R-eTACs mediate oral tolerance to food antigen. (A) Flow cytometry of cells pre-gated on single, live, Lin⁻ from AireDTR⁻ and AireDTR⁺ mouse LNs. All mice were treated with DT every other day for five doses. (B) Schematic of AireDTR oral tolerance experimental design. AireDTR⁻ and AireDTR⁺ mice were pretreated with three doses of DT every other day to deplete Aire-expressing cells. After OT-II transfer and OVA gavage, mice are treated with three more doses of DT before harvest. (C and D) Quantification of RORγt⁺ Tregs, RORγt⁻ Tregs, and Tfh out of CPD^lo OT-IIs from AireDTR⁻ and AireDTR⁺ mLNs by flow cytometry after 2 days (n = 9 from each group) (C) and 7 days (n = 7 from each group) (D) of oral OVA. (E) Quantification of RORγt⁺ Tregs and RORγt⁻ Tregs out of CPD^lo OT-IIs from mixed BMC generated from WT, MHCII^ΔRORγt, and AireDTR mLN after 2 days of OVA gavage (n = 7 for WT and MHCII^ΔRORγt, n = 8 for MHCII^ΔRORγt+AireDTR). (F and G) BMCs were generated with either CD45.2⁺ AireDTR⁻×RORγt^Cre×Rosa26-LSL-tdTmt (herein referred to as AireDTR⁻ for simplicity) or CD45.2⁺ AireDTR⁺×RORγt^Cre×Rosa26-LSL-tdTmt (AireDTR⁺) mice. 8 wk after reconstitution, LN cells from AireDTR⁻ and AireDTR⁺ mixed BMCs were isolated, sorted (tdTmt⁺MHCII⁺), and subjected to scRNA-seq. DT was administered to all mice five times every other day before harvest. (F) UMAP from single-cell analysis of RORγt-lineage APCs present in AireDTR⁻ and AireDTR⁺ BMC mice after DT depletion. (G) Ratio (AireDTR⁺/AireDTR⁻) of different subsets of RORγt-lineage APCs from the single-cell analysis. (H) Oral tolerance experimental setup is the same as Fig. 1 A. Quantification of RORγt⁺ Tregs and RORγt⁻ Tregs out of CPD^lo OT-IIs isolated from mLN of WT versus Itgb8^ΔRORγt mice 2 days after OVA gavage (n = 7 from each group). Data in C, D, and H are pooled from two to three independent experiments. Data in E are representative of three independent experiments. Error bars: mean ± SEM; statistics were calculated by one-way ANOVA with Tukey’s multiple comparisons test (E) and unpaired two-sided t test (C, D, and H); *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001.