Extended Data Fig. 5. Phenotypic characterization of Thetis cells.

a, Flow cytometry of index sorted mLN RORγt⁺MHCII⁺ cells for DC markers, CD11c and CD11b. b, Coverage track for smart-seq2 single cell sequencing reads mapping to the Rorc locus, demonstrating expression of the Rorgt isoform by TCs. c, Flow cytometry of Lin^–CXCR6^–MHCII⁺ cells from mLN of Rorgt^CreR26^lsl-tdTomatoRorc^{Venus-CreERT2} mice and summary graph of frequency of tdTomato⁺ cells amongst Lin^–CXCR6^–Venus(YFP)⁺ cells (n = 3 mice). d, Index sorting flow cytometric analysis of all RORγt⁺MHCII⁺ cells (left panel) and cells identified as ILC3 (right panel). e, Electron microscopy of CCR7^– DCs, CCR7⁺ DCs, Aire⁺ mTECs, TC I, TC IV and MHCII⁺ ILC3 cells. Far right panel: arrows indicate distinctive mitochondrial cristae in TCs. f, Representative immunofluorescence imaging of TC and Treg markers in mLN sections from 2-week-old Aire^GFP mice. Arrowheads indicate Aire⁺ TC I/III or Cd11b⁺ TC IV. Images are representative of two independent experiments with similar results. g-h, Immunofluorescence analysis of mLN from P17 Rorc^Venus mice. Representative histo-cytometry plot for identification of RORγt⁺Foxp3⁺ pTreg cells and RORγt⁺MHCII⁺CD11c⁺CD11b⁺ TC IV (g) and representative immunofluorescence imaging demonstrating distribution of indicated cell types (h). n = 3 mice, 2 lymph nodes per mouse. Data in c are representative of 2 independent experiments.

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