Increased autoreactivity and maturity of EBI2+ antibody-secreting cells from nasal polyps

Abstract

Elevated numbers of antibody-secreting cells (ASCs) and anti-double-stranded DNA (anti-dsDNA) antibodies are found in nasal polyp (NP) tissue. The presence of anti-dsDNA IgG in tissue prospectively predicts recurrent NP but the characteristics of the source ASCs are unknown. Here, we investigated whether NP B cells expressing the extrafollicular marker EBI2 have increased propensity for autoantibody production and evaluated the molecular characteristics of NP ASCs. NPs showed increased frequencies of anti-dsDNA IgG and total IgG ASCs compared with tonsils, with more pronounced differences among EBI2+ cells. In NPs, EBI2+ cells were frequently double negative (IgD-CD27-) and ASCs. Single-cell RNA-Seq analysis of tonsils and NPs revealed substantial differences in B lineage composition, including differences in percentages of ASCs, germinal centers, proliferative cells, and non-ASCs. NPs exhibited higher expression of specific isotypes (IGHE, IGHA1, IGHA2, and IGHG4) and mature plasma genes, including SDC1 and XBP1, than tonsils. Gene Ontology biological processes indicated upregulated NF-κB and downregulated apoptosis pathways in NP ASCs. Together, these data indicate that NP EBI2+ ASCs secret increased total and anti-dsDNA IgG compared with those from tonsils and had molecular features of mature plasma cell differentiation.

Keywords: Adaptive immunity; Immunoglobulins; Immunology; Inflammation; Respiration.

Figure 1. NP tissue contained increased anti-dsDNA IgG and total IgG-secreting cells.

(A) Bar plot showing the frequency of B cells in all cells in tonsils (n = 11) and NPs (n = 6). (B) Pie charts showing the frequency of B subsets in B cells derived from tonsils (n = 11) and NPs (n = 6) enumerated by flow cytometry. (C) Frequencies of dsDNA-specific (10 μg/mL) and total IgG-expressing ASCs were quantified by ELISpot. (D–F) The frequencies of ASCs were compared between tonsils (n = 10) and NPs (n = 8): (D) IgG ASCs per B cell, (E) anti-dsDNA IgG ASCs per B cell, and (F) anti-dsDNA IgG ASCs per IgG ASC. The Mann-Whitney U test was used for the comparisons. ***P <.001, ****P < .001. Median with IQR is indicated.

Figure 2. Frequencies of IgG ASCs and anti-dsDNA–specific IgG ASCs are enriched in sorted EBI2⁺ cells.

(A) Bar plot showing the frequency of EBI2⁺ cells in B cells in tonsils and NPs. (B) Pie charts showing the frequency of EBI2⁺ cells in B cell subsets derived from tonsils (n = 11) and NPs (n = 6) determined using flow cytometry. sMemory, class-switched memory. (C–E) Comparisons of total and dsDNA-specific ASCs in B cells from tonsils (n = 7) and NPs (n = 5): (C) IgG ASCs in total B cells, (D) anti-dsDNA IgG ASCs in total B cells, and (E) anti-dsDNA IgG ASCs in total IgG ASCs determined by ELISpot. The Mann-Whitney U test was used for the 2-group comparisons. The Kruskal-Wallis test with Dunn’s correction was used for the multiple group comparisons. If P > 0.05, the result is not significant (ns). *P < 0.05, **P < 0.01. Median with IQR is indicated.

Figure 3. Characterization of B cell subsets using scRNA-Seq and CITE-Seq analyses.

(A) Single-cell analysis of cells from patients with tonsil (n = 5) and CRSwNP (n = 5). Two-dimensional UMAP plots of 28,192 cells. (B) Feature plots of characteristic genes in different cell types.

Figure 4. Two-dimensional UMAP plots of B lineage cells.

(A) UMAP plots of B lineage cells separated by tissue type. (B) Dot plot of marker genes of B lineage cells: expression levels across B cell subsets. (C) Discrete ASC signatures by scRNA-Seq (left) and CITE-Seq (right) analysis.

Figure 5. Comparison of NP- and tonsil-derived ASCs.

(A) Violin plots of genes upregulated in NP- and tonsil-derived ASCs. (B) GO biological process performed on most variable genes in ASCs derived from NPs and tonsils. Wilcoxon’s test was used for gene comparisons. (C) GSEA was performed on the top 2,000 most variable features in ASCs derived from NPs and tonsils. NF-κB ranked as the most-enriched signaling pathway in NPs.