Abstract
Ammonia is thought to be a cytotoxin and its increase in the blood impairs cell function. However, whether and how this toxin triggers cell death under pathophysiological conditions remains unclear. Here we show that ammonia induces a distinct form of cell death in effector T cells. We found that rapidly proliferating T cells use glutaminolysis to release ammonia in the mitochondria, which is then translocated to and stored in the lysosomes. Excessive ammonia accumulation increases lysosomal pH and results in the termination of lysosomal ammonia storage and ammonia reflux into mitochondria, leading to mitochondrial damage and cell death, which is characterized by lysosomal alkalization, mitochondrial swelling and impaired autophagic flux. Inhibition of glutaminolysis or blocking lysosomal alkalization prevents ammonia-induced T cell death and improves T cell-based antitumour immunotherapy. These findings identify a distinct form of cell death that differs from previously known mechanisms.
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Source data are provided with this paper. All of the other data supporting the findings of this study are available from the corresponding author on reasonable request.
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Acknowledgements
This work was supported by the National Key Research and Development Program of China (2022YFA1206000), National Natural Science Foundation of China (82388201 and 32322030) and Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2021-I2M-1-021 and 2023-I2M-2-005).
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B.H. and H.Z. conceived of and designed the study. B.H., H.Z., K.T., J.M., J.C., Y.Z., J. Lv and N.Z. conducted the experiments. H.Z., J. Liu, W.Y., X. Luo, J.F., X. Liu, Q.Z., Y.P and Y.L. performed the experiments and/or analysed the data. B.H. and H.Z. wrote the manuscript. All authors read and approved the article.
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Extended data
Extended Data Fig. 1 Ammonia induces effector CD8+ T cell death.
a, Spleen cells isolated from OT-I mice were stimulated with OVA257-264 peptides in vitro. Propidium iodide (PI) staining was analyzed by flow cytometry at indicated time point (n=3 independent experiments). b, Spleen cells isolated from OT-I mice were stimulated with SIINFEKL peptide in vitro. Ammonia levels in activated CD8+ OT-I Teff cells (n=3 independent experiments). c, Immunoblot of CPS1 in CD8+ OT-I Teff cells transduced with NC or OE-CPS1 retrovirus (n=3 independent experiments). d, Propidium iodide staining of CD8+ OT-I Teff cells transduced with NC or OE-CPS1 (n=3 independent experiments). e, The percentages and numbers of CD45.1+CD8+ OT-I Teff cells in spleen were analyzed by flow cytometry 14 days post Lm-OVA infection (n=6 mice per group). f,g, Spleen cells isolated from OT-I mice were stimulated with SIINFEKL peptide in vitro. Cells were treated with Z-VAD-fmk (20 μM), Nec-1(10 μM) or Fer-1(10 μM) 7 days later. Propidium iodide staining (f) (n=4 independent experiments) and ammonia levels (g) were analyzed 10 days after activation (n=5 independent experiments). h, CD8+ Teff cells (3 days’ SIINFEKL stimulation) were treated with 10 mM NH4Cl in vitro in the presence or absence of Z-VAD-fmk, ferrostatin-1, and necrostatin-1. CD8+ Teff cells induced apoptosis with etoposide, ferroptosis with erastin, and necroptosis with TNF-α/SM-164/Z-VAD-fmk were used as positive controls. Propidium iodide staining of CD8+ OT-I Teff cells were analyzed 24 h later (n=3 independent experiments). i,j, The levels of ROS (i) and lipid ROS (j) in CD8+ OT-I T cells in the spleen were analyzed on day 2 to12 post infection (n=3 independent experiments). k, The percentage and numbers of CD45.1+ CD8+ OT-I Teff cells in spleen (n=6 mice per group). l, Morphology images of CD8+ Teff cells treated with chloroquine (5μM) for 16 h (n=3 independent experiments). m, CD8+ Teff cells were treated with NH4Cl in the presence or absence of 3-MA for 24 h. Propidium iodide staining was measured (n=3 independent experiments). Data are representative of as mean ± SD. Statistical significance was determined by two-tailed unpaired Student’s t-test. Experiments were repeated three times (l) with similar results.
Extended Data Fig. 2 Glutamine-derived ammonia contributes to effector T cell death.
a,b, Spleen cells isolated from OT-I mice were stimulated with SIINFEKL peptide in vitro. Cells were treated with DON (1 μM), CB839 (2 μM) or cultured in glutamine free medium 7 days later. Ammonia levels (a) and propidium iodide staining (b) were measured 10 days after activation (n=4 independent experiments). c-e, C57BL/6J mice (CD45.2+) were transferred with 1 × 105 naive CD45.1+ CD8+ OT-I T cells. 1 day later, mice were i.v. infected with Lm-OVA. Mice were then treated with JHU083 or CB839 7 days after Lm-OVA infection. (c,d) The percentages (c) and numbers (d) of CD45.1+ CD8+ OT-I Teff cells in spleen (n=10 mice per group). (e) Mean fluorescent intensity (MFI) of CD25, IFN-γ and TNF-α in CD45.1+CD8+ OT-I T cells (n=10 mice per group). f,g, CD8+ Teff cells were treated with DON (1μM) or CB839 (1μM) for 24 h. (f) the expression of CPS1 in CD8+ Teff cells were analyzed by western blot (n=3 independent experiments). (g) Urea levels in the culture medium were measured. h, Percentage of KLRG1-CD127+ MPEC in CD45.1+CD8+ OT-I T cells (n=3 independent experiments). i, Ammonia levels in splenic CD8+ OT-I Teff cells were measured 14 days after Lm-OVA infection (n=4 independent experiments). j, The percentages and numbers of CD45.1+ CD8+ OT-I Teff cells in spleen (n=5 mice per group). k,l, mRNA levels from real-time PCR results (k) and immunoblot (l) of GLS1 in CD8+ OT-I Teff cells transduced with shNC or shGLS1 retrovirus (n=3 independent experiments). m, Ammonia levels in splenic CD45.1+ CD8+ OT-I Teff cells isolated 7 days after Lm-OVA infection (n=3 independent experiments). n, The percentages and numbers of CD45.1+ CD8+ OT-I Teff cells in spleen (n=6 mice per group). o, Immunoblot of GLS1 in CD8+ OT-I Teff cells transduced with Tet-On-shNC- or Tet-On-shGLS1 (n=3 independent experiments). Data are representative of as mean ± SD. Statistical significance was determined by two-tailed unpaired Student’s t-test. Experiments were repeated three times (f, l and o) with similar results.
Extended Data Fig. 3 Ammonia is stored in and damages lysosomes in effector CD8+ T cells.
a-c, Ammonia levels in the lysosome (a), endoplasmic reticulum (b) and nucleus (c) of CD8+ Teff cells stimulated by anti-CD3/28 in vitro (n=3 independent experiments). d, Western blotting against Histone H3 (Nucleus, Nuc), VDAC1 (mitochondria, Mito), Lamp1 (lysosome, Lyso), Endoplasmic reticulum (ER) and β-actin (homogenate, HM) (n=3 independent experiments). e, Lysosomal pH value in CD8+ Teff cells stimulated by anti-CD3/28 in vitro were measured at indicated time point (n=3 independent experiments). f, mRNA expression of Rhag, Rhbg and Rhcg in CD8+ OT-I Teff cells (n=3 independent experiments). g-i, Immunofluorescence staining of RhCG and TOM20 (g), Calnexin (h) or Syntaxin 6 (i) in splenic CD8+ OT-I Teff cells. Scale bar, 10 μm (n=3 independent experiments). j, Western blot of RhCG in CD8+ OT-I Teff cells isolated on day 2 to12 post infection (n=3 independent experiments). k, Immunoblot of RhCG in CD8+ OT-I Teff cells transduced with shNC or shRhCG retrovirus (n=3 independent experiments). l,m, ammonia levels in the lysosome (l) or mitochondria (m) of activated CD8+ Teff cells stimulated by anti-CD3/28 antibodies in vitro (n=3 independent experiments). n, Immunoblot of RhCG in CD8+ OT-I Teff cells transduced with NC or OE-RhCG retrovirus (n=3 independent experiments). o, Ammonia levels in CD8+ OT-I Teff cells transduced with NC or OE-RhCG retrovirus (n=3 independent experiments). p, Propidium iodide staining was analyzed 10 days after activation (n=3). Data are representative of as mean ± SD. Statistical significance was determined by two-tailed unpaired Student’s t-test. Experiments were repeated three times (d, g-k, n) with similar results.
Extended Data Fig. 4 Ammonia retention results in mitochondrial damage.
a, Relative fold changes of TMRE, MTG (Mito-tracker Green) and the ratio of TMRE to MTG in CD8+ OT-I Teff cells (n=3 independent experiments). b,c, CD8+ T cells were activated with anti-CD3/28 antibodies for 48 h and subsequently exposed to NH4Cl treatment for 24 h (n=3 independent experiments). (b) Representative electron microscope images and quantitative plots of mitochondrion number in CD8+ Teff cells were analyzed (n=13 cells), scale bar, 2 μm. (c) Representative electron microscope images and quantitative plots of crista number per mitochondrion in CD8+ Teff cells (n=26), scale bar, 500 nm. d-h, C57BL/6J mice (CD45.2+) were transferred with 1 × 105 naive CD45.1+ CD8+ OT-I T cells. 1 day later, mice were i.v. infected with Lm-OVA. Mice were then treated with JHU083 7 days after Lm-OVA infection. (d) Relative copy number of mtDNA in splenic CD8+ OT-I Teff cells isolated form mice treated with JHU083 or PBS (n=4 independent experiments). (e) Relative fold changes of TMRE, MTG (Mito-tracker Green) and the ratio of TMRE to MTG in splenic CD8+ OT-I Teff cells (n=4 independent experiments). (f) Representative electron microscope images and quantitative plots of mitochondrion number in splenic CD8+ OT-I Teff cells (n=20 cells), scale bar, 2 μm. (g) Representative electron microscope images and quantitative plots of crista number per mitochondrion in splenic CD8+ OT-I Teff cells (n=23), scale bar, 500 nm. (h) Mitochondrial ammonia levels were measured in splenic CD8+ OT-I Teff cells isolated form mice treated with JHU083 or PBS (n=4 independent experiments). Data are representative of as mean ± SD. Statistical significance was determined by two-tailed unpaired Student’s t-test. Experiments were repeated three times (b, c, f and g) with similar results.
Extended Data Fig. 5 Damaged mitochondria cannot be cleared via autophagy.
a, Immunoblots of NIX, BNIP3 and FUNDC1 in splenic CD8+ OT-I Teff cells (n=3 independent experiments). b, Immunofluorescence staining of Parkin and TOM20 in splenic CD8+ OT-I Teff cells, scale bar, 10 μm (n=3 independent experiments). c, Immunofluorescence staining of NIX and TOM20 in splenic CD8+ OT-I Teff cells, scale bar, 10 μm (n=3 independent experiments). d, CD8+ T cells (3 days’ SIINFEKL stimulation) were exposed to NH4Cl or chloroquine treatment for 24 h. Representative electron microscope images of autolysosomes in CD8+ OT-I Teff cells, scale bar, 1 μm (n=3 independent experiments). e-h, C57BL/6J mice (CD45.2+) were transferred 1 × 105 naive CD45.1+ CD8+ OT-I T cells, and then infected with Lm-OVA. 7 days later, mice were treated with C381 or PBS once per day. (e) Lysosomal pH value in splenic CD8+ OT-I Teff cells (n=5 independent experiments). (f) Immunoblots of Atg5, Atg12, LC3I/II and P62 in splenic CD8+ OT-I Teff cells (n=3 independent experiments). (g) Relative LMP value in splenic OT-I Teff cells (n=5 independent experiments). (h) Enzymatic activities of CTSB, CTSD, β-Gal, and α-Man in splenic CD8+ OT-I Teff cells (n=5 independent experiments). (i) Spleen cells isolated from OT-I mice were stimulated with SIINFEKL peptide in vitro. Cells were treated with C381 7 days later. Propidium iodide staining were measured 10 days after activation (n=3 independent experiments). j-n, C57BL/6J mice (CD45.2+) were transferred 1 × 105 naive CD45.1+ CD8+ OT-I T cells, and then infected with Lm-OVA. 7 days later, mice were treated with Torin1 or PBS once per day. (j) Experimental design. (k) The percentages and numbers of CD45.1+ CD8+ OT-I Teff cells in spleen (n=6 mice per group). Lysosomal pH value (l), LMP value (m) and Enzymatic activities of CTSB, CTSD, β-Gal, and α-Man (n) in splenic CD8+ OT-I Teff cells (n=5 independent experiments). Data are representative of as mean ± SD. Statistical significance was determined by two-tailed unpaired Student’s t-test. Experiments were repeated three times (a-d and f) with similar results.
Extended Data Fig. 6 Ammonia death blockade enhances adoptive T cell therapy against cancer.
a, The percentage of CD45.1+ CD8+ OT-I Teff cells in spleen, lymph node and lung were analyzed (n=10 mice per group). (b) Ki-67 levels in tumour-infiltrating CD8+ OT-I Teff were analyzed (n=5 independent experiments). c-h, CD45.1+ CD8+ OT-I Teff cells were activated by SIINFEKL peptide and IL-2 (100 U/ml) for 24 h and then treated with C381, DON or PBS for 24 h. B16-OVA-bearing C57BL/6J mice received 1 × 107 activated OT-I Teff cells pretreated with C381, DON or PBS on day 7. (c) Experimental design. The percentage (d) and number (e) of CD45.1+ CD8+ OT-I Teff cells per gram of tumour tissue were quantified (n=10 mice per group). Tumour growth curve (f) (n=5 mice per group), tumour weight (g) (n=5 mice per group) and survival curve (h) (n=9 mice per group) are shown. i,j, PBS-, DON- or C381-pretreated CD45.1+ CD45.2+ OT-I Teff cells were mixed with untreated CD45.1- CD45.2+ OT1 OT-I Teff cells (1:1 ratio). B16-OVA-bearing mice (CD45.1+) received 1 × 107 activated CD8+ OT-I Teff cells on day 7. The percentages of CD45.1+ (untreated, C381- or DON-pretreated) OT-I Teff and CD45.1- (PBS-treated) OT-I Teff in total CD8+ OT-I Teff cells were analyzed in tumour (i) and other tissues (spleen, lymph node and lung) (j) (n=6 mice per group). Data are representative of as mean ± SD. Statistical significance was determined by two-tailed unpaired Student’s t-test (a, b-e, g, i and j), two-way ANOVA followed by Tukey’s test (f) or log-rank (Mantel-Cox) test (h).
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Zhang, H., Liu, J., Yuan, W. et al. Ammonia-induced lysosomal and mitochondrial damage causes cell death of effector CD8+ T cells. Nat Cell Biol (2024). https://doi.org/10.1038/s41556-024-01503-x
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DOI: https://doi.org/10.1038/s41556-024-01503-x
