Fig. 1.

Intestinal ILC3s are maintained independently of bone marrow replenishment. (A) Experimental design for intestinal shielded bone marrow chimeras. Recipient (CD45.2⁺) adult C57BL/6 mice received sub-lethal whole-body irradiation with a lead shield covering the intestines followed by injection of donor (CD45.1⁺) bone marrow. Mice were culled ≥ 10 weeks post-irradiation. (B) Representative flow cytometry plots of CD45.1⁺ and CD45.2⁺ CD4⁺ T cells, DN ILC3, NCR⁺ ILC3 and LTi-like ILC3 in SiLP at ≥ 10 weeks post-irradiation. (C) Frequency of CD45.1⁺ CD4⁺ T cells, DN ILC3, NCR⁺ ILC3 and LTi-like ILC3 in SiLP pooled from three independent experiments at >10 weeks post-irradiation. (D and E) Frequency of Cxcr4-^{CreERT2-tdTomato} labeled blood monocytes (CD11b⁺, Ly6C⁺), microglia (CD11b⁺, CD64⁺, F4/80⁺), CD4⁺ T cells, NCR⁺ ILC3 and LTi-like ILC3 12 weeks post-labeling (Microglia, CD4⁺ T cell and ILC3 are normalized to blood monocytes). Panel B-C gated as in Supplementary Fig. S1A, panels D–E gated as in Supplementary Fig. S1B. Data shown as mean ± standard error of mean and represents two (Microglia in E) or three independent experiments (n = 10–14). Numbers in flow plot indicate percentage of cells in the respective gate. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 using unpaired t test or Tukey’s (C) or Holm-Šídák’s (E) multiple comparisons test. CD = clusters of differentiation; DN = double negative; ILC3 = group 3 innate lymphoid cell; LTi-like = lymphoid tissue-inducer-like; mLN = mesenteric lymph nodes; NCR = natural cytotoxicity receptor-expressing; ns = not significant.