Session Title: ACR Plenary Session III: Discovery 2013
Session Type: Plenary Sessions
Background/Purpose: Rheumatoid arthritis (RA) is characterised by breach of self-tolerance towards citrullinated proteins. Around 40% of patients display synovial tertiary lymphoid structures (TLS) with functional B cell follicles supporting a germinal-centre response and local autoantibody production (1). However, the nature of the main (auto)antigenic reactivity of synovial B cells is unknown. Here we characterized the autoreactive B cell response of lesional B cells isolated from TLS+ RA synovium.
Methods: Single CD19+ B cells were FACS sorted from synovial cell suspension of 4 TLS+ RA patients. RNA was used to amplify Ig VH and VL genes and PCR products were cloned and expressed as recombinant monoclonal antibodies displaying identical specificity of the original B cells (2). Recombinant antibodies were then tested 1) to determine the frequency of polyreactive clones and 2) to define their immunoreactivity towards native and citrullinated antigens using a synovial antigen microarray platform (3).
Results: We obtained 139 individual VH sequences of which 33% were IgM, 40% IgG, 27% IgA and 175 VL sequences and we generated a total of 66 complete (H+L chains) recombinant monoclonal antibodies. Analysis of the VH gene somatic mutation rate showed evidence of antigen selection and intra-synovial clonal diversification. No skewed distribution of the VH and VL gene usage was observed. Around 30% of synovial monoclonal antibodies were reactive towards citrullinated histones in the antigen microarray, in particular citH2A and citH2B. This reactivity was confirmed by citH2A and citH2B ELISA. Moreover, reactivity towards the citrullinated form of fibrinogen was observed.
Conclusion: Here we provided novel evidence that highly mutated, locally differentiated B cells within RA synovial germinal centre-like structures display a strong immunoreactive bias towards citrullinated histones. This suggests that citrullinated histones are the main antigens driving in situ B cell activation and differentiation sustaining the humoral autoimmune response within the RA joints.
1. Humby F, Bombardieri M, Manzo A, Kelly S, Blades MC, Kirkham B, Spencer J, Pitzalis C. Ectopic lymphoid structures support ongoing production of class-switched autoantibodies in rheumatoid synovium. PLoS Med. 2009 Jan 13;6(1):e1
2. Wardemann H, Yurasov S, Schaefer A, Young JW, Meffre E, Nussenzweig MC. Predominant autoantibody production by early human B cell precursors. Science. 2003 Sep 5;301(5638):1374-7
3. Robinson WH, DiGennaro C, Hueber W, Haab BB, Kamachi M, Dean EJ, Fournel S, Fong D, Genovese MC, de Vegvar HE, Skriner K, Hirschberg DL, Morris RI, Muller S, Pruijn GJ, van Venrooij WJ, Smolen JS, Brown PO, Steinman L, Utz PJ. Autoantigen microarrays for multiplex characterization of autoantibody responses. Nat Med. 2002 Mar;8(3):295-301.
W. H. Robinson,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/recombinant-monoclonal-antibodies-derived-from-single-cd19-synovial-b-cells-of-ra-patients-with-tertiary-lymphoid-structures-display-a-strong-immunoreactivity-towards-citrullinated-histones/