Aleksandra Kusowska

Supplementary Fig. 7 shows that conditioned medium decreases expression and secretion of perforin by NK cells

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 NH4Cl (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 NH4Cl, 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 NH4Cl 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 NH4Cl 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 NH4Cl. 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 NH4Cl at the time of cell death (n = 6). G, Time required to kill a reporter-expressing cell by NK cells in different concentrations of NH4Cl 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 NH4Cl 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 NH4Cl (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.

Ammonia concentration is increased in cancer cell–conditioned medium and TIF. Ammonia concentration in the tumor-conditioned medium, collected after 48 hours of incubation, was measured using a Dimension Ammonia assay (Siemens; n = 3). Cells were cultured at different densities: lymphoma (0.5 × 106, 1.0 × 106, 1.5 × 106/mL; A), multiple myeloma (1 × 106, 2 × 106, 4 × 106/mL; B), and breast cancer cell lines (0.5 × 106, 1 × 106, 1.5 × 106/mL; C). In A–C, empty medium incubated for 48 hours (without the cells) at 4°C or 37°C is presented as a control. D, Schematic presentation of the TIF and SCF isolation from mice. TIF was collected from tumors not exceeding 1,500 mm3. SCF was isolated at the same time from the contralateral tight as a control tissue fluid. E–G, The concentration of ammonia in TIF and SCF isolated from Raji tumor–bearing NSG (n = 5; E), MM.1s tumor–bearing SCID mice (n = 5; F), and breast cancer–bearing mice (EMT6 in BALB/c mice, n = 5; E0771 in BALB/c mice, n = 5; 4T1 in BALB/c mice; n = 5; MCF7 in NSG mice, n = 4; MDA-MB-231 in NSG mice, n = 3; G). P values were calculated using a paired t test. Data show individual values and means ± SEM. n values are the number of biological replicates in in vitro experiments or the number of mice used to obtain the data. D, Created with BioRender.com. Winiarska, M. (2025) https://BioRender.com/x56a982.

Supplementary Fig. 4 shows that ammonia accumulates in NK cells, affects their cytotoxic potential but not their viability.

Supplementary Fig. 3 shows changes in pH and ammonia concentration in conditioned media

Supplementary Fig. 11 shows that ammonia increases the number of LAMP1+ vesicles but decreases their volume and LAMP1 level

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.

Supplementary Fig. 1 shows that culture-conditioned medium suppresses cytotoxicity of NK cells

Supplementary Fig. 5 shows ammonium chloride’s impact on the efficiency of rituximab therapy in vivo

Supplementary Fig. 6 shows that ammonia is rapidly excreted or metabolized in TME