ALBERT

Description: 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.

Description: Transfection with tumor antigen RNA is one of the promising tools not only because of a possible sufficient amplification of tumor antigen RNA but also because of the absence of antigen peptides-associated MHC restriction. Several succeeded experiments about generation of CTLs using DCs transfeced in vitro transcribed (IVT) cancer specific antigen mRNA such as PSA, CEA, hTERT and MUC-1 have been reported in these a few years. In addition, recent reports about the simultaneous presentation of peptides in both MHC class I and class II molecules on DCs after mRNA electroporation show another superiority of mRNA transfection into DCs. In this presentation, we demonstrate successful generation of tumor antigen specific CTLs using with DCs transfected with IVT mRNA such as SART-1 and WT-1 by electroporation. This is the first report about the generation of SART-1 and WT-1 specific CTLs by using mRNA transfected DCs. [Methods] HLA-A24 positive human PB CD14+ cell-derived DCs were transfected with IVT mRNA (SART-1and WT-1) by electroporation. MRNA transfected DCs were co-cultured with autologous lymphocytes. The bulk co-cultures were re-stimulated several times with same DCs. CD8+ cells were separated and CTL activity was evaluated by 51chromium release assay. To determine whether the induced CTL cells could recognize the target cells in an HLA class I restricted manner, anti-HLA class I monoclonal antibodies were utilized to block the cytotoxicity of effectors. [Results] Electroporation of mRNA showed no effect on the surface phenotypes and antigen presenting ability of DCs. In addition to the demonstration of efficient transfection of M1 mRNA into DCs by using RT-PCR, which eliminated the amplification of transfected mRNA by the treatment with RNase before RNA extraction from the transfected cells, we identified the definite expression of WT-1 protein in the cytoplasm of DCs by using immunoblotting. CTL assay indicated that 1) DCs transfected with mRNA stimulated the generation of antigen-specific CTLs which are capable of lysing autologous DCs transfected with the same mRNA. 2) CTLs also demonstrated cytotoxic ability against cell lines such as KE-4 presenting SART-1 peptides on HLA-A24, MEGO1 presenting WT-1 peptides on MHC class I, and HLA-A24 cDNA transfected T2 which were used as target cells after co- incubation with 9 mer SART-1 peptides with strong affinity to HLA-A24. 3) Each cytotoxicities were markedly blocked after co-incubation of target cells with anti-MHC class I antibody and not inhibited with anti-MHC class II antibody. [Conclusion] Our results showed that IVT mRNA-transfected DCs which is constructed non-virally have a highly efficient ability to stimulate specific T-cell immunity against tumor. Unlike peptide- or tumor cells extract-pulsed DCs based vaccines, anti-tumor immunotherapy using the DCs transfected with antigen mRNA could be extended to a wide range of patients who have previously been excluded from clinical trials for the reason of the un-identification of tumor specific antigens, for the reason of the impossibility of obtaining sufficient tumor specimens, or for the reason of MHC restriction of the tumor specific antigens.

Description: Heparin is bound with heparin-binding sites on certain cells, which induces proliferation and differentiation signals. In addition, heparin is bound with heparin-binding domains of various cytokines, which enhances the interaction between cytokines and target cells. Monocytes have been demonstrated to posses heparin-binding sites on cell surfaces. In the present study, we investigated the effects of heparin (including low molecular weight heparin) and heparan sulfate on antigen presentation and antigen-specific CTL induction of monocyte-derived DCs. Peripheral blood CD14+ cells were cultured to generate immature and mature DCs with various concentrations of heparin, low molecular weight heparin or heparan sulfate. Cultured cells were analyzed for DC-associated surface phenotypes by flow cytometry and evaluated for allogeneic antigen presenting ability by mixed leukocyte culture. In order to evaluate the effects of heparin on monocyte-derived DCs to generate antigen-specific CTL, DCs were generated from HLA-A2402 donors by serum-free culture with heparin, transfected with in vitro transcribed WT-1 mRNA on day 6 and cultured with the addition TNF-α/IL-1α/IL-6/IFN-γ/PGE2 for further 1 day. WT-1 mRNA-transfected DCs were used for priming autologous lymphocytes in co-culture at the stimulator:responder ratio of 1:10. Lymphocytes were primed with the same DCs 2-3 times in the interval of 5-7 days. CD8+ T cells were separated and used as effector cells in 51Cr-release assay. WT-1 expressing and HLA-A24+ cell line MegO1 was used as target cells. In order to evaluate the association of MHC molecules in the cytotoxicity, 51Cr-lebelled target cells were treated with anti-MHC class I or class II monoclonal antibody before cytotoxicity assay. In order to evaluate the antigen specificity of the generated CTL, unlabelled target cells were added to the cytotoxicity assay. By the addition of heparin, the expression of CD1a and CD80 on both immature and mature DCs was markedly enhanced and the allogeneic antigen presenting ability was elevated in both immature and mature DCs. By the addition of low molecular weight heparin, the expression of CD1a was enhanced and antigen presenting ability was elevated also. By the addition of heparan sulfate, similar results of elevated antigen presentation were obtained. By the priming of lymphocytes with WT-1 mRNA transfected DCs generated from monocytes by the serum-free culture with heparin, cytotoxic capability against WT-1 expressing target cells was demonstrated in the primed lymphocytes. The cytotoxic capability of the lymphocytes was blocked by the treatment of the target cells with anti-MHC class I monoclonal antibody and the addition of unlabelled target cells in the cytotoxicity reaction. The present study demonstrated that heparin/low molecular weight heparin/heparan sulfate could enhance the antigen presentation and antigen-specific CTL induction of monocyte-derived DCs. These findings suggest the usefulness of heparin for generating efficient DCs for DC-based immunotherapy and the involvement of heparan sulfate in immunological defense mechanism.