Study of autoimmune reactions in rheumatoid arthritis

Rheumatoid arthritis (RA) is a systemic autoimmune disease that affects approximately 1% of the population worldwide. Despite being rather common, the etiopathology of RA remains unclear. Approximately two-thirds of patients have antibodies to citrullinated proteins (ACPAs), generally detected using the 2nd generation cyclic citrullinated peptide (CCP2) ELISA. A wealth of evidence implicates ACPA in the etiology of RA: the major risk factors - a group of HLA-DRB1 alleles referred to as the shared epitope (SE), and smoking - have been described to associate primarily with the ACPA-positive subset of RA. Moreover, ACPAs can be detected years before clinical onset, and their presence are highly predictive of progression to RA. However, the mechanism by which ACPAs might contribute to disease, as well as the definitive in vivo target, is not understood. The aim of this thesis was therefore to better characterize the ACPA response in RA, in terms of antigen specificity, association with genetic and environmental risk factors, cross-reactivity with carbamylated antigens, presence in CCP2-negative RA, and finally the antigen specificity and functional characteristics of ACPA-positive B cells.

Through studies using affinity purified anti-CCP2 IgG, we could demonstrate that the CCP2 ELISA directly captures the ACPA response, and that ACPA purified in this manner bound a variety of citrullinated peptide epitopes and exhibited binding to RA synovial tissue and immune cells (Study I). We also discovered that purified ACPA could bind both carbamylated and citrullinated proteins, and peptide absorption experiments confirmed extensive cross-reactivity between ACPA and anti-carbamylated protein (CarP) antibodies in the context of the candidate autoantigen α-enolase, casting doubt on the specificity of the anti-CarP response, which we posit may be cross-reactive ACPA (Study II). By screening 2,836 serum samples from the population-based case-control cohort EIRA on an autoantigen multiplex array, we then showed that “seronegative” RA is not truly a seronegative disease subset. Autoantibodies - including ACPA and rheumatoid factor (RF) - were present in a substantial proportion, and this subset resembled seropositive RA in terms of associations with risk factors (Study III). This study highlights the need for new biomarkers, better classification of seronegative RA, and more sensitive clinical tests for seropositive RA. Finally, we utilized a method of B cell immortalization to derive ACPA-producing B cell clones from RA synovial fluid that retained surface immunoglobulin expression. We successfully generated a CEP-1-positive B cell clone from a SE-positive RA patient, and visualized surface binding to citrullinated (but not native) protein (Study IV). This pilot study lays the ground for in-depth investigation of the characteristics of the ACPA lymphocyte population, specifically in regards to HLA-DRB1 SE-mediated antigen presentation.

It is my hope that the data presented in this thesis can provide a basis for future studies into the putative specificity and mechanism of the ACPA response, in order to elucidate disease processes in RA and ultimately improve the diagnosis and treatment of the disease.

List of scientific papers

I. Ossipova E, Cerqueira CF, Reed E, Kharlamova N, Israelsson L, Holmdahl R, Nandakumar KS, Engström M, Harre U, Schett G, Catrina A, Malmström V, Sommarin Y, Klareskog L, Jakobsson PJ, Lundberg K. Affinity purified anti-citrullinated protein/peptide antibodies target antigens expressed in the rheumatoid joint. Arthritis Res Ther. 2014;16(4):R167.
https://doi.org/10.1186/ar4683

II. Reed E, Jiang X, Kharlamova N, Ytterberg AJ, Catrina A, Israelsson L, Mathsson-Alm L, Hansson M, Alfredsson L, Rönnelid J, Lundberg K. Antibodies to carbamylated α-enolase epitopes in rheumatoid arthritis also bind citrullinated epitopes and are largely indistinct from anti-citrullinated protein antibodies. Arthritis Res Ther. 2016;18(1):96.
https://doi.org/10.1186/s13075-016-1001-6

III. Reed E, Hedström AK, Hansson M, Mathsson-Alm L, Brynedal B, Saevarsdottir S, Jakobsson PJ, Holmdahl R, Skriner K, Serre G, Alfredsson L, Rönnelid J, Lundberg K. How seronegative is ”seronegative” RA – Characterization of autoantibody responses in anti-cyclic citrullinated peptide antibody positive and –negative rheumatoid arthritis. [Manuscript]

IV. Reed E, Germar K, van Uden N, Hansson M, Israelsson L, Rizzi T, Kwakkenbos M, de Vries N, Spits H, Lundberg K, Baeten D, Malmström V. Isolation, immortalization and characterization of an anti-citrullinated protein antibody producing B cell clone from rheumatoid arthritis synovial fluid. [Manuscript]