶: ۴۲۲۵-۳۳. ۱۷- Feili-Hariri M, Dong X, Alber SM, Watkins SC, Salter RD, Morel PA. Immunotherapy of NOD mice with bone marrow–derived dendritic cells. Diabetes. ۱۹۹۹ ۴۸: ۲۳۰۰-۰۸. ۱۸- Karimi MH, Ebadi P, Pourfathollah AA, et al. Immune modulation through RNA interference-mediated silencing of CD۴۰ in dendritic cells. Cellular Immunol. ۲۰۰۹ ۲۵۹: ۷۴-۸۱. ۱۹- Mei S, Jin-Dong LI, Rui J, et al. Construction and characterization of lentiviral shRNA expression vector targeting rat CD۴۰ gene in dendritic cells. Chem Res. ۲۰۰۹ ۲۵(۵): ۶۶۶-۷۲. ۲۰- Jantsch J, Turza N, Volke M, et al. Small interfering RNA (siRNA) delivery into murine bone marrow-derived dendritic cells by electroporation. J of Immunological Methods. ۲۰۰۸ ۳۳۷: ۷۱-۷. ۲۱- Giannoukakis N, Trucco M. Dendritic cell therapy for type ۱ diabetes suppression. Immunother. ۲۰۱۲ ۴(۱۰): ۱-۱۲. ۲۲- Giannoukakis N. Tolerogenic dendritic cells for type ۱ diabetes. Immunother. ۲۰۱۳ ۵(۶): ۱-۳. ۲۳- Lee CN, Lew AM, Wu L. The potential role of dendritic cells in the therapy of Type۱ diabetes. Immunother. ۲۰۱۳ ۵(۶): ۵۹۱-۶۰۶.

Keywords: CD۴۰ Gene, Lentiviral vector, Murine Dendritic Cells