The research described in this thesis focused on the use of bioorthogonal antigens to investigate immunological processes in antigen presenting cells. Bioorthogonal antigens are antigenic proteins... Show moreThe research described in this thesis focused on the use of bioorthogonal antigens to investigate immunological processes in antigen presenting cells. Bioorthogonal antigens are antigenic proteins produced through recombinant expression in a methionine auxotrophic E. coli strain. This allows for the replacement of methionine residues with the bioorthogonal non-canonical amino acid, azidohomoalanine (Aha), that resembles methionine. Aha contains an azide group that enables the selective and rapid visualization or enrichment of the antigen after a biological experiment using alkyne-modified fluorophores or alkyne-containing resins, respectively, via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The research involved studying the effects of post-translational modifications (PTMs), antigen complexation and glycosylation of antibodies in immune complexes on the uptake, proteolysis, and T cell activation by dendritic cells (DCs) of Aha-containing antigens. Additionally, a new method was developed to enrich low abundant bioorthogonal antigenic fragments from complex mixtures. This method can be used in future studies to identify processed Aha-containing fragments from immune cells that are preserved for T cell presentation. Show less
Rheumatoid arthritis (RA) is an auto-inflammatory disease, affecting ~1% of the world population. RA is hallmarked by the presence of autoantibodies, one of the well-known autoantibodies in RA are... Show moreRheumatoid arthritis (RA) is an auto-inflammatory disease, affecting ~1% of the world population. RA is hallmarked by the presence of autoantibodies, one of the well-known autoantibodies in RA are the anti-citrullinated protein antibodies (ACPA). ACPA is highly specific for RA and about 70-80% of the RA patients are positive for ACPA. Previously, we discovered that ACPA-IgG are extensively glycosylated in the variable (V) region. In this thesis, we determined that over 90% of the ACPA-IgG carries glycans in the v-domain were as only 17% of the conventional IgG carries additional glycans. Additionally, the glycans were highly sialylated. Intriguingly, we also showed that the glycans are introduced via somatic hyper mutations in the germinal center reaction. Furthermore, we discovered that ACPA-IgG v-domain glycosylation is a predictive marker for the development of ACPA positive RA. Lastly, we found that the binding strength is not influenced by the glycans but it influences the amount of available binding sites. Nevertheless, the ACPA-BCR still overcome negative selection which might suggest that the glycans can trigger an alternative way for positive selection in the germinal center. This will be subject for further studies regarding the role of ACPA-IgG v-domain glycosylation. Show less
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... Show moreRheumatoid 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. Show less
