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  1. Abstract

    Cytometry is an important technique widely used in medicine and biological research. Biologists traditionally analyze single‐cell cytometry data by manual gating, which can be subjective and labor intensive. To address this issue, many automated and semiautomated methods have been developed. These advanced methods are designed to speed up and standardize the analysis of cytometry data, but their popularity is limited by their visualizations which are not intuitive to biologists who are accustomed to the conventional biaxial gating plots. In this article, we present a new method called Cluster‐to‐Gate (C2G) that can take clustering results as input, and automatically generate a nested two‐dimensional gating hierarchy, which is a visualization representation that biologists are familiar with. This method can generate gating sequences for multiple target populations simultaneously and summarize them in one hierarchical tree that represents the gating hierarchy. We have tested this method on target populations defined by manual gating, automated clustering algorithms (k‐means for example), and visualization‐assisted methods (SPADE and tSNE). We have demonstrated that C2G is able to generate gating sequences that capture cell populations defined by the various clustering strategies, and robust to over‐clustered and overlapping target populations. © 2018 International Society for Advancement of Cytometry

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  2. Objective

    To obtain the comprehensive transcriptome profile of human citrulline‐specific B cells from patients with rheumatoid arthritis (RA).


    Citrulline‐ and hemagglutinin‐specific B cells were sorted by flow cytometry using peptide–streptavidin conjugates from the peripheral blood ofRApatients and healthy individuals. The transcriptome profile of the sorted cells was obtained byRNA‐sequencing, and expression of key protein molecules was evaluated by aptamer‐basedSOMAscan assay and flow cytometry. The ability of these proteins to effect differentiation of osteoclasts and proliferation and migration of synoviocytes was examined by in vitro functional assays.


    Citrulline‐specific B cells, in comparison to citrulline‐negative B cells, from patients withRAdifferentially expressed the interleukin‐15 receptor α (IL‐15Rα) gene as well as genes related to protein citrullination and cyclicAMPsignaling. In analyses of an independent cohort of cyclic citrullinated peptide–seropositiveRApatients, the expression ofIL‐15Rα protein was enriched in citrulline‐specific B cells from the patients’ peripheral blood, and surprisingly, all B cells fromRApatients were capable of producing the epidermal growth factor ligand amphiregulin (AREG). Production ofAREGdirectly led to increased migration and proliferation of fibroblast‐like synoviocytes, and, in combination with anti–citrullinated protein antibodies, led to the increased differentiation of osteoclasts.


    To the best of our knowledge, this is the first study to document the whole transcriptome profile of autoreactive B cells in any autoimmune disease. These data identify several genes and pathways that may be targeted by repurposing severalUSFood and Drug Administration–approved drugs, and could serve as the foundation for the comparative assessment of B cell profiles in other autoimmune diseases.

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