Chronic airway inflammation provides a unique environment for B cell activation and antibody production

Abstract

Background: B cells play many roles in health and disease. However, little is known about the mechanisms that drive B cell responses in the airways, especially in humans. Chronic rhinosinusitis (CRS) is an inflammatory disease of the upper airways that affects 10% of Europeans and Americans. A subset of CRS patients develop nasal polyps (NPs), which are characterized by type 2 inflammation, eosinophils and group 2 innate lymphoid cells (ILC2s). We have reported that NP contain elevated levels of B cells and antibodies, making NP an ideal system for studying B cells in the airways.

Objective: We sought to determine the mechanisms that drive B cell activation and antibody production during chronic airway inflammation.

Methods: We analysed B cells from NP or tonsil, or after ILC2 coculture, by flow cytometry. Antibody production from tissue was measured using Luminex assays and the frequency of antibody-secreting cells by ELISpot. Formation of B cell clusters was assessed using immunohistochemistry. Expression of genes associated with B cell activation and class switch recombination was measured by qRT-PCR.

Results: NP contained significantly elevated frequencies of plasmablasts, especially those that expressed the extrafollicular marker Epstein-Barr virus-induced protein 2 (EBI2), but significantly fewer germinal centre (GC) B cells compared with tonsil. Antibody production and the frequency of antibody-secreting cells were significantly elevated in NP, and there was evidence for local class switch recombination in NP. Finally, ILC2s directly induced EBI2 expression on B cells in vitro.

Conclusions and clinical relevance: Our data suggest there is a unique B cell activation environment within NP that is distinct from classic GC-mediated mechanisms. We show for the first time that ILC2s directly induce EBI2 expression on B cells, indicating that ILC2s may play an important role in B cell responses. B cell-targeted therapies may provide new treatment options for CRSwNP.

Keywords: ENT; B cells; IgE; lymphocytes.

Figure 1

The frequency of B cell subsets in NP and tonsil. A. Identification of B cell subsets by flow cytometry. All cells were identified after gating on single alive cells. Total CD19⁺ frequency was calculated from the CD3neg gate. The frequency of naïve, memory, GC, and plasmablasts (PB) are expressed as a % of total CD19⁺ cells. B. Tonsils contained elevated levels of total B cells and naïve B cells. C. The frequency of activated B cell subsets was distinct between NP and tonsil. NP contained a significantly higher frequency of plasmablasts, while tonsil contained a higher frequency of GC B cells. D. Representative 20× images of CD20 staining in a control UT and NP sample. Immunohistochemical staining of CD20⁺ cells revealed no increase in the frequency of B cell follicles (group of >300 CD20⁺ cells in a 200µm×200µm area) or clusters (group of 100–299 CD20⁺ cells in a 200µm×200µm area) in NP compared to normal sinus tissue from non-CRS patients. Data represent mean ± SEM; *p<0.05, **p<0.01; ***p<0.001 by Mann-Whiney U test.

Figure 2

Antibody production in NP and tonsil. A. Tissue explants from NP or tonsil were cultured in vitro for 4 days, and total antibody production was measured from the culture supernatants. NP produced significantly higher levels of all antibody isotypes compared to tonsil. B. The frequency of antibody-secreting cells was determined by ELISpot assays. Representative triplicate ELISpot wells from NP are shown; arrows in the IgE wells indicate representative positive spots. NP contained significantly elevated frequencies of IgG-, IgA-, and IgE-secreting cells compared to tonsil. C. Expression of AID and germline transcripts was measured using qRT-PCR. AID expression was highest in tonsil, but detectible in NP. Expression of germline transcripts for IgE, IgG₁, IgG₄, IgA₁ and IgA₂ was detectible in NP. Data represent mean ± SEM; *p<0.05, **p<0.01; ***p<0.001 by Mann-Whiney U test.

Figure 3

EBI2 expression on B cell subsets. A. Representative histograms from one NP sample showing EBI2 expression on plasmablast (PB), naïve and memory B cells. The frequency of EBI2 expressing cells was highest in the plasmablasts in NP. B. The frequency of EBI2⁺ plasmablasts, as a percentatge of total plasmablasts, was significantly higher in NP compared to tonsil, but the frequency of EBI2⁺ cells was not different in naïve or memory B cells. Data represent mean ± SEM; *p<0.05, **p<0.01; ***p<0.001 by Kruskal-Wallis test with Dunn’s correction (A) or Mann-Whiney U test (B).

Figure 4

ILC2s induce EBI2 expression on B cells. A. Gating strategy for the identification of ILC2s in peripheral blood by flow cytometry. Data are representative of n=6 samples. B. The frequency of EBI2⁺ cells was significantly enhanced in B cells co-cultured with autologous ILC2s. Data represent mean ± SEM; *p<0.05, **p<0.01; ***p<0.001 by Mann-Whiney U test.