Fig. 3: Tregs can be induced in the lung without cDC migration.

a, b Migration of cDC2 subsets. All C57BL/6 mice received PKH26 dye and some mice received OVA or OVA/HDE by o.p. aspiration. The frequencies of each subset in PKH⁺ migratory cDC2s in mLNs of the recipient mice were evaluated by flow cytometry (n = 6 biological replicates). Gating strategy depicted in Fig. S5a. c Treg generation in the lung of WT mice treated with PTX. All mice received naïve CD4⁺ T cells isolated from CD45.1 OT-II mice by intravenous injection. OVA and PTX were given by o.p. aspiration to the indicated groups. The phenotype of CD45.1⁺ donor-derived CD4⁺ T cells was analyzed by flow cytometry of surface proteins and intracellular Foxp3 (Control: CD44^hi n = 8, CD25⁺ n = 6, Foxp3⁺ n = 6 CD25⁺Foxp3⁺ n = 7; OVA n = 8; PTX + OVA n = 8 biological replicates). d Treg generation in the lung of WT and Ccr7^–/– mice. All mice received naïve CD4⁺ T cells isolated from CD45.1 OT-II mice by intravenous injection, and OVA by o.p. aspiration. The phenotype of CD45.1⁺ donor-derived CD4⁺ T cells was analyzed by flow cytometry of surface proteins and intracellular Foxp3 (No OVA n = 8, Ccr7^+/+ OVA n = 10, Ccr7^–/– n = 11 biological replicates). e Timeline for mouse model of asthma to test tolerance induction. Some WT and Ccr7^–/– mice received OVA by o.p. aspiration and were sensitized with OVA/HDE. After OVA aerosol challenge, cells in BALF were analyzed. f Cell numbers of the indicated leukocytes in BALF (Ccr7^+/+ no treatment n = 8, Ccr7^+/+ sensitization n = 12, Ccr7^+/+ OVA pretreatment n = 12, Ccr7^–/–no treatment n = 9, Ccr7^–/– sensitization n = 12, Ccr7^–/– OVA pretreatment n = 11 biological replicates). a–d, f Each dot represents an individual mouse. Combined results from two (a–c, f) or three (d) independent experiments are shown. Data are presented as mean values ± SEM. (c, d) Data is presented in the fold change of each subset frequency within the CD45.1⁺CD4⁺ or within CD45.1⁺CD44^hiCD4⁺ population. Gating strategy depicted in Fig. S5c. a–c, f Data were analyzed by one-way ANOVA with Fisher’s LSD test. d Data were analyzed by Kruskal-Wallis test with Dunn’s multiple comparisons. P values are indicated above the graphs. Source data are provided as a Source Data file. Treg regulatory CD4⁺ T cells, cDC conventional dendritic cells, OVA ovalbumin, HDE house dust extract, OP oropharyngeal, WT wildtype, PTX pertussis toxin, BALF bronchoalveolar lavage fluid.