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. RA synovial rmAbs display frequent immunoreactivity towards citrullinated histones To investigate the autoantigenic immunoreactivity of the synovial B-cell clones, we cloned matching VH+VL Ig genes from individual B cells into specific expression vectors and produced 66 in vitro whole rmAbs as complete IgG1 displaying identical VH+VL pairing and specificity of the parental B cells.21 26 Sufficient yield (>5?g/mL) was obtained from 59?rmAbs (RA015/11=12; RA056/11=26; RA057/11=21), which were used for downstream analysis. 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)-, 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: Rheumatoid Arthritis, B cells, Autoantibodies Introduction Rheumatoid arthritis (RA) is characterised by breach of self-tolerance towards citrullinated proteins (anti-citrullinated peptide/proteins antibodies (ACPA)), which can occur years prior to clinical onset of RA at extra-articular sites.1C6 Several post-translationally deiminated proteins have been indicated as a potential source of citrullinated antigens in the RA joints,3 but to date their cellular source and specific contribution to the lesional ACPA response is unknown. Around 40%C50% of patients with RA display synovial ectopic lymphoid structures (ELS) characterised by B-cell follicles supporting a germinal centre (GC) response.7 8 Synovial ELS are self-sustained niches whereby autoreactive B cells undergo antigen-driven selection/differentiation with local antibody diversification through Ig genes somatic hypermutation (SHM)9 and class switching.10 Citrullination, or arginine deimination, is catalysed by the enzyme peptidyl-arginine-deiminase (PAD). In the RA synovium, monocyteCmacrophages are the main source of this enzyme.11 12 As a result, citrullination of fibrinogen, vimentin and -enolase, among others, has been observed within the RA joints and associated with circulating ACPA.13C15 Accordingly, monoclonal antibodies Proglumide sodium salt generated from synovial fluid B cells frequently react against citrullinated antigens.16 PAD-mediated deimination of core histones (H2A/H2B/H3/H4) has been described in neutrophils during the neutrophil extracellular traps (NETs) formation, or NETosis, a form of cell death which enhances the antimicrobial properties of activated neutrophils.17 18 Interestingly, RA synovial fluid neutrophils display an enhanced NETosis in the absence of microbial stimuli due to the RA proinflammatory milieu19 and RA sera Proglumide sodium salt react against citrullinated H4 from NETs.2 At present, direct evidence that synovial B cells from ELS+RA recognise citrullinated proteins and the specific contribution Proglumide sodium salt of different citrullinated antigens in fuelling the lesional ACPA production is missing. To this aim, we investigated the immunoreactivity of recombinant monoclonal antibodies (rmAbs) generated from single synovial B-cell clones obtained from patients with Proglumide sodium salt ELS+/ACPA+RA. Materials and methods A full list of methods is reported in the online supplementary methods. Patients Three synovial tissues from total joint replacement were obtained after informed consent (National-Research-Ethics-ServiceCCommittee-London-LREC05/Q0703/198) from patients with ACPA+ RA (all females, age range 66C75, all on combination Disease-Modifying AntiRheumatic Drug (DMARD) therapy including methotrexate) diagnosed according to the revised American College of Rheumatology (ACR) criteria.20 This board specifically approved the collection of the synovial tissue. Synovial tissue was dissected and processed as previously described.10 Synovial mononuclear cell isolation and CD19+ cell FACS sorting Mononuclear cells were isolated from fresh synovial tissue specimens obtained as above. Detailed method is reported in the online supplementary methods. Generation of recombinant monoclonal antibodies Single-cell real time-PCR reactions and IgV gene amplification were performed as described in refs. 21 and 22. Briefly, cDNA from CD3-CD19+B cells was amplified using reverse primers that bind the C/C or C constant region in three independent nested-PCR. The complete sequence of primers is reported in online supplementary table S1. Aliquots Rabbit Polyclonal to EPN1 of Variable Heavy (VH)/V/V chains second PCR products were sequenced with the respective reverse primer and analysed by IgBlast. IgH complementary determining region (CDR)3 amino acids and length were determined as described.21 The V gene somatic mutations analysis was performed using IMmunoGeneTics/Variable (IMGT/V)-QUEry and STandardization (QUEST) to characterise the silent versus non-silent mutation in each Framework Region (FR)/CDR region to determine the R:S ratio. The expression vector cloning strategy and the monoclonal antibody production were performed as described in ref. 21. Immunoglobulin Analysis Tool (IgAT) software was used to calculate the probability of antigen-driven selection within the Ig repertoire of the RA-rmAbs,23 as previously described.22 Multiplex autoantibody assay The multiplex autoantibodies assay containing 20 citrullinated RA-associated antigens (see online supplementary table S2) was performed as previously published.5 Briefly, rmAbs were added at 10?g/mL to custom Bio-Plex beads associated with RA putative autoantigens and incubated at room temperature (RT) for 1?h. After washing, PhycoErythrin.