Age-associated B cells (ABCs) are an immune cell subset linked to autoimmunity, infection and ageing, and whose pathophysiological importance was recently highlighted using single cell synovial tissue profiling. To elucidate their pathophysiological relevance, peripheral blood (PB) ABCs from early rheumatoid arthritis (eRA) patients naïve to disease-modifying anti-rheumatic drugs (DMARDs) were compared with their synovial fluid (SF) counterparts, and to PB ABCs from psoriatic arthritis patients and healthy controls. PB and SF B-cell subsets were phenotyped by multi-parameter flow cytometry, sorted and subjected to gene expression profiling (NanoString nCounter ® Immunology V2 Panel) and functional characterization (stimulated cytokine measurements by immunoassay). PB ABCs of eRA patients, which are transcriptionally distinct from those of control cohorts, express chemokine receptors and adhesion molecules, such as CXCR3, that favour homing to inflammatory sites over lymphoid tissue. These cells are an activated, classswitched B-cell subset expressing high levels of HLA-DR, co-stimulatory molecules and T-bet. Their secretion profile includes IL-12p70 and IL-23 but low levels of IL-10. High surface expression of FcRL family members, including FcRL3, furthermore suggests a role for these cells in autoimmunity. Finally, and unlike in the periphery where they are rare, ABCs are the predominant B-cell subsets in SF. These observations indicate the predilection of ABCs for inflammatory tissue in RA, where their propensity for antigen presentation and pro-inflammatory phenotype may support autoimmune pathology. Their potential as a therapeutic target therefore warrants further study.
Background:Rheumatoid arthritis (RA) is a chronic inflammatory disease of immune dysregulation affecting the joints. While T cells play a recognised role in disease pathogenesis, the presence of autoantibodies years before the clinical onset of disease, and the efficacy of the B cell-depleting therapy rituximab, highlight a pathogenic role for these functionally diverse lymphocytes. A novel subset, termed age-associated B cells (ABCs), are described as CD19high CD21- CD11c+, with a high proportion expressing the transcription factor T-bet; they are elevated in murine models of autoimmunity and produce autoantibodies characteristic of autoimmune disease. A detailed characterisation of ABCs in early, drug naïve RA has not yet been conducted.Objectives:We aimed to characterise peripheral blood (PB) and synovial fluid (SF) ABCs in patients suffering from early drug naïve RA. As a secondary objective we sought to determine whether this population differs between RA patients and age-matched early psoriatic arthritis (PsA; disease controls) and healthy controls.Methods:Newly presenting early RA and other inflammatory arthritis patients, naïve to immunomodulatory treatment, were recruited from the Newcastle Early Arthritis Clinic. Patients with established RA (≥1 year duration on treatment and age-matched healthy controls were recruited in parallel. B cell subsets (naïve (CD19+IgD+CD27-), switched memory (CD19+IgD-CD27+) and ABC (CD19+CD21-CD11c+)) in PB and SF were characterised by flow cytometry. FACS sorted PB B cell subset gene expression profiles were assessed using a customised NanoString nCounter Human Immunology v2 Panel.Results:Transcriptionally, ABC differed from both naïve and switched memory B cells. In keeping with published studies ABCs had an activated memory phenotype, exemplified by elevated expression of CD69, CD80, and CD86, as well as Ki67, HLA-DR, IgG and T-bet. Interestingly, ABC had high expression of the Fc Receptor Like (FcRL) family (FcRL1-5), and an inflammatory homing profile (high levels of CXCR3 and low levels of CXCR4 and CXCR5). We found no difference in the proportion of PB ABC between RA patients and control groups, and no association of ABC frequencies with age. Focussing on RA ABCs specifically, we observed elevated ABC frequencies in females, but no association with disease activity. In keeping with their chemokine receptor profile, cross-sectional analysis also demonstrated ABC enrichment in SF compared to PB, with SF frequencies higher in established than early disease. The transcriptome of ABCs from early RA patients differed from both age-matched disease controls and healthy donors (Figure 1 next page).Conclusion:These data demonstrate that ABCs have a unique, activated, class-switched proliferative phenotype that is transcriptionally distinct from switched memory and naïve B cells. Interestingly, at the transcriptome level early RA ABCs differ to their counterparts in health and other inflammatory arthritides, suggesting they may differ functionally and contribute to pathogenesis. Of note, their high expression of MHC class II (HLA-DR) and co-stimulatory (CD80 and CD86) molecules suggests an important antigen-presentation function of ABC, which together with their unique FcRL family expression pattern, warrants further functional characterisation.Figure 1.Differential gene expression in ABCs from early RA patients compared to early PsA patients (A) and healthy controls (B). A NanoString nCounter Technologies chip was used to assess gene expression. Raw counts were normalised to the housekeeping genes. Sample quality was then assessed using the arrayQualityMetrics package. Gene expression profiles between different donor groups was assessed using the DESeq2 R package. In both hierarchical clustering heatmaps gene expression intensities were log2 transformed and their z-scores are displayed as colours ranging from yellow (low expression) to red (high expression) as shown in the key.Disclosure of Interests:Gemma Vidal-Pedrola: None declared, Najib Naamane: None declared, Dagmar Scheel-Toellner: None declared, Arthur Pratt Grant/research support from: GSK, Pfizer and Janssen, Andrew Mellor: None declared, John D Isaacs Speakers bureau: Abbvie, Gilead, Roche, UC, Consultant of: Abbvie, Gilead, Roche, UC, Grant/research support from: Pfizer, GSK and Janssen, Amy E. Anderson Grant/research support from: Pfizer, GSK and Janssen
Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterised by joint inflammation. Studies in mice and patients suffering from autoimmune diseases described a novel subset of B cells named age-associated B cells (ABCs). Moreover, a subset of synovial fluid B cells with a similar phenotype and FcRL4 expression contributes to RA pathogenesis. Newly diagnosed patients were recruited from the Newcastle Early Arthritis Clinic. B cell subsets in blood were phenotyped using flow cytometry. NanoString Immunlogy Panel was used to asses gene expression from sorted ABCs, naïve, memory and CD5+ B cells from RA patients, age-matched healthy controls and disease controls. Our work showed that there are no significant differences in the frequency of ABCs between RA patients, and disease and healthy controls. Our results also show that ABCs resemble the memory B cell phenotype regarding class-switch immunoglobulins expression. Interestingly, the FcRL4+, the Ki67+ and the T-bet expressing B cells were enriched in the ABC population. ABCs expressed high levels of HLA-DR and co-stimulatory molecules, as well as the activation marker, CD69. Gene expression analysis showed a specific gene signature for the ABCs when compared to the other B cells. KEGG analysis results showed that differentially expressed genes were enriched in the antigen presentation and the cytokine-cytokine receptor interaction pathways. Moreover, 13 genes were overexpressed in ABCs from the RA patients compared to the other control groups. This data supports the idea that ABCs have a pathogenic role in RA, potentially via T cell stimulation and migration to sites of inflammation. However, further characterisation of this subset and functional studies are needed.
Career situation of first and presenting authorStudent for a master or a PhD.IntroductionIncreasing evidence points to a mucosal origin of the autoimmune process in the development of rheumatoid arthritis. A B cell subset identified by surface expression of Fc receptor-like 4 (FcRL4) associates with inflammation in the mucosa associated lymphoid tissue.1 Our group identified these cells in the synovium of RA patients.2 They express high levels of RANKL and participate in the autoimmune response to citrullinated proteins.3 4 Recent in vitro work suggested that FcRL4 may be a low-affinity receptor for aggregated IgA.5 FcRL4 +B cells in RA SF express low levels of CD21 and high levels of CD11c, a phenotype they share with Age-Associated B cells (ABC). Very low numbers of ABC are found in blood of healthy individuals but their frequency increases with age however their exact role in RA is not yet understood.6 Objectives1) Investigate the binding activity of IgA immune complexes to FcRL4+ B cells in tonsil, RA synovial fluid, and the peripheral blood of healthy individuals and RA patients. 2) Determine if FcRL4 can specifically capture IgA from the RA SF. 3) Examine the specificity of FcRL4 for secreted and serum IgA and for different IgA isotypes.MethodsSF, peripheral blood and tonsil mononuclear cells were labelled for FcRL4, IgA, IgG, and CD19 (PBMCs were also labelled for CD21 and CD11c) and analysed by flow cytometry. Cells pre-treated with a pH3 buffer to remove receptor-bound immunoglobulins were compared with untreated cells to distinguish IgA B cell receptor expression from receptor- bound IgA Immune complexes. An FcRL4 expressing cell line was incubated with RA synovial fluid and purified human IgA to assess FcRL4s specificity.ResultsReceptor-bound IgA Immune complexes were observed on ex vivo RA SF and tonsil FcRL4+ B cells but not on FcRL4- B cells (p=0.001). Intriguingly, this IgA capture was not observed in peripheral blood FcRL4 +ABC from HC or RA patients. Furthermore, the proportion of B cells with IgA BCRs was increased in tonsil and SF FcRL4 +B cells but not in peripheral FcRL4+ B cells, compared to FcRL4- B cells. Using a cell line, we show that FcRL4 specifically binds IgA immune complexes from SF and shows a preference for IgA1 over IgA2 (p=0.02).ConclusionsFcRL4 is a receptor for IgA immune complexes in RA joints. RA SF and tonsil FcRL4 +B cells RA SF differ from peripheral blood FcRL4 +ABC in their in vivo capture of IgA immune complexes and in their frequency of IgA BCRs, suggesting distinct function and origin of these cells.ReferencesEhrhardt GR, et al. J Exp Med 2005.Yeo L, et al. Ann Rheum Dis 2011.Yeo L, et al. Ann Rheum Dis 2015.Amara K, et al. J Autoimmun 2017.Wilson TJ, et al. J Immunol 2012; 188.Marrack P, et al. J Immunol 2015.Disclosure of InterestNone declared.
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