SignificanceThe presence and development of autoantibodies to citrullinated proteins (ACPAs) are highly associated with rheumatoid arthritis (RA). The mechanisms leading to the production of ACPAs are unknown. Here, we propose a model to explain the emergence of anticitrullinated protein autoantibodies in RA. Indeed, we could trigger the development of anticitrullinated fibrinogen autoantibodies in normal mice by immunization with peptidyl arginine deiminase (PAD), most likely through a hapten/carrier mechanism in which the carrier is the PAD enzyme that performs citrullination and the hapten is any protein being citrullinated (hence bound) by PAD. Our results allow us to understand the birth of anticitrullin autoimmunity.

Objective. The presence of autoantibodies to citrullinated proteins (ACPAs) often precedes the development of rheumatoid arthritis (RA). Citrullines are arginine residues that have been modified by peptidylarginine deiminases (PADs). PAD4 is the target of autoantibodies in RA. ACPAs could arise because PAD4 is recognized by T cells, which facilitate the production of autoantibodies to proteins bound by PAD4. We previously found evidence for this haptencarrier model in mice. This study was undertaken to investigate whether there is evidence for this model in humans.Methods. We analyzed antibody response to PAD4 and T cell proliferation in response to PAD4 in 41 RA patients and 36 controls. We tested binding of 65 PAD4 peptides to 5 HLA-DR alleles (DRB1and selected 11 PAD4 peptides for proliferation studies using samples from 22 RA patients and 27 controls. Peripheral blood lymphocytes from an additional 10 RA patients and 7 healthy controls were analyzed by flow cytometry for CD3, CD4, CD154, and tumor necrosis factor expression after PAD4 stimulation.Results. Only patients with RA had both antibodies and T cell responses to PAD4. T cell response to peptide 8, a PAD4 peptide, was associated with RA (P = 0.02), anti-PAD4 antibodies (P = 0.057), and the shared epitope (P = 0.05).Conclusion. ACPA immunity is associated with antibodies to PAD4 and T cell responses to PAD4 and PAD4 peptides. These findings are consistent with a hapten-carrier model in which PAD4 is the carrier and citrullinated proteins are the haptens.

IntroductionAutoantibodies to citrullinated proteins (ACPAs) are highly associated with rheumatoid arthritis (RA). ACPAs are produced in the absence of T cell responses to citrullinated proteins.ObjectivesPeptidyl arginine deiminase 4 (PAD4), which binds numerous different proteins and citrullinates them, is the target of autoantibodies in early RA. This suggests a model to explain the production of ACPAs in the absence of T cells to citrullinated proteins. ACPAs could arise because, at first, PADs are recognised by T cells, which, in turn help the production of autoantibodies to proteins being citrullinated by PADs, through a hapten/carrier model. Here, we test this model in mice.MethodsHLA-DRB1*04:01 transgenic mice were immunised subcutaneously with PADs or phosphate buffered saline (PBS) in Freund’s complete adjuvant (CFA). Three booster injections of PAD or PBS in Freund’ incomplete adjuvant (IFA) were given subcutaneously 15, 35 and 55 days later. Mice were:tested for anti-PAD antibodies by ELISA.tested for T cell responses to PADs, native or citrullinated fibrinogen 65 days after PAD immunisation.tested for anti-citrullinated fibrinogen antibodies by ELISA using fibrinogen peptides under citrullinated and native form. ResultsHLA-DRB1*04:01 transgenic mice immunised with PADs developed antibodies and T cells to PADs and IgG antibodies to citrullinated peptides from fibrinogen, in the absence of T cell response to native or citrullinated fibrinogen.ConclusionsT cell immunisation to PAD proteins triggers ACPAs through a hapten carrier mechanism in which the carrier is PAD which performs citrullination and the hapten any protein being citrullinated by PAD.Disclosure of interestNone declared