Peptidylarginine Deiminase Autoimmunity and the Development of Anti-Citrullinated Protein Antibody in Rheumatoid Arthritis: The Hapten-Carrier Model

Abstract

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 hapten-carrier 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 (DRB1*04:01, *04:02, *04:04, *01:01, and *07:01) and 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.

Figure 1

Antibody responses to human peptidylarginine deiminase 4 (PAD4) in patients with anti–citrullinated protein antibody–positive rheumatoid arthritis (RA; n = 41), patients with psoriatic arthritis (PsA; n = 25), and healthy controls (n = 11). Subjects were tested for IgM anti‐PAD4 and IgG anti‐PAD4 by enzyme‐linked immunosorbent assay. Each antibody assay was performed in duplicate. The OD ratio was the ratio of the OD for a well with serum and PAD4 protein to the OD for a well with serum but without PAD4 protein. Positivity was defined as an OD ratio of >3 for IgM and an OD ratio of >2 for IgG (dotted lines). Symbols represent individual subjects; red lines indicate the mean OD ratio.

Figure 2

Proliferation of T cells in response to human PAD4, native fibrinogen (FB), citrullinated fibrinogen (cit FB), and phytohemagglutinin (PHA) in patients with RA, patients with PsA, and healthy controls. Proliferative response was evaluated by bromodeoxyuridine incorporation (n = 4 replicates per protein). The OD ratio was the ratio of the OD for a well with cells and protein to the OD for a well with cells but without protein. Positivity was defined as an OD ratio of >2 (dotted lines). Symbols represent individual subjects; red lines show the mean OD ratio. See Figure 1 for other definitions.

Figure 3

Antibody response to PAD4 and proliferation of T cells in response to PAD4 in patients with RA (n = 41), patients with PsA (n = 25), and healthy controls (n = 11). Subjects were tested for antibody response to PAD4 and proliferative response to PAD4 and classified into the following 4 groups: negative for PAD4 antibody (Ab−) and positive for T cell proliferation (Tc+), positive for both PAD4 antibody and T cell proliferation, negative for both PAD4 antibody and T cell proliferation, or positive for PAD4 antibody and negative for T cell proliferation. Values are the number of shared epitope (SE)–positive subjects/total number of subjects in the indicated group. See Figure 1 for other definitions.

Figure 4

Binding of peptidylarginine deiminase 4 (PAD4) peptides to 5 different HLA–DR alleles. The binding of each purified HLA–DR allele was assayed on enzyme‐linked immunosorbent assay plates coated with 65 PAD4 peptides (each in duplicate wells). Two control wells were coated with a positive binder, hemagglutinin peptide. Positive binding was defined as an OD value equal to that for hemagglutinin peptide. Open boxes indicate no binding, red boxes indicate binding to a shared epitope–positive allele, and blue boxes indicate binding to a shared epitope–negative allele.

Figure 5

Proliferation of T cells in response to PAD4 peptides in patients with RA, patients with PsA, and healthy controls. Proliferative response was evaluated by bromodeoxyuridine incorporation (n = 4 replicates per peptide). The OD ratio was the ratio of the OD for a well with cells and peptide to the OD for a well with cells but without peptide. Positivity was defined as an OD ratio of >2 (dotted lines). Symbols represent individual subjects; red lines show the mean OD ratio. See Figure 1 for definitions.

Figure 6

Antibody response to PAD4 and proliferation of T cells in response to peptide 8 in patients with RA (n = 22), patients with PsA (n = 16), and healthy controls (n = 11). Subjects were tested for antibody response to PAD4 and proliferative response to peptide 8 and classified into the following 4 groups: negative for PAD4 antibody (Ab−) and positive for T cell proliferation (Tc+), positive for both PAD4 antibody and T cell proliferation, negative for both PAD4 antibody and T cell proliferation, or positive for PAD4 antibody and negative for T cell proliferation. Values are the number of shared epitope (SE)–positive subjects/total number of subjects in the indicated group. See Figure 1 for other definitions.