Background/Purpose: Rheumatoid arthritis (RA) often has a progressive and debilitating course, with significant impact on the patient’s quality of life. Despite the long-known association with autoantibodies, knowledge of the role of B cells and their potential direct contribution to RA pathogenesis remains elusive.

Characterization of B cell subpopulations in the periphery and synovial tissue.

Identification of chemokine receptors responsible for the synovial accumulation of B cells.

Characterization of B cell function and metabolism under the hypoxic conditions of the RA joint.

Methods: Synovial tissue biopsies from RA patients were obtained through key-hole arthroscopy followed by single cell flow cytometric analysis of B cell subpopulations and chemokine receptor expression. SPICE analysis was performed for the chemokine receptor expression of synovial B cells and B cell invasion assays. Functional characterization of sorted RA B cells, cultured in vitro under hypoxia, simulating the inflamed joint environment. Characterization of B cell metabolism and transcriptional regulation of pSTAT3 by flow cytometry. Notably, novel, non-invasive Fluorescent Lifetime Imaging Microscopy (FLIM) assay for the characterisation of the metabolic status of sorted RA patient PD-1 B cells coupled with RNAseq analysis was performed.

Results: Extensive flow-cytometric characterisation and SPICE algorithm analyses of single-cell synovial tissue from RA-patients revealed the accumulation of switched and double negative memory PD-1 expressing B cells at the site of inflammation. Accumulation of memory B cells is mediated by CXCR3, evident by the observed increase in CXCR3 expressing synovial B cells compared to the periphery, differential regulation by key synovial cytokines, and restricted B cell invasion demonstrated in response to CXCR3 blockade. Notably, under 3% O₂ hypoxic conditions that mimic the joint-microenvironment, RA B cells maintain marked expression of MMP-9,-TNF and IL-6, with PD-1⁺ B-cells demonstrating higher expression of CXCR3, CD80, CD86, IL-1β and GMCSF than their PD-1^– counterparts. Following functional analysis and flow cell sorting of RA PD-1⁺ vs PD-1^– B-cells, we demonstrate using RNAseq and novel FLIM visualisation of cellular NADH, a significant shift in metabolism of RA PD-1⁺ B-cells towards glycolysis, associated with an increased transcriptional signature of key cytokines and chemokines that are strongly implicated in RA pathogenesis.

Conclusion: Highly polyfunctional pro-inflammatory T cell responses pre-date disease onset as demonstrated by the accumulation of polyfunctional T cells in the synovial tissue of pre-RA arthralgia subjects. These data highlight a key early pathogenic role for T cell plasticity and specific synovial T cell clusters including, DP T cells in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/pathogenic-glycolytic-pd-1-b-cells-accumulate-in-the-hypoxic-ra-joint/