Wei2007 - DN Memory B Cells in SLE

Full citation: Wei C, Anolik J, Cappione A, Zheng B, Pugh-Bernard A, Brooks J, Lee EH, Milner ECB, Sanz I. A New Population of Cells Lacking Expression of CD27 Represents a Notable Component of the B Cell Memory Compartment in Systemic Lupus Erythematosus. J Immunol. 2007;178(10):6624–6633. doi:10.4049/jimmunol.178.10.6624

Raw file: [[raw/Wei2007.pdf]]

Summary

Wei et al. provide the first detailed characterisation of IgD⁻CD27⁻ “double-negative” (DN) B cells as a distinct memory subset in human peripheral blood. While prior reports had noted their existence in SLE, this paper formally establishes their memory credentials — somatic hypermutation of VH genes, inability to extrude Rhodamine 123, and CpG-driven proliferation without BCR crosslinking — and distinguishes them from conventional CD27⁺ memory cells and from the tissue-resident FcRH4⁺ CD27⁻ population described by Ehrhardt et al. in tonsil.

The study documents a striking disease-specific expansion in SLE: healthy subjects carry ~4.6% DN B cells among total PBL B cells, whereas 50% of SLE patients exceed 10% (mean 19.4% in that subgroup), and in some patients DN cells constitute the single largest B cell compartment. This expansion is not observed in rheumatoid arthritis or chronic hepatitis C. Within SLE, DN-high patients show significantly elevated nephritis rates, anti-dsDNA and anti-RNP/Sm autoantibodies, 9G4 autoreactive B cells, and higher disease activity scores.

Mechanistically, the authors propose that DN cells develop outside conventional germinal centre reactions — via extrafollicular pathways — because CD27 is normally acquired during GC-dependent B–T cell cognate interactions, and because the lower somatic hypermutation rate in DN vs. CD27⁺ memory cells is consistent with abortive or GC-independent differentiation. This extrafollicular origin hypothesis provides the conceptual scaffolding for interpreting analogous DN/atypical B cell expansions in acute infections including dengue.

Study Design

• Type: Cross-sectional
• Sample size: 36 SLE patients, 29 healthy controls, 45 RA patients, 7 chronic hepatitis C patients; tonsil samples from healthy subjects undergoing routine tonsillectomy
• Setting: University of Rochester Medical Center; PBMCs isolated by Ficoll-Hypaque density gradient centrifugation
• Population: Adults ≥18 years meeting ACR revised classification criteria for SLE or RA; chronic hepatitis C and healthy subjects as disease controls; tonsil from routine tonsillectomy donors

Key Findings

• DN (IgD⁻CD27⁻ CD19⁺) B cells are present in all healthy PBL samples at mean 4.6 ± 1.8% of CD19⁺ cells (n=29); always <10%
• DN cells represent >10% of CD19⁺ PBL B cells in 50% of SLE patients (18/36); mean 19.4% in the DN-high subgroup; >20% in 14% of patients (5/36)
• In 25% of SLE patients, DN cells outnumber CD27⁺ memory cells
• DN frequency is not elevated in RA (n=45, mean 4.24%) or chronic hepatitis C (n=7, mean 3.57%) vs. healthy controls (mean 4.59%), indicating SLE specificity
• Somatic hypermutation (VH3 family IgG⁺ cells): DN cells carry 3.2% nucleotide mutation rate in healthy donors (n=28) and 2.6% in SLE (n=24); CD27⁺ memory cells carry 5.4% (healthy, n=28) and 5.1% (SLE, n=23) — DN cells are hypermutated but less so than CD27⁺ counterparts
• Isotype composition: ~44% IgG⁺, ~15% IgM⁺ in healthy DN cells; similar proportions in SLE; DN IgM⁺ cells lack IgD co-expression (unlike the majority of CD27⁺ IgM⁺ memory cells)
• DN cells fail to extrude Rhodamine 123 (R123), identical to CD27⁺ memory cells; naive B cells extrude R123 readily (ABCB1 transporter absent in memory cells)
• DN cells proliferate in response to CpG2006 DNA without BCR crosslinking; naive B cells require BCR co-stimulation for equivalent proliferation
• Proliferating DN cells upregulate CD27 after CpG stimulation
• DN cells are FcRH4⁻ in peripheral blood (both healthy and SLE); tonsil DN (Bm5 CD27⁻) cells contain a FcRH4⁺ fraction alongside FcRH4⁻ cells
• CD38 expression on DN cells parallels Bm5/early-Bm5 memory levels — distinctly below transitional cells, pre-GC cells, and plasmablasts
• Clinical correlations (DN-high ≥10% vs DN-low <10% of PBL B cells, n=46 SLE patients):
  • Nephritis: 15/26 (DN-high) vs 4/20 (DN-low), p=0.025
  • Anti-dsDNA Abs: p=0.001
  • Anti-RNP/Sm Abs: p=0.009
  • SLAM disease activity score (n=23 with available data): p=0.02
  • 9G4 autoantibodies: significantly elevated in DN-high group
• 9G4 autoreactive B cells (VH4-34 encoded) are present at similar frequencies in DN and CD27⁺ switched memory compartments within individual SLE patients
• Panel: FACSCalibur; 8-color: CD19, IgD, CD27, CD38, IgG or IgM or B220 or CD10; 9-color protocol adds CD24, CD138, CD3; FcRH4 detected with biotinylated goat anti-FcRH4 + streptavidin

Methods Used

• Conventional Flow Cytometry (8–9 color, FACSCalibur; IgD/CD27 quadrant gating; Bm1–Bm5 classification)
• FACS Sorting (FACSAria; sorted CD27⁺ and CD27⁻ IgG⁺ CD19⁺CD20⁺IgD⁻ cells for VH gene analysis)
• BCR Sequencing (VH3 family-specific PCR → pCR4Blunt-TOPO cloning → BigDye Terminator sequencing)
• In Vitro B Cell Stimulation (CpG2006 2.5 µg/mL + IL-2 + IL-10, 4-day culture; CFSE proliferation assay; ± anti-IgM BCR crosslinking)

Entities Mentioned

Double-Negative B Cell, CD27, IgD, FcRH4, CD38, IgG, IgM, IgA, CD19, CD10, CD24, B220

Concepts Addressed

Extrafollicular Response, Germinal Center, Somatic Hypermutation, Class Switch Recombination, Memory B Cell

Relevance & Notes

This is an SLE paper with no dengue data. Its value in this wiki is as the foundational characterisation of IgD⁻CD27⁻ double-negative memory B cells and their proposed extrafollicular origin — the conceptual ancestor of what later literature calls atypical B cells, age-associated B cells (ABCs), or T-bet⁺ B cells in acute infections including dengue. The IgD/CD27 quadrant gating scheme introduced here remains the reference framework for identifying this population across disease contexts.

The FcRH4-negative, circulating phenotype is important for comparing with later papers that use FcRH5/FCRL5 as an atypical B cell marker. Wei et al.’s PBL DN cells lack FcRH4, whereas the tonsillar tissue-resident population carries it — a distinction with practical implications for panel design in dengue flow cytometry studies.

The clinical correlations in SLE (nephritis, anti-dsDNA, disease activity) establish that DN B cell expansion is not epiphenomenal but tracks with immunopathology. Whether analogous clinical correlations exist in dengue (e.g., with severe disease or secondary infection) is an open question this wiki is positioned to address.

Limitations: Cross-sectional design; no longitudinal tracking through disease flares or treatment. VH mutation analysis used only VH3 family sequences (n=28 per group), limiting generalisability to other families. No transcriptomic or epigenomic data to compare DN cells with later-characterised atypical B cell populations.

Questions Raised

• Do DN B cells in dengue share transcriptional programs (T-bet, FcRL5, CD11c) with atypical B cells characterised in later acute infection studies?
• Is the lower SHM rate (~3% vs ~5%) a consistent discriminating feature between DN and CD27⁺ memory B cells across disease contexts, or does it vary with antigen specificity and chronicity?
• Do DN B cells expand in acute dengue with similar kinetics to the plasmablast response, or on a different timescale?
• Can the IgD⁻CD27⁻ gate from this paper be used as a retrospective reference when re-analysing older dengue datasets that included IgD and CD27 staining?
• The SLE-specificity finding (no expansion in RA or hepatitis C) raises the question: which features of the SLE immune environment drive DN expansion — chronic TLR stimulation, type I IFN, or loss of tolerance checkpoints? How do these compare to the acute viral environment of dengue?