Fig. 1: STING signaling in mucosal DCs induces T_H17 cells.

a, b Cytokines expression by CLN and MLN cells obtained from wild-type mice at 7 days after the last immunization with 20 μg OVA without or with 25 μg c-di-GMP. a Cells were cultured for 48 h ex vivo with 20 μg/ml OVA, and cytokines were measured by ELISA. n = 4. ***p < 0.001. b Representative data (left) depicting the frequency (%) of IL-17A-GFP⁺ CD4⁺ cells from Il17a-GFP mice or Tmem173^−/−;Il17a-GFP mice. n = 4. **p < 0.01. c Analysis of DCs migration from SI to MLNs upon c-di-GMP stimulation. Small intestines (SI) of KikGR mice and KikGR;Tmem173^−/− mice were exposed with a 405 nm laser and i.p. injected with c-di-GMP (200 μg/mouse) 18 h before sacrifice. Migrated cells were defined in MLN by gating CD45⁺ KikGR-Red⁺ cells using flow cytometry. Graph (left) shows the percentage of CD11c⁺ MHCII⁺ DC in CD45⁺ KikGR-Red⁺ migrated cells. Graph (right) shows the percentage of CD103⁺CD11b⁺ or CD103⁺CD11b⁻ DCs in CD11c⁺MHCII⁺ KikGR-Red⁺ migrated DCs. n = 3. ***p < 0.001. d Analysis of MHCII⁺ Zbtb46⁺ or Cx3cr1⁺ LP-DCs. LP-DCs were isolated from Zbtb46-GFP or Cx3cr1-GFP mice and analyzed for CD103 and CD11b expressions. e Analysis of Il17a mRNA expression after antigen-specific T cell activation by Zbtb46⁺ or Cx3cr1⁺ LP-DCs. Zbtb46-GFP and Cx3cr1-GFP mice were injected (i.p.) with c-di-GMP (200 μg/mouse) on −5, −3 and −1 day prior to sacrifice. CD11c⁺ GFP⁺ DCs were sorted from SI-LP and incubated with naive T cells isolated from spleen of OT-II transgenic mice with OVA (50 μg/ml) for 5 days (DC:T cell = 1:5). n = 4. ***p < 0.001. f mRNA expression was analyzed by qRT-PCR in Zbtb46⁺ LP-DCs. CD11c⁺ Zbtb46-GFP⁺ cells were sorted by flow cytometry and incubated with c-di-GMP (50 μg/ml) for 4 h. n = 3, **p < 0.01. p-values, one-way a, two-way c, e ANOVA followed by Tukey’s multiple-comparisons test or two-tailed unpaired Student’s t-test. b, f Data are representative of two independent experiments a–c or are from three indpendent experiments d–f.