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

Kunisawa J, Gohda M, Hashimoto E, Ishikawa I, Higuchi M, Suzuki Y, Goto Y, Panea C, Ivanov II, Sumiya R, Aayam L, Wake T, Tajiri S, Kurashima Y, Shikata S, Akira S, Takeda K, Kiyono H - Nat Commun (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan. kunisawa@nibio.go.jp

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.

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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).
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f6: 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).

Mentions: To examine the immunological importance of proliferating IgA+ PCs present mainly in CD11b+ IgA+ PCs, mice were orally immunized with OVA plus CT. In this assay, one group received CPM treatment during immunization and the second group received CPM treatment 4 days after the final immunization (Fig. 6a). Because of the high cell-proliferation activity, CPM treatment during oral immunization resulted in efficient killing of peanut agglutinin (PNAhi) B220+ GC B cells and thus a reduction in the numbers of IgA+ IgM– plasmablasts in the PPs (Supplementary Fig. S10). Thus, treatment with CPM during oral immunization led to an ~90% reduction in the numbers of OVA-specific IgA AFCs (Fig. 6b); this was associated with almost complete disappearance of faecal IgA produced against OVA (Fig. 6c). On the other hand, when mice were treated with CPM 4 days after the final immunization to remove proliferating cells mainly present in CD11b+ IgA+ cells in the iLP, the reduction in numbers of OVA-specific IgA AFCs in the iLP was only about 50% (Fig. 6b). This finding was consistent with our current finding that CD11b+ IgA+ PCs accounted for half the number of OVA-specific IgA AFCs (Fig. 3c). Thus, CPM treatment after the last immunization preferentially depleted CD11b+ IgA+ cells, with little influence on CD11b− IgA+ cells. Of note, these mice showed ~90% reduction in OVA-specific IgA content in the faeces compared with mice not treated with CPM (Fig. 6c). We also confirmed that CPM treatment 4 days after final immunization induced a reduction in the production of IgA specific to the B subunit of CT (that is, CTB), which was associated with the halving of the abundance of CTB-specific IgA AFCs in the intestine (Fig. 6d). Like OVA-specific IgA responses (Figs. 3c and 6b), similar levels of reduction of CTB-specific IgA AFCs were noted when CD11b+ IgA+ cells were depleted before ELISPOT assay (Fig. 6e). These mice showed reduced resistance to oral challenge with CT and developed watery diarrhoea (Fig. 6f and Supplementary Fig. S11).


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

Kunisawa J, Gohda M, Hashimoto E, Ishikawa I, Higuchi M, Suzuki Y, Goto Y, Panea C, Ivanov II, Sumiya R, Aayam L, Wake T, Tajiri S, Kurashima Y, Shikata S, Akira S, Takeda K, Kiyono H - Nat Commun (2013)

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).
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f6: 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).
Mentions: To examine the immunological importance of proliferating IgA+ PCs present mainly in CD11b+ IgA+ PCs, mice were orally immunized with OVA plus CT. In this assay, one group received CPM treatment during immunization and the second group received CPM treatment 4 days after the final immunization (Fig. 6a). Because of the high cell-proliferation activity, CPM treatment during oral immunization resulted in efficient killing of peanut agglutinin (PNAhi) B220+ GC B cells and thus a reduction in the numbers of IgA+ IgM– plasmablasts in the PPs (Supplementary Fig. S10). Thus, treatment with CPM during oral immunization led to an ~90% reduction in the numbers of OVA-specific IgA AFCs (Fig. 6b); this was associated with almost complete disappearance of faecal IgA produced against OVA (Fig. 6c). On the other hand, when mice were treated with CPM 4 days after the final immunization to remove proliferating cells mainly present in CD11b+ IgA+ cells in the iLP, the reduction in numbers of OVA-specific IgA AFCs in the iLP was only about 50% (Fig. 6b). This finding was consistent with our current finding that CD11b+ IgA+ PCs accounted for half the number of OVA-specific IgA AFCs (Fig. 3c). Thus, CPM treatment after the last immunization preferentially depleted CD11b+ IgA+ cells, with little influence on CD11b− IgA+ cells. Of note, these mice showed ~90% reduction in OVA-specific IgA content in the faeces compared with mice not treated with CPM (Fig. 6c). We also confirmed that CPM treatment 4 days after final immunization induced a reduction in the production of IgA specific to the B subunit of CT (that is, CTB), which was associated with the halving of the abundance of CTB-specific IgA AFCs in the intestine (Fig. 6d). Like OVA-specific IgA responses (Figs. 3c and 6b), similar levels of reduction of CTB-specific IgA AFCs were noted when CD11b+ IgA+ cells were depleted before ELISPOT assay (Fig. 6e). These mice showed reduced resistance to oral challenge with CT and developed watery diarrhoea (Fig. 6f and Supplementary Fig. S11).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan. kunisawa@nibio.go.jp

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.

Show MeSH
Related in: MedlinePlus