Indeed, the article that was supposed to have demonstrated the point studied the binding of one unique citrullinated vimentin peptide to three shared epitope-positive and five shared epitope-negative HLA-DRB1 alleles (11). early RA. This suggests a model for the emergence of ACPAs in the absence of detectable T cells specific for citrullinated antigens: ACPAs could arise because PADs are recognized by T cells, which help the production of autoantibodies to proteins bound by PADs, according to a hapten/carrier model. Here, we tested this model in normal mice. C3H are healthy mice whose IEk chain is highly homologous to the 1 chain HLA-DRB1*04:01, the allele most strongly associated with RA in humans. C3H mice immunized with PADs developed antibodies and T cells to PAD and IgG antibodies to citrullinated fibrinogen peptides, in the absence of a T cell response to fibrinogen. To analyze the MHC background effect on hapten/carrier immunization, we immunized DBA/2 mice (whose IEd chain is similar to that of HLA-DRB1*04:02, an HLA-DR4 subtype not associated with RA). DBA/2 mice failed to develop antibodies to citrullinated fibrinogen peptides. Thus, T cell immunization to PAD proteins may trigger ACPAs through a hapten/carrier mechanism. This may constitute the basis for a new mouse model of ACPA-positive RA. Rheumatoid arthritis (RA), a chronic inflammatory joint disease, is associated with HLA-DRB1 alleles expressing the shared epitope-like HLA-DRB1*04:01 and HLA-DRB1*01:01 (1C4). RA is usually preceded by the development of anticitrullinated protein autoantibodies (ACPAs) (5, 6). ACPAs recognize citrullins [citrullin is an amino acid obtained by posttranslational modification of arginin by enzymes called Peptidyl Arginyl Deiminases (PADs)] on many different proteins like fibrin, vimentin, enolase, collagen, and so forth (7C10). The function Rabbit Polyclonal to CLIP1 of HLA-DR molecules is to present peptides to helper T cells to help the production of IgG antibodies by B cells. Therefore, an obvious explanation for the association of RA with HLA-DR and ACPAs Posaconazole could be that HLA-DRCrestricted T cells might help antibody responses to the many different citrullinated proteins known to be recognized by ACPAs. However, T cells specific for citrullinated proteins have not been identified so far. Furthermore, this explanation Posaconazole implies that RA-associated HLA-DR alleles bind citrullinated peptides to allow their presentation to T cells. This citrullinated peptide binding hypothesis is controversial, supported by very limited binding data (11) and contradicted by extensive binding data on fibrinogen, vimentin, collagen, and EpsteinCBarr virus peptides under native and citrullinated forms (12C14). While trying to identify new autoantibodies in patients with early or late RA by using human protein chips, we found that IgG antibodies to PAD4, one of the five isotypes of PAD, are present in 20% of patients with early and 40% of patients with late RA (15). This finding led us to propose the hypothesis that PAD4 is the T cell target whose recognition provides help for the production of autoantibodies to citrullinated proteins (ACPAs), according to a classical hapten/carrier model. Here, we Posaconazole demonstrate this model in normal mice. Results Anti-PAD Antibodies in C3H Mice Immunized with PADs. C3H mice were immunized with murine or human PAD2, or murine or human PAD4, or PBS in Freunds complete adjuvant (CFA). Three booster injections with PAD or PBS in Freunds incomplete adjuvant (IFA) Posaconazole were given s.c. 15, 35, and 55 d later (Fig. 1). IgG responses to PADs were analyzed by ELISA. Sera were diluted at 1/40. Positive sera were defined by an OD value higher than twice the background OD (obtained by adding each serum to a well without PAD). Open in a separate window Fig. 1. Model and methods. Shown are the immunization protocol (= 6 10?14) (Fig. 2). In C3H mice immunized with murine PAD2, the average background OD was 0.06, and the average test OD was 1 after the first immunization, 1.4 after the second immunization, 1.8 after the third Posaconazole immunization, and 1.7 after the fourth immunization (Fig. 2= 6 10?6) (Fig. 3). Open in a separate window Fig. 3. T cell responses to PADs in C3H mice. Spleen and lymph nodes were obtained at 65 d postimmunization for PBS-immunized mice (= 0.014). Open in a separate window Fig. 4. Citrullinated peptides recognized by the sera of C3H mice immunized with PADs. ELISA plates were coated with 46 citrullinated peptides and blocked with BSA. Sera from primed mice obtained at 55 and 65 d postimmunization were diluted at 1/80. After washing, peroxidase-conjugated antimurine IgG was added. The OD was read at 405 nm. The background OD was obtained by adding each.