Figure 4.
Cytotoxic trNK cells emerge later in life and follow a Hobit-dependent differentiation program. (A and B) High-dimensional flow cytometry of group 1 ILCs from SGs at indicated age, shown as UMAP with FlowSOM clustering and corresponding heatmap displaying relative marker expression in identified cell populations. (B) Cluster frequency at indicated timepoint. (C) Representative flow cytometry plots showing emergence of cytotoxic (GzmB+GzmC+) population. (D and E) Immunofluorescence staining of Ncr1CreR26RAi14 SGs at indicated age. Quantification of morphology (D) and representative images (E) showing DAPI (blue), NKp46-TdTomato (magenta), GzmB (yellow), and EpCAM (teal). Scale bars, 15 μm. (F and G) Histogram showing expression (F) and quantification (G) of Hobit-TdTomato in indicated SG group 1 ILC subsets. (H) Representative flow cytometry plots and quantification of cytotoxic trNK cells in WT and HobitKO mice. (I and J) Flow cytometric analysis of sorted splenic NK cells after in vitro culture (I) for 24 h (pulse) with IL-15 (50 ng/ml) only, or 24 h or 7 days with TGF-β (5 ng/ml) and minimal IL-15 (required for NK cell survival; 10 ng/ml), separated into immature (CD27+) and mature (CD11b+) subsets or (J) all NK cell subsets together for 72 h with indicated concentrations of TGF-β (5 ng/ml) and minimal IL-15. (K and L)Ncr1CreERT2 (Ctrl) and Ncr1CreERT2Tgfb1fl mice were treated with tamoxifen via oral gavage for 3 days and analyzed 1 wk later by flow cytometry. Representative plots (K) and quantification (L) of SG GzmB+CD103+/− trNK cells. (M and N) Flow cytometric analysis of sorted splenic NK cells after in vitro culture with IL-15 (50 ng/ml) or TGF-β (5 ng/ml) with minimal IL-15 for 24 h (pulse) or 3 days (continuous; cont.), or after 24 h pulse with TGF-β (5 ng/ml) followed by culture in IL-15 (20 ng/ml) for 3 or 7 days (indicated as TGF-β → IL-15). (O) Flow cytometric quantification of GzmB+CD103+/− SG trNK after two injections (48 h apart) of IL-15/IL-15Rα complex. (P) Hobit-TdTomato Reporter+ SG cNK cells after 72 h culture with IL-15 (low: 25 ng/ml; high: 100 ng/ml) and TGF-β (5 ng/ml). (A and B) Data from one representative experiment with total n = 4–5 mice per timepoint, or (C, F–H, K, and L) from one representative of two to three independent experiments with n = 3–6 mice, (D and E) representative for n = 2–4 mice per timepoint, or (I, J, and M–P) representative for one of two independent experiments. Error bars display means ± SD. Statistical significance was calculated using one-way ANOVA or two-tailed t test; *P < 0.05, **P < 0.01, and ****P < 0.0001. ns, not significant.

Cytotoxic trNK cells emerge later in life and follow a Hobit-dependent differentiation program. (A and B) High-dimensional flow cytometry of group 1 ILCs from SGs at indicated age, shown as UMAP with FlowSOM clustering and corresponding heatmap displaying relative marker expression in identified cell populations. (B) Cluster frequency at indicated timepoint. (C) Representative flow cytometry plots showing emergence of cytotoxic (GzmB+GzmC+) population. (D and E) Immunofluorescence staining of Ncr1CreR26RAi14 SGs at indicated age. Quantification of morphology (D) and representative images (E) showing DAPI (blue), NKp46-TdTomato (magenta), GzmB (yellow), and EpCAM (teal). Scale bars, 15 μm. (F and G) Histogram showing expression (F) and quantification (G) of Hobit-TdTomato in indicated SG group 1 ILC subsets. (H) Representative flow cytometry plots and quantification of cytotoxic trNK cells in WT and HobitKO mice. (I and J) Flow cytometric analysis of sorted splenic NK cells after in vitro culture (I) for 24 h (pulse) with IL-15 (50 ng/ml) only, or 24 h or 7 days with TGF-β (5 ng/ml) and minimal IL-15 (required for NK cell survival; 10 ng/ml), separated into immature (CD27+) and mature (CD11b+) subsets or (J) all NK cell subsets together for 72 h with indicated concentrations of TGF-β (5 ng/ml) and minimal IL-15. (K and L)Ncr1CreERT2 (Ctrl) and Ncr1CreERT2Tgfb1fl mice were treated with tamoxifen via oral gavage for 3 days and analyzed 1 wk later by flow cytometry. Representative plots (K) and quantification (L) of SG GzmB+CD103+/− trNK cells. (M and N) Flow cytometric analysis of sorted splenic NK cells after in vitro culture with IL-15 (50 ng/ml) or TGF-β (5 ng/ml) with minimal IL-15 for 24 h (pulse) or 3 days (continuous; cont.), or after 24 h pulse with TGF-β (5 ng/ml) followed by culture in IL-15 (20 ng/ml) for 3 or 7 days (indicated as TGF-β → IL-15). (O) Flow cytometric quantification of GzmB+CD103+/− SG trNK after two injections (48 h apart) of IL-15/IL-15Rα complex. (P) Hobit-TdTomato Reporter+ SG cNK cells after 72 h culture with IL-15 (low: 25 ng/ml; high: 100 ng/ml) and TGF-β (5 ng/ml). (A and B) Data from one representative experiment with total n = 4–5 mice per timepoint, or (C, F–H, K, and L) from one representative of two to three independent experiments with n = 3–6 mice, (D and E) representative for n = 2–4 mice per timepoint, or (I, J, and M–P) representative for one of two independent experiments. Error bars display means ± SD. Statistical significance was calculated using one-way ANOVA or two-tailed t test; *P < 0.05, **P < 0.01, and ****P < 0.0001. ns, not significant.

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