Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a prototypic autoimmune disease, with a subset of AAV patients manifesting anti-myeloperoxidase (MPO) IgG. Patients with AAV respond positively to B cell-targeting and complement-targeting therapies, but disease flares are not uncommon. Here, by comparing samples from healthy individuals and MPO+ AVV patients, we show that B cell autoreactivity against MPO in the circulation of patients is dominated by CD27+IgM+ B cells whereas MPO-specific IgG+ cells are infrequent. Additionally, while naive anti-MPO-IgM B cells are present in both patients and controls and produce anti-MPO IgM upon stimulation, anti-MPO-IgM memory B cells and serum anti-MPO IgM are features of patients. Our results thus hint that defective elimination of B cell reactivity to MPO in the human repertoire, the presence of activated IgM+ anti-MPO B cells in disease, and a dominant role for anti-MPO IgM in complement activation, may all contribute to MPO+ AAV etiology and thereby serve as potential target for therapy.

Background: Rheumatoid arthritis (RA) is a prevalent autoimmune disease characterized by B-cells that produce autoantibodies against post translationally modified proteins, such as anti-citrullinated protein antibodies (ACPA), anti-acetylated protein antibodies (AAPA) and anti-carbamylated protein antibodies (a-CarP), together called anti-modified protein antibodies (AMPA). What initially leads to the breach in tolerance and eventually causes rheumatoid arthritis is still unknown, but there are indications, such as changes in the fecal microbiome composition of RA patients, that this tolerance breach takes place in the intestines. Objectives: Since antibody responses in the intestines are dominated by IgA, particularly in dimeric form, we set out to characterize the AMPA IgA response in detail. Methods: Sera from ACPA-positive RA, ACPA-negative RA and healthy donors were fractionated using size exclusion chromatography, separating proteins on size. Next, all fractions were tested by ELISA for ACPA IgA, AAPA IgA and anti-CarP IgA, as well as total IgM, IgA and IgG. Additionally, AMPA were purified from serum to determine their size by IgA western blot and to investigate the IgA tailpiece by tandem mass spectrometry (MS/MS). Lastly, monoclonal IgA was produced with both tailpiece variants, after which its ability to polymerize was analyzed. Results: Intriguingly, of all AMPA IgA, ~70% was found to be polymeric, while total IgA contained only ~20% polymeric IgA. MS/MS analysis furthermore showed a difference in the AMPA IgA tailpiece: in the dimeric fraction ~60% had the complete tailpiece, while in the monomeric fraction this was ~20%. Recombinant IgA with such a complete tailpiece formed up to ~90% polymers, while this was ~40% in IgA with a truncated tailpiece. Conclusion: These data indicate that AMPA IgA responses are differently regulated compared to the total IgA antibody response and that the tailpiece might be related to a difference in polymer formation. Moreover, the high abundance of polymeric AMPA IgA suggests a mucosal origin. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests: Anouk G. van Mourik: None declared, Sanne Reijm: None declared, Alice Bacon: None declared, Myrthe A.M van Delft: None declared, Nivine Levarht: None declared, Rayman Tjokrodirijo: None declared, Peter van Veelen: None declared, Leendert A Trouw Research funding from INOVA diagnostics, part of Werfen, Rene E.M. Toes Research funding from INOVA diagnostics, part of Werfen, Karin A. van Schie: None declared, Diane van der Woude Galapagos, Galapagos.

Background ANCA-associated vasculitides (AAV) are characterized by recurrent, chronic small vessel inflammation and deleterious organ damage. The main targets of ANCA are myeloperoxidase (MPO) and proteinase 3 (PR3). ANCA, B cells and the complement system are crucial to AAV pathogenesis, as evidenced by the clinical benefit of B cell depletion with rituximab and, more recently, the C5a receptor antagonist avacopan. While ANCA in serum have been studied extensively, phenotypic and functional characteristics of the underlying B cell responses remain largely unknown. Objectives To develop a flow cytometry-based technique for identifying MPO-specific B cells in the circulation of MPO-positive AAV patients in order to characterize this B cell response and its potential contribution to disease pathogenesis. Methods Human neutrophil-derived MPO was conjugated to two different fluorochromes and used to identify MPO-specific B cells by flow cytometry. An antigen-specific staining protocol was developed and validated using MPO- and PR3-specific hybridoma cells. MPO-specific B cells were phenotypically characterized and isolated from the peripheral blood of AAV patients by fluorescence activated cell sorting (FACS) and cultured as single cells. MPO-specificity was confirmed by ELISA on culture supernatants. B cell receptor (BCR) sequences were obtained from MPO-positive clones by full length ARTISAN-PCR and Sanger sequencing. MPO-specific IgG and IgM monoclonal antibodies (mAb) were produced to validate specificity and to examine their ability to activate complement. Finally, MPO-positive AAV patient plasma and plasma depleted of IgG or IgM was tested in in vitro complement assays. Results The newly developed, differential antigen labelling approach successfully identified MPO-specific but not PR3-specific hybridoma cells. Subsequently, we detected MPO-specific B cells in the circulation of MPO-positive AAV patients at a frequency of up to 1:1000 B cells. FACS sorting and single cell culture yielded an enrichment of MPO-specificity of ~80%. Notably, the majority of isolated, MPO-specific B cells (60-95%) displayed an IgM memory phenotype, which corresponded to the presence of anti-MPO IgM in plasma. The remainder of the MPO-specific cells were mainly IgG memory B cells and few naive cells. BCR sequencing revealed a polyclonal IgM response with diverse V-gene usage, consisting of both germline and highly mutated clones. Generation of mAb (n=5) confirmed MPO specificity by inhibition ELISA for both germline and somatically mutated clones. Interestingly, anti-MPO IgM mAb showed a high capacity for complement factor deposition upon MPO binding. MPO-specific complement assays with IgG- and IgM-depleted patient plasma showed that anti-MPO IgM activated complement much more efficiently than anti-MPO IgG. Conclusion We demonstrate the direct ex-vivo identification, isolation and characterization of MPO-specific B cells in human AAV. Intriguingly, we observed a remarkable expansion of MPO-specific, IgM-expressing memory B cells in patients. This so far unrecognized, active IgM-compartment may be clinically relevant, as both mAb and plasma-derived polyclonal MPO-specific IgM strongly activated complement, a pathway thought to play a central role in AAV. Next to these novel insights into autoreactive B cell biology in AAV, our findings now provide new opportunities for studying auto-reactive B cell responses in different clinical phases of AAV, amongst which active disease, remission and (imminent) flares. Disclosure of Interests None declared.

Background:Fc neonatal receptor (FcRn) is crucial for IgG half-life and transplacental transport. Different sites of IgG carry glycans which may affect binding to FcRn. While the effect of Fc-glycans has been investigated, the impact of Fab-glycosylation (~14% IgG) on IgG-FcRn interaction remains unclear. Anti-citrullinated protein antibodies (ACPA) of rheumatoid arthritis patients exhibit remarkably high Fab-glycosylation (~90%). This makes ACPA an ideal model to investigate how Fab-glycosylation influences IgG-FcRn interaction.Objectives:To investigate the potential impact of IgG Fab-glycosylation on IgG transplacental transfer and interaction with FcRn.Methods:To investigate transplacental transport of ACPA and total IgG, serum of ACPA-positive RA patients (mothers) as well as of healthy mothers and their respective newborns was analyzed. IgG Fab- and Fc-glycosylation was investigated with liquid chromatography and mass-spectrometry. Furthemore, ACPA monoclonal IgG were produced and glycoengineered to acquire several variants of the same monoclonal antibody differing only in their glycosylation profile. These glycovariants were then used to investigate the impact of Fab-glycans on the affinity of IgG for FcRn. Surface plasmon resonance (SPR) and affinity chromatography were implemented.Results:When measured in mothers’ serum and cord blood samples, Fab-glycosylation of IgG antibodies was ~20% lower in newborns compared to their mothers, which was observed for ACPA IgG, non-ACPA IgG in RA patients and total IgG of healthy controls (Figure 1). This may indicate that transplacental transfer of Fab-glycosylated antibodies is impaired. SPR results suggested that presence of Fab-glycans slightly lowered the affinity of IgG for FcRn. However, presence of Fab-glycans did not have a significant effect on the results of FcRn affinity chromatography. Together, these results suggest that Fab-glycans may impair association of IgG with FcRn, while dissociation likely stays intact.Conclusion:Our results suggest that Fab-glycans inhibit IgG-FcRn binding which negatively affects the transplacental transfer of Fab-glycosylated IgG. The impact of Fab-glycosylation on IgG half-life requires further investigation.Figure 1.Disclosure of Interests:Mikhail Volkov: None declared, Karin van Schie: None declared, Albert Bondt: None declared, Theresa Kissel: None declared, Maximilian Brinkhaus Grant/research support from: argenx, Arthur Bentlage: None declared, Carolien Koeleman: None declared, Steven de Taeye Grant/research support from: Genmab, Radboud Dolhain: None declared, Manfred Wuhrer: None declared, Rene Toes: None declared, Gestur Vidarsson: None declared, Diane van der Woude: None declared

Background Besides anti-citrullinated protein antibodies (ACPA), rheumatoid arthritis patients (RA) often display autoantibody reactivities against other post-translationally modified (PTM) proteins, more specifically carbamylated and acetylated proteins. Immunizing mice with one particular PTM results in an anti-modified protein antibody (AMPA) response recognizing different PTM-antigens. Furthermore, human AMPA, isolated based on their reactivity to one PTM, cross-react with other PTMs. However, it is unclear whether the AMPA-reactivity profile is “fixed” in time or whether consecutive exposure to different PTMs can shape the evolving AMPA response towards a particular PTM. Methods Longitudinally collected serum samples of 8 human individuals at risk of RA and 5 with early RA were tested with ELISA, and titers were analyzed to investigate the evolution of the AMPA responses over time. Mice (13 per immunization group in total) were immunized with acetylated (or carbamylated) protein (ovalbumin) twice or cross-immunized with an acetylated and then a carbamylated protein (or vice versa) and their serum was analyzed for AMPA responses. Results Human data illustrated dynamic changes in AMPA-reactivity profiles in both individuals at risk of RA and in early RA patients. Mice immunized with either solely acetylated or carbamylated ovalbumin (AcOVA or CaOVA) developed reactivity against both acetylated and carbamylated antigens. Irrespective of the PTM-antigen used for the first immunization, a booster immunization with an antigen bearing the other PTM resulted in increased titers to the second/booster PTM. Furthermore, cross-immunization skewed the overall AMPA-response profile towards a relatively higher reactivity against the “booster” PTM. Conclusions The relationship between different reactivities within the AMPA response is dynamic. The initial exposure to a PTM-antigen induces cross-reactive responses that can be boosted by an antigen bearing this or other PTMs, indicating the formation of cross-reactive immunological memory. Upon subsequent exposure to an antigen bearing another type of PTM, the overall reactivity pattern can be skewed towards better recognition of the later encountered PTM. These data might explain temporal differences in the AMPA-response profile and point to the possibility that the PTM responsible for the initiation of the AMPA response may differ from the PTM predominantly recognized later in time.

We read with a great interest the report by Van Schie et al 1 which highlighted that the antibody response against human and chimeric anti-TNF therapeutic antibodies primarily targets the TNF binding region. The results described in this report confirm very clearly the results that we obtained recently. This study adds to the one we performed, which shows the neutralising capacity of antidrug antibodies (ADAs) against infliximab (IFX) in patients with inflammatory bowel disease (IBD) treated by IFX. We assessed ADAs’ biological activity by a functional assay using a reporter HEK-Dual TNF cell line developed for TNF detection (InvivoGen). Briefly, sera of patients with IBD were first incubated …