title: Carboxyl-terminal fusion of E7 into Flagellin shifts TLR5 activation to NLRC4/NAIP5 activation and induces TLR5-independent anti-tumor immunity.
abstract: Flagellin has the capacity to activate both Toll-like receptor 5 (TLR5) and Nod-like receptor C4 (NLRC4)/neuronal apoptosis inhibitory protein 5 (NAIP5) inflammasome signaling. We fused E7m (the inactivated E7 of human papillomavirus) to either end of the flagellin protein, and the resulting recombinant flagellin-E7m proteins (rFliCE7m and rE7mFliC) were used as immunogens. Both fusion proteins activated receptor signaling to different degrees. rE7mFliC-induced TLR5 activity was 10-fold higher than that of rFliCE7m, whereas rFliCE7m activated the NLRC4/NAIP5 pathway more strongly. Therefore, these recombinant proteins provided a tool to investigate which signaling pathway is critical for the induction of antigen-specific T cell responses and anti-tumor immunity. We demonstrated that rFliCE7m induced higher levels of E7-specific IFN-gamma-secreting cells and cytotoxic T lymphocytes (CTLs) than rE7mFliC, and a single injection with rFliCE7m but not rE7mFliC inhibited E7-expressing tumor growth in vivo. Furthermore, we confirmed that CD8(+) T cells played a major role in the anti-tumor immunity induced by rFliCE7m. These findings suggested that the NLRC4/NAIP5 intracellular signaling pathway was critical for the induction of anti-tumor immunity. These observations provide important information for the rational design of flagellin-based immunotherapy.
authors.affiliation: National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan Town, Miaoli 350, Taiwan ROC.
authors.forename: Kuo-Hsing
authors.initials: KH
authors.lastname: Lin
authors.affiliation: National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan Town, Miaoli 350, Taiwan ROC.|Institute of Biotechnology and Department of Life Science, National Tsing-Hua University, Hsinchu 305, Taiwan ROC.
authors.forename: Li-Sheng
authors.initials: LS
authors.lastname: Chang
authors.affiliation: National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan Town, Miaoli 350, Taiwan ROC.
authors.forename: Chun-Yuan
authors.initials: CY
authors.lastname: Tian
authors.affiliation: National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan Town, Miaoli 350, Taiwan ROC.
authors.forename: Yi-Chen
authors.initials: YC
authors.lastname: Yeh
authors.affiliation: National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan Town, Miaoli 350, Taiwan ROC.|Institute of Biotechnology and Department of Life Science, National Tsing-Hua University, Hsinchu 305, Taiwan ROC.
authors.forename: Yu-Jie
authors.initials: YJ
authors.lastname: Chen
authors.affiliation: Immunology Research Center, National Health Research Institutes, Zhunan Town, Miaoli 350, Taiwan ROC.
authors.forename: Tsung-Hsien
authors.initials: TH
authors.lastname: Chuang
authors.affiliation: National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan Town, Miaoli 350, Taiwan ROC.|Graduate Institute of Immunology, China Medical University, Taichung 402, Taiwan ROC.
authors.forename: Shih-Jen
authors.initials: SJ
authors.lastname: Liu
authors.affiliation: National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan Town, Miaoli 350, Taiwan ROC.|Graduate Institute of Immunology, China Medical University, Taichung 402, Taiwan ROC.
authors.forename: Chih-Hsiang
authors.initials: CH
authors.lastname: Leng
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D053468:CARD Signaling Adaptor Proteins / Q000378:metabolism*; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D002460:Cell Line; D002942:Circular Dichroism; D058501:Enzyme-Linked Immunospot Assay; D005260:Female; D005408:Flagellin / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D007113:Immunity, Innate; D007371:Interferon-gamma / Q000032:analysis / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D051616:Neuronal Apoptosis-Inhibitory Protein / Q000378:metabolism*; D050725:Papillomavirus E7 Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D051198:Toll-Like Receptor 5 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
chemical_list: D053468:CARD Signaling Adaptor Proteins; D051616:Neuronal Apoptosis-Inhibitory Protein; D050725:Papillomavirus E7 Proteins; D011993:Recombinant Fusion Proteins; D051198:Toll-Like Receptor 5; C059731:oncogene protein E7, Human papillomavirus type 16; D005408:Flagellin; D007371:Interferon-gamma