Figure 7 from Ammonia Suppresses the Antitumor Activity of Natural Killer Cells and T Cells by Decreasing Mature Perforin

<p>Ammonia decreases perforin levels and inhibits the cytotoxicity of CAR T cells. <b>A,</b> Viability of unmodified T cells and CD19 CAR T cells incubated with different concentrations of NH₄Cl for 6 hours was assessed using propidium iodide staining and flow cytometry. Shown are data from technical replicates (<i>n</i> = 2). <b>B,</b> CD19 CAR T-cell cytotoxicity against Raji cells in the presence of different concentrations of NH₄Cl. Cytotoxicity was determined after 6 hours using flow cytometry. Shown are data from technical replicates (<i>n</i> = 3). <b>C,</b> CD20 CAR T-cell cytotoxicity against luciferase-expressing CD20⁺ Nalm6 cells in the presence of different concentrations of NH₄Cl. Cytotoxicity was determined after 16 hours and normalized to CD20⁺ Nalm6 cells without CAR T cells. Shown are data from one donor (<i>n</i> = 2). <b>D,</b> CD22 CAR T-cell cytotoxicity against luciferase-expressing Nalm6 cells in the presence of different concentrations of NH₄Cl. Cytotoxicity was determined after 16 hours and normalized to Nalm6 cells without CAR T cells. Shown are data from one donor (<i>n</i> = 2). <b>E,</b> PD-L1 CAR T-cell cytotoxicity against luciferase-expressing PD-L1 knockout or PD-L1⁺ MDA-MB-231 cells in the presence of different concentrations of NH₄Cl. Cytotoxicity was determined after 16 hours and normalized to MDA-MB-231 cells without CAR T cells (<i>n</i> = 3). <b>F,</b> EGFRvIII CAR T-cell cytotoxicity against GFP-expressing EGFRvIII⁺ U87 cells in the presence of different concentrations of NH₄Cl. Cytotoxicity was measured using live-cell microscopy (IncuCyte) for 20 hours. Shown are data from one donor (<i>n</i> = 2). <b>G,</b> The level of perforin detected in human T cells stimulated with anti-CD3/CD28 and incubated with NH₄Cl for 4 hours determined by intracellular staining using an anti-perforin antibody (δG9 clone) and flow cytometry (<i>n</i> = 4). MFI, mean fluorescence intensity. <b>H,</b> The level of total perforin in T cells stimulated with αCD3/CD28 and incubated with ammonia determined by Western blot methods using an anti-perforin antibody (Prf-344 clone). β-Actin is presented as a loading control. Representative blot from one donor is shown. <b>I,</b> Raji tumor–bearing mice were intratumorally injected with 5 × 10⁶ human CD19 CAR T cells. After 4 hours, tumors were dissected and enzymatically dissociated, followed by CAR T-cell analysis for perforin levels using intracellular staining with an anti-perforin antibody (δG9 clone) and flow cytometry (<i>n</i> = 6). CD19 CAR T cells incubated with Raji cells for 4 hours in control medium <i>in vitro</i> were used as controls. <b>J,</b> Level of perforin protein in the CD8⁺ T cells and NK cells in the blood and tumors of patients with HCC analyzed from a published dataset (<a href="#bib20" target="_blank">20</a>). <b>K,</b> Ammonia increases pH in secretory lysosomes, which results in the dissociation of perforin from proteoglycans. Subsequently, dissociated perforin is susceptible to inactivation and proteolytic degradation. Data show means ± SEM or median ±95% CI for <b>J</b>. <i>n</i> values are the number of biological replicates in <i>in vitro</i> experiments or the number of mice used to obtain the data. <b>K,</b> Created with BioRender.com. Winiarska, M. (2025) <a href="https://BioRender.com/t34e524" target="_blank">https://BioRender.com/t34e524</a>.</p>