title: Loss of CD4 T-cell-dependent tolerance to proteins with modified amino acids.
abstract: The site-specific incorporation of the unnatural amino acid p-nitrophenylalanine (pNO(2)Phe) into autologous proteins overcomes self-tolerance and induces a long-lasting polyclonal IgG antibody response. To determine the molecular mechanism by which such simple modifications to amino acids are able to induce autoantibodies, we incorporated pNO(2)Phe, sulfotyrosine (SO(3)Tyr), and 3-nitrotyrosine (3NO(2)Tyr) at specific sites in murine TNF-α and EGF. A subset of TNF-α and EGF mutants with these nitrated or sulfated residues is highly immunogenic and induces antibodies against the unaltered native protein. Analysis of the immune response to the TNF-α mutants in different strains of mice that are congenic for the H-2 locus indicates that CD4 T-cell recognition is necessary for autoantibody production. IFN-γ ELISPOT analysis of CD4 T cells isolated from vaccinated mice demonstrates that peptides with mutated residues, but not the wild-type residues, are recognized. Immunization of these peptides revealed that a CD4 repertoire exists for the mutated peptides but is lacking for the wild-type peptides and that the mutated residues are processed, loaded, and presented on the I-A(b) molecule. Overall, our results illustrate that, although autoantibodies are generated against the endogenous protein, CD4 cells are activated through a neo-epitope recognition mechanism. Therefore, tolerance is maintained at a CD4 level but is broken at the level of antibody production. Finally, these results suggest that naturally occurring posttranslational modifications such as nitration may play a role in antibody-mediated autoimmune disorders.
authors.affiliation: Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: Varun
authors.initials: V
authors.lastname: Gauba
authors.forename: Jan
authors.initials: J
authors.lastname: Grünewald
authors.forename: Vanessa
authors.initials: V
authors.lastname: Gorney
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Deaton
authors.forename: Mingchao
authors.initials: M
authors.lastname: Kang
authors.forename: Badry
authors.initials: B
authors.lastname: Bursulaya
authors.forename: Weijia
authors.initials: W
authors.lastname: Ou
authors.forename: Richard A
authors.initials: RA
authors.lastname: Lerner
authors.forename: Christian
authors.initials: C
authors.lastname: Schmedt
authors.forename: Bernhard H
authors.initials: BH
authors.lastname: Geierstanger
authors.forename: Peter G
authors.initials: PG
authors.lastname: Schultz
authors.forename: Teresa
authors.initials: T
authors.lastname: Ramirez-Montagut
mesh_terms: D019943:Amino Acid Substitution; D000596:Amino Acids / Q000235:genetics / Q000276:immunology*; D000818:Animals; D001323:Autoantibodies / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D000939:Epitopes / Q000276:immunology / Q000378:metabolism; D005260:Female; D007108:Immune Tolerance / Q000276:immunology*; D007114:Immunization / Q000379:methods; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D050505:Mutant Proteins / Q000235:genetics / Q000276:immunology; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000235:genetics / Q000276:immunology; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000235:genetics / Q000276:immunology
chemical_list: D000596:Amino Acids; D001323:Autoantibodies; D000939:Epitopes; D050505:Mutant Proteins; D014409:Tumor Necrosis Factor-alpha; C040239:4-nitrophenylalanine; C005662:tyrosine O-sulfate; C002744:3-nitrotyrosine; D014443:Tyrosine; D010649:Phenylalanine; D004815:Epidermal Growth Factor