Figure S4.
Autocrine TGF-β drives the emergence of cytotoxic trNK cells in the SG but is redundant in the tumor microenvironment. (A) Color-coded, normalized marker expression of SG group 1 ILCs, referring to Fig. 4 A. (B) Immunofluorescence staining of Ncr1CreR26RAi14 SGs at indicated age displaying DAPI (blue), NKp46-TdTomato (magenta), GzmB (yellow), and EpCAM (teal) with arrowheads pointing to NKp46+ group 1 ILCs of flat (f) or round (r) morphology, with undetectable/low or high (*) GzmB expression. Scale bars, 15 μm. (C) Flow cytometric analysis of sorted splenic NK cells after in vitro culture 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), referring to Fig. 4 I. (D)Ncr1CreERT2 (Ctrl) and Ncr1CreERT2Tgfb1fl mice were treated with tamoxifen via oral gavage for 3 days and analyzed 1 wk later by flow cytometry. Quantification of indicated markers on trNK cells. gMFI, geometric mean fluorescence intensity. (E and F) 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). (G) Experimental scheme for H-K. Mice were injected subcutaneously (s.c.) with 5 × 105 MC38 tumor cells and analyzed 15 days later. (H and I) Representative flow cytometry plots (H) of group 1 ILCs in the tumors of WT, Ncr1CreTgfb1fl, and Ncr1CreTgfbr2fl mice and quantification (I) of trNK cell percentage at the endpoint (15 days). (J) Tumor size (mm3) over time in indicated mice. (K) Quantification of indicated immune cell populations. Mφ, Macrophage. APC, antigen-presenting cell. (A) Data from one representative experiment with total n = 4–5 mice per timepoint, or (B) representative for n = 2–4 mice per timepoint, or (C–F) representative for one of two independent experiments, or (G–K) representative for one of three independent experiments with n = 5–8 mice per group. Error bars display means ± SD. Statistical significance was calculated using one-way ANOVA or two-tailed t test; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns, not significant.

Autocrine TGF-β drives the emergence of cytotoxic trNK cells in the SG but is redundant in the tumor microenvironment. (A) Color-coded, normalized marker expression of SG group 1 ILCs, referring to Fig. 4 A. (B) Immunofluorescence staining of Ncr1CreR26RAi14 SGs at indicated age displaying DAPI (blue), NKp46-TdTomato (magenta), GzmB (yellow), and EpCAM (teal) with arrowheads pointing to NKp46+ group 1 ILCs of flat (f) or round (r) morphology, with undetectable/low or high (*) GzmB expression. Scale bars, 15 μm. (C) Flow cytometric analysis of sorted splenic NK cells after in vitro culture 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), referring to Fig. 4 I. (D)Ncr1CreERT2 (Ctrl) and Ncr1CreERT2Tgfb1fl mice were treated with tamoxifen via oral gavage for 3 days and analyzed 1 wk later by flow cytometry. Quantification of indicated markers on trNK cells. gMFI, geometric mean fluorescence intensity. (E and F) 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). (G) Experimental scheme for H-K. Mice were injected subcutaneously (s.c.) with 5 × 105 MC38 tumor cells and analyzed 15 days later. (H and I) Representative flow cytometry plots (H) of group 1 ILCs in the tumors of WT, Ncr1CreTgfb1fl, and Ncr1CreTgfbr2fl mice and quantification (I) of trNK cell percentage at the endpoint (15 days). (J) Tumor size (mm3) over time in indicated mice. (K) Quantification of indicated immune cell populations. Mφ, Macrophage. APC, antigen-presenting cell. (A) Data from one representative experiment with total n = 4–5 mice per timepoint, or (B) representative for n = 2–4 mice per timepoint, or (C–F) representative for one of two independent experiments, or (G–K) representative for one of three independent experiments with n = 5–8 mice per group. Error bars display means ± SD. Statistical significance was calculated using one-way ANOVA or two-tailed t test; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns, not significant.

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