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.loss of tolerance | autologous antibody response | protein nitration P reviously we showed that the site-specific incorporation of the unnatural amino acid p-nitrophenylalanine (pNO 2 Phe) into a protein can induce autoantibodies against the endogenous, unmodified protein (1, 2). The murine proteins TNF-α and retinolbinding protein (RBP4) were modified genetically to incorporate pNO 2 Phe at several surface-exposed positions and were used as immunogens. Vaccination of mice with these altered proteins resulted in an IgG polyclonal antibody response against the endogenous proteins that lasted for at least 40 wk. Experiments involving hybridomas generated from the TNF-α-immunized mice revealed that the IgG antibody response was directed against different regions of the protein and not exclusively against the region containing the pNO 2 Phe residue. The autoantibodies generated by the mutated proteins were tested in an endotoxemia model for their ability to neutralize wild-type TNF-α. Protection from endotoxemia indicated that vaccination with pNO 2 Phemodified self-protein is able to elicit physiologically relevant autoantibody responses. Although these initial studies implicated a role for CD4 T cells in autoantibody production, the exact mechanism by which the site-specific incorporation of pNO 2 Phe breaks immunological self-tolerance remained to be elucidated.Recent studies have indicated that postt...

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