Fig 2: Dectin-1 induces TNFSF15 and OX40L expression through Syk and Raf1.(a) iDCs generated from mice (n=3–5) were stimulated with TNF-a/IL-1ß (T/I) or Curdlan (Cur) for 0.5 or 1 h. Cell lysates were prepared and subjected to western blot analysis using indicated antibodies. (b) iDCs were prepared from WT and dectin-1-/- mice (n=3) and stimulated with TNF-a/IL-1ß or Curdlan for 0.5 h. Cell lysates were subjected to western blot analysis using indicated antibodies. iDCs were matured by TNF-a/IL-1ß (BMDCs) or Curdlan (CurDCs) in the presence of piceatannol (Pic), GW5074 (GW) or DMSO as control for 48 h. (c) qPCR assessed Tnfsf15 and Ox40l expression in DCs. (d) Flow cytometry of TNFSF15 and OX40L expression in DCs. Right, summarized results of three independent experiments obtained as at left. MFI, mean fluorescence intensity. (e) Mouse (n=3) iDCs were treated by Syk (si-Syk), Raf1 (si-Raf1) or control siRNA (Si-CT) and subjected to the stimulation of TNF-a/IL-1ß (BMDC) or Curdlan (CurDCs) for48 h. qPCR assessed mRNA levels of Syk and Raf1 in DCs. (f,g) qPCR (f) and flow cytometry (g) analysed TNFSF15 and OX40L expression in DCs generated from mice (n=3). Data are representative of three (a,b) independent experiments or presented as mean±s.d. of at least three (c–g) independent experiments. *P<0.05; **P<0.01 (Student's t-test).

Fig 3: TNFSF15 and OX40L contribute to Th9 cell differentiation primed by dectin-1-activated DCs.(a,b) Pooled splenic CD4+ naive T cells from mice (n=3–5) were differentiated under Th0 or Th9 polarizing conditions with (TNFSF15) or without addition of TNFSF15 for 3 days. (a) qPCR assessed IL-9 expression in CD4+ T cells. (b) ELISA assessed IL-9 secretion in the culture. (c,d) Pooled splenic naive CD4+ T cells from mice (n=3) were cocultured with BMDCs or CurDCs under Th9 polarizing conditions with the addition of a TNFSF15-neutralization antibody (aTNFSF15) or isotype control IgG (IgG). Cells were cultured for 3 days. CD4+ T cells and culture supernatants were collected and subjected to qPCR (c) and ELISA (d) for IL-9 expression. (e,f) Pooled splenic naive CD4+ T cells from mice (n=3) were isolated from WT or OX40-/- mice and differentiated under Th9 polarizing conditions in the presence of BMDCs or CurDCs for 3 days. CD4+ T cells and culture supernatants were collected for IL-9 expression by qPCR (e) and ELISA (f). Similar as c and d, pooled splenic naive CD4+ T cells from mice (n=3) were cocultured with BMDCs or CurDCs under Th9 polarizing conditions with the addition of an anti-OX40L blocking antibody (aOX40L) or control IgG (IgG). IL-9 expression was examined by qPCR (g) and ELISA (h). Results shown are the mean±s.d. of at least three independent experiments. *P<0.05; **P<0.01 (Student's t-test).

Fig 4: Dectin-1 induces TNFSF15 and OX40L expression through NF-?B signalling pathway.(a) Mouse (n=3–5) iDCs were stimulated with TNF-a/IL-1ß (T/I) or Curdlan (Cur) for 1 and 3 h. Whole-cell lysates were prepared and subjected to western blot analysis using indicated antibodies. (b) Mouse (n=3) iDCs were stimulated with TNF-a/IL-1ß (T/I) or Curdlan (Cur) for 6 or 12 h. Nuclear extracts were subjected to western blot analysis using indicated antibodies. HDAC1 was used as loading control. (c,d) Mouse (n=3) iDCs were matured by TNF-a/IL-1ß (BMDC) or curdlan (CurDC) in the absence (DMSO) or presence of NF-?B inhibitor bortizomib (Bor) for 48 h. (c) qPCR analysed Tnfsf15 and Ox40l expression in DCs. (d) Flow cytometry analysis of TNFSF15 and OX40L expression in DCs. Right, summary of results of three independent experiments obtained as at left. MFI, mean fluorescence intensity. (e,f) 293T cells were transfected with vectors contained Tnfsf15 or Ox40l promoter or empty vector, followed by transfecting with vectors expressing the indicated NF-?B molecules. Luciferase reporter assay showed NF-?B-dependent activation of Tnfsf15 (e) and Ox40l (f) promoter in 293T cells. Data are representative of at least three (a,b) independent experiments or presented as mean±s.d. of at least three (c–f) independent experiments. *P<0.05; **P<0.01 (Student's t-test).

Fig 5: ILC2-Expressed OX40L Is Essential for Airway Adaptive Type 2 Immune Response to Helminth InfectionMice of the specified genotypes were infected with Nippostrongylus brasiliensis (N.b.) on day 0, followed by analysis on day 28 (or day 5) of:(A–C) Lung Th2, GATA3+ Treg, and GATA3- Treg cell numbers.(D) Representative lung histology (Mason’s trichrome).(E and F) Lung (E) and bronchoalveolar lavage (F) eosinophil numbers.(G) Lung RELMa+ M2 macrophage (MF) numbers.(H) Bronchoalveolar lavage IL-4 and IL-5 cytokine concentrations.(I) Whole lung cell suspensions were re-stimulated with PMA and ionomycin, followed by quantification of lung IL-13+ Th2 cell numbers by intracellular staining.(J–L) Mediastinal lymph node Th2, GATA3+ Treg, and GATA3- Treg cell numbers.(M) Concentration of IgE present in lung homogenate, normalized for total protein content.(N) Intestinal worm burden of indicated mouse genotypes 5 days post infection.Bar graphs indicate mean (±SEM). (A)–(C), ANOVA, three repeat experiments; (D), two repeat experiments; (E)–(I), ANOVA, two repeat experiments; (J)–(L), ANOVA, three repeat experiments; (M), ANOVA, two repeat experiments; (N), two-tailed Student’s t test, two pooled experiments. ns = not significant, *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001. See also Figure S7.