SUPPLEMENTARY INFORMATION

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Elijah Hoover
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GP2 Type I-piliated bacteria FAE M cell M cell pocket idc T cell mdc Generation of

Supplementary Figure 1 Schematic diagram showing GP2-dependent antigen transcytosis

GP2 expressed on the apical surface of M cells functions as a transcytotic receptor

The bacteria transcytosed were further transferred to immature DC (idc) accumulating

1 GP2 Type I-piliated bacteria FAE M cell M cell pocket idc T cell mdc Generation of antigenspecific T cells Induction of antigen-specific mucosal immune response Supplementary Figure 1 Schematic diagram showing GP2-dependent antigen transcytosis in PP. GP2 expressed on the apical surface of M cells functions as a transcytotic receptor for type1-piliated bacteria. The bacteria transcytosed were further transferred to immature DC (idc) accumulating underneath M cells. The antigen-primed DCs undergo maturation (mdc) and subsequently migrate to the T-cell-rich region in PP for antigen presentation. Consequently, antigen-specific T cells were generated in PP. 1

2 Supplementary Figure 2 Expression of GP2 in mouse PP M cells. (a) Whole mount specimens of mouse PP were stained with anti-gp2 mab (green), UEA-1 (red) and phalloidin (blue), and were analyzed by a confocal laser microscopy. An X-Y view from the luminal side is shown. (b) lighmagnification images of the FAE region depicted with a square in panel a were shown (upper panel). The X-Z image at the position indicated by a dotted line in the X-Y image is shown (lower panel). M cells characterised by M-cell pocket formation due to invagination of the basolateral plasma membrane (an arrow in the lower panel) were double positive for GP2 and UEA-1. In contrast, goblet cells that lack M-cell pocket structure are single positive for UEA-1 (an arrowhead in the lower panel). (c) Single-color images of GP2 and UEA-1 staining indicate restricted expression of GP2 in UEA-1-positive cells. Scale bars: 50 (a) or 10 (b) μm. 2

3 GP2 Clusterin Supplementary Figure 3 Expression of GP2 in human PP M cells. Serial tissue sections of human PP were stained with the antibody specific for GP2 (upper) or Clusterin (lower), a marker of human M cells. Similar expression patterns of GP2 and Clusterin were observed (arrowheads). Note that Clusterin is also expressed by follicular dendritic cells in the germinal center. Similar staining was observed in two other human PP specimens. Scale bars: 100 µm. 3

e Number of colloidal gold particle per region Number of cell and region counted Apical plasma membrane Basolateral plasma membrane Golgi/endosomal structures 4.9 ± 3.3 (8 cells, 31 regions) 1.0 ± 1.

a-d) Immunoelectron microscopy were performed on Cryo-ultrathin sections of mouse PP fixed with 4% paraformaldehyde-0.1% glutaraldehyde.

4 e Number of colloidal gold particle per region Number of cell and region counted Apical plasma membrane Basolateral plasma membrane Golgi/endosomal structures 4.9 ± 3.3 (8 cells, 31 regions) 1.0 ± 1.3 (6 cells, 24 regions) 3.9 ± 1.3 (3 cells, 11 regions) Others 0.2 ± 0.5 (7 cells, 31 regions) Supplementary Figure 4 Analysis of subcellular localisation of GP2 by immunoelectron microscopy. a-d) Immunoelectron microscopy were performed on Cryo-ultrathin sections of mouse PP fixed with 4% paraformaldehyde-0.1% glutaraldehyde. GP2 (arrowheads) were detected intensely on the apical plasma membrane of M cells (a), whereas they were absent in the staining with control IgG (b). The positive signals were also observed in intracellular organelles such as Golgi apparatus (Go) (c) and endosomal structures (Fig. 1g in the main text) as well as the basolateral plasma membrane (d). Scale bars: 0.5 µm. e) For quantitative analysis, 0.25 x 0.25 µm square regions were randomly selected in the apical and basolateral plasma membrane domains, Golgi apparatus/endosomal structures, and other intracellular regions (nucleus, mitochondria and cytosol), and the number of colloidal gold particles in each area was counted. The values represent means and s.d. 4

0051 0 mgp2-fc hgp2-fc higg-fc Supplementary Figure 5 Inhibitory effect of mannose on the

5 n.s. p = n.s. p = mgp2-fc hgp2-fc higg-fc Supplementary Figure 5 Inhibitory effect of mannose on the binding of E. coli to GP2. In vitro binding assay with recombinant GP2-Fc or control Fc proteins in the presence or absence of mannnose and galactose. Data are means and s.e.m. (n = 3). n.s.: not significant. 5

6 Supplementary Figure 6 Binding assay of GP2 with various bacterial components. Total membrane fraction that contains FimH was prepared from E. coli. Highly purified LPS, peptideglycan and flagellin were purchased from Invivogen. These bacterial components were immobilized on microtiter plates. After blocking, the microtiter wells were incubated with recombinant GP2-Fc or control Fc proteins. The amount of bound proteins was detected by horseradish peroxidase-conjugated anti-human IgG antibody and visuarized using 3,3',5,5'-tetramethylbenzidine as a substrate. Data are means and s.e.m. (n = 3). 6

Typhimurium was colocalized with GP2 on the apical plasma membrane and in the subapical cytoplasmic vesicles (arrowheads).

7 GP2 GFP-S. typhimurium 90 o Apical Basal Supplementary Figure 7 Colocalizaion of GP2 and S. Typhimurium in M cells. The interaction of GP2 and S. Typhimurium was examined in vivo by the ligated intestinal loop assay. GFP-expressing S. Typhimurium was colocalized with GP2 on the apical plasma membrane and in the subapical cytoplasmic vesicles (arrowheads). Top, apical view. Bottom, lateral view. For the 3D-imaging view, see Supplementary Information, Movie3. Scale bars: 10 μm. 7

a GFP-E. coli GP2 F-actin E. coli WT E. coli ΔFimH b GFP + M cells/pp dome area (cells/mm 2 ) 80 70 60 50 40 30 20 10 p = 0.

coli wild-type (WT) or FimH-deficient (ΔFimH) strains by M cells was evaluated by the ligated intestinal loop assay in BALB/c mice.

coli (green) for one hour, whole-mount specimens of PPs were stained with anti-gp2 mab (red) to detect M cells, followed by phalloidin

coli taken up by M cells. Scale bars: 80 µm. (b) The number of M cells taking up GFP-E.

8 a GFP-E. coli GP2 F-actin E. coli WT E. coli ΔFimH b GFP + M cells/pp dome area (cells/mm 2 ) p = WT ΔFimH Supplementary Figure 8 FimH is necessary for efficient uptake of E. coli by M cells. (a) The uptake of GFP-E. coli wild-type (WT) or FimH-deficient (ΔFimH) strains by M cells was evaluated by the ligated intestinal loop assay in BALB/c mice. After incubating with GFP-E. coli (green) for one hour, whole-mount specimens of PPs were stained with anti-gp2 mab (red) to detect M cells, followed by phalloidin (blue) for F-actin counterstaining. The specimens were analyzed under a confocal laser microscope. Arrowheads represent GPF-E. coli taken up by M cells. Scale bars: 80 µm. (b) The number of M cells taking up GFP-E. coli WT or ΔFimH strain in PPs was counted and is normalized to surface area of FAE. M cells internalising the bacteria were determined by visually analyzing the X-Y and X-Z images of FAE region. Data are means and s.e.m. (Student s t-test; n = 4 or 5 samples per WT or ΔFimH group, respectively). 8

a GP2 +/+ GP2 -/- b Merge GFP-E. coli GP2 UEA-1 Bacterial uptake/fae area 80 70 60 50 40 30 20 10 p = 0.0463 0 GP2 +/+ GP2 -/- Supplementary Figure 9 Uptake of E.

microscopy. The X-Y image of FAE (surrounded by dotted lines) shows that a number of E.

9 a GP2 +/+ GP2 -/- b Merge GFP-E. coli GP2 UEA-1 Bacterial uptake/fae area p = GP2 +/+ GP2 -/- Supplementary Figure 9 Uptake of E. coli by M cells is markedly reduced in GP2 -/- mice. (a) GFP-E. coli (green) were injected into the ligated intestinal loop prepared in GP2 +/+ or GP2 -/- mice. After incubating with GFP- E. coli (green) for one hour, PPs excised from the small intestine were stained with anti-gp2 mab (blue) and UEA-1 (red) to detect M cells, and were analyzed by confocal laser microscopy. The X-Y image of FAE (surrounded by dotted lines) shows that a number of E. coli are engulfed by M cells (cyan, some are indicated by arrowheads) in GP2 +/+ mice, whereas bacterial uptake is markedly diminished in GP2 -/- mice. (b) The number of bacteria taken up by M cells was visually counted in FAE of GP2 +/+ and GP2 -/- mice. Three different FAE domes obtained from different mice for each group were examined. Data are means and s.e.m. (Mann-Whitney U test). Scale bars: 80 µm. 9

Supplementary Figure 10 Transfer of S. Typhimurium from M cells to DC in the SED region. GP2 +/+ and GP2 -/- mice were orally administrated with S. Typhimurium (5 x 10 8 CFU).

10 Supplementary Figure 10 Transfer of S. Typhimurium from M cells to DC in the SED region. GP2 +/+ and GP2 -/- mice were orally administrated with S. Typhimurium (5 x 10 8 CFU). After 3 h, the mice were killed to collect one or two PPs located in the terminal ileum. Tissue sections of the PPs were stained with antibodies specific for Salmonella (Red) and CD11c (Green) to visualize DCs, followed by UEA-1 lectin (blue) to visualize M cells (arrowheads), and DAPI (cyan). Translocation of S. Typhimurium into the SED region was abundantly observed in PP of GP2 +/+ mice, where most of the bacteria were colocalized with DCs (upper right panel). Quantitative analysis demonstrated that 92.83% of the translocated bacteria were colocalized with DCs (n = 107, observed in 5 PP specimens from 3 different mice). In GP2 -/- mice, the bacteria were frequently observed on the luminal surface of epithelial layer rather than the SED region (lower right panel). The same experiments were repeated three times and reproducible results were obtained. The representative data are shown here. Scale bar: 100 μm 10

Anti-Salmonella IgA titer (log 2 dilution) p = 0.

GP2 +/+ or GP2 -/- C57BL/6 mice were orally immunized with 5 10 7 CFUs of rsalmonella-toxc.

11 Anti-Salmonella IgA titer (log 2 dilution) p = Oral immunization Supplementary Figure 11 GP2 -/- mice shows impaired Salmonella-specific IgA response. GP2 +/+ or GP2 -/- C57BL/6 mice were orally immunized with CFUs of rsalmonella-toxc. The presence of TT-specific IgA in feces at 2 and 3 weeks after oral immunization were evaluated by ELISA. Data are means and s.d. (n = 4; Student s t test). 11

GP2 +/+ and GP2 -/- mice received intraperitoneal injection with 5 x 10 5 CFU of rsalmonella-toxc for systemic immunization.

12 [ 3 H] thymidine incorporation (x10 4 cpm) Day0 Day9 Systemic immunization Supplementary Figure 12 GP2 -/- mice show normal immune response to systemic challenge with rsalmonella-toxc. GP2 +/+ and GP2 -/- mice received intraperitoneal injection with 5 x 10 5 CFU of rsalmonella-toxc for systemic immunization. To evaluate TT-specific T-cell response, CD4 + T cells were prepared from spleen 9 days after systemic immunization and were cocultured for 4 days with irradiated splenic antigen-presenting cells obtained from C57BL/6 mice. Data are means and s.d. (n = 3). 12

13 Movie Legends Movie 1 Three-dimensional image of M cells taking up anti-mouse GP2 mab in the ligated intestinal loop assay. The intestinal tissue containing FAE was excised and wholemount staining was performed. Internalized mab was visualized with Cy3-labeled antirat IgG antibody after fixation and permeabilization. The 3D image was processed with a DeltaVision restoration deconvolution microscope. The initial projection of the 3D image is a view from the luminal side of M cells and the image is rotated by 180º. Movie 2 Visualization of an M cell taking up E. coli. After the ligated intestinal loop assay using GFP-E. coli (green), the intestinal tissue containing FAE was excised and whole-mount staining of M cells was done by GP2 staining (red). The 3D image of an M cell was processed with a DeltaVision Restoration deconvolution microscope. The initial projection of the 3D image is a view from the luminal side of the M cell and the image is rotated by 180º. Movie 3 Transcytosis of S. Typhimurium by an M cell. After the intestinal loop assay for uptake of GPF-S. Typhimurium (green) cells, the intestinal tissue containing FAE was excised and whole-mount staining of M cells was done using anti-gp2 mab (red). The initial projection of the 3D image is a view from the luminal side of the M cell and the image is rotated by 180º. Movie 4 Transport of E. coli from an M cell to underlying DC. After the intestinal loop assay, the intestinal tissue containing FAE was excised and whole-mount staining of M cells was performed. M cells and DCs were stained with anti-gp2 (red) and anti-cd11c (blue) mabs, respectively. The initial projection of the 3D image is a view from the luminal side of the M cell and the image is rotated by 180º. 13

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION doi:10.1038/nature11419 Supplementary Figure 1 Schematic representation of innate immune signaling pathways induced by intracellular Salmonella in cultured macrophages. a, During the infection Salmonella