Fig 2: Cell Surface Protein Expression Levels Track the Dynamics of Naive-to-Primed PSC Transition(A) Overview of the experimental design. Shown is a time course experiment of PSCs undergoing a transition from the naive state to the primed state, with flow cytometry analysis every 48 hr.(B) Phase contrast images of H9 PSCs reveal the morphological changes that occur during naive state-to-primed state transition under t2i/L+PKCi conditions. Scale bars, 100 µm.(C) Flow cytometry dotplots of pairwise antibody combinations over the time course. Shown are primed-specific markers on the y axis (CD57, top; CD90, bottom) and naive-specific markers on the x axis (CD75, top; CD130, bottom).(D) FlowSOM visualization of the flow cytometry time course data for H9 PSCs. The minimal spanning tree of the self-organizing map displays an unsupervised clustering of the samples based on their cell surface protein expression levels (right). The results reveal a progressive change in cell surface protein expression during conversion from the naive state to the primed state. The heatmap shows the expression level of each cell surface protein marker in the cell clusters (left).

Fig 3: The inflammatory environment shapes the fate of IL-18Rα+ ILCs(A) Representative dot plots of T-bet and IL18Rα expression after intranasal administration of PBS and IL-12+IL-18 in Rag2−/− mice in Lin−CD45.2+CD90.2+NK1.1−RORγt− cells.(B) Absolute numbers of IL-18Rα− ILC2 (green), IL-18Rα+ ILC (yellow), and ILC1-like cells (blue) after cytokine (IL-12+IL-18) or control (PBS) treatment.(C) Percentage of TCF-1 in lung IL-18Rα− ILC2 after intranasal administration of PBS or IL-12+IL-18.(D) Percentages of cells expressing IL-5 or IFN-γ among the indicated ILC subsets after PMA/ionomycin stimulation.(E) Percentages of IFN-γ + cells in the indicated ILC subsets after ex vivo stimulation with IL-12+IL-18, PMA/ionomycin, or not from IL-12+IL-18-treated C57BL/6 mice.(F) Representative dot plot of T-bet and IL-18Rα expression in IL-12+IL-18-treated Stat1+/+versus Stat1−/− mice.(G) Absolute numbers of ILC1-like cells (left) and IL-18Rα+ ILC (right) in Stat1+/+versus Stat1−/− mice treated as in (F).(H) Representative dot plots of T-bet and IL18Rα expression after IL-33, IL-12+IL-18, and IL-12+IL-18+IL-33-treated Rag2−/− mice in Lin−CD45.2+CD90.2+NK1.1−RORγt− cells.(I–K) Absolute numbers of IL-18Rα− ILC2 (I), IL-18Rα+ ILC (J), and ILC1-like cells (K) in IL-33, IL-12+IL-18, or IL-12+IL-18+IL-33-treated mice.(L) Percentage of IL-5+IFN-γ− (right), IL-5−IFN-γ+ (center), and IL-5+IFN-γ+ (left) in IL-18Rα+ ILC in IL-33, IL-12+IL-18, and IL-12+IL-18+IL-33-treated mice. Each symbol represents an individual mouse.Statistical analysis was performed using Mann-Whitney (B, C, and G) and 1-way (I–L) and 2-way (D and E) ANOVA tests. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Graphs depict data as means ±SEMs. Data are representative of 3 (B, D, and I–K) and 2 (C, E, G, and L) independent experiments.

Fig 4: ILC1-like cells confer protection against Mtb(A) Mycobacterial loads at days 14 and 21 post-infection in Rag2−/−γc−/− mice adoptively transferred with ST2+ILC (black) or not (gray).(B) T-bet expression (%) at different days post-infection in transferred ILC2 in Rag2−/−γc−/− mice.(C) Mycobacterial loads at day 21 post-infection in Rag2−/−γc−/− mice adoptively transferred with ST2+IL-18Rα− ILC (green) versus ST2+IL-18Rα+ ILC.(D) Schematic representation of the in vivo expansion of ILC1-like cells in Rag2−/− mice treated with IL-12+IL-18+IL-33, cell sorting of ILC1-like cells (Lin−CD45.2+CD90.2+NK1.1−ST2−CD49a+IL-18Rα+), and adoptive transfer in Rag2−/−γc−/− 1 day before infection with Mtb by the intratracheal route.(E) Representative histograms of T-bet, GATA3, and RORγt expression in sorted ILC1-like cells (gray) versus ILC2 (Lin−CD45.2+CD90.2+NK1.1−ST2+ cells).(F) Bacterial loads at day 21 post-infection in Rag2−/−γc−/− mice having received (gray) or not (blue) an adoptive transfer of ILC1-like cells from IL-12+IL-18+IL-33-treated Rag2−/− mice 1 day before Mtb infection.(G) C57BL/6 were vaccinated by intranasal administration of BCG or treated intranasally with IL-12+IL-18 or not (PBS) 60 days before Mtb infection. After 14 days post-infection, mice were euthanized.(H) Mycobacterial loads at day 14 post-infection in BCG-vaccinated (gray), IL-12+IL-18-treated (black), or control mice (white).(I) Percentages of total lung ILCs expressing T-bet.(J) Absolute numbers of NK, ILC1, IL-18Rα− ILC2, IL-18Rα+ ILC, ILC3, and ILC1-like cells at day 14 post in BCG-vaccinated (gray), IL-12+IL-18-treated (black), or control mice (white).(K) Percentages of IFN-γ + cells among NK (left), ILC1 (center), and ILC1-like cells (left) in BCG-vaccinated (gray), IL-12+IL-18-treated (black), or control mice (white). after ex vivo stimulation with IL-12+IL-18. Each symbol represents an individual mouse.Statistical analysis was performed using the Mann-Whitney test (B–F) and 1-way (H–K) and 2-way (A) ANOVA tests. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Graphs depict data as means ±SEMs. Data are representative of 2 (A, B, and F–K) independent experiments and a pool of 2 experiments (C).

Fig 5: ILC3-Expressed GPR183 Is Required for the Formation of Colonic Lymphoid Tissues(A) Distribution of GFP+ cells in the small intestine and colon of Gpr183GFP/+ mice. Tissue sections were co-stained with a-CD90.2 and a-B220 Abs. Scale bars (white) represent 100 µm.(B) Number of CPs and ILFs in the small intestine and colon of Rorc(?t)GFPGpr183+/+ and Rorc(?t)GFPGpr183-/- mice (n = 3–5). The upper panel shows representative images of a CP and an ILF from Rorc(?t)GFPGpr183+/+ mice. Scale bars (red) represent 100 µm.(C) Number of peripheral and mesenteric lymph node cells (n = 6–8), Peyer’s patches (n = 10), and colonic patches (n = 3) from Gpr183+/+ and Gpr183-/- mice.(D) Number of ROR?t+ clusters in the colon of bone marrow chimeras (n = 5–9). Bone marrow cells from Gpr183+/+ or Gpr183-/- mice were injected into either Gpr183+/+ or Gpr183-/- irradiated recipient mice.(E) Number of CPs in the colon of Rag1-deficient Gpr183+/+ and Gpr183-/- mice (n = 5).(F) Number of CPs, ILFs, Peyer’s patches, and colonic patches in Rorc-cre Gpr183flox/flox mice and Gpr183flox/flox or Gpr183flox/+ controls (n = 3–6).Data are represented as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Student’s t test or one-way ANOVA with Tukey’s post-test (D). Data are representative of or combined from two (F) or three (A–E) experiments. See also Figures S3 and S4.