Fig. 7.

ILC3-derived OX40L is important for Treg homeostasis in the intestine. a, b Rag1^–/– mice were lethally irradiated and reconstituted with bone marrow mixed at 1:1 ratio from Rag1^–/–Rorc^gfp/gfp mice plus Rag1^–/–Tnfsf4^+/– or Rag1^–/–Tnfsf4^–/– mice. After 6 weeks, 4 × 10⁵ Tregs (CD4⁺Foxp3-YFP⁺) were sorted from splenocytes of Foxp3^Cre-YFP mice and transferred to bone marrow chimeric mice. c, d Rag1^–/–Rorc^gfp/gfp mice were transferred with 3 × 10⁵ Tregs (CD4⁺Foxp3-YFP⁺) together with 2×10⁵ ILC3s (Lin^–Thy1.2^highCD45.2^intermediate) purified from small intestinal LPLs of Rag1^–/–Tnfsf4^+/– or Rag1^–/–Tnfsf4^–/– hosts. a, b, d Small and large intestinal LPLs from recipient mice were isolated for analysis 5 weeks after the transfer of Tregs. Expressions of CD3, CD4, and Foxp3 were analyzed by flow cytometry. a, d Percentages of Tregs (CD3⁺CD4⁺Foxp3⁺ cells) in live lymphocytes were calculated and shown. b Total number of Tregs of indicated groups was calculated and shown. e, f Approximately 4 × 10⁵ Tregs (CD4⁺Foxp3-YFP⁺) were sorted from the spleen of Foxp3^Cre-YFP mice and transferred to Rag1^–/–Rorc^gfp/+ mice. Mice were sacrificed for analysis 6 weeks after the transfer of Tregs. g Eight-week-old Rorc^gfp/+ mice were sacrificed for analysis. e, g Expression of CD3 (red), Foxp3 (yellow), and RORγt-GFP (green) in cryptopatches was analyzed by immunofluorescence. f Percentages of Tregs (CD3-Red⁺, Foxp3-Yellow⁺) with location associated with ILC3s (CD3-Red^–, RORγt-GFP-Green⁺) among all Tregs observed per CP were calculated and shown. Data were pooled from 12 (SI) and 15 (LI) cryptopatches of four mice. a–g Data are representative of two independent experiments. CP, cryptopatch; LPLs, lamina propria lymphocytes