The Elmezzi School of Molecular Medicine Manhasset, New York, United States
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Background/Purpose: Studying antinuclear antibody (ANA) producing B cells at the single-cell level offers critical insights into their fine specificity and functional characteristics. However, conventional techniques including bulk B cell cultures, ELISpot assays, and single-cell sequencing present several limitations. To overcome some of these limitations, we developed an integrated platform that combines flow cytometric detection of ANA⁺ B cells with an optimized single-B cell culture system (Nojima). Methods: We employed a flow cytometry assay using biotinylated nuclear extracts to detect ANA reactive B cells, followed by single cell sorting and culture in the presence of feeder cells expressing CD40L and adding BAFF, IL-2, IL-21 (Figure 1). Results: This approach enabled the isolation and in vitro expansion of ANA-producing B cells coming from patients with SLE and healthy donors from naïve, marginal zone and memory B cell subsets. Following 26 days of culture, a high proportion of wells produced significant immunoglobulin levels: 82% of naïve, 67% of marginal zone, and 57% of IgG⁺ memory B cell cultures exceeded 1 µg/mL. ANA reactivity in culture supernatants was confirmed by HEp-2 cell staining (shown in figure 2), yielding a sensitivity of 95% and specificity of 68%, validating the functional identity of the expanded B cells. Studying previously sorted ANA+ cells significantly increases the efficiency of the assay. In order to study the immunoglobulin form 100 ANA⁺ cells would ordinarily require screening 2,857 B cells (assuming ANA prevalence of 5%), whereas our approach reduces this to just 150 cells. Conclusion: This method provides a robust platform for isolating and expanding ANA-producing B cells, enabling the production of autoantibodies for further study. The approach has potential applications in elucidating SLE pathogenesis.
