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Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by systemic and persistent inflammation of the joints. Around 50-80% of the RA patients harbour either one or multiple types of autoantibodies, under which also Anti-Citrullinated Protein Antibodies (ACPA). ACPA are highly specific for RA and are strongly associated with the severity of arthritis. The studies described in this thesis aim to explore the structure and biological function of ACPA and the B cells producing these ACPA.The studies highlight the importance of the synovial environment for the survival of ACPA B cells. Moreover, we demonstrate that the variable region of ACPA-IgG differs from that of ACPA-IgM by the introduction of glycans, and that glycans in the constant part of antibodies can be modulated by influences from the micro-environment. This may affect the biological functions of the antibodies. Furthermore, we show that ACPA can bind to so-called Fc gamma receptor I on immune cells which could be a potential effector mechanisms of ACPA. These findings demonstrate that the structure and environment of ACPA play an important role in the ACPA immune response and provide multiple arguments for the active contribution of ACPA in the chronic inflammation of RA.

Although quite some data is available on anti-CarP antibodies, several questions remain unanswered, including the reproducibility of the clinical data on anti-CarP antibodies, such as their presence before disease onset and association with joint damage. It is also unknown whether these findings can be expanded to non-caucasian populations. Furthermore, it is unclear how anti-CarP antibodies are induced, what proteins they recognize, whether they are able to recognize multiple carbamylated proteins and what the characteristics of these autoantibodies are. Here, some of these questions will be answered. In this thesis, I will first discuss the clinical implications of the presence of anti-CarP antibodies compared to RA-specific autoantibodies in both RA patients other relevant conditions (chapters 2 till 7). This is followed by more detailed investigations into the characteristics of anti-CarP antibodies and their antigens (chapters 8 till 12).

Rheumatoid arthritis (RA) is a chronic inflammatory disease with an unknown aetiology characterized by joint destruction. Both genetic and environmental factors contribute to the disease development with HLA-DRB1* alleles and smoking identified as most important. The disease is characterized by the presence of autoantibodies, originally by rheumatoid factor (RF) and more recently by anti citrullinated protein/peptide antibodies (ACPA) and antibodies against carbamylated peptides (CarP). These autoantibodies are present, not only after the onset of disease, but also prior to the onset of symptoms. The development of RA is a gradual process lasting several years before the onset of any joint symptom, but when and if there is a temporal difference in the development both between and within the different antibody systems is currently unknown. B-cells produce the antibodies, and a subset of B-cells, i.e., B-regulatory (Breg) cells, produces interleukin-10, and thus have the ability to down-regulate pro-inflammatory cytokines. Whether the Breg cells are involved in the pathogenesis of RA is, as yet, unknown. The aim of this thesis was to increase knowledge of the pathophysiological processes in the development of RA through identification of factors involved. The analyses involved detection of autoantibodies to post-translationally modified peptides/proteins in addition to RF isotypes, cell surface markers on immune cells in asymptomatic individuals, who have an increased risk of developing RA. In a co-analysis of the registers of patients with RA attending the Department of Rheumatology, with the registers from population based screening programmes within the Biobank of Northern Sweden, blood samples collected from individuals prior to the onset of symptoms were identified, as were those from population control subjects. A cohort of pre-symptomatic individuals also donated samples at the time of receiving a diagnosis of RA. First-degree relatives (FDR) of patients with RA were also identified and included for analyses. The levels of ten different ACPAs, i.e., (fibrinogen (Fib) α563-583(573), Fibα580-600(591), Fibβ62-81a(72), Fibβ62-81b(74), Fibβ36-52, a-enolase (CEP-1), triple helical collagen type II (citC1III), filaggrin (Fil307-324), vimentin (Vim) 2-17, and Vim60-75) were measured using the ImmunoCAP ISAC system (Phadia/ThermoFischer, Uppsala, Sweden) in blood samples from individuals before the onset of symptoms and when diagnosed with RA in comparison with those in population based controls. In a subset of samples, the levels of anti-CarP antibodies were measured using ELISA coated with anti-CarP-FCS, as well as analysis of RF of IgM, IgG and IgA isotype using the EliA assay (Phadia, Uppsala, Sweden). Breg cells were analysed both with and without stimulation ex vivo along with other cell types using flow cytometry in samples from patients with RA, their first degree relatives (FDR) and healthy controls. In paper I it was shown that levels of ACPA were initially restricted to a few antibodies but disseminated over time to involve additional different antibodies. The levels of antibodies to CEP-1, Fibß36-52, and filaggrin were significantly increased. In paper II, anti-CarP antibodies were positive in 5-13% of the individuals negative for the various ACPA studied. The presence of anti-CarP antibodies was significantly related to radiological destruction of joints at baseline, at follow-up after 24 months and to the radiological progress between baseline and 24months. In paper III, the relationships between the frequencies of RF isotypes, the ten different ACPA, anti-CCP2 and anti-CarP antibodies before the onset of any symptoms and the presence of certain combinations of antibodies were associated with a very high risk of developing RA. In paper IV Breg cells from patients with RA are functionally impaired and FDR showed a similar pattern by responding less to stimulation ex vivo than cells from healthy controls. In conclusion, individuals who subsequently develop RA have an increased number and amount of ACPAs, anti-CarP antibodies and RF of IgM, IgG and IgA isotype, several years before symptom onset. Most of the different antibodies analysed remain associated with disease development after adjustments for each separate antibody. In FDRs, Breg cells were functionally altered in that they produce less IL-10 and consequently contribute to a more inflammation-prone status, as in their relatives with RA. These findings contribute to information about the development of RA as well as a given individual’s risk(s) of developing RA and its progression.

Rheumatoid Arthritis (RA) is a chronic inflammatory disorder that typically affects cartilage and bone of small and middle-sized joints. Infiltration of the synovium by inflammatory cells causes destruction of cartilage, erosion of the adjacent bone and ultimately loss of function of the affected joint. Systemic inflammation, often going in parallel, can affect several organs and has long-term impact on organ function. This thesis presents work that investigates several aspects of basic immunological disease mechanisms with relevance to the inflammatory immune response in RA. Specifically, three main research questions triggered the experiments presented and form the outline of this thesis: 1. Do regulatory T cells feature anti-inflammatory properties besides the inhibition of effector T cells, which could help explain their therapeutic effectiveness in a murine model of established arthritis? 2. Are there specific features of the immune response to citrullinated antigens that could contribute to inflammation in RA, and can analysis of these features help in understanding the characteristics of anti citrullinated protein antibody producing B cells and their development? 3. Do certain genetic variants that associate with RA susceptibility contribute also to disease progression, as evidenced by the rate of joint destruction in RA?

The ACPA response is different from conventional antibody responses. The ACPA response is generally of a much lower avidity than recall responses. Avidity maturation is limited and occurs before disease onset. The presence of low avidity ACPA is associated with higher rate of joint destruction. In vitro, the binding of low avidity ACPA to citrullinated epitopes was weakly inhibited by citrullinated antigens and low avidity ACPA have a higher ability to activate the complement system. In addition, there are certain antigens which continuously drive ACPA (IgM) response in RA.