ILCs are dispensable for oral tolerance to food antigen. (A) Schematic of mixed BMC generation plus oral tolerance experimental design. Congenically distinct (CD45.2⁺) BM cells were isolated from WT, MHCII^ΔRORγtor Il7r^−/− BM and subsequently reconstituted in lethally irradiated recipient WT mice (CD45.1⁺) to generate three groups of BMC mice: WT, MHCII^ΔRORγt, and MHCII^ΔRORγt+Il7r^−/− (mixed in 1:2 ratio). 6–8 wk after reconstitution, mice were subjected to OT-II adoptive transfer and two OVA gavages. (B) Flow cytometry quantification of RORγt⁺ Tregs, RORγt⁻ Tregs, and Tfh out of CPD^lo OT-IIs in BMC mouse mLNs after 2 days of oral OVA (n = 10 from each group). (C) Quantification of OVA-specific IgG1 (left) and IgG2c (right) in serum isolated from mice subjected to a delayed-type hypersensitivity model (n = 10 from each group). The experimental design was as follows: reconstituted mice were gavaged with PBS (control) or tolerized with OVA (days 0–1), sensitized with subcutaneous CFA-OVA (day 9), challenged with subcutaneous OVA (days 23 and 33), and harvested for serum (day 35). (D and E) BM cells were isolated from congenically distinct CD45.2⁺RORγt^Cre×Rosa26-LSL-tdTmt (referred to as WT for simplicity) mice or Il7r^−/−×RORγt^Cre×Rosa26-LSL-tdTmt (Il7r^−/−) mice. These cells were then mixed at a 2:1 ratio with WT non–lineage-tracing CD45.1⁺CD45.2⁺ BM and transferred into lethally irradiated CD45.1⁺ recipients. 8 wk after reconstitution, LN cells from WT or Il7r^−/− mixed BMCs were isolated, sorted (tdTmt⁺MHCII⁺), and subjected to scRNA-seq. (D) UMAP from single-cell analysis of RORγt-lineage APCs present in WT and Il7r^−/− mice. (E) Ratio (Il7r KO/WT) of different subsets of RORγt-lineage APCs from the single-cell analysis. Data in B and C are pooled from two independent experiments. Error bars: mean ± SEM; statistics were calculated by one-way ANOVA with Tukey’s multiple comparisons test (B and C); *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001.