Integrin CD11b provides a new marker of pre-germinal center IgA+ B cells in murine Peyer's patches

Abstract

Activated B cells can enter germinal centers (GCs) for affinity maturation to produce high-affinity antibodies. However, which activated B cells will enter GCs remains unknown. Here, we found a small population of CD11b+IgA+ B cells located outside of GCs in murine Peyer's patches (PPs). After injection of the CD11b+IgA+ PP B cells into a PP of a recipient mouse, they entered GCs forty hours later. They expressed GC surface markers and pre-GC B cell genes, suggesting that CD11b provides a novel surface marker of pre-GC IgA+ B cells in murine PPs. Furthermore, independently of dendritic cell activation, CD11b expression on B cells can be induced by bacterial antigens, such as pam3CSK4 and heat-killed Escherichia coli in vitro. In addition, mice orally administered with pam3CSK4 or heat-killed E. coli increased the number of PP GC B cells within two days, and enhanced the mucosal antigen-specific IgA response. Our results demonstrate that the induction of CD11b on B cells is a promising marker for selecting an effective mucosal vaccine adjuvant.

Keywords: B cell stimulation; CD11b; affinity maturation; pre-GC B cell marker.

Graphical Abstract

Fig. 1.

The CD11b⁺IgA⁺ PP B cells are located outside GCs migrating towards GCs. (A) Representative flow cytometry data of IgA and CD11b expression in the PP B220⁺ B cells from WT mice. (B, C) Flow cytometry of GC B cells in CD11b⁺IgA⁺ and CD11b⁻IgA⁺ PP B cells, stained with B220 and PNA (B) or B220 and Fas (C). (A–C) Numbers indicate the percentage of cells. Representative data are from at least 20 experiments. (D) Microscopic images of PP section stained with PNA (cyan), CD11b (green), IgA (red) and DAPI (blue). Two CD11b⁺IgA⁺ double positive cells (yellow arrows) were found outside the GC (dashed circle). Scale bar, 200 μm. (E) Images show the SED, IF, GC areas stained with ICAM-1 (red), CD11c (yellow), CD4 (green, in the left photo), MAdCAM-1 (green, in right photos) and DAPI. Each area is marked in the middle schema. Scale bar, 500 μm. (F) Images show that CD11b⁺IgA⁺ B cells (white arrows, marked as 1–7) are located in the indicated areas. The GC and IF areas are marked by dashed lines. Scale bar, 200 μm. (G) CD11b⁻IgA⁺ B cells and CD11b⁺IgA⁺ B cell were directly injected into a PP of IgA-Cre/YC3.60^flox mice, respectively. One hour after injection, representative images show the location of transferred CD11b⁺IgA⁺ PP B cells (red, lower images) or CD11b⁻IgA⁺ PP B cells (red, upper images) in the PP of IgA-Cre/YC3.60^flox mice. The GC boundary (dashed white line) was identified as an IgA-YC3.60 (green) enriched area. Scale bar, 200 μm. (H) Forty hours after injection, the PP with injected CD11b⁺IgA⁺ B cells (red) was observed by microscopy. Scale bar, 200 μm. Data are representative from three independent experiments.

Fig. 2.

CD11b⁺IgA⁺ B cells are pre-GC B cells interacting with CD4⁺ T cells. (A) Heatmap indicating the log fold change (CD11b⁺IgA⁺ B cells/CD11b⁻IgA⁺ B cells) in expression of indicated genes by microarray analysis. (B) Expression levels of indicated GC specific genes and non-GC specific genes and a pre-GC gene were analyzed by qPCR and normalized to those from the β-actin gene. The mean of relative expression levels of CD11b⁻IgA⁺ B cells is taken as 1. Each spot represents the mean of technical triplicates. Bar graphs show the mean values (±SD) of three or four independent measurements. Data were analyzed by ANOVA followed by Tukey’s multiple comparisons. (C) Flow cytometry histograms depicting expression of surface markers of CXCR4 and CD86 in CD11b⁺IgA⁺ B cells and CD11b⁻IgA⁺ B cells. Representative data are from at least five independent experiments. (D) Flow cytometry data of singlet IgA⁺ B cells and conjugated CD4⁺ IgA⁺ cells. (E) Flow cytometry data of CD11b⁻IgA⁺ and CD11b⁺IgA⁺ PP B cells with CD4⁺ T cells in the conjugated gate. (F) Percentage of the CD4⁺ T cells in conjugation with CD11b⁺IgA⁺ and CD11b⁻IgA⁺ PP B cells. Bar graphs show the mean values (±SD) of six independent measurements. Data were compared by two-tailed unpaired Student’s t test. (G) Imaging of sorted CD11b⁺IgA⁺ PP B cells conjugating with CD4⁺ T cells. Scale bar, 20 μm.

Fig. 3.

Bacterial antigens induce CD11b expression on B cells. (A, F) Flow cytometry data of the CD11b expression of the spleen naive B cells stimulated with indicated stimulations for 3 days. (B, G) Percentages of CD11b⁺ B cells of each stimulation. (C, H) qPCR analysis of itgam with the sorted B cells after indicated stimulations. The mean of relative expression levels of spleen naive B cells was taken as 1. (D) Flow cytometry data of sorted CD11b⁺IgA⁺ and CD11b⁻IgA⁺ PP B cells after one day culture on 40LB cells. (E) Pam3CSK4-induced CD11b expression was lost by culture on 40LB cells. (I) Percentages of CD11b⁺ B cells of each stimulation with indicated inhibitors. (B, C, G, H, I), Bar graphs show the mean values (±SD) of at least three independent measurements. (B, C, G, H) Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons. (I) Data were analyzed by two-way ANOVA followed by Tukey’s multiple comparisons.

Fig. 4.

CD11b inducible bacterial antigens enhance the existing GC reaction in PPs. (A) Flow cytometry data of the CD11b expression of the spleen naive B cells stimulated with indicated stimulations for three days. (B) Percentages of CD11b⁺ B cells for each stimulation. (C–E) Balb/c mice were orally administered with indicated heat-killed bacteria, pam3CSK4 or PBS (ctrl). Two days later, flow cytometry data (C) of PNA^hiB220⁺ GC B cells, percentages (D) and absolute numbers (E) of PNA^high B220⁺ PP GC B cells from each mouse were calculated. (F) Indicated iGB cells were sorted, labeled (cyan) and then intravenously injected to mice, independently. One of the PPs was selected to analyze the injected cell localization. To identify the GC boundary (dashed white line), anti-IgA was directly injected into the selected PP. Scale bar, 500 μm. (G) Schedule for oral immunization. (H) OVA-specific IgA after immunization. (B, D, E, H) Bar graphs show the mean values (±SD) of at least three independent measurements. (D, E) Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons. (H) Data were analyzed by two-way ANOVA followed by Tukey’s multiple comparisons.