FIGURE LEGENDS
Fig 1. CXCL13 expression in NPs with and without eLT formation. A, The mRNA expression levels of CXCL13 in different study groups as detected by quantitative RT-PCR. B, The protein levels of CXCL13 in different study groups as detected by ELISA. C,Representative photomicrographs showing immunofluorescence staining of CXCL13 on vimentin+ stromal cells and CD20+ B cells in polyp tissues with and without eLTs. Original magnification ×200. Insets show a higher magnification of the outlined area, and arrows denote representative positive cells.D, Reduction of CXCL13 mRNA expression after depletion of stromal cells in relation to total polyp cells from NPs with and without eLTs. Data are expressed as means ± SEMs. E, Reduction of CXCL13 mRNA expression after depletion of B cells in relation to total polyp cells from NPs with and without eLTs. Data are expressed as means ± SEMs. NPs, nasal polyps; eLTs+, with ectopic lymphoid tissues; eLTs-, without ectopic lymphoid tissues; DAPI, 4, 6-Diamidino-2-phenylindole dihydrochloride. *P< 0.05; ***P < 0.001.
Fig 2. Phenotypic changes of stromal cells in NPs A, The mRNA expression levels of CXCL13 in nasal stromal cells purified from different types of tissues as detected by quantitative RT-PCR.B, The gating strategy and representative flow plots showing nasal stromal cells in tissues. C, The frequencies of different stromal cell subpopulations in different study groups as detected by flow cytometry. D, The composition of stromal cell subsets in different study groups. Mean percentages are shown. E, The gating strategy of CXCL13+ stromal cells. F,The composition of different types of stromal cells for CXCL13+ stromal cells in different types of tissues as detected by flow cytometry. Mean percentages of different stromal cell types are shown. NPs, nasal polyps; eLTs+, with ectopic lymphoid tissues; eLTs-, without ectopic lymphoid tissues; EpCAM, epithelial cell adhesion molecule; Pdpn, podoplanin; FRCs, fibroblastic reticular cells; LECs, lymphoid endothelial cells; BECs, blood endothelial cells; DNs, double negative cells. **P < 0.01; ***P < 0.001.
Fig 3. IL-17A induces CXCL13 production and FRC expansion in nasal stromal cells. A, The mRNA expression levels of IL-17A in different study groups as detected by quantitative RT-PCR. B,The correlation between IL-17A and CXCL13 mRNA expression levels in NPs with and without eLTs. C, Purified nasal stromal cells from control tissues were stimulated with various doses of IL-17A for 12 hours and mRNA expression of CXCL13 were measured by quantitative RT-PCR (n = 6). D, Purified nasal stromal cells from control tissues were stimulated with IL-17A at 100 ng/mL for 36 hours and CXCL13 positive cells were quantified by flow cytometry (n = 6). Representative flow plots are shown. E, Purified nasal stromal cells from control tissues were stimulated with IL-17A at 100 ng/mL for 36 hours, and CXCL13 positive cells were analyzed for the composition of different types of stromal cells by flow cytometry (n = 6). Mean percentages of different stromal cell types are shown. F, After treatment with IL-17A at 100 ng/mL for different time points, the frequencies of different types of stromal cells were detected by flow cytometry (n = 6). NPs, nasal polyps; eLTs+, with ectopic lymphoid tissues; eLTs-, without ectopic lymphoid tissues; Pdpn, podoplanin; FRCs, fibroblastic reticular cells; LECs, lymphoid endothelial cells; BECs, blood endothelial cells; DNs, double negative cells. **P < 0.01; ***P < 0.001.
Fig 4. A positive feedback loop between CXCL13 and LTα1β2 on B cells. A, The mRNA expression levels of LTα and LTβ in different study groups as detected by quantitative RT-PCR. B, Representative photomicrographs showing immunofluorescence staining of LTα and LTβ on CD20+ B cells in eLTs in NPs. Original magnification ×200. Insets show a higher magnification of the outlined area, and arrows denote representative positive cells. C, Reduction of LTα and LTβ mRNA expression after depletion of B cells in relation to total polyp cells from NP with and without eLTs. Data are expressed as means ± SEMs. D, B cells purified from polyp tissues with eLTs were stimulated with CXCL13 at various doses for 12 hours, and the mRNA expression of LTβ in B cells was detected by RT-PCR (n = 6). E,B cells purified from poly tissues with eLTs were stimulated with CXCL13 at 1000 ng/mL for 36 hours, the membrane expression of LTα on B cells was detected by flow cytometry (n = 6). The representative flow plots are shown. F, B cells purified from polyp tissues with eLTs were stimulated with LTα1β2 at various doses for 12 hours, and the mRNA expression of CXCL13 in B cells was detected by RT-PCR (n = 6). G, B cells purified from polyp tissues with eLTs were stimulated with LTα1β2 at 1000 ng/mL for 48 hours, the protein levels of CXCL13 in culture supernatants were detected by ELISA (n = 6). NPs, nasal polyps; eLTs+, with ectopic lymphoid tissues; eLTs-, without ectopic lymphoid tissues; LT, lymphotoxin. **P < 0.01; ***P< 0.001.
Fig 5. LTα1β2 induces FRC and LEC expansion and CXCL13 production in nasal stromal cells. A, Nasal stromal cells purified from control tissues were treated with LTα1β2 at various doses for 12 hours, and the mRNA expression of CXCL13 in stromal cells were detected by RT-PCR (n = 6). B, After stimulation with LTα1β2 at 100 ng/mL for 36 hours, CXCL13 positive nasal stromal cells were analyzed by flow cytometry (n = 6). Representative flow plots are shown. C, After stimulation with LTα1β2 at 100 ng/mL for 36 hours, CXCL13 positive stromal cells were analyzed for the composition of different stromal cell types by flow cytometry (n = 6). Mean percentages of different types of stromal cells are shown. D, After stimulation with LTα1β2 at 100 ng/mL for different time points, the frequencies of different stromal cell populations were detected by flow cytometry (n = 6). NPs, nasal polyps; eLTs+, with ectopic lymphoid tissues; eLTs-, without ectopic lymphoid tissues; LT, lymphotoxin; Pdpn, podoplanin; FRCs, fibroblastic reticular cells; LECs, lymphoid endothelial cells; BECs, blood endothelial cells; DNs, double negative cells. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig 6. IL-17A is required for de novo nasal follicle formation in an IL-17A high mouse model. A, Histologic studies showed lymphoid aggregates in WT and il17a-/- mice challenged by curdlan at 100 μg or PBS (control). B, The number of eLTs and lymphocyte clusters in different study groups. C,The mRNA levels of CXCL13 and LTβ in nasal mucosa in different study groups. WT, wild type; eLTs, ectopic lymphoid tissues; LT, lymphotoxin. *P < 0.05; **P < 0.01.
Fig 7. Nasal eLT formation is diminished by CXCL13 and LTβR blockage in the mouse model. A, Histologic studies showed lymphoid aggregates in WT mice challenged by curdlan at 100 μg and treated with anti-CXCL13 or rat control IgG1. B, The number of eLTs and lymphocyte clusters in WT mice with and without anti-CXCL13 treatment.C, The mRNA levels of CXCL13 and LTβ in nasal mucosa in WT mice with and without anti-CXCL13 treatment. D, Histologic studies showed lymphoid aggregates in WT mice challenged by curdlan at 100 μg and treated with LTβR-Ig or mouse control IgG1. E, The number of eLTs and lymphocyte clusters in WT mice with and without LTβR-Ig treatment. F, The mRNA levels of CXCL13 and LTβ in WT mice with and without LTβR-Ig treatment. WT, wild type; eLTs, ectopic lymphoid tissues; LT, lymphotoxin. *P < 0.05; **P< 0.01.