Extended Data Fig. 8. Antigen presentation by ILC3s or dendritic cells is not required for extra-thymic intestinal pTreg differentiation.

a–d, Flow cytometry of mLN from P14 Rorc^{Venus-creERT2} (a,c) or H2-Dma^–/– and littermate wild-type mice (b) or BALB/c x B6 F1 Rorc^{Venus-creERT2} mice (d). demonstrating expression of indicated antigen processing and presenting molecules. MHCII⁺ ILC3s: (Lin^–CXCR6⁺RORγt⁺MHCII⁺, TCs: (Lin^–CXCR6^–RORγt⁺MHCII⁺ and DC2s: (Lin^–RORγt^–CD11c⁺MHCII^hiCD11b⁺. Representative of n = 4 mice. e, frequency of Foxp3⁺ T cells amongst CD4⁺ T cells following co-culture of naïve CD4⁺ C7 T cells with indicated TC or DC subset. f, tdTomato labeling in MHCII⁺ILC3 (Lin^–CXCR6⁺MHCII⁺) from mLN of IL22 fate-mapping (Il22^Cre/+R26^lsl-tdtomato) mice at P18. Representative flow cytometry and summary bar graph, n = 4 mice. g, Frequency of MHCII⁺ ILC3s and RORγt⁺ pTreg amongst CD4⁺Foxp3⁺ cells in indicated tissues from 3-week-old MHCII^ΔIL22 (n = 4) and control (H2-Ab1^fl/fl) (n = 4) mice. h, tdTomato expression by MHCII⁺ cell types in mLN of Rorc^VenusRora^creR26^lsl-tdTomato fate-mapped mice at P14; n = 4 mice. i-j, Immune cell composition of 3-week-old MHCII^ΔRORα (n = 3) and Rora^creH2-Ab1^fl/wt (n = 3) mice. (i) Frequency of MHCII⁺ ILC3s and TCs in mLN. (j), Frequency of CD4⁺Foxp3^–CD44^hi T_eff and RORγt⁺ Th17 cells, (k) Total number of RORγt⁺Foxp3⁺ pTreg and RORγt⁺ Th17 cells. l-m, Immune cell composition in mLN of 3-week-old MHCII^ΔDC (n = 4) and control (Clec9a^creH2-Ab1^fl/wt) (n = 8) mice from 2 independent experiments. Frequency of MHCII expressing DCs or TCs within mLN (l). Frequency of total Foxp3⁺ Treg cells, pTreg cells amongst CD4⁺Foxp3⁺ cells, and Th17 cells in mLN and LI (m). Data in a-k are representative of 2-3 independent experiments, data in l,m are pooled from 2 independent experiments. Error bars: means ± s.e.m. ***P < 0.01, ****P < 0.0001; unpaired two-sided t-test.

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