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. 2024 Jan 17;10(3):eadh5520. doi: 10.1126/sciadv.adh5520

Fig. 2. NTOC-derived ILC1s are cytokine-producing and have cytotoxic functionality.

Fig. 2.

(A) Transmission electron microscopy pictures of sorted NTOC-derived CD4 T cells (a + b; vehicle supernatant) or ILC1s (c + d; IL-12 + IL-18 supernatant); representative cells are shown from two independent experiments. Red arrows in ILC1 pictures indicate black organelles identified as dense cytotoxic–like granules. Scale bars, 2 μm. (B) YAC-1 killing assay, showing dose-dependent killing of YAC-1 target cells following increasing cell numbers of four different types of sorted effector cells: (i) ILC1s (NTOC IL-12 + IL-18), (ii) cNK cells (adult spleen IL-12 + IL-18), (iii) cNK cells (adult spleen), and (iv) noncytotoxic CD4+ thymocytes (NTOC vehicle). Killing was shown as 4′,6-diamidino-2-phenylindole–positive (DAPI+) YAC-1 cells after 4 hours of coculture. Data are pooled from three independent experiments showing n = 4 to 6 biological replicates per condition. Error bars display mean with SEM. (C) Histograms of intracellular flow cytometry, visualizing active expression of IFN-γ, TNF-α, GM-CSF, and Granzyme B in different NTOC-derived ILC1s compared to ex vivo P0.5 ILC1s. Expression is shown 4 hours after brefeldin A and monensin block and no further stimulation. Histograms are representative of n = 6 to 9 biological replicates per condition from two to three independent experiments. FMO, fluorescence minus one. (D) Bar plot of protein measurements of cytokines in the supernatant after 6 days of NTOC, measured by multiplex enzyme-linked immunosorbent assay. Data are pooled from three independent experiments showing n = 6 biological replicates per condition. Each dot represents a replicate, and error bars show mean with SEM. Statistical significance was calculated by (B) one-way analysis of variance (ANOVA) between cell types; (D) one-way ANOVA (TNF-α and CCL3). Nonparametric Kruskal-Wallis test (IFN-γ and GM-CSF). **P < 0.01, ***P < 0.001, and ****P < 0.0001. (B) One-way ANOVA between NTOC-derived ILC1 and noncytotoxic CD4+ thymocytes: #P < 0.05 and ###P < 0.001.