A Comprehensive Evaluation of the Relationship Between Different IgG and IgA Anti-Modified Protein Autoantibodies in Rheumatoid Arthritis.

Seropositive rheumatoid arthritis (RA) is characterized by the presence of rheumatoid factor (RF) and anti-citrullinated protein autoantibodies (ACPA) with different fine-specificities. Yet, other serum anti-modified protein autoantibodies (AMPA), e.g. anti-carbamylated (Carb), -acetylated (KAc), and malondialdehyde acetaldehyde (MAA) modified protein antibodies, have been described. In this comprehensive study, we analyze 30 different IgG and IgA AMPA reactivities to Cit, Carb, KAc, and MAA antigens detected by ELISA and autoantigen arrays in N=1985 newly diagnosed RA patients. Association with patient characteristics such as smoking and disease activity were explored. Carb and KAc reactivities by different assays were primarily seen in patients also positive for anti-citrulline reactivity. Modified vimentin (mod-Vim) peptides were used for direct comparison of different AMPA reactivities, revealing that IgA AMPA recognizing mod-Vim was mainly detected in subsets of patients with high IgG anti-Cit-Vim levels and a history of smoking. IgG reactivity to acetylation was mainly detected in a subset of patients with Cit and Carb reactivity. Anti-acetylated histone reactivity was RA-specific and associated with high anti-CCP2 IgG levels, multiple ACPA fine-specificities, and smoking status. This reactivity was also found to be present in CCP2+ RA-risk individuals without arthritis. Our data further demonstrate that IgG autoreactivity to MAA was increased in RA compared to controls with highest levels in CCP2+ RA, but was not RA-specific, and showed low correlation with other AMPA. Anti-MAA was instead associated with disease activity and was not significantly increased in CCP2+ individuals at risk of RA. Notably, RA patients could be subdivided into four different subsets based on their AMPA IgG and IgA reactivity profiles. Our serology results were complemented by screening of monoclonal antibodies derived from single B cells from RA patients for the same antigens as the RA cohort. Certain CCP2+ clones had Carb or Carb+KAc+ multireactivity, while such reactivities were not found in CCP2- clones. We conclude that autoantibodies exhibiting different patterns of ACPA fine-specificities as well as Carb and KAc reactivity are present in RA and may be derived from multireactive B-cell clones. Carb and KAc could be considered reactivities within the "Cit-umbrella" similar to ACPA fine-specificities, while MAA reactivity is distinctly different.

Grönwall C ，Liljefors L ，Bang H ，Hensvold AH ，Hansson M ，Mathsson-Alm L ，Israelsson L ，Joshua V ，Svärd A ，Stålesen R ，Titcombe PJ ，Steen J ，Piccoli L ，Sherina N ，Clavel C ，Svenungsson E ，Gunnarsson I ，Saevarsdottir S ，Kastbom A ，Serre G ，Alfredsson L ，Malmström V ，Rönnelid J ，Catrina AI ，Lundberg K ，Klareskog L ... -  《Frontiers in Immunology》

Different Hierarchies of Anti-Modified Protein Autoantibody Reactivities in Rheumatoid Arthritis.

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. 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 histone-derived peptides with different posttranslational modifications (JPT Histone Code), covering >207,000 peptides (>7,800 gene products). 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 citrullinated peptides. 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-carbamylated and anti-citrullinated 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.

Sahlström P ，Hansson M ，Steen J ，Amara K ，Titcombe PJ ，Forsström B ，Stålesen R ，Israelsson L ，Piccoli L ，Lundberg K ，Klareskog L ，Mueller DL ，Catrina AI ，Skriner K ，Malmström V ，Grönwall C ... -  《-》

Differential ACPA Binding to Nuclear Antigens Reveals a PAD-Independent Pathway and a Distinct Subset of Acetylation Cross-Reactive Autoantibodies in Rheumatoid Arthritis.

Lloyd KA ，Wigerblad G ，Sahlström P ，Garimella MG ，Chemin K ，Steen J ，Titcombe PJ ，Marklein B ，Zhou D ，Stålesen R ，Ossipova E ，Lundqvist C ，Ekwall O ，Rönnelid J ，Mueller DL ，Karlsson MCI ，Kaplan MJ ，Skriner K ，Klareskog L ，Wermeling F ，Malmström V ，Grönwall C ... -  《Frontiers in Immunology》

Presence of autoantibodies in "seronegative" rheumatoid arthritis associates with classical risk factors and high disease activity.

Reed E ，Hedström AK ，Hansson M ，Mathsson-Alm L ，Brynedal B ，Saevarsdottir S ，Cornillet M ，Jakobsson PJ ，Holmdahl R ，Skriner K ，Serre G ，Alfredsson L ，Rönnelid J ，Lundberg K ... -  《-》

Carbamylated vimentin represents a relevant autoantigen in Latin American (Cuban) rheumatoid arthritis patients.

Martínez G ，Gómez JA ，Bang H ，Martínez-Gamboa L ，Roggenbuck D ，Burmester GR ，Torres B ，Prada D ，Feist E ... -  《-》