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: Anti-tumoral effector responses are supported by CD8+ T cells and NK cells activation and cytotoxicity. (a–e) Flow cytometry was performed in HIF-1a?Nf orthotopic transplanted (OT) and littermate HIF-1afl/fl control mice to assess cytotoxic T lymphocytes (CTL) and natural killer (NK) cell populations. CTL are gated based on their expression of CD45 + CD90.2 + CD3 + CD8+ and NK cells are gated based on their expression of CD45 + CD90.2-CD19-NK1.1+. Shown are scatter plots of (a) the % CD3 + CD8+ T cells, and (b) % NK cells of the total CD45+ population in the pancreata of HIF-1a?Nf OT (red clear) and control OT (black clear) mice (HIF-1a?Nf n = 22, control n = 14 mice per group, pooled from three independent experiments). CTL and NK cells were stimulated with 10 ng/mL PMA and 1 µM ionomycin for 4 h. Scatter plots indicate the percentage (c) Perforin+, (d) Granzyme B+, and (e) IFN-?+ cells of total CTL and NK cells in the pancreata of HIF-1a?Nf OT (red clear) and control OT (black clear) mice (HIF-1a?Nf n = 22, control n = 14 mice per group, pooled from three independent experiments). Data represent the means ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001 by unpaired Student’s t-test with a 95% confidence interval.

Fig 4: 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 5: GPR183 and 7a,25-OHC Promote ILC3 Localization to CPs and ILFs(A) Distribution of Gpr183+/+ and Gpr183-deficient hematopoietic cells in the colon of mixed bone marrow chimeras. Bone marrow cells from Gpr183+/+ or Gpr183-/- mice (CD45.2+) were mixed 9:1 with B6 cells (CD45.1+) and injected into irradiated Rag1-/- recipients (CD45.1+) for the generation of bone marrow chimeras. Sections were stained for detection of Gpr183+/+ and Gpr183-/- cells (CD45.2, red) or B6 cells (CD45.1, green). Nuclei were visualized by DAPI staining (blue). Scale bars on the right (white) represent 100 µm.(B) Distribution of Gpr183+/+ and Gpr183-deficient ILCs in the colon of mixed bone marrow chimeras. Bone marrow cells from Gpr183+/+ or Gpr183-/- mice (CD90.2+) were mixed 9:1 with B6 cells (CD90.1+) and injected into irradiated B6 recipients (CD90.1+) for the generation of bone marrow chimeras. Colon sections were stained for detection of Gpr183+/+ and Gpr183-/- (CD90.2, red) or B6 (CD90.1, green) ILCs. Scale bars (red) represent 100 µm. The lower panel shows the number of clusters in Gpr183+/+-B6 and Gpr183-/--B6 chimeras consisting of CD90.2+ (red) or CD90.1+ (green) ILCs. Data are represented as means ± SEM. p value by two-way ANOVA.(C and D) Distribution of donor-derived ILC3s (ROR?t-GFP+, green) in the colon (C) and small intestine (D) of bone marrow chimeras. Bone marrow cells from Rag1-deficient Rorc(?t)GFP transgenic mice were injected into irradiated Ch25h+/+ or Ch25h-/- recipients for the generation of bone marrow chimeras. Sections were co-stained for detection of B cells (B220+, red). Scale bars (red) represent 100 µm. The lower panel in (C) shows the number of donor-derived ILC3s in the colon of Ch25h+/+ or Ch25h-/- hosts. Data are representative of or combined from two (B–D) or three (A) experiments. See also Figure S5.