Supplemental Information The Sensing of Environmental Stimuli by Follicular Dendritic Cells Promotes Immunoglobulin A Generation in the Gut
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1 Immunity, Volume 33 Supplemental Information The Sensing of Environmental Stimuli by Follicular Dendritic Cells Promotes Immunoglobulin A Generation in the Gut Keiichiro Suzuki, Mikako Maruya, Shimpei Kawamoto, Katarzyna Sitnik, Hiroshi Kitamura, William W. Agace, and Sidonia Fagarasan 1
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3 Figure S1, related to Figure 1. FDC Characteristics (A) Immunofluorescent microscopy analysis of consecutive sections from PP, naive pln and pln after immunization with TNP-KLH in IFA, stained as indicated. Note higher expression of FDC-M1 (Mfge8) in FDCs from PP and pln after immunization. Inset: Tingible body macrophages in PP and pln GCs are FDC-M1 hi CD21 or CD35 -. (B) Validation of purity of FDC preparations. Q-PCR analyses of Mfge8, Prnp and Clu gene expression (relative to housekeeping gene Gapdh) in indicated cells purified from the PPs or gut LP. (C and D) Q-PCR analyses of indicated Tlr gene expression in freshly purified FDCs and in whole tissues of the indicated organs (C) and overlay histograms of flow cytometry showing intracellular expression of TLR2 and TLR4 on FDCs prepared from PPs and plns (D). Shaded histograms represent isotype control antibodies. Geometric mean intensities are shown. (E) Q-PCR analyses of indicated Adh and Raldh gene expression in freshly purified FDCs, in vitro cultured FDCs and in whole tissues or CD11c + DCs isolated from the indicated tissues. 3
4 Figure S2, related to Figure 2. Expression Profile of pln FDCs after Immunization with TNP-KLH with Innate Stimuli (A) Comparison of probe-expression values in ex vivo FDCs isolated from plns without immunization (x axis) and plns after immunization with TNP-KLH in CFA (left panel) and TNP-KLH in CFA with Pam2CSK4 (right panel) (y axis). The number of transcripts that have differential levels of expression (cut off; 2-fold) is shown in the parenthesis. (B) The probes up-regulated upon pln immunization with TNP-KLH in CFA without or with Pam2CSK4 (cut off; 2-fold) were selected and re-plotted on the panel showing probe-expression values of ex vivo FDCs purified from PPs (y axis) and nonimmunized plns (x axis); the number of probes is shown in parenthesis. (C) PP signature genes encoding cytokines, chemokines and adhesion molecules (see legend for Figure 2) were selected and re-plotted on the panels showing probe-expression values of FDCs isolated from naive plns and plns after immunization with TNP-KLH in CFA with Pam2CSK4. Transcripts highly expressed in PP FDCs and apparently induced by immunization are shown in red. 4
5 Figure S3, related to Figure 3. Stimulation of pln FDCs with RA and TLR Ligands Reconstitutes Partially the Expression of Genes Characteristic for PP FDCs (A) Heat-map analyses for the PP FDC genomic signature for in vitro pln FDCs stimulated for 4 days with RA and indicated TLR ligands. Shown are the probes selected in Figure 3B. Up-regulated and down-regulated transcripts are indicated in red and green, respectively. (B) Q-PCR analyses showing expression of Cxcl13, Tnfsf13b (BAFF), Mmp2 and Mmp9 relative to housekeeping gene Gapdh by pln FDCs stimulated as indicated. Note that Tnfsf13b (BAFF) was induced mainly by stimulation of FDCs with RA and LPS, while Cxcl13, Mmp2 and Mmp9 were induced mainly by stimulation with RA and TLR2 ligands, particularly Pam3CSK4. No significant changes were detected after FDCs stimulation with TGF-β1 or IL-21 and CD40L (stimuli also abundantly present in the GC environment). 5
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7 Figure S4, related to Figure 4. Reduced GC Formation and IgA Synthesis in PPs of Myd88 -/- and VAD Mice (A) Representative flow cytometric profiles of cells stained for the indicated markers. Numbers on plots indicate the frequency of cells in the gate. (B) Immunofluorescent microscopy analyses of AID (red), CD3 (green) and DAPI staining for DNA in horizontal sections of PPs from indicated mice. (C and D) B cell follicles from PPs of WT, Myd88 -/- and VAD mice analyzed for the AID and FDC-M2-positive staining cells by automated image analyses (C) and the number of FDCs isolated from PPs of 2-month-old WT, Myd88 -/- and VAD mice (D). Figure S5, related to Figure 5. Trace Amounts of RA Induce TGF-β1 Production by Mucosal FDCs Isolated from WT Mice but Not from VAD Mice Total amount of TGF-β1 determined by ELISA in conditioned media of FDCs cultured in VA-depleted media prepared from plns, mlns and PPs of WT or VAD mice, as indicated. 7
8 Figure S6, related to Figure 6. pln FDCs Stimulated with Pam2CSK4 and RA Support IgA Generation Similar to Nonstimulated PP FDCs (A) RT-PCR of AID, αgt, IgA (Iμ-Cα) and αct in IgA depleted spleen cells cultured 3 days with conditioned media from pln, mln FDCs stimulated as indicated, or from nonstimulated PP FDCs. Media with RA, Pam2CSK4 and a combination of the two without FDCs served as control. IgA depleted spleen cells stimulated with LPS without or with IL-5 and TFG-β1 are also shown. (B) Total amount of secreted IgA in the 5 day supernatants from IgA-depleted spleen cells cultured in conditions indicated in (A). 8
9 Figure S7, related to Figure 7. Predominant Class Switching to IgA of GC B Cells from PPs and mlns Representative flow cytometric profiles of cells isolated from PPs, mlns, pooled plns or cervical LNs (clns) stained for the indicated markers. Numbers on plots indicate the frequency of cells in the gate. Data are representative from three experiments with similar results. 9
Supplemental Figure 1
Supplemental Figure 1 A IL-12p7 (pg/ml) 7 6 4 3 2 1 Medium then TLR ligands MDP then TLR ligands Medium then TLR ligands + MDP MDP then TLR ligands + MDP B IL-12p4 (ng/ml) 1.2 1..8.6.4.2. Medium MDP Medium
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Practice Test Test 2 1. A pathogen invades a tissue. Which cell of the immune system is more likely to respond first? a. Neutrophil b. T Cell c. B Cell d. Macrophage 2. The receptor for C3b is? a. CR1