POS0006 AUTOREACTIVE B CELLS IN RHEUMATOID ARTHRITIS ARE RECENTLY ACTIVATED AND SHOW LARGE EXPANSIONS OF CXCR3+ ANTIBODY-SECRETING CELLS

Background

Many autoimmune diseases (AIDs) are characterized by aberrant, autoreactive B cell responses and autoantibody production. Rheumatoid arthritis (RA) is a common AID in which autoantibodies recognizing post-translational modifications (PTMs), collectively called anti-modified protein antibodies (AMPA), are intimately associated with disease pathogenesis. Previously, we observed that memory B cells (mBC) against one of these PTM-antigens, citrulline, are highly activated. This phenotype persists for years in patients with established disease [1]. The evolution, dynamics and more detailed phenotypic characteristics of the PTM-directed B cell compartment are still ill defined, however, partly owing to challenges linked to the visualization of such cells in human samples and the limitations of conventional flow cytometry. Here, we visualized the autoreactive B cell response against three different PTM antigens (citrulline, homocitrulline, acetyllysine) by spectral flow cytometry, allowing us to address cross-reactivity on the B cell level and to perform deep phenotypic profiling of individual B cell compartments.

Objectives

To develop a detailed understanding of the autoreactive B cell response against PTM-antigens in RA with the aim to elucidate features associated with its cross-reactivity, state of activation in the disease context, and its remarkable persistence for years without signs of exhaustion or decay.

Methods

We developed a spectral flow cytometry staining approach using differentially labelled streptavidin molecules coupled to individual PTM-antigens. B cells reactive to each antigen were identified together in individual samples by double staining for each antigen, while at the same time excluding cells reactive to either arginine or lysine control peptides. This combinatorial staining was further extended by the concomitant visualization of tetanus-toxoid specific B cells and expression levels of various activation and homing markers, and applied to peripheral blood samples of patients with established RA. Importantly, we performed intracellular stainings, allowing us to additionally enumerate and characterize circulating plasmablasts and plasmacells.

Results

We successfully visualized autoreactive B cells directed against different PTM-antigens and their subset distribution in individual patient samples. We observed extensive cross-reactivity against all three PTM antigens with citrulline as dominant antigen. Unsupervised clustering revealed several memory B cell populations, with most autoreactive B cells populating the most recently activated, mostly class-switched, CD80^highCD24^lowCD21^low mBC compartment. We also noted large expansions of CXCR3⁺ IgG or IgA-expressing antibody secreting cells (ASC), comprising up to 50% of the autoreactive B cell compartment. Anti-tetanus toxoid B cells clustered with a different, more resting mBC subset.

Conclusion

Our results identify citrulline as the most prominent antigen recognized by AMPA-expressing B cells. The study highlights the recent and persistent activation state of PTM-reactive mBC and their continuous differentiation towards ASC. Such ASC may home towards CXCR3 ligands known to be abundant in the synovial compartment. This degree of mBC activation was also found in patients with low clinical disease activity scores, indicating that conventional therapeutic interventions may suppress inflammation but fail to silence the most disease-specific autoreactive B cell response in RA. Targeting this compartment may therefore be relevant for future therapeutic interventions aiming at the induction of tolerance and/or permanent cure. Finally, the combinatorial staining approach presented will be a valuable tool to delineate the development of PTM-directed autoimmunity in the pre-disease phase as well as its state of activation in phases of sustained drug-free remission and/or during tolerogenic interventions.

Reference

[1]Kristyanto et al., Sci Transl Med. 2020 Nov 18;12(570):eaaz5327

Acknowledgements:

NIL.

Disclosure of Interests

Sanne Reijm: None declared, Joanneke Kwekkeboom: None declared, Nienke Blomberg: None declared, Jolien Suurmond Grant/research support from: Janssen, Diane van der Woude: None declared, Rene Toes: None declared, Hans Ulrich Scherer Speakers bureau: BMS, Lilly, Pfizer, Consultant of: Galapagos, Grant/research support from: BMS, Lilly, Pfizer, Janssen.