{
  "abstract": "Objectives Anti-dsDNA IgG antibodies are characteristic for systemic lupus erythematosus (SLE). Evidence suggests that these antibodies contribute to various disease manifestations, particularly lupus nephritis. Although the overall expansion of plasma cells is well established in SLE, surprisingly little is known about the phenotype of antigen-specific, dsDNA-reactive B cells and the mechanisms underlying tolerance break. As insights into these mechanisms are important to understand the emergence of autoantibodies, we aimed to disentangle the cellular phenotype and B cell differentiation pathways involved in the production of anti-dsDNA antibodies.Methods A novel flow cytometry staining using fluorescent dsDNA-tetramers was developed and validated using supernatants of single-cell sorted dsDNA-reactive B cells. Phenotyping of dsDNA-reactive B cells was performed using spectral flow cytometry on PBMCs from healthy subjects (n=10), and anti-dsDNA+ (n=10) and anti-dsDNA- (n=11) SLE patients. Anti-dsDNA positivity in serum and culture supernatants was determined using ELISA.Results By index sorting and single cell culture we show that naïve dsDNA-reactive B cells are present in both healthy donors and SLE patients. Using spectral flow cytometry, we show that these B cells selectively became class-switched and activated (IgG+/CD95+/CD69+/Ki67) only in anti-dsDNA+ SLE patients, and exhibit a phenotype resembling DN2 cells (CD11c+/CXCR5-/CD21-) and plasmablasts. Most strikingly, whereas most DN2 cells in the total B cell compartment co-expressed T-bet, CD11c+ dsDNA-reactive B cells exhibited minimal T-bet expression. In addition, one patient had received anti-CD19 CAR-T therapy, and upon B cell repopulation, only naïve dsDNA-reactive B cells with a resting phenotype re-emerged, resembling those in healthy subjects.Conclusions We conclude that dsDNA-reactive B cells are present within resting naïve B cells in healthy subjects and anti-dsDNA IgG-negative SLE, pointing to defective elimination of dsDNA-reactivity in the human naïve B cell repertoire. In anti-dsDNA+ SLE patients, we identify a novel, unconventional, extrafollicular pathway of CD11c+ B cell differentiation, thereby licensing the human dsDNA-IgG responses to differentiate. These findings offer potential targets for selectively inhibiting these autoreactive B cells in SLE.",
  "authors": [
    {
      "affiliations": [
        "Leiden University Medical Centre, Leiden, The Netherlands"
      ],
      "name": "Lars Van Vliet"
    },
    {
      "affiliations": [
        "Leiden University Medical Centre, Leiden, The Netherlands"
      ],
      "name": "Annemarie Dorjee"
    },
    {
      "affiliations": [
        "Leiden University Medical Centre, Leiden, The Netherlands"
      ],
      "name": "Rene Toes"
    },
    {
      "affiliations": [
        "Leiden University Medical Centre, Leiden, The Netherlands"
      ],
      "name": "Jolien Suurmond"
    },
    {
      "affiliations": [
        "Leiden University Medical Centre, Leiden, The Netherlands"
      ],
      "name": "Jacqueline S Dekkers"
    }
  ],
  "title": "PT1:04 Differential expression of activation markers on dsDNA-reactive B-cells between healthy subjects and SLE patients reveals unconventional extrafollicular activation in SLE",
  "uid": "56824ca9-3506-5337-a753-3d93f6742259"
}