Journal: Cancer Research

Article Title: Ammonia Suppresses the Antitumor Activity of Natural Killer Cells and T Cells by Decreasing Mature Perforin

doi: 10.1158/0008-5472.CAN-24-0749

Figure Lengend Snippet: Ammonia inhibits NK cell serial killing. A, The percentage of NK cells that underwent serial degranulation in response to stimulation with target cells (K562) in different concentrations of NH 4 Cl ( n = 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, The level of CD56 in NK cells incubated with NH 4 Cl, with or without target cells, determined by surface staining and flow cytometry ( n = 2). C, The level of CD95 (Fas) in NK cells incubated with NH 4 Cl with or without target cells determined by surface staining and flow cytometry ( n = 2). D, The level of CD178 (FasL) in NK cells incubated with NH 4 Cl with or without target cells determined by surface staining and flow cytometry ( n = 2). E–K, 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. E, Activation of granzyme B (GrzB) and caspase-8 reporters in target HeLa cells incubated with NK cells in different concentrations of NH 4 Cl. Time point 0 corresponds to the time of cell death ( n = 15). Images were acquired every 3 minutes. F, Activation of granzyme B and caspase-8 reporters in target HeLa cells incubated with NK cells in different concentrations of NH 4 Cl at the time of cell death ( n = 6). G, Time required to kill a reporter-expressing cell by NK cells in different concentrations of NH 4 Cl through the activation of granzyme B ( n = 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. H, Time required to kill a reporter-expressing cell by NK cells in different concentrations of NH 4 Cl by activation of caspase-8 ( n = 15). I, 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, n = 26; 1.25 mmol/L, n = 26; 2.5 mmol/L, n = 25; 5 mmol/L, n = 28; 10 mmol/L, n = 27). J, Percentage of serial killers among NK cells in different concentrations of NH 4 Cl (controls, n = 26; 1.25 mmol/L, n = 26; 2.5 mmol/L, n = 26; 5 mmol/L, n = 28; 10 mmol/L, n = 27). Label-free count (LFC) was obtained from label-free quantification. P values were calculated using two-way ANOVA with Tukey post hoc test. Data show means ± SEM. n values are the numbers of biological replicates in in vitro experiments. MFI, mean fluorescence intensity.

Article Snippet: Cells were imaged using Opera Phenix live cell microscopy (PerkinElmer).

Techniques: Staining, Incubation, Flow Cytometry, Transfection, Activation Assay, Fluorescence, Time-lapse Microscopy, Expressing, Sequencing, Quantitative Proteomics, In Vitro

Journal: Cancer Research

Article Title: Ammonia Suppresses the Antitumor Activity of Natural Killer Cells and T Cells by Decreasing Mature Perforin

doi: 10.1158/0008-5472.CAN-24-0749

Figure Lengend Snippet: Ammonia decreases perforin levels and inhibits the cytotoxicity of CAR T cells. A, Viability of unmodified T cells and CD19 CAR T cells incubated with different concentrations of NH 4 Cl for 6 hours was assessed using propidium iodide staining and flow cytometry. Shown are data from technical replicates ( n = 2). B, CD19 CAR T-cell cytotoxicity against Raji cells in the presence of different concentrations of NH 4 Cl. Cytotoxicity was determined after 6 hours using flow cytometry. Shown are data from technical replicates ( n = 3). C, CD20 CAR T-cell cytotoxicity against luciferase-expressing CD20 + Nalm6 cells in the presence of different concentrations of NH 4 Cl. Cytotoxicity was determined after 16 hours and normalized to CD20 + Nalm6 cells without CAR T cells. Shown are data from one donor ( n = 2). D, CD22 CAR T-cell cytotoxicity against luciferase-expressing Nalm6 cells in the presence of different concentrations of NH 4 Cl. Cytotoxicity was determined after 16 hours and normalized to Nalm6 cells without CAR T cells. Shown are data from one donor ( n = 2). E, 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 NH 4 Cl. Cytotoxicity was determined after 16 hours and normalized to MDA-MB-231 cells without CAR T cells ( n = 3). F, EGFRvIII CAR T-cell cytotoxicity against GFP-expressing EGFRvIII + U87 cells in the presence of different concentrations of NH 4 Cl. Cytotoxicity was measured using live-cell microscopy (IncuCyte) for 20 hours. Shown are data from one donor ( n = 2). G, The level of perforin detected in human T cells stimulated with anti-CD3/CD28 and incubated with NH 4 Cl for 4 hours determined by intracellular staining using an anti-perforin antibody (δG9 clone) and flow cytometry ( n = 4). MFI, mean fluorescence intensity. H, 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. I, Raji tumor–bearing mice were intratumorally injected with 5 × 10 6 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 ( n = 6). CD19 CAR T cells incubated with Raji cells for 4 hours in control medium in vitro were used as controls. J, 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 . K, 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 J . n values are the number of biological replicates in in vitro experiments or the number of mice used to obtain the data. K, Created with BioRender.com. Winiarska, M. (2025) https://BioRender.com/t34e524 .

Article Snippet: Cells were imaged using Opera Phenix live cell microscopy (PerkinElmer).

Techniques: Incubation, Staining, Flow Cytometry, Luciferase, Expressing, Knock-Out, Microscopy, Fluorescence, Western Blot, Control, Injection, Cell Analysis, In Vitro