title: [Enhanced translational efficacy of internal ribosomal entry site-mediated mRNA in dendritic cells contributes to efficient induction of antitumor immunity].
abstract: OBJECTIVE
  To develop a more economic and applicable substitute for modification of dendritic cells (DCs) by capped mRNA in induction of anti-tumor immunity.
  
  
  METHODS
  Four DNA plasmids as templates of mRNA in vitro transcription were constructed: pmRNA luciferase (Luc), pmRNA internal ribosomal entry site (IRES)-Luc, pmRNA ovalbumin (OVA), and pmRNA IRES-OVA. The translational efficiency of uncapped-Luc, capped-Luc, or IRES-Luc mRNA in murine DCs was detected via a Luc reporter system. Nine C57BL/6 mice were divided into 3 equal groups to be injected intraperitoneally with DCs transfected with IRES-OVA or capped-OVA mRNA and untreated DCs respectively, 1 weeks later the mice were injected with splenocytes wrapped with the target peptide or control peptide labeled by CFSE, and 4 hours later the mice were killed and suspension of splenocytes was made to test the activity of cytotoxic lymphocytes (CTLs). Another 30 mice were divided into 3 equal groups to undergo immunization by DCs as mentioned above, 1 week later mice melanoma cells of the line MO5 stably expressing OVA were injected the caudal vein, and 3 weeks later the mice were killed and their lungs were taken out to observe the metastasis of tumor, using OVA as a target antigen.
  
  
  RESULTS
  Insertion of IRES into upstream of gene of interest in mRNA transcriptional templates didn't affect the yield of mRNA in vitro transcription. The level of Luc activity expressed by IRES-Luc mRNA in the DCs was 20 times higher than that by expressed by Uncapped Luc mRNA, and one time higher than that by Capped-Luc mRNA 8 h after transfection of DC. Expression of Luc could be detected up to 96 h after the transfection with IRES-Luc and Capped-Luc mRNA. Four hours after the injection of peptides the CTL activity of the mice immunized with DCs pulsed with Capped-OVA mRNA was (28 +/- 3)%, not significantly different from that of the mice immunized with DCs pulsed with IRES-OVA mRNA [(32 +/- 4)%, P > 0.05]. Mata static melanoma nodules could be seen in all control mice, only one mouse of the Capped-OVA mRNA group, and none of the mice of the IRES-OVA mRNA group.
  
  
  CONCLUSIONS
  IRES-containing tumor-associated antigen (TAA) mRNA as a more economic and applicable substitute fully replaces Capped-TAA mRNA for mRNA-based DC vaccines and DCs pulsed with IRES-containing TAA mRNA induces the same effective antigen-specific cellular response as the DCs pulsed with Capped-TAA mRNA.
authors: Hematology Department, General Hospital of Beijing Military Area, Beijing 100700, China. xiaohua_t@hotmail.com
authors: Xiao-Hua
authors: XH
authors: Tan
authors: Yong-Hong
authors: YH
authors: Wan
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology* / Q000378:metabolism*; D005260:Female; D016219:Immunotherapy, Adoptive / Q000379:methods*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008546:Melanoma, Experimental / Q000276:immunology / Q000473:pathology / Q000517:prevention & control; D051379:Mice; D008810:Mice, Inbred C57BL; D009362:Neoplasm Metastasis; D010047:Ovalbumin / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D013154:Spleen / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology / Q000276:immunology; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D012333:RNA, Messenger; D010047:Ovalbumin; D008156:Luciferases