R-eTACs are antigen-presenting cells of putative myeloid lineage. (A) Flow cytometry plots showing the gating of R-eTAC1/3, R-eTAC2, R-DC, and ILCs/LTis isolated from LNs of RORγt-lineage tracing with Aire reporter mice (RORγt^Cre×Aire-GFP reporter×Rosa26-LSL-tdTmt). (B) Quantification of the composition of R-eTACs, R-DCs, and ILCs/LTis among Aire reporter (top) or Aire protein (bottom)-positive cells out of tdTmt⁺MHCII⁺ gate in the RORγt-lineage tracing with Aire reporter mice (n = 8). (C) Flow cytometry of cells isolated from LNs in RORγt lineage–tracing mice pre-gated on single, live, Lin⁻ (SiglecF⁻CD3⁻B220⁻Ly6G⁻NK1.1⁻F4/80⁻). (D) Distribution of R-eTAC subsets across different organs in our adult scRNA-seq data. (E) Flow cytometry quantification of percentage (left) and absolute count per mouse (right) of R-eTACs across different organs in RORγt lineage–tracing adult mice from Fig. 2 (n = 7). (F) Flow cytometry plots of OT-II proliferating Tregs (FoxP3⁺CPD^lo of CD90.1⁺CD90.2⁺ T cells) after 72-h coculture with R-eTACs (GFP⁺tdTmt⁺) or DCs (GFP⁻CD11c⁺) isolated and sorted from mouse LNs. (G) Quantification of OT-II proliferation (CPD^lo of CD90.1⁺CD90.2⁺ T cells) and OT-II proliferating Tregs after coculture with no APCs, R-eTACs, or DCs (left to right) (n = 3 for each group). (H) Congenically distinct (CD45.2⁺) BM cells were isolated from WT or Il7r^−/− BM and mixed in 1:2 ratio and subsequently reconstituted in lethally irradiated recipient WT mice (CD45.1⁺) to generate mixed BMCs. 8 wk after reconstitution, LNs (cervical, inguinal, axillary, brachial, mesenteric, and para-aortic) were isolated. Quantification of ILCs (CD90⁺CD127⁺), DCs (CD11c⁺MHCII⁺), and R-eTACs (Aire⁺MHCII⁺) from LNs of WT⁺Il7r^−/− mixed BMC mice (n = 5 for each group). (I)Cx3cr1^CreER×Rosa26-LSL-tdTmt mice were treated with tamoxifen three times a week for 4 wk for complete labeling. LNs were harvested, and tdTmt⁺ cells were quantified by flow cytometry (n = 3). (J) LNs were harvested from adult Zbtb46^Cre×Rosa26-LSL-tdTmt mice, and tdTmt⁺ cells were quantified by flow cytometry (n = 10). Data in C and H are representative of three independent experiments. Data in E are representative of two independent experiments. Data in B, C, F, G, I, and J are pooled from two to three independent experiments. Error bars: mean ± SEM; statistics were calculated by one-way ANOVA with Tukey’s multiple comparisons test (G); *P < 0.05, **P < 0.005.