EFB Dengue Literature Review

DN3 B Cell

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DN3 B Cell

Overview

DN3 B cells are a subset of IgD⁻CD27⁻ (double-negative) B cells defined by the phenotype CXCR5⁻, CD21⁻, CD11c⁻, T-bet⁻. They were described in acute COVID-19 and active SLE and represent pre-plasmablasts that accumulate in tissues in autoimmune fibrosis and severe COVID-19 infection. DN3 cells are distinct from DN2 B Cells (which are CD11c⁺⁺ T-bet⁺⁺) and from DN1 cells (which are CXCR5⁺ CD21⁺).

DN3 cells should not be classified as atypical B cells or age-associated B cells (ABC) despite their CD21lo phenotype, because they lack the defining ABC markers CD11c and T-bet.

Key Points from Literature

  • Phenotype: IgD⁻, CD27⁻, CXCR5⁻, CD21⁻, CD11c⁻, T-bet⁻. The key distinguishing feature from DN2 is the absence of both CD11c and T-bet — the defining markers of ABC/DN2 identity. DN3 cells also express low CD19 (see Sanz2025 - Human Atypical B Cells Overview, review citing Woodruff et al. 2020, Allard-Chamard et al. 2023, Perugino/Pillai et al.).
  • Pre-plasmablast identity: DN3 cells convincingly represent pre-plasmablasts that accumulate in tissues in autoimmune fibrosis and severe COVID-19. They correspond to the CD27⁻ CXCR5⁻ CD19⁻low plasmablast-low population described in SLE (Szelinski et al. 2022), and likely to the early pre-plasmablasts or ASC population 1 reported in tetanus vaccination responses in healthy subjects (see Sanz2025 - Human Atypical B Cells Overview, review).
  • Expanded in acute COVID-19 and SLE: DN3 cells were first reported in the Sanz lab’s acute SARS-CoV-2 study (Woodruff et al. 2020) and subsequently confirmed in active SLE. They represent a large fraction of DN cells in some SLE patients (see Sanz2025 - Human Atypical B Cells Overview).
  • Tissue infiltration: Allard-Chamard et al. (2023) demonstrated that extrafollicular IgD⁻CD27⁻CXCR5⁻CD11c⁻ DN3 B cells infiltrate inflamed tissues in autoimmune fibrosis and severe COVID-19, consistent with their pre-plasmablast identity and tissue-homing capacity (see Sanz2025 - Human Atypical B Cells Overview, review citing Allard-Chamard et al.).
  • Durability post-vaccination: Antigen-specific DN2 and DN3 cells persist for over a year post-SARS-CoV-2 mRNA vaccination, accounting for >50% of all spike/RBD⁺ cells (see Sanz2025 - Human Atypical B Cells Overview, review citing Faliti et al. 2024).
  • Not ABC despite CD21lo phenotype: The expression of low levels of both CD21 and the defining ABC/DN2 markers (CD11c, T-bet) emphasises the importance of comprehensive phenotyping. CD21lo alone cannot be used to identify ABC/AtB (see Sanz2025 - Human Atypical B Cells Overview).
  • Primary data: DN3 significantly expanded in ICU COVID-19: DN3 cells (CD11c⁻CD21⁻ within IgD⁻CD27⁻) were significantly expanded in ICU-C patients compared with HD (P ≤ 0.01). In hierarchical clustering of B cell population frequencies, DN3 consistently grouped with aN and DN2 populations — not with DN1 — supporting its association with the EF effector pathway. CD38 expression on DN3 cells from ICU patients was observable but heterogeneous (see Woodruff2020 - EF B Cell Responses in COVID-19, 24-marker spectral FCM, Extended Data Fig. 2).
  • DN3 first described in this paper: Although the Sanz2025 review cites DN3 as a known entity, Woodruff2020 is the original description. The formal gating definition — IgD⁻CD27⁻CD11c⁻CD21⁻ within the CD19⁺CD24⁻CD38⁻ DN gate — was established here. The paper notes that DN3 was “previously unreported in other conditions” at the time of publication (see Woodruff2020 - EF B Cell Responses in COVID-19).
  • UMAP heterogeneity within DN3: UMAP projections indicated a split in the DN3 compartment driven by HLA-DR and CD19 expression — suggesting internal heterogeneity that requires further interrogation (see Woodruff2020 - EF B Cell Responses in COVID-19, Fig. 2e).

Contradictions & Debates

  • The relationship between DN3 cells and the plasmablast maturation continuum (CD138⁻ → CD138⁺ ASC) described in Tipton2015 - ASC Diversity and Origin in SLE is not fully resolved. DN3 cells may represent the earliest circulating pre-plasmablast stage — before CD27 and CD38 upregulation — but this requires direct comparison with sorted populations.
  • Whether DN3 cells are an obligate intermediate in the EF pathway (aNAV → DN2 → DN3 → PB) or arise independently of DN2 is unclear.

Double-Negative B Cell, DN2 B Cell, Plasmablast, Extrafollicular Response, CXCR5, CD11c, T-bet

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