Stephen Edward Rapecki

Identification of a novel chemokine-dependent molecular mechanism underlying rheumatoid arthritis-associated autoantibody-mediated bone loss

Objectives Rheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) appear before disease onset and are associated with bone destruction. We aimed to dissect the role of ACPAs in osteoclast (OC) activation and to identify key cellular mediators in this process. Methods Polyclonal ACPA were isolated from the synovial fluid (SF) and peripheral blood of patients with RA. Monoclonal ACPAs were isolated from single SF B-cells of patients with RA. OCs were developed from blood cell precursors with or without ACPAs. We analysed expression of citrullinated targets and peptidylarginine deiminases (PAD) enzymes by immunohistochemistry and cell supernatants by cytometric bead array. The effect of an anti-interleukin (IL)-8 neutralising antibody and a pan-PAD inhibitor was tested in the OC cultures. Monoclonal ACPAs were injected into mice and bone structure was analysed by micro-CT before and after CXCR1/2 blocking with reparixin. Results Protein citrullination by PADs is essential for OC differentiation. Polyclonal ACPAs enhance OC differentiation through a PAD-dependent IL-8-mediated autocrine loop that is completely abolished by IL-8 neutralisation. Some, but not all, human monoclonal ACPAs derived from single SF B-cells of patients with RA and exhibiting distinct epitope specificities promote OC differentiation in cell cultures. Transfer of the monoclonal ACPAs into mice induced bone loss that was completely reversed by the IL-8 antagonist reparixin. Conclusions We provide novel insights into the key role of citrullination and PAD enzymes during OC differentiation and ACPA-induced OC activation. Our findings suggest that IL8-dependent OC activation may constitute an early event in the initiation of the joint specific inflammation in ACPA-positive RA.

Rheumatoid synovial fluid interleukin-17- producing CD4 T cells have abundant tumor necrosis factor-alpha co-expression, but little interleukin-22 and interleukin-23R expression

IntroductionTh17 cells have been implicated in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to systematically analyse the phenotype, cytokine profile and frequency of interleukin-17 (IL-17) producing CD4-positive T cells in mononuclear cells isolated from peripheral blood, synovial fluid and synovial tissue of RA patients with established disease, and to correlate cell frequencies with disease activity.MethodsFlow cytometry was used to analyse the phenotype and cytokine production of mononuclear cells isolated from peripheral blood (PBMC) (n = 44), synovial fluid (SFMC) (n = 14) and synovium (SVMC) (n = 10) of RA patients and PBMC of healthy controls (n = 13).ResultsThe frequency of IL-17-producing CD4 T cells was elevated in RA SFMC compared with RA PBMC (P = 0.04). However, the frequency of this population in RA SVMC was comparable to that in paired RA PBMC. The percentage of IL-17-producing CD4 T cells coexpressing tumor necrosis factor alpha (TNFα) was significantly increased in SFMC (P = 0.0068). The frequency of IFNγ-producing CD4 T cells was also significantly higher in SFMC than paired PBMC (P = 0.042). The majority of IL-17-producing CD4 T cells coexpressed IFNγ. IL-17-producing CD4 T cells in RA PBMC and SFMC exhibited very little IL-22 or IL-23R coexpression.ConclusionsThese findings demonstrate a modest enrichment of IL-17-producing CD4 T cells in RA SFMC compared to PBMC. Th17 cells in SFMC produce more TNFα than their PBMC counterparts, but are not a significant source of IL-22 and do not express IL-23R. However, the percentage of CD4 T cells which produce IL-17 in the rheumatoid joint is low, suggesting that other cells may be alternative sources of IL-17 within the joints of RA patients.

Generation of Recombinant Monoclonal Antibodies from Immunised Mice and Rabbits via Flow Cytometry and Sorting of Antigen-Specific IgG+ Memory B Cells

Single B cell screening strategies, which avoid both hybridoma fusion and combinatorial display, have emerged as important technologies for efficiently sampling the natural antibody repertoire of immunized animals and humans. Having access to a range of methods to interrogate different B cell subsets provides an attractive option to ensure large and diverse panels of high quality antibody are produced. The generation of multiple antibodies and having the ability to find rare B cell clones producing IgG with unique and desirable characteristics facilitates the identification of fit-for-purpose molecules that can be developed into therapeutic agents or research reagents. Here, we describe a multi-parameter flow cytometry single-cell sorting technique for the generation of antigen-specific recombinant monoclonal antibodies from single IgG+ memory B cells. Both mouse splenocytes and rabbit PBMC from immunised animals were used as a source of B cells. Reagents staining both B cells and other unwanted cell types enabled efficient identification of class-switched IgG+ memory B cells. Concurrent staining with antigen labelled separately with two spectrally-distinct fluorophores enabled antigen-specific B cells to be identified, i.e. those which bind to both antigen conjugates (double-positive). These cells were then typically sorted at one cell per well using FACS directly into a 96-well plate containing reverse transcriptase reaction mix. Following production of cDNA, PCR was performed to amplify cognate heavy and light chain variable region genes and generate transcriptionally-active PCR (TAP) fragments. These linear expression cassettes were then used directly in a mammalian cell transfection to generate recombinant antibody for further testing. We were able to successfully generate antigen-specific recombinant antibodies from both the rabbit and mouse IgG+ memory B cell subset within one week. This included the generation of an anti-TNFR2 blocking antibody from mice with an affinity of 90 pM.

A2.24 Plasma cell derived monoclonal anti-citrulline antibodies from ra synovial fluid are multireactive

Background and objectives Human recombinant monoclonal antibodies have previously been generated at our laboratory from isolated patient-derived memory B cells to assess the repertoire and specificity of human anti-citrulline antibodies. The aim of the current study was to access joint-derived antibody secreting plasma cells and explore the proportion of citrulline reactivity, the immunoglobulin mutation patterns and antigen reactivity patterns. Clearly these antibodies can be utilised as tools and can help dissect the contribution antibodies make towards disease manifestations. Materials and methods Spontaneously IgG antibody-secreting cells (referred to as plasma cells) from synovial fluid of the inflamed joints of RA patients were isolated by a fluorescent foci method. The cells were identified and selected utilising a micromanipulator-equipped microscope. Recombinant monoclonal antibodies were generated from the isolated cells by amplification of the heavy and light chain variable antibody regions and were subsequently cloned into expression vectors. The monoclonal antibodies were screened for citrulline-reactivity by in-house ELISA or by a multiplex peptide array of RA associated antigens. Results We generated 375 IgG sequences and 101 recombinant antibodies from both ACPA+ (n = 3) and ACPA- (n = 1) patients. Four of the antibodies from ACPA+ synovial fluid displayed strong reactivity towards a citrullinated RA antigen peptide, but not to the cognate native peptide. The antibodies were multireactive to a diverse backbone with only the citrulline residue as common denominator, however with different reactivity patterns. The majority of the anti-citrulline clones are from the VH4 gene family. We found clonality within the plasma cells (including within the anti-citrulline antibodies), but also between different B cell populations. Conclusions Only a minority of the local plasma cells in the synovial fluid were autoreactive as measured by citrulline reactivity. However, the citrulline reactive plasma cells in the inflamed joint of RA patients where shown to be multireactive to several different citrulline peptides, with unique reactivity patterns. Further studies are needed to distinguish whether these plasma cells have differentiated locally in the joint. Still, it is tempting to speculate that the antibody characteristics that lead to multireactivity are driven by interaction towards several modified antigens, as has been suggested in the setting of influenza.

Human plasma cell derived monoclonal antibodies to post-translationally modified proteins recognize amino acid motifs rather than specific proteins

Antibodies against posttranslationally modified proteins are a hallmark of rheumatoid arthritis (RA), but the emergence and pathogenicity of these autoantibodies are still incompletely understood. The aim of this study was to analyze the antigen specificities and mutation patterns of monoclonal antibodies (mAb) derived from RA synovial plasma cells and address the question of antigen cross‐reactivity.

08.24 Monoclonal acpa antibodies recognising a common citrulline motif are mainly dependent on light chain hypermutations for antigen recognition

Background Anti-citrullinated protein antibodies (ACPA) can be detected many years before disease onset, implicating that not all ACPAs are pathogenic and that the autoantibodies undergo critical maturation steps closer to disease onset. We have studied the ACPA profile of synovial fluid and of monoclonal antibodies generated from plasma cells of the same synovial fluid to address these questions. Materials and methods IgG-secreting cells from RA synovial fluid were isolated, the variable Ig regions were amplified, and recombinant monoclonal antibodies were expressed. The antibody reactivity to citrullinated peptides were addressed both by a planar multi array of RA associated peptides, as well as on a 1 75 000 peptide micro-array of arginine/citrulline peptides from extracellular matrix proteins. The matching synovial fluid was also analysed on the peptide arrays. Chimeric monoclonal antibodies were produced based on IMGT predicted germline sequences of the ACPAs, to address the importance of hypermutations in the heavy versus the light chain. The induction of osteoclastogenesis by the ACPAs were tested in an in vitro assay. Results Sera from RA-patients display wide cross-reactivity between many citrullinated proteins and peptides. Synovial fluid replicates this pattern, and a glycine-rich consensus sequence could be deduced. The monoclonal ACPAs recognised different consensus sequences, of which some replicated the dominant polyclonal pattern. To dissect the contribution of the heavy and light chain to antigen recognition, chimeric variants were expressed where either the light or the heavy chain was exchanged with the respective germline sequence, or to an unrelated chain. Surprisingly, the ACPAs with the common consensus epitope were highly dependent on the light chain mutations for citrulline reactivity, while the heavy chain appeared primarily important for stability and conformation, independent on the acquired mutations. Even if these ACPA were highly cross-reactive, they did not cause osteoclastogenesis. The pathogenic ACPA was instead dependent on hypermutations in both heavy and light chains for citrulline reactivity, and had a more restricted citrulline peptide reactivity. Conclusions Our data lend support to the observed high cross-reactivity of ACPA between citrullinated peptides. We also find differences in citrulline-reactivity patterns and light chain contribution between pathogenic and non-pathogenic ACPA.