Single cell cloning and recombinant monoclonal antibodies generation from RA synovial B cells reveal frequent targeting of citrullinated histones of NETs

Abstract

Objectives: Rheumatoid arthritis (RA) is characterised by breach of self-tolerance towards citrullinated antigens with generation of anti-citrullinated peptide/proteins antibodies (ACPA). Currently, the nature and source of citrullinated antigens driving the humoral autoimmune response within synovial ectopic lymphoid structures (ELS) is a crucial unknown aspect of RA pathogenesis. Here we characterised the autoreactive B-cell response of lesional B cells isolated from ELS+RA synovium.

Methods: Single synovial tissue CD19+cells were Fluorescence Activated Cell Sorting (FACS)-sorted and VH/VL Ig genes cloned to generate recombinant monoclonal antibodies (rmAbs) from patients with ELS+/ACPA+RA.

Results: RA-rmAbs immunoreactivity analysis provided the following key findings: (1) in a chIP-based array containing 300 autoantigens and in a 'citrullinome' multiplex assay, a strong reactivity against citrullinated histones H2A/H2B (citH2A/H2B) was observed in ∼40% of RA-rmAbs, followed by cit-fibrinogen and cit-vimentin; (2) anti-citH2A/H2B-reactive RA-rmAbs (but not anti-citH2A/H2B negative) selectively recognised neutrophil extracellular traps (NETs) from peripheral blood and/or RA joint neutrophils; (3) anti-citH2A/citH2B and anti-NET immunobinding was dependent on affinity maturation and was completely abrogated following reversion of hypermutated IgVH/VL genes to germline sequences; (4) ELS+ (not ELS-) RA synovial tissues engrafted into Severe Combined ImmunoDeficiency (SCID) mice released human anti-citH2A/citH2B and anti-NET antibodies in association with the intra-graft expression of CXCL13 and lymphotoxin (LT)-β, two master regulators of ELS.

Conclusion: We provided novel evidence that B cells differentiated within synovial ELS in the RA joints frequent target deiminated proteins which could be generated during NETosis of RA synovial neutrophils including histones. Thus, NETs could represent a source of citrullinated antigens fuelling the ACPA autoimmune response within the RA synovium.

Keywords: Autoantibodies; B cells; Rheumatoid Arthritis.

Figure 1

Histological characterisation of synovial ectopic lymphoid structures (ELS), single synovial CD19+ cell sorting and V_H/V_L Ig gene analysis. (A) Representative immunohistological characterisation of synovial tissue samples from patients with rheumatoid arthritis (RA) used in this study (RA015/11, RA056/11 and RA057/11). To assess the presence of ELS, sequential paraffin tissue sections were stained for CD20 (B cells, left-panel), CD3 (T cells, central-panel) and CD138 (plasma cells, right-panel), respectively. (B) Isolation strategy of single CD19+ RA synovial B cells is shown. Mononuclear cells were surface labelled with fluorochrome-coupled anti-CD19 and anti-CD3 antibodies; the sorting gate strategy for single CD19+CD3− B cell is shown. A total of 50 000 events is shown in the FACS plot. (C) The frequencies of μ, γ and α heavy chain among all CD19+ B cells for which VH sequences were obtained are shown. (D) Frequencies of μ, γ and α heavy chain among all CD19+ B cells for each synovial tissue are shown. (E) The VH and JH gene repertoire of single CD19+ synovial B cells for each individual chain isotype, μ (white), α (grey) and γ (black) is shown. (F) The Vκ and (G) Vλ gene repertoire are shown. The red bars indicate the V(H+L) gene repertoire of peripheral blood-naïve B cells. The absolute number of sequences analysed is reported over each graph. Error bars in bar graph indicate SEM for individual patients. p Values compare data from RA synovial B cells with peripheral blood-naïve B cells; *p<0.05; ***p<0.001.

Figure 2

VH/VL Ig gene analysis demonstrating intra-synovial antigen-driven B-cell affinity maturation and clonal diversification. Ig gene sequences of CD19+ synovial B cells were analysed for the absolute numbers of somatic mutations in VH genes (FRs+CDRs) (shown separately for IgM, IgG and IgA clones in (A) as well as VL genes (κ and λ shown separately in (B)). (C) Frequency of replacement (R) and silent (S) mutation ratio in FR (white) and CDR (black) regions for IgM, IgG and IgA is shown. Significant differences between the R/S ratio in FR versus CDR regions of IgG and IgA clones are shown: (D) IgH CDR3 aa length and (E) number of positively charged aa in the CDR3 is shown for each heavy chain isotype separately. (F) Lineage trees generated by comparison of Ig VH sequences of synovial B cells are shown. Clonally related B cells were identified based on cells derived from a single germline rearrangement characterised by the same V, D and J gene usage, CDR3 length and sequence. The synovial B-cell clones are depicted as white circle, the putative common progenitor as grey circle and the germline sequences as black circle. The number inside the circle corresponds to the name of the clone and the number beside the line represents the additional mutation acquired. At the bottom of each clone the correspondent Ig VL sequence is indicated; na=not available and it indicates those genes for the light chain only that could not be amplified; *p<0.05 and **p<0.01.

Figure 3

Characterisation of the binding of the rheumatoid arthritis (RA) synovial recombinant monoclonal antibodies (rmAbs) towards citrullinated antigens demonstrates biased immunoreactivity towards citrullinated histones. (A) Luminex heatmap is shown. Heatmap tiles reflect the amount of IgG autoantibody binding reactivity based on the fluorescence intensity scale as indicated on the top-right. rmAb-IDs (individual columns, top labelling) and the location of each citrullinated antigen in the assay (individual rows, right side legend) are shown. (B) Column bar graph representation of the mean fluorescence intensity (FI+SEM) of the luminex heatmap for each citrullinated antigen. (C) Pie charts showing the general percentage of reactivity towards citH2A (top) and citH2B (bottom) histones of the RA-rmAbs after correction of background signal and breakdown of the prevalence in each individual synovial tissue. (D and E) Binding of the RA and control rmAbs (30 naïve/memory B-cell clones from patients with Sjögren's syndrome (SS)) to native and in vitro citrullinated histone H2A and H2B tested by ELISA. Results are grouped according to tissue donors and shown as increased percentage of binding comparing native versus citrullinated histones. A flu control rmAb is shown in red. The dotted horizontal line represents the cut-off for positivity of the rmAbs which was determined as the mean+2SD of the 30 SS rmAbs reactivity (right panel). (F) Binding of the RA-rmAbs (black circles=non-reactive; coloured circles=reactive) and control rmAbs (open circles) to citrullinated histone H2A peptides tested by ELISA (H2A1-21Cit; H2A27-47Cit; H2A69-90Cit; H2A79-98Cit; H2A94-113Cit) is shown. Results are expressed as absorbance at 405 nm. Each coloured circle represents an individual RA-rmAb.

Figure 4

Rheumatoid arthritis (RA) synovial recombinant monoclonal antibodies (rmAbs) display selective immunoreactivity towards neutrophil extracellular traps (NETs). Representative pictures of PMA-stimulated neutrophils incubated with RA synovial (A) versus control Sjögren's syndrome (SS) rmAbs (B), demonstrating selective immunoreactivity of RA-rmAbs towards NETs. NETs are clearly evident as web-like structures rich in nuclear material stained by DAPI (blue, left columns) and are strongly bound by RA synovial (but not SS) rmAbs (green, middle columns, with overlap staining in the right columns). Corresponding multiplex tiles reporting the binding of the same rmAb towards citH2A and citH2B histones are reported beside each ImmunoFluorescence (IF) staining (C), demonstrating good accordance with anti-NET staining. (D) Binding of the RA synovial rmAbs to NETs is confirmed also in using synovial fluid neutrophils. Top image: RA SF-neutrophils seeded untreated onto cell culture cover slides for 30 min before fixation with 4% ParaFormAldehyde (PFA). A representative picture is shown. (E) Pie chart displaying the percentage of synovial rmAbs reacting towards NETs within individual synovial tissue demonstrated that up to 42% of the intra-synovial humoral response is directed towards NETs. (F) Subanalysis of the ELISA immunoreactivity towards citH2A and citH2B histones demonstrates significantly higher binding in anti-NET+ versus anti-NET− clones.

Figure 5

Immunoreactivity towards neutrophil extracellular traps (NETs) is dependent on somatic hypermutation. (A) Subanalysis of the anti-citH2A (top) and citH2B (bottom) histone reactivity in ELISA according to the number of somatic mutations in the VH regions of IgM (left), IgG (central) and IgA (right) clones demonstrates progressive increase in immunoreactivity according to the mutational load in all isotypes. (B) Reversal to germline (GL) sequences by overlapping PCR in representative individual anti-NET+ rheumatoid arthritis (RA) recombinant monoclonal antibody (rmAb) invariably abrogated the binding to NETs. The family usage, CDR3 sequence and the total number of somatic mutations in the FR and CDR regions of VH and VL Ig genes prior to reversal to GL sequences is shown beside each IF staining; *p<0.05; **p<0.01.

Figure 6

Ectopic lymphoid structures (ELS)+ rheumatoid arthritis (RA) synovia are self-maintained and release anti-neutrophil extracellular traps (NETs) and anti-citrullinated histone antibodies in vivo when engrafted in the Hu-RA/SCID chimeric model. (a) RA ELS+ synovial tissues RA015/11 and RA056/11 transplanted into SCID mice (arrow depict site of transplant) displayed persistent ELS after 4 weeks post-engraftment as shown in representative pictures of sequential analysis of paraffin-embedded sections stained for H&E and for CD20 (B cells), CD3 (T cells) and CD138 (plasma cells) (B). (C) Serum from Hu-RA SCID mice engrafted with RA015/11 and RA056/11 synovia reproduced the reactivity towards NETs in PMA-stimulated neutrophils. Representative pictures with NETs visualised by DAPI (blue) and the binding of human IgG in green are shown. (D) Binding of the human IgG in Hu-RA SCID mice to citrullinated versus unmodified H2A and H2B histones by ELISA confirmed the immunoreactivity observed with the recombinant monoclonal antibodies from the same patients. Results are shown as increased percentage in immunoreactivity in citrullinated versus native H2A and H2B histones. (E) Stratification of synovial RA grafts based on citH2A and citH2B immunoreactivity versus non-reactive demonstrated increased synovial expression of mRNA transcripts for CXCL13 and LTβ in anti-citH2A and citH2B reactive versus non-reactive samples; *p<0.05.