Different Hierarchies of Anti–Modified Protein Autoantibody Reactivities in Rheumatoid Arthritis

Abstract

Objective. Anti-citrullinated protein antibodies (ACPAs) are a hallmark of seropositive rheumatoid arthritis (RA). Yet, the precise disease-relevant autoantigens that are targeted by ACPAs remains a matter of debate. This study utilized patient-derived monoclonal ACPAs, rather than serum autoantibody analysis, to characterize the multireactivity to different protein modifications and to reveal autoantibody subsets in patients with RA. Methods. Twelve human monoclonal ACPAs (positive by the second-generation cyclic citrullinated peptide test) were generated from 6 RA patients, and a head-to-head comparison of their reactivities was performed. For profiling, we used a complementary DNA-based protein array (Engine GmbH) and 3 peptide-screening platforms with RA autoantigens (Thermo Fisher Scientific), citrullinated and carbamylated peptides (NimbleGen/Roche), or histonederived peptides with different posttranslational modifications (JPT Histone Code), covering >207,000 peptides (>7,800 gene products). Results. The fine-specificity profiles of the investigated ACPAs varied, but all of the monoclonal ACPAs displayed multireactivity to a large number of citrullinated peptides/proteins, each characterized by specific binding properties. ACPA subsets could be defined by clone-distinct consensus binding motifs (e.g., Cit-Gly, Gly-Cit, or Arg-Cit-Asp), with the most common ACPA recognition being that of a Gly in the +1 flanking position, but with additional amino acid preferences. For ACPA protein recognition, we observed a preference for citrullinated RNA-binding proteins with high Arg/Gly content. Six of the 12 ACPA clones also bound acetylated-lysine (KAc) or homocitrulline peptide motifs, displaying a similar affinity or higher apparent affinity than that for Cit peptides. Conclusion. ACPAs and anti-modified protein autoantibodies represent overlapping facets of RA autoimmunity and bind to a wide variety of modified proteins, extending well beyond the historically recognized set of RA autoantigens. So far, KAc reactivity has been detected only in the context of anti-Carb and anti-Cit peptide autoantibody responses, postulating the existence of hierarchies of autoreactivity in RA. Future investigations of ACPA fine specificities and functionality should take into consideration the presence of consensus Cit/Carb/KAc motifs and the multireactivity of these autoantibodies in patients with RA.

Shared recognition of citrullinated tenascin-C peptides by T and B cells in rheumatoid arthritis

“…Since it has been recently shown that ACPA can cross-react against multiple peptides (especially those that contain a common citrulline-glycine motif; ref. 34), we examined the specificity and potential cross-reactivity between cit-TNC17-and cit-TNC56-reactive antibodies by performing inhibition experiments using sera from subjects that were double-reactive for cit-TNC17 and cit-TNC56 antibodies. Absorption by the homologous peptide was efficient for both cit-TNC17 and cit-TNC56 ( Figure 5, left panels), while cross-reactivity was limited; only partial inhibition was observed in serum 2 (maximum inhibition: 24.8% for cit-TNC56 versus cit-TNC17) and serum 6 (maximum inhibition: 22% for cit-TNC17 versus cit-TNC56 and 43% for citTNC56 versus cit-TNC17) ( Figure 5, middle panels).…”

“…ACPA and rheumatoid factor are diagnostic in RA, can precede disease by up to 10 years, and are thought to participate in joint pathology (2)(3)(4). ACPA have been shown to target multiple citrullinated antigens, with some exhibiting narrow or single-antigen specificity and others demonstrating substantial cross-reactivity toward shared consensus motifs (34). Notably, the number of ACPA specificities expands as individuals progress from pre-RA to develop inflammatory arthritis (4).…”

“…In some sense, the positive relationship between cit-TNCreactive antibodies and anti-CCP positivity is expected, given that polyclonal anti-CCP pools reacted to cit-TNC17, cit-TNC56, and cit-TNC5. Indeed, one confounding factor for cit-TNC17 in particular is the presence of a highly conserved citrulline-glycine "shared motif " within this peptide sequence that has been shown be recognized by highly multireactive ACPA (7,34), such that these antibodies cannot be strictly defined as being cit-TNC specific. Therefore, it could be argued that an unknown proportion of cit-TNC17-reactive antibodies are multireactive ACPA with no discernible primary specificity.…”

Anti-carbamylated protein antibody isotype pattern differs between palindromic rheumatism and rheumatoid arthritis

“… 22 However, even a highly specific ACPA monoclonal antibody can cross-react toward various post translational modifications (citrulline, homocitrulline and acetylide). 23 , 24 Likewise, immunization with a specific antigen can generate different AMPA. 25 An individual patient may have various AMPA clones with distinct reactivity profiles capable of recognizing multiple amino acid motifs, rather than specific proteins.…”

“… 25 An individual patient may have various AMPA clones with distinct reactivity profiles capable of recognizing multiple amino acid motifs, rather than specific proteins. 23 – 25 It might be speculated that PR AMPA clones have a narrowed amino acid motif recognition profile, ensuing a lower cross reactivity.…”