Rheumatoid arthritis (RA) is the most common autoimmune inflammatory arthritis leading to chronic and severe systemic inflammation. There is currently no cure for RA and only a small proportion of patients ever experience prolonged disease remission. B cells are key drivers of chronic inflammation in RA, shown by the success of B cell depletion therapies. There is limited understanding of the relationship between synovial B cell subsets and antibody secreting cells (ASCs), despite this knowledge being crucial for the development of more targeted B-cell depleting therapies. A CD11c⁺ᵛᵉ double-negative B cell population, DN2 B cells, have recently been shown to be increased in patients with systemic lupus erythematosus (SLE). While DN2 B cells have been suggested as an ASC precursor in SLE, to date there is no proven link between the two subsets in RA. To address this, I used full spectrum flow cytometry to explore significant changes in the B cell populations in RA patients. I have used a combination of manual gating and unbiased computational methods to characterise both circulating and synovial B cells. This revealed that DN2 B cells and their precursors, called activated naive B cells, were nearly twice as frequent in RA patients compared to healthy age matched controls. Moreover, DN2 B cells were further enriched in the synovial tissue of RA patients. These DN2 B cells exhibited elevated CD11c, CD19, and FcRL5 expression, alongside reduced levels of CD21, CD24, and CD38, matching previous observations in SLE. Remarkably, RA DN2 B cells displayed lower CD95 expression compared to healthy DN2 B cells, which may allow autoreactive cells to evade tolerance mechanisms. Next, I have used single-cell sequencing with paired BCR sequencing to study synovial B cells from patients with established RA. This revealed 12 distinct B cell clusters within the synovial tissue, including naive, memory, and DN2 B cells, as well as a large population of ASCs. A novel subset of heat shock protein expressing B cells were also identified that showed significant enrichment for pathways related to incorrect protein folding. Investigation into the differentially expressed transcription pathways in DN2 B cells highlighted the activation of numerous pathways which could participate in the disease process, including those involved in processing and presenting antigens. The BCR sequences of synovial B cells showed attributes that have previously been linked to autoreactivity, including increased N-linked Fab glycosylation and reduced somatic hypermutation. To better understand the differentiation patterns within the diseased tissue, a combination of RNA-based trajectory inference and clonal lineage analysis of BCR relationships were used. Both forms of analysis indicated that DN2 B cells serve as major iii precursors to synovial ASCs with examples of the clusters sharing exact heavy and light chain CDR3 sequences. Finally, I have cloned and expressed antibodies from the BCR sequencing data to ascertain the specificity of six BCR sequences, four from DN2-derived ASCs and two from large clonal expansions. Sequences were cloned using the Polymerase Incomplete Primer Extension (PIPE) cloning method and expressed using HEK293T cells. Once purified the antibodies were screened using a custom peptide microarray, which suggested that histones H2A and H2B, citrullinated albumin, and citrullinated clusterin may be important self-antigens in these samples. The novel findings of this thesis advance our understanding of B cells in RA and reveals the origin of pathogenic ASCs in the RA synovial tissue. Given the significant role of DN2 B cells as a progenitor to ASCs in RA, it is important to conduct additional research to investigate the origins of DN2 B cells in RA and explore their potential as therapeutic targets in place of the less specific pan-B cells depletion therapies currently in use.
