Background/Purpose: Rheumatoid arthritis is an autoimmune disease characterized by immune cell infiltration in synovial tissue and progressive bone damage. B cells are abundant in the synovial tissue of a subset of RA patients, where they play critical roles in shaping local RA pathogenesis. The unique microenvironment in RA synovial tissue may drive pathogenic B cell function, but current approaches to interrogate single cells rely on disaggregation of tissue with loss of information about the spatial structure and paracrine interactions between cells. To better understand the role of B cells in RA-driven synovial pathogenesis, we are taking the novel approach of examining RNA signatures of B cells isolated from discrete anatomical areas in synovial tissue using laser capture microdissection (LCM).

Methods: Synovial tissue from RA patients was obtained from arthroplasties and immediately flash frozen in OCT freezing medium. 8 µm tissue sections were prepared under strict RNAse free conditions. RA synovial sections were stained with anti-CD20 in the presence of three RNase inhibitors at 4^oC. Using a PALM MicroBeam Zeiss microscope, individual B cell were marked and catapulted from the tissue section into a cap with adhesive coating. Targeted qPCR for CD19, CD3 and CD14 was performed to assess the specificity of LCM and RNA integrity numbers (RIN) of samples after LCM were quantitated. RNA sequencing was performed on captured B cells using low input SmartSeq2 methodology.

Results: In the presence of three RNAse inhibitors, high quality RNA was obtained after cell capture from synovial tissue (RIN values 6.3 ±0.4). Targeted qPCR for CD19, CD3 and CD14 showed >600-fold enrichment of CD19 transcript in captured B cells. In contrast, CD3 and CD14 were minimally detected. We performed RNA sequencing of captured synovial B cells (400 B cells, n=3). High sensitivity gene detection was achieved with over 11,000 genes at >1 CPM mapped reads. Compared to sequential sections of whole tissue, we observed higher expression of CD138 and Blimp1 in captured B cells suggesting enrichment for antibody secreting cells. Further examination of differential gene expression revealed higher expression of pro-inflammatory cytokines (IL6 400-fold, IFNg 39-fold, TNF 10-fold) in captured B cells, suggesting that B cells contribute significantly to the pro-inflammatory synovial microenvironment. In addition, captured B cells had higher expression of CD27 and FCRL4, markers for activated memory B cells.

Conclusion: These data suggest that LCM is a valuable tool to interrogate the transcriptome of discrete cell populations in the synovial target tissue. Ongoing studies are underway to further characterize the impact of anatomical region and neighboring cell interactions on cell function.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/laser-capture-microdissection-to-interrogate-b-cells-in-ra-synovial-tissue/