Ectopic germinal centers in the nasal turbinates contribute to B cell immunity to intranasal viral infection and vaccination

Abstract

The nasal mucosa is the first immunologically active site that respiratory viruses encounter and establishing immunity at the initial point of pathogen contact is essential for preventing viral spread. Influenza A virus (IAV) in humans preferentially replicates in the upper respiratory tract (URT) but mouse models of infection result in lower respiratory tract infection. Here, we optimize IAV inoculation to enhance replication in the nasal turbinate (NT) and study local B cell immunity. We demonstrate that URT-targeted IAV infection stimulates robust local B cell responses, including germinal center (GC) B cell formation in the NT, outside of classical nasal-associated lymphoid tissues. NT GC contributes to local tissue-resident B cell generation and enhances local antibody production. Furthermore, URT-focused immunization also induces significant GC formation in the NT. Finally, we detect steady-state GC in the NT of both mice and healthy humans, suggesting continuous immune surveillance triggered by environmental stimuli. These findings highlight the pivotal role of the NT in local and systemic immunity, with important implications for future mucosal vaccines targeting the upper airways.

Keywords: B cell; germinal center; influenza A virus; mucosal immunity; upper respiratory tract.

Fig. 1.

URTI induces robust virus-specific B cell responses within the NT. (A) Scheme of the differential method of PR8 infection. URTI consisted in 10 μL of viral inoculum containing 10⁵ TCID₅₀. LRTI inoculum contained 500 TCID₅₀ diluted in 25 μL. Created with BioRender.com. (B) Representative microscopic frontal section of NT stained with Hoechst (light blue) with detection of infected cells after PR8-mCherry (red) infection along the nasal cavities. (C) Log TCID₅₀ calculation on infected NT and lungs lysates after differential infection methods. Mean ± SEM; one-way ANOVA test. Shown is one representative of three independent experiments (n = 5). (D and E) Representative flow cytometry plot of total (D) or IAV-specific (E) GC B cell gating in URTI vs LRTI in mLN and cLN. On the Right are represented on bar graphs total (D) or IAV-specific (E) GC B cells percentages among total B cells after either URTI or LRTI at 14- and 21-d post infection. Mean ± SEM. Shown is one representative of two independent experiments (n = 5); one-way ANOVA test. (F) Serial dilution curves of NP-specific Ab from nasal washes (NW) and sera of mice at steady state or 28 d post infection (URTI or LRTI). Mean ± SD; one-way ANOVA test was conducted between URTI and LRTI conditions. AUC were calculated and plotted from detected anti-NP Abs in NW and sera. Mean ± SEM; Student’s t test. One representative of three independent experiments (n = 6). (G) Representative flow plot of HA-specific B cell gates 28 d post URTI or LRTI. Plotted populations on bars with Mean ± SEM. (H) Bar graph representation of antigen-specific CD138⁻CD38⁻ B cells, comparison of both populations following URT or LRT infection methods. Mean ± SEM, (G and H) shown is one representative of two independent experiments (n = 9). ns P >0.05; *P < 0.05; **P < 0.005; ***P < 0.0005.

Fig. 2.

The nasal tissue hosts Influenza-specific germinal center B cells after infection. (A) Scheme of experiment strategy. Created with BioRender.com. (B) Dilution curves of detected NP-specific Abs in NW from mice at steady-state or 28 d after URTI with or without FTY720 treatment. Mean ± SD; Bar graphs comparison of calculated AUC from above-mentioned groups. Mean ± SEM; one-way ANOVA test. One representative experiment of two independent ones (n = 5). (C) Bar graphs of HA-specific NT and NALT CD45iv⁻ IgD⁻ B cells total count 28 d after URTI with or without FTY720 treatment. Mean ± SEM, (n = 5); Student’s t test. (D and E) Representative flow gates of CD45iv⁻ GC B cells at day 0 and 21 post URTI along with bar graphs of total or HA-specific GC B cells temporal dynamic percentages in NT (D) and NALT (E). Mean ± SEM; (n = 8 and 9), from two independent experiments; one-way ANOVA test. (F and G) NT frontal (F) and sagittal (G) microscopy sections of infected mice 21 d post URTI with close-up to the middle turbinates (MD) with indicated staining colors. (H) Scheme of experiment strategy with S1pr2-mice. Created with BioRender.com. (I) Representative histogram plots of NT TdTomato intensity in CD138⁻IgD⁻ GC B cells at the indicated timepoints post URTI. (J) Bar graph temporal dynamic comparison of TdTomato labeled GC B cells percentages in the NT. Mean ± SEM; (n = 4 and 5); one-way ANOVA test. (K) Curve plot representation of TdTomato labeled CD138⁻IgD⁻ GC B cells total count dynamic in NT, NALT, and lungs at indicated timepoints post URTI. Mean ± SEM; (n = 4 and 5). (L) Switched (IgM⁻IgD⁻) B cell proportions among TdTomato labeled CD138⁻IgD⁻ GC B cells from day 21 post URTI. Mean ± SEM; Student’s t test (n = 4 and 5). ns P > 0.05; *P < 0.05; **P < 0.005; ***P < 0.0005.

Fig. 3.

Germinal center B cells in NT exhibit canonical features. (A) UMAP visualization of single-cell RNA sequencing data from sorted TdTomato⁺ GC B cells in the NT, cLN, and NALT. Cells are grouped either based on cell types according to signatures identified in B or to organ of origin. (B) Density Plot showing gene expression of Cd38, Ighd, Aicda, Bcl6, and DZ and LZ signatures (37) on the UMAP. (C) Box plot showing the BCR nucleotide mutation frequency among B cell types, as identified in A, across tissues (NT, blue; NALT, violet; cLN, green). Data are presented as median and interquartile range. (D) Representative flow cytometry plot of gated GC B cells and their division into DZ and LZ GC B cells, based on CXCR4 and CD86 expression, across NT, NALT, cLN, and mLN. (E) Dot plot comparing the frequencies of DZ and LZ GC B cells across NT, NALT, cLN, and mLN. Mean ± SD. Statistical analysis performed using a t test. Data represent two independent experiments (n = 10). (F) Dot plot displaying the ratio of DZ to LZ GC B cells in NT, NALT, cLN, and mLN. Mean ± SD. Statistical analysis performed using one-way ANOVA. Data represent two independent experiments (n = 10).

Fig. 4.

URT-restricted immunizations promote GC B1-8^hi cell responses in the NT. (A) Scheme of experiment strategy. Created with BioRender.com. (B) Representative flow plots of NT, NALT, and lungs EGFP⁺ cells among CD19iv⁻ B cells along with their percentages plotted on bars. Mean ± SEM; (n = 5). (C) NT frontal section of a mouse immunized with NP-CT after adoptive transfer with B1-8^hi cells as indicated in Fig. 3A. (D) Representative flow gates for GC B cells gated from CD19iv⁻ B cells in NT and NALT with or without immunization. GC B cells and ASC percentages are plotted per organs and conditions and differences are evaluated between immunized and nonimmunized groups. Mean ± SEM; (n = 5), unpaired multiple t tests. (E) Microscopic NT frontal section of a mouse immunized with NP-CT in a URT-restricted manner after adoptive transfer with B1-8^hi cells showing CD95⁺GL7⁺ B1-8^hi cells in NT niches. Quadrants represent single color positive staining and merge duplicates of a close-up on a B cell cluster of the NT. (F) Representative flow gates of B1-8^hi labeled GC B cells populations in NT and NALT (Left) and the quantification in percentages among CD19iv⁻IgD⁻GC B cells (Right). Mean ± SEM; (n = 5). ns P >0.05; *P < 0.05; **P < 0.005; ***P < 0.0005. ****P < 0.00005.

Fig. 5.

Human nasal tissue retains germinal center reaction in the absence of detectable active infection or challenge. (A) Representative flow cytometry plot showing the gating strategy for GC B cells from human nasopharyngeal swabs collected from deep nasal cavity tissue (Deep NT) and peripheral blood mononuclear cells (PBMC) as a negative control. (B) GC B cell frequencies among IgD⁻ B cells displayed as dot plots, comparing human Deep NT and PBMC. Data is from three combined independent experiments. Mean ± SD. Statistical analysis performed using a t test (n = 36). (C) (Left) Frequencies of IgD⁻ B cells among total B cells, plotted as dot plots and detected in human Deep NT and PBMC. (Right) Frequencies of IgA⁺ B cells among IgD⁻ B cells, also plotted as dot plots, in Deep NT and PBMC. Data is from three combined independent experiments. Mean ± SD. Statistical analysis performed using a t test (n = 36). (D) Schematic representation of human nasal swab collection methods, illustrating the collection from Deep NT and higher nasal cavities (High NT). Created with BioRender.com. (E) Dot plot comparison of the total count of B cells collected using the high and deep nasal swab methods in healthy individuals. Data represent one of four independent experiments. Mean ± SD. Statistical analysis performed using a t test (n = 12). (F) Representative flow cytometry plot showing the gating strategy for human GC B cells based on CD10 expression from tonsils (positive control), Deep NT, High NT, and PBMC (negative control). (G) GC B cell frequencies among CD38⁺ IgD⁻ B cells (Top) and among total B cells (Bottom), shown as dot plots for human tonsils, High NT, Deep NT, and PBMC. Data represent two independent experiments. Mean ± SD. Statistical analysis performed using a one-way ANOVA test (3 < n < 12). (H) Ratio of IgG⁺ to IgA⁺ B cells displayed in dot plots, comparing tonsils, High NT, and Deep NT. Data represent two independent experiments. Mean ± SD. Statistical analysis performed using a one-way ANOVA test (3 < n < 12). (I) Representative flow cytometry plot showing the gating strategy for IgA⁺ and IgG⁺ B cells from human tonsils and nasopharyngeal swabs collected from Deep and High NT.