Microbe-dependent CD11b+ IgA+ plasma cells mediate robust early-phase intestinal IgA responses in mice

Abstract

Intestinal plasma cells predominantly produce immunoglobulin (Ig) A, however, their functional diversity remains poorly characterized. Here we show that murine intestinal IgA plasma cells can be newly classified into two populations on the basis of CD11b expression, which cannot be discriminated by currently known criteria such as general plasma cell markers, B cell origin and T cell dependence. CD11b(+) IgA(+) plasma cells require the lymphoid structure of Peyer's patches, produce more IgA than CD11b(-) IgA(+) plasma cells, proliferate vigorously, and require microbial stimulation and IL-10 for their development and maintenance. These features allow CD11b(+) IgA(+) plasma cells to mediate early-phase antigen-specific intestinal IgA responses induced by oral immunization with protein antigen. These findings reveal the functional diversity of IgA(+) plasma cells in the murine intestine.

Figure 1. Intestinal CD11b⁺ IgA⁺ cells require microbial stimulation.

(a–c) Mononuclear cells were isolated from the small intestines of SPF or GF mice (a), mock- or antibiotic-treated SPF mice (b), or MyD88 WT or knockout (KO) mice (c) for analysis of IgA and CD11b expression by flow cytometry. Graphs show data from individual mice, and bars indicate median. Statistical analyses were performed with Mann–Whitney’s U-test. (d) Specimens of small intestinal tissues of WT and MyD88 KO mice were stained for IgA and CD11b, and counterstained with 4′,6-diamidino-2-phenylindole. Data are representative of three independent experiments. Scale bars, 50 μm.

Figure 2. Both CD11b⁺ and CD11b⁻ IgA⁺ cells in the intestine are categorized as plasma cells.

(a) Cells were purified by cell sorting from the iLP, and their morphology was examined by haematoxylin and eosin staining after cytospin. Data are representative of three independent experiments. (b) Cells were isolated from the iLP for the analysis of F4/80, CD11c and CCR3 expression on CD11b⁻ IgA⁺, CD11b⁺ IgA⁺ and CD11b⁺ IgA⁻ cells. Grey indicates isotype control. Similar results were obtained from three separate experiments. (c) Cells were isolated from the iLP for comparisons between CD11b⁺ and CD11b⁻ IgA⁺ cells in terms of cell size (FSC) and density (SSC), and expression of CD19, B220, CD138, CD38, CD40 and Blimp1. Grey indicates isotype control. Similar results were obtained from five separate experiments.

Figure 3. CD11b⁺ IgA⁺ cells require the lymphoid structure of Peyer’s patches.

(a) Mice were treated with FTY720 every day for 5 days. The day after the final treatment, the proportions of CD11b⁺ and CD11b⁻ IgA⁺ cells were measured by flow cytometry. Data are presented as means±s.d. from four mice. Similar results were obtained from three separate experiments. (b) Proportions of CD11b⁺and CD11b⁻ IgA⁺ cells in the iLP of WT and TCRβδ KO mice were measured by flow cytometry. Data are presented as means±s.d. from four mice. Similar results were obtained from three separate experiments. (c) After three oral immunizations with OVA plus cholera toxin, cells were isolated from the iLP and used in an ELISPOT assay to enumerate OVA-specific IgA AFCs. In some groups of mice, CD11b⁺ or CD11b⁻ IgA⁺ cells were depleted by cell sorting before application of ELISPOT assay. Phosphorylcholine-specific IgA AFCs were measured. Graphs show data from individual mice, and bars indicate median. Statistical analyses were performed with Mann–Whitney’s U-test. (d) Mononuclear cells were isolated from the iLP of Peyer’s patch (PP)-normal (control Ab) and -null (anti-IL-7Rα Ab) mice for analysis of IgA and CD11b expression by flow cytometry. Graphs show data from individual mice. Statistical analyses were performed with Mann–Whitney’s U-test. (e) Mononuclear cells were isolated from PPs for analysis of CD11b⁺ and CD11b⁻ IgA⁺ cells by flow cytometry. Similar results were obtained from three separate experiments.

Figure 4. CD11b⁺ IgA⁺ cells are proliferating cells.

(a) mRNA was purified from small intestinal CD11b⁺ and CD11b⁻ IgA⁺ cells and used for microarray analysis. Data related to the cell cycle and proliferation are shown. Data are representative of two independent experiments. (b) Mice were treated with BrdU, and uptake of BrdU by CD11b⁺ and CD11b⁻ IgA⁺ cells was determined by flow cytometry. Data are representative of four independent experiments. (c) Cells were isolated from the intestinal lamina propria of mice receiving CPM to analyse CD11b⁺ IgA⁺ cells. Similar results were obtained from four separate experiments. Graphs show data from individual mice. Statistical analyses were performed with Mann–Whitney’s U-test.

Figure 5. Role of IL-10 in the maintenance of CD11b⁺ IgA⁺ cells in the iLP.

(a) Mice were treated with antibodies to block IL-5, IL-6, IL-10 or antagonistic TACI-immunoglobulin (TACI-Ig) fusion protein. Mononuclear cells were isolated from the iLP and used for analysis of CD11b⁺ and CD11b⁻ IgA⁺ cells by flow cytometry. Data are presented as means±s.d. (n=4). (b) Mononuclear cells were isolated from the iLP of WT or IL-10 KO mice for analysis of IgA and CD11b expression by flow cytometry. Graphs show data from individual mice. Statistical analyses were performed with Mann–Whitney’s U-test.

Figure 6. Proliferating IgA⁺ cells mediate early-phase IgA responses to oral antigen.

(a) Experimental schedule for oral immunization and CPM treatment. Mice were orally immunized with OVA plus CT on days 0, 7 and 14. One group received CPM during oral immunization (days 0, 7 and 14) and another received CPM after the last immunization (days 18, 19 and 20). (b,c) One week after the final immunization (day 21), mononuclear cells were isolated from the iLP to quantify OVA-specific IgA-forming cells by ELISPOT (b). Simultaneously, faeces (c,d) were collected and were used for the detection of the (c) OVA-or (d) B subunit of CT (CTB)-specific IgA by enzyme-linked immunosorbent assay. Data are from individual mice and bars indicate median (b) and represent means±s.d. (n=10) from two separate experiments (c,d). *P<0.001, **P<0.01, ***P<0.05 (two tailed unpaired t-test). (e) Mononuclear cells were isolated from the iLP of mock- or CPM-treated mice 1 week after the final immunization to quantify CTB-specific IgA-forming cells by ELISPOT. In some groups of mock-treated mice, CD11b⁺ or CD11b⁻ IgA⁺ cells were depleted by cell sorting before application of ELISPOT assay. Graphs show data from individual mice, and bars indicate median. (f) On day 21, mice were orally challenged with 100 μg CT. After 15 h, the volume of intestinal fluid was measured. Graphs show data from individual mice, and bars indicate median. Similar results were obtained from two separate experiments. (g) Spot sizes of CTB-specific IgA AFCs were measured by Zeiss KS ELISPOT software. Graphs show data from individual mice, and bars indicate median. Statistical analyses were performed with Mann–Whitney’s U-test (e–g).