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 NH4Cl 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 NH4Cl. 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 NH4Cl. 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 NH4Cl. 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 NH4Cl. 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 NH4Cl. 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 NH4Cl 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 × 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 (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 (20). 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.