title: The anergy induction of M3 muscarinic acetylcholine receptor-reactive CD4+ T cells suppresses experimental sialadenitis-like Sjögren's syndrome.
abstract: OBJECTIVE
  Autoreactive CD4+ T cells are involved in the pathogenesis of Sjögren's syndrome (SS). The aim of the present study was to clarify the dominant T cell epitopes of M3 muscarinic acetylcholine receptor (M3R) and to establish a new antigen-specific therapy for SS using an experimental mouse model.
  
  
  METHODS
  Production of cytokines from M3R-reactive CD4+ T cells, after culture with various M3R peptides, was analyzed by enzyme-linked immunosorbent assay. Adoptive cell transfer was performed using splenocytes from M3R(-/-) mice that were immunized with M3R peptides or phosphate buffered saline plus H37Ra as a control. Rag1(-/-) mice were inoculated with the splenocytes and examined for the development of sialadenitis. Altered peptide ligands (APLs) of the T cell epitopes, with substitutions in amino acid residues at T cell receptor contact sites, were synthesized, and the ability of the APLs to suppress sialadenitis was evaluated. The mechanisms underlying such effects were assessed.
  
  
  RESULTS
  CD4+ M3R-reactive T cells produced interleukin-17 (IL-17) and interferon-γ (IFNγ) in response to the N-terminal 1 (N1) and 1st extracellular loop peptides of M3R, and Rag1(-/-) mice that received N1- and/or 1st peptide-immunized splenocytes developed sialadenitis. Among the designed APLs, N1-APL7 (N→S at amino acid 15) significantly suppressed IFNγ production in vitro, and also suppressed sialadenitis in vivo. Levels of early growth response 2 in CD4+ T cells from the cervical lymph nodes of N1-APL7-treated mice were significantly higher than those of control mice, and cell proliferation was reversed by administration of exogenous IL-2. Levels of the anergy-related molecules itchy homolog E3 ubiquitin-protein ligase, Casitas B-lineage lymphoma b, gene related to anergy in lymphocytes, and Deltex-1 were significantly higher in CD4+ T cells cultured with N1-APL7.
  
  
  CONCLUSIONS
  The major T cell epitopes were from the N1 and 1st peptide regions. Moreover, N1-APL7, selected as the antagonistic APL in vitro, also suppressed sialadenitis through the induction of anergy. This is a potentially useful strategy for regulating pathogenic T cell infiltration in SS.
authors.affiliation: University of Tsukuba, Tsukuba, Japan.
authors.forename: Hiromitsu
authors.initials: H
authors.lastname: Asashima
authors.affiliation: University of Tsukuba, Tsukuba, Japan.
authors.forename: Hiroto
authors.initials: H
authors.lastname: Tsuboi
authors.affiliation: University of Tsukuba, Tsukuba, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Takahashi
authors.affiliation: University of Tsukuba, Tsukuba, Japan.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Hirota
authors.affiliation: University of Tsukuba, Tsukuba, Japan.
authors.forename: Mana
authors.initials: M
authors.lastname: Iizuka
authors.affiliation: University of Tsukuba, Tsukuba, Japan.
authors.forename: Yuya
authors.initials: Y
authors.lastname: Kondo
authors.affiliation: Fureai Higashitotsuka Hospital, Yokohama, Japan.
authors.forename: Minoru
authors.initials: M
authors.lastname: Matsui
authors.affiliation: University of Tsukuba, Tsukuba, Japan.
authors.forename: Isao
authors.initials: I
authors.lastname: Matsumoto
authors.affiliation: University of Tsukuba, Tsukuba, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Sumida
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D019264:Adoptive Transfer; D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D017635:Clonal Anergy / Q000276:immunology; D004268:DNA-Binding Proteins / Q000378:metabolism; D004195:Disease Models, Animal; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D018398:Homeodomain Proteins / Q000235:genetics; D007371:Interferon-gamma / Q000378:metabolism; D020381:Interleukin-17 / Q000378:metabolism; D007376:Interleukin-2 / Q000494:pharmacology; D008024:Ligands; D051379:Mice; D018345:Mice, Knockout; D050721:Proto-Oncogene Proteins c-cbl / Q000378:metabolism; D043587:Receptor, Muscarinic M3 / Q000235:genetics / Q000276:immunology*; D012793:Sialadenitis / Q000276:immunology*; D012859:Sjogren's Syndrome / Q000276:immunology*; D013154:Spleen / Q000166:cytology; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C118387:Cblb protein, mouse; D004268:DNA-Binding Proteins; D018984:Epitopes, T-Lymphocyte; D018398:Homeodomain Proteins; D020381:Interleukin-17; D007376:Interleukin-2; D008024:Ligands; D043587:Receptor, Muscarinic M3; C064658:RAG-1 protein; D007371:Interferon-gamma; C471637:Itch protein, mouse; C104035:Dtx1 protein, mouse; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases