Panel A shows flow cytometry plots identifying ILC populations in the small intestine and colon under WT and Lingo4 knockout conditions. Panel B and C show scatter bar plots of the absolute numbers of ILC subsets in the small intestine and colon, respectively, with individual data points and mean values. Panel D shows a scatter bar plot of ILC3 subsets in the small intestine, while Panel E shows a scatter bar plot of ILC3 subsets in the colon. Panel F shows scatter plots of RORγt geometric mean fluorescence intensity in ILC3s from different tissues. Panel G shows flow cytometry plots and corresponding scatter bar plots of IL-22 production in small intestine ILC3s under mock and IL-23 stimulation conditions. Panel H shows scatter bar plots of cytokine production, including IL-22 and IL-17, across small intestine ILC3 subsets under mock and IL-23 stimulation. Panel I shows flow cytometry plots and scatter bar plots of IL-22 production in colonic ILC3s under mock and IL-23 stimulation. Panel J illustrates a mixed bone marrow chimera experimental setup along with representative flow cytometry plots of spleen reconstitution. Panel K and L show flow cytometry plots and scatter bar plots of ILC3 frequencies and IL-22 production in the small intestine. Panel M shows a flow cytometry plot and scatter bar plot comparing WT and Lingo4 knockout cells, and Panel N shows scatter bar plots of ILC3 numbers and subset distribution in the small intestine. Panel O shows flow cytometry plots and scatter bar plots of IL-22 and IL-17 production in ILC3s following IL-23 stimulation.
LINGO4 deficiency reduces ILC3 abundance and IL-22 production in a cell-intrinsic manner. (A–C) Representative flow cytometry plots (A) and absolute numbers of ILCs in the si (B) and colonic (C) LP of WT and Lingo4−/− mice. Mean ± SEM, n = 7–12; pooled from three independent experiments. (D and E) Numbers of ILC3 subsets (NKp46+, CCR6+, and DN) in the siLP (D) and colon (E) of WT and Lingo4−/− mice. Mean ± SEM, n = 7–12; pooled from four experiments. (F) Geometric mean fluorescence intensity (gMFI) of RORγt in ILC3s from siLP and colon of WT and Lingo4−/− mice. Mean ± SEM, n = 6–7; two experiments combined. (G) Representative flow cytometry plots and frequencies of IL-22+ and IL-17+ siLP ILC3s after ex vivo stimulation with IL-23 (10 ng/ml). Mean ± SEM, n = 7; two experiments combined. (H) Frequencies of IL-22+ cells across siLP ILC3 subsets after ex vivo stimulation with IL-23. Mean ± SEM, n = 7; two experiments combined. (I) Representative flow cytometry plots and frequencies of IL-22+ and IL-17+ colonic ILC3s after ex vivo stimulation with IL-23 (10 ng/ml). Mean ± SEM, n = 9; two experiments combined. (J) Mixed BM chimeras: irradiated CD45.1 recipients (1,100 cGy) were reconstituted with a 1:1 mixture of CD45.1/2+ WT and CD45.2+Lingo4−/− BM cells. Representative reconstitution profile in spleen at 8 wk. n = 9; two experiments combined. (K and L) Frequencies of WT (CD45.1/2+) and Lingo4−/− (CD45.2+) ILC3s in siLP (K) and IL-22+ ILC3s following IL-23 stimulation (L). Mean ± SEM, n = 9 (K) and n = 4 (L); two experiments combined. (M and N) Representative flow cytometry plots and numbers of ILC3s (M) and ILC3 subsets (N) in siLP of Lingo4f/f and Lingo4ΔILC3 mice. ILC3s were defined as live lymphocyte-sized Lin– (CD3− CD19−) Thy1.2hi CD45int cells. Mean ± SEM, n = 4; representative of two experiments. (O) Frequencies of IL-22+ and IL-17+ ILC3s after IL-23 stimulation. Mean ± SEM, n = 4; representative of two experiments. (A–O) Statistical significance was assessed using unpaired two-tailed t tests or one-way ANOVA with Tukey’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.