Bestowing Transient Cytotoxic Capability on Dendritic Cells by Transfection with In Vitro Transcribed (IVT) TRAIL mRNA.
Authors: Watanabe Norihiro , Narita Miwako , Yano Toshio , Sato Naoko , Sekiguchi Asuka , Saito Anri , Yokoyama Ayumi , Ayres Flavio , Alldawi Louie , Furukawa Tatsuo , Toba Ken , Watanabe Yoko , Takahashi Yoshiaki , Takahashi Masuhiro 
Published: April 2020

Abstract
Bestowing transient cytotoxic capability on dendritic cells could expand the applicability of dendritic cells in the fields of the treatment for autoimmune disorders and anti-tumor immunotherapy. If it would be possible to construct dendritic cells, which present antigens associated with autoimmune disorders and possess transient cytotoxic ability, these dendritic cells could be used for the therapy of the disorders by eliminating active lymphocytes reactive to autologous etiological antigens specifically. As to the applicability for anti-tumor immunotherapy, dendritic cells with transient cytotoxicity could be administered into the tumor region directly for efficient tumor antigen presentation by effective killing tumor cells and up taking apoptotic/necrotic tumor cells. In the present study, the transfection of IVT TRAIL mRNA into dendritic cells was applied to induce a transient cytotoxic ability in functional dendritic cells. By using microbeads with anti-CD34 monoclonal antibody, human CD34+ cells were separated from cord blood with informed consent. In order to generate mature dendritic cells, CD34+ cells were cultured with GM-CSF, SCF and TNF-α for 8-10 days. CD34+ cell-derived dendritic cells were transfected with TRAIL mRNA by square wave pulse-electroporation and cultured in the medium containing the same cytokines for 1 day. TRAIL mRNA was in vitro transcribed from the T7 promoter transcription vector containing TRAIL cDNA by using mMESSAGE mMACHINE kit. As a positive control of TRAIL protein expression, CD34+ cell-derived dendritic cells were cultured with IFN-γ at the concentration of 1,000 U/ml for 1 day, which was the best cytokine/culture condition to induce the expression of TRAIL protein in dendritic cells. The expression of TRAIL protein and dendritic cell-associated surface phenotypes on the dendritic cells transfected with IVT TRAIL mRNA or cultured with IFN-γ was evaluated by flow cytometry using anti-TRAIL monoclonal antibody and the other antibodies. Cytotoxic capability of dendritic cells transfected with IVT TRAIL mRNA or cultured with IFN-g was evaluated by 51Cr-release assay in which TRAIL receptor (DR4 and/or DR5) -expressing cell lines such as HL-60 and Jurkat were used as target cells. By the transfection of IVT TRAIL mRNA, CD34+ cell-derived dendritic cells were demonstrated to express TRAIL proteins and showed no change of dendritic cell-associated surface phenotypes. The dendritic cells cultured with IFN-γ also showed the similar results. The expression of TRAIL protein on dendritic cells lasted only for a few days. Dendritic cells gained the cytotoxic capability against cell lines like HL-60 after the transfection with IVT TRAIL mRNA. The present study demonstrated that trans-membrane protein like TRAIL could be expressed and function as a molecule generating death signals transiently in the dendritic cells by the transfection with IVT mRNA. These findings revealed that the dendritic cells with bestowed transient cytotoxic capability by RNA transfection could be applied for the treatment of autoimmune disorders and anti-tumor immunotherapy.