Fig 2: Lung-resident Mgl2+/IDO1+ R2D2 population generates T-regs in the lung.a Flow cytometry plots (left) and quantification (right) of CD4+Foxp3+ T-reg cells in in WT, Batf3-/-, and IRF4fl/flCD11ccre mice treated with one dose of OVA (1 µg) intranasally (i.n.). Lungs were harvested on day 14; n = 4–6 mice/group. Data are representative of two independent experiments. b, c Flow cytometry plots (left) and quantification (right) of T-regs in TNFR2fl/flCD11ccre b and RelBfl/flCD11ccre mice c treated with one dose of OVA (1 µg) i.n. Lungs were harvested on day 14; n = 3–5 mice/group. Data are representative of two independent experiments. d IRF4fl/flCD11ccre mice were adoptively transferred (i.n.) with lung TNFR2+ and TNFR2- cDC2 from WT mice lung and treated with one dose of OVA (1 µg) i.n. Flow cytometry analysis (left) and quantification (right) of T-regs at day 14; n = 3–6 mice/group. Data are representative of two independent experiments. e, f Flow cytometry analysis of TNFR2+ cDC2 at steady state. Data are representative of three independent experiments; n = 3 mice/group. g IRF4fl/flCD11ccre mice were adoptively transferred (i.n.) with lung Mgl2+TNFR2+ and Mgl2-TNFR2+ cDC2 and treated with one dose of OVA (1 µg) i.n. Flow cytometry analysis (left) and quantification (right) of T-regs at day 14; n = 3–4 mice/group. Data are representative of two independent experiments. Graphs represent the mean with error bars indication s.e.m. P values determined by one-way ANOVA Tukey’s multiple comparison test a, d, g or unpaired student t-test b, c. *P < 0.05, **P < 0.001, and ***P < 0.0001.

Fig 3: IFNß-IFNAR1-TGFß1 signaling axis in iR2D2 promotes lung T-regs induction.a Flow cytometry analysis of TNFR2+ cDC2 treated i.n. with PBS or H7N7-HA (1 µg). Lungs were harvested 24 h later; n = 3 mice/group. Data are representative of two independent experiments. b Flow cytometry analysis (left) and numbers (right) of T-regs in mice treated with PBS, OVA (1 µg)/isotype control, or OVA (1 µg) and anti-TGFß1 neutralizing antibody (75 µg); n = 3–4 mice/group. Lungs were harvested on day 14. Data are representative of two independent experiments. c Flow cytometry analysis of TGFß1 production by TNFR2+ cDC2 in mice treated i.n. with PBS, OVA (1 µg), or OVA (1 µg) and IFNß (200 ng). Lungs were harvested 24 h later; n = 3 mice/group. Data are representative of three independent experiments. d Flow cytometry analysis of TGFß1 production by TNFR2+ cDC2 in mice treated with PBS, OVA (1 µg)/isotype control, or OVA (1 µg) and anti-IFNAR1 blocking antibody (20 µg). Lungs were harvested 24 h later; n = 3 mice/group. Data are representative of two independent experiments. e Experimental design for adoptive transfer. f–g IFNAR1-/- mice were adoptively transferred (i.n.) with lung TNFR2+ cDC2 and treated with PBS or IFNß (200 ng). Flow cytometry analysis of TGFß1 production by TNFR2+ cDC2 f and T-regs g; n = 3 mice/group. Data are representative of two independent experiments. Graphs represent the mean with error bars indication s.e.m. P values determined by unpaired student t-test b, f, g. *P < 0.05, **P < 0.001, and ***P < 0.0001.

Fig 4: IFNß-IFNAR1 signaling in iR2D2 cells promotes lung T-regs induction.a Flow cytometry analysis (left) and quantification (right) of lung TNFR2+ cDC2 in WT mice treated i.n. with isotype (20 µg) or anti-IFNAR1 blocking antibody (20 µg). Lungs were harvested 24 h later; n = 3–4 mice/group. Data are representative of three independent experiments. b Flow cytometry analysis (left) and quantification (right) of lung T-regs in WT treated i.n. with isotype (20 µg) or anti- IFNAR1 blocking antibody (20 µg) and one dose of OVA (1 µg) i.n. Lungs were harvested on day 14; n = 4–5 mice/group. Data are representative of three independent experiments. c Flow cytometry analysis of TNFR2+ cDC2 at steady state; n = 3 mice/group. Data are representative of two independent experiments. d IFNAR1-/- mice were adoptively transferred (i.n.) with lung TNFR2+ cDC2 and one dose of OVA (1 µg) i.n. Flow cytometry analysis of T-regs on day 14; n = 3 mice/group. Data are representative of two independent experiment. e Flow cytometry analysis (left) and numbers (right) of T-regs in mice treated i.n. with OVA (1 µg) or IFNß (200 ng). Lungs were harvested on day 14; n = 3–5 mice/group. Data are representative of two independent experiments. f Flow cytometry analysis (left) and absolute numbers (right) of T-regs in IRF4fl/fl and IRF4fl/flCD11ccre mice treated i.n. with OVA (1 µg) or OVA (1 µg) and IFNß (200 ng). Lungs were harvested on day 14; n = 3 mice/group. Data are representative of two independent experiments. g Numbers of Mgl2+TNFR2+ and Mgl2-TNFR2+ cDC2 in mice treated i.n. with PBS or IFNß (200 ng); n = 3 mice/group. Data are representative of two independent experiment. h–i Flow cytometry analysis of IFNß expression at steady state by the anti-IFNß mAb (clone D2J1D); n = 3 mice/group. Data are representative of two independent experiments. Graphs represent the mean with error bars indication s.e.m. P values determined by one-way ANOVA Tukey’s multiple comparison test f or unpaired student t-test a, b, e. *P < 0.05, **P < 0.001, and ***P < 0.0001.

Fig 5: The lung TNFR2+ cDC2 population maintains lung tolerance and prevents lung inflammation at steady state.a Numbers of lung CD4+ T cells at steady state in C57BL/6 J (n = 7), Batf3-/- (n = 6), CCR2-/- (n = 4), and IRF4fl/flCD11ccre (n = 7) mice. Data were compiled from two independent experiments. b Numbers of lung CD4+ T cells in WT (n = 12), IRF4fl/flCD11ccre (n = 10), RelBfl/flCD11ccre (n = 7), and TNFR2fl/flCD11ccre (n = 5) mice at steady state. Data were representative of two independent experiments. c Image of mediastinal lymph nodes (mLNs; top) and spleens (bottom) in of 7- to 8-week-old knockout strains at steady state; n = 3 mice/group. Data are representative of two independent experiments. d Numbers of CD45+ cells in the lungs (left), mLNs (center), and spleens (right) in the indicated strains at steady state; n = 3 mice/group. Data were representative of two independent experiments. e Numbers of CD4+ T cells in IRF4fl/flCD11ccre mice 14 days post cell adoptive transfer. TNFR2+ or TNFR2- cDC2 were sorted from WT mice lung and intranasally (i.n.) transferred into IRF4fl/flCD11ccre recipient mice; n = 4–6 mice/group. Data are representative of two independent experiments. f Numbers of lung Foxp3+CD4+ T-reg cells at steady state in C57BL/6 J, Batf3-/-, CCR2-/-, and IRF4fl/flCD11ccre mice; n = 4–5 mice/group. Data were representative of two independent experiments. Graphs represent the mean with error bars indication s.e.m. P values determined by one-way ANOVA Tukey’s multiple comparison test. *P < 0.05, **P < 0.001, and ***P < 0.0001.