Figure 6 from Ammonia Suppresses the Antitumor Activity of Natural Killer Cells and T Cells by Decreasing Mature Perforin

<p>Ammonia inhibits NK cell serial killing. <b>A,</b> The percentage of NK cells that underwent serial degranulation in response to stimulation with target cells (K562) in different concentrations of NH₄Cl (<i>n</i> = 4). Cells were stained with anti-CD107a after 2 hours of incubation, and then after an additional 2 hours, they were stained with an anti-CD107a antibody conjugated with a different fluorochrome and analyzed using flow cytometry. NK cells that underwent serial degranulation were defined as CD107a-double positive NK cells. <b>B,</b> The level of CD56 in NK cells incubated with NH₄Cl, with or without target cells, determined by surface staining and flow cytometry (<i>n</i> = 2). <b>C,</b> The level of CD95 (Fas) in NK cells incubated with NH₄Cl with or without target cells determined by surface staining and flow cytometry (<i>n</i> = 2). <b>D,</b> The level of CD178 (FasL) in NK cells incubated with NH₄Cl with or without target cells determined by surface staining and flow cytometry (<i>n</i> = 2). <b>E–K,</b> HeLa cells were transfected with NES-ELQTD-GFP-T2A-NES-VGPD-mCherry and CD48. In these cells, NES-RIEADS-mCherry (activation of GrzB) and NES-VGPD-mGFP [activation of caspase-8 (Casp8)] reporter cleavage can be detected by the appearance of fluorescence inside the nucleus. GrzB cleaves the reporter, resulting in an increase in the red fluorescent signal in the nucleus, whereas the caspase-8 reporter is specifically activated by death receptor–mediated apoptosis and results in an increase of green fluorescence in the nucleus. Confocal time-lapse microscopy started immediately after NK cell exposure. <b>E,</b> Activation of granzyme B (GrzB) and caspase-8 reporters in target HeLa cells incubated with NK cells in different concentrations of NH₄Cl. Time point 0 corresponds to the time of cell death (<i>n</i> = 15). Images were acquired every 3 minutes. <b>F,</b> Activation of granzyme B and caspase-8 reporters in target HeLa cells incubated with NK cells in different concentrations of NH₄Cl at the time of cell death (<i>n</i> = 6). <b>G,</b> Time required to kill a reporter-expressing cell by NK cells in different concentrations of NH₄Cl through the activation of granzyme B (<i>n</i> = 15). SYTOX Blue dye was used to assess the viability of the target cell. Early or late cell death was evaluated based on the kinetics of the target cell nucleus staining after contact with NK cells. <b>H,</b> Time required to kill a reporter-expressing cell by NK cells in different concentrations of NH₄Cl by activation of caspase-8 (<i>n</i> = 15). <b>I,</b> The diagram displays target cell death in single NK cell–target cell contacts. Each row displays the contact sequence of one individual NK cell imaged using time-lapse microscopy (controls, <i>n</i> = 26; 1.25 mmol/L, <i>n</i> = 26; 2.5 mmol/L, <i>n</i> = 25; 5 mmol/L, <i>n</i> = 28; 10 mmol/L, <i>n</i> = 27). <b>J,</b> Percentage of serial killers among NK cells in different concentrations of NH₄Cl (controls, <i>n</i> = 26; 1.25 mmol/L, <i>n</i> = 26; 2.5 mmol/L, <i>n</i> = 26; 5 mmol/L, <i>n</i> = 28; 10 mmol/L, <i>n</i> = 27). Label-free count (LFC) was obtained from label-free quantification. <i>P</i> values were calculated using two-way ANOVA with Tukey post hoc test. Data show means ± SEM. <i>n</i> values are the numbers of biological replicates in <i>in vitro</i> experiments. MFI, mean fluorescence intensity.</p>