title: Recombinant attenuated Salmonella harboring 4-1BB ligand gene enhances cellular immunity.
abstract: OBJECTIVE
  To transfect antigen presenting cells (APCs) with 4-1BB ligand DNA by attenuated Salmonella enterica serovar Typhimurium in vivo, and to observe the effects of ectogenous 4-1BBL on the immune functions of infected rats.
  
  
  METHODS
  Attenuated Salmonella typhimurium (vaccine strain) carrying plasmids pIRES2-EGFP-4-1BBL was constructed and used to infect HepG2 hepatoma cells. The expression of reporter gene, green fluorescent protein (GFP) and rat 4-1BBL in the transfected cells was detected by double-immunofluorescence staining. Rats were fed with the recombinant bacteria intragastrically on three occasions in 2 weeks, and were then sacrificed. The transcription and expression of GFP and 4-1BBL genes in splenocytes were measured by RT-PCR and flow cytometry. The phenotypes of T cells in peripheral blood and splenocytes were determined by flow cytometry. The content of IFN-gamma in the cultural supernatant of splenocytes stimulated by PHA was measured by ELISA.
  
  
  RESULTS
  The recombinant bacteria harboring 4-1BBL had the same invasive abilities as the original bacteria, and it was able to deliver exogenous genes into HepG2 cells, where the GFP and 4-1BBL were successfully expressed. There were significant upregulations of CD3(+)CD8(+) T cells (P=0.018) and CD3(+)CD25(+) T cells (P=0.019) in the peripheral blood cells as well as CD3(+)CD8(+) T cells (P=0.022), and CD3(+)CD25(+) T cells (P=0.008) in splenocytes of the infected rats. The rats had more 4-1BBL expression detected in the spleen. IFN-gamma released by PHA-stimulated splenocytes increased significantly by the recombinant bacteria as compared with controls (P=0.002).
  
  
  CONCLUSIONS
  Salmonella serovar Typhimurium containing 4-1BBL can transfect target genes into antigen presenting cells in vivo, and the expression of exogenous 4-1BBL enhances cellular immunity markedly.
authors.affiliation: Department of Gastroenterology, The First Affiliated Hospital of Soochow University, Key Laboratory of Clinical Immunology of Jiangsu Province, Suzhou 215006, China.
authors.forename: Jian-Xin
authors.initials: JX
authors.lastname: Ye
authors.forename: Yuan-Ting
authors.initials: YT
authors.lastname: Zhang
authors.forename: Xue-Guang
authors.initials: XG
authors.lastname: Zhang
authors.forename: Da-Ming
authors.initials: DM
authors.lastname: Ren
authors.forename: Wei-Chang
authors.initials: WC
authors.lastname: Chen
mesh_terms: D053330:4-1BB Ligand / Q000235:genetics*; D000818:Animals; D000938:Antigen-Presenting Cells / Q000276:immunology; D017252:CD3 Complex / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D007111:Immunity, Cellular / Q000187:drug effects*; D007371:Interferon-gamma / Q000096:biosynthesis; D008297:Male; D010957:Plasmids; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D022562:Salmonella Vaccines / Q000235:genetics* / Q000494:pharmacology*; D012486:Salmonella typhimurium / Q000235:genetics*; D013154:Spleen / Q000166:cytology / Q000276:immunology; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology; D014613:Vaccines, Attenuated
chemical_list: D053330:4-1BB Ligand; D017252:CD3 Complex; D022562:Salmonella Vaccines; D014613:Vaccines, Attenuated; D049452:Green Fluorescent Proteins; D007371:Interferon-gamma