Fig. 2. Microbiota disruption causes migration of intestinal ILCs to a circulation system.

(A) 3D Immunofluorescence staining of CD69⁺ and S1PR1⁺ ILCs in SI from Ctrl and Abx-treated mice. White arrows denote CD69⁺ and S1PR1⁺ ILCs. Scale bars, 100 μm. (B to D) Mice were intraperitoneally injected FTY720 every other day and treated with Abx for 2 weeks. ILC1s, ILC2s, and ILC3s in SI (B), mLN (C), and spleen (D) derived from Ctrl, Abx-treated, and Abx + FTY720–treated mice were analyzed by flow cytometry. n = 5. (E to G) PE-labeled anti-CD45 antibody (2 μg) was intravenously injected into mice. Ten minutes later, PE-labeled intestinal ILC1s (E), ILC2s (F), and ILC3s (G) from Ctrl and Abx-treated mice were detected by flow cytometry. Frequencies of PE-labeled ILC1s, ILC2s, and ILC3s were calculated. n = 4. (H) Lin⁻ intestinal lymphocytes (1 × 10⁷) from CD45.2⁺ donor mice were sorted and intravenously injected into lethally irradiated CD45.1⁺ Ctrl or Abx-treated recipients. Eighteen hours later, donor-derived ILC subsets homing to intestine were analyzed by flow cytometry. Frequencies and numbers of intestinal CD45.2⁺ ILC subsets in indicated recipients are shown in the right panel. n = 4. (I) Bone marrow (BM) cells (5 × 10⁶) from CD45.2⁺ donor mice were intravenously injected into lethally irradiated CD45.1⁺ Ctrl or Abx-treated recipients. Eight weeks later, homing and stable localization of donor-derived ILC subsets in the intestine were analyzed by flow cytometry. Frequencies and numbers of intestinal CD45.2⁺ ILC subsets in indicated recipients are shown in the right panel. n = 4. Data are representative of at least three independent experiments and are shown as the means ± SD. Statistical analysis was performed by using unpaired two-tailed Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant). SSC, side scatter; W, width.