Figure 1: WASH deficiency reduces the cell pool of NKp46+ ILC3s.
From: WASH maintains NKp46+ ILC3 cells by promoting AHR expression
(a) ILCs from Washflox/flox and Washflox/floxMx1-Cre mice 3 weeks post poly(I:C) administration were immunoblotted with the indicated antibodies (left panel). Numbers of ILCs in small intestines of Washflox/flox (Wash+/+) and Washflox/floxMx1-Cre (Wash−/−) mice were calculated (right panel). (b) Numbers of NKp46− and NKp46+ ILC3s in small intestines of the indicated mice were calculated. (c) Numbers of ILC progenitor cells in BM of the indicated mice. Gating stratedies: Lin− cKitlow CD127+α4β7+ for αLP, Lin−CD127+α4β7+ PLZF+ for ILCP, Lin− CD127+α4β7+ CD25−CD244+Id2+ for CHILP. For a–c, n=4. (d) NKp46+ ILC3s sorted from small intestine lamina propria lymphocyte populations (SI LPL) of Washflox/flox and Washflox/floxNcr1-Cre mice were immunoblotted with the indicated antibodies. (e) Numbers of cryptopatch (CP) clusters in the small intestine (left panel) or large intestine (right panel) of Washflox/flox and Washflox/floxNcr1-Cre mice. n=6. (f) Flow cytometry analysis of SI LPL of Washflox/flox and Washflox/floxNcr1-Cre mice. Cells were gated from CD45+CD19−CD3− cells (left panel). Numbers of ILC3s and the indicated subpopulations of ILC3s were calculated (right panel). (g) ILC3s sorted from SI LPL of Washflox/flox and Washflox/floxRorc-Cre mice were immunoblotted with the indicated antibodies. (h) Representative images of SI CPs from Washflox/flox and Washflox/floxRorc-Cre mice staining with RORγt, NKp46 and DAPI (upper panel). Numbers of cryptopatch (CP) clusters in the small intestine (lower left) or large intestine (lower right) of Washflox/flox and Washflox/floxRorc-Cre mice. n=6. Scale bar, 50 μm. (i) Small intestine sections from Washflox/flox and Washflox/floxRorc-Cre mice were stained with antibodies against NKp46, RORγt and CD3ɛ, followed by nuclear staining with DAPI. Cells with NKp46 and RORγt staining were annotated with white arrow heads. Scale bar, 100 μm. (j) Live NKp46+ ILC3 cells were sorted from SI LPL of Washflox/flox and Washflox/floxNcr1-Cre mice carrying RORγt-GFP reporter through identifying surface markers, followed by Hoechst33342 and pyronin Y staining (upper panel). Percentages of S/G2/M cells in the indicated cells were calculated (lower panel). n=7. Data are shown as means±s.d. *P<0.05; **P<0.01; ***P<0.001. Data are representative of at least three independent experiments.
