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Ammonia detoxification promotes CD8+ T cell memory development by urea and citrulline cycles

Nature Immunology volume 24, pages 162–173 (2023)Cite this article

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Abstract

Amino acid metabolism is essential for cell survival, while the byproduct ammonia is toxic and can injure cellular longevity. Here we show that CD8+ memory T (TM) cells mobilize the carbamoyl phosphate (CP) metabolic pathway to clear ammonia, thus promoting memory development. CD8+ TM cells use β-hydroxybutyrylation to upregulate CP synthetase 1 and trigger the CP metabolic cascade to form arginine in the cytosol. This cytosolic arginine is then translocated into the mitochondria where it is split by arginase 2 to urea and ornithine. Cytosolic arginine is also converted to nitric oxide and citrulline by nitric oxide synthases. Thus, both the urea and citrulline cycles are employed by CD8+ T cells to clear ammonia and enable memory development. This ammonia clearance machinery might be targeted to improve T cell-based cancer immunotherapies.

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Fig. 1: Urea cycle is active in CD8+ TM cells.
Fig. 2: Urea cycle is required for CD8+ T cell memory development.
Fig. 3: CD8+ TM cells mobilize arginase 2 for urea formation.
Fig. 4: SLC25A29 and SLC14A1 are utilized for urea cycle.
Fig. 5: CD8+ TM cells dispose ammonia by the citrulline cycle.
Fig. 6: CPS1 is crucial for nitrogen disposal and memory development.
Fig. 7: β-Hydroxybutyrylation of Cps1 triggers the CP metabolic cascade.

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Data availability

The data supporting this study are available within main article and Supplementary Information files or are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported by the Natural Science Foundation of China (81788101 to B.H. and 82071864, 82150103 to K.T.), CAMS Innovation Fund for Medical Sciences (2021-1-I2M-021 to B.H.), Program for HUST Academic Frontier Youth Team (2018QYTD01 to K.T.), the Key R&D program of Hubei Province (2020BCA068 to K.T.) and the Young Top-notch Talent Cultivation Program of Hubei Province (K.T.).

Author information

Author notes

  1. These authors contributed equally: Ke Tang, Huafeng Zhang, Jinghui Deng.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    Ke Tang, Jinghui Deng, Shichuan Liu, Shuya Lu, Qingfa Cui, Chen Chen, Jincheng Liu, Zhuoshun Yang & Bo Huang

  2. Cell Architecture Research Center, Huazhong University of Science and Technology, Wuhan, China

    Ke Tang

  3. Department of Pathology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    Huafeng Zhang

  4. Department of Immunology & National Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (CAMS) & Peking Union Medical College, Beijing, China

    Dianheng Wang, Jie Chen, Jiadi Lv & Bo Huang

  5. Hubei Provincial Key Laboratory for Applied Toxicology, Hubei Provincial Center for Disease Control and Prevention, Wuhan, China

    Yonggang Li

  6. Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    Jingwei Ma

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Contributions

B.H. conceived the project. K.T., H.Z., J.D., D.W., S.L., S.L., Q.C., C.C., J. Liu, J.C., J. Lv and J.M. conducted the experiments. K.T. and B.H. analyzed the data. B.H. and K.T. wrote the manuscript.

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Correspondence to Bo Huang.

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Nature Immunology thanks Ping-Chih Ho and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: N. Bernard, in collaboration with the Nature Immunology team.

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Extended data

Extended Data Fig. 1 Urea cycle is active in CD8+ Tm cells.

a, Standard curve of urea cycle substances. b, The levels of hydroxyurea (CP derivant), ornithine, citrulline, argininosuccinate and arginine in CD4+ OVA specific Tn, Te and Tm cells from mice were analyzed by LC-MS/MS through standard curve. C, Te (IL-2 derived) cells treated with BPTES (10 μM) for 12 hours, then cultured with 15N-glutamine (2 mM). 24 hours later, the incorporation of 15N-glutamine into urea cycle intermediates including CP, citrulline, argininosuccinate, arginine and urea were analyzed by LC-MS/MS. In b and c, n = 3 independent experiments. Two-tailed unpaired Student’s t-tests followed by Bonferroni’s test (b and c). The data represent mean ± s.d.

Source data

Extended Data Fig. 2 Urea cycle is required for CD8+ T cell memory development.

a, IL-15-derived Tm cells were treated with NH4Cl (20 mM) or erastin (10 μM) for 24 hours, lipid peroxidation was determined by flow cytometry, erastin treatment group was as a positive control. b, IL-15-derived Tm cells were treated with NH4Cl for 24 hours or TNF-α for 3 hours, the expression were analyzed by western blot, all samples were pre-treated with 20 μM Z-VAD for 30 min, TNF-α treatment group was as a positive control. c, IL-15-derived Tm cells were treated with 10 mM NH4Cl for 24 hours, the expression were detected by western blot. d, IL-2-derived Teff cells and IL-15-derived Tm cells were treated with different doses of NH4Cl for 24 hours, the expression were detected by western blot. e, The same as d, except that the LysoSensor fluorescence intensity was measured by flow cytometry. f, IL-15-derived Tm were treated with 3MA (5 mM) or NH4Cl (10 mM) for 24 hours, and then PI was analyzed. g, The knockdown efficiency of shCps1 in IL-15-derived Tm cells. h, CD8+ Teff (IL-2) and Tm (IL-15) cells were cultured with 15N-glutamine (2 mM), the incorporation of N-acetylgutamate (m + 1) were analyzed by LC-MS/MS. i, The knockdown efficiency of shGDH in IL-15-derived Tm cells. j, IL-15-derived Tm cells transduced with shGDH were cultured with 15N-glutamine (2 mM), LC-MS/MS analysis was performed for m + 1 N-acetylgutamate. k, The levels of ammonia in IL-15-derived CD8+ Tm cells transduced with shGDH were analyzed. l, The same as k, except that the cell number was analyzed. m, The gating strategy of flow cytometry analysis on Tetramer+CD45.1+CD8+ T cells. n, The same as Fig. 2k, except that on day 7, the CD45.1+Tetramer+CD8+ T cells were analyzed by flow cytometry. o, The gating strategy of flow cytometry analysis on Tetramer+ CD8+ T cells. p, CD8+ Tn (IL-7), Teff (IL-2) or Tm (IL-15) cells were cultured with 15N-glutamine (2 mM). the incorporation of NH3 (m + 1), N-acetylgutamate were analyzed by LC-MS/MS. In a-i and p, n = 3 independent experiments; n, n = 5 mice. Two-tailed unpaired Student’s t-tests(a, g-l and n),or one-way ANOVA followed by Bonferroni’s test (f and p). The data represent mean ± s.d.

Source data

Extended Data Fig. 3 CD8+ Tm cells mobilize arginase 2 for urea formation.

a, Western blot analysis of the expression of ARG1 in CD8+ Tn, Teff and Tm cells from mice or IL-2-derived Teff cells and IL-15 derived Tm cells, M2 type macrophages was as a positive control. b, The knockdown efficiency of shArg2 in IL-15-derived Tm cells. c, C57BL/6 mice were transferred with CD45.1+CD8+ OT-I T cells and infected with Lm-OVA. 7 days later, the mice were injected with nor-NOHA (10 mg/kg) or control saline once per day. On day 30, the CD45.1+CD8+ T cells in spleen were sorted, and then the ammonia and urea were detected. the level of ammonia and urea in GFP+CD8+ Tm cells from spleen were analyzed. d, The gating strategy of flow cytometry analysis on CD45.1+CD8+ T cells. e, The gating strategy of flow cytometry analysis on GFP+CD8+ T cells. f, C57BL/6 mice were transferred with GFP-shArg2 transduced CD8+ OT-I T cells, and then infected with Lm-OVA. On day 30, the GFP+CD8+ T cells in the LNs, PBMCs and spleen were analyzed by flow cytometry. In a and b,n = 3 independent experiments; c and f, n = 5 mice. Two-tailed unpaired Student’s t-tests followed by Bonferroni’s test (b and c). The data represent mean ± s.d.

Source data

Extended Data Fig. 4 SLC25A29 and SLC14A1 are utilized for urea cycle.

a, The knockdown efficiency of shSlc25a29 in IL-15-derived Tm cells. b, The knockdown efficiency of shSlc14a1 in IL-15-derived Tm cells. In a and b, n = 3 independent experiments. Two-tailed unpaired Student’s t-tests followed by Bonferroni’s test (a and b).The data represent mean ± s.d.

Source data

Extended Data Fig. 5 CD8+ Tm cells dispose ammonia by citrulline cycle.

a, The knockdown efficiency of shnNOS, shiNOS and sheNOS in IL-15-derived Tm cells. b, IL-15-derived CD8+ Tm treat with 100 μM LNMMA for 48 hours, then cultured with 15N-NH4Cl for 24 hours and LC-MS/MS analysis was performed for m + 1 citrulline, m + 1 argininosuccinate, m + 1 arginine and m + 1 urea. c, d, IL-15-derived CD8+ Tm were treated with 10 μM nor-NOHA (c) or 100 μM L-NMMA (d) for 48 hours, then were cultured with [U4] 15N-arginine for 6 hours and LC-MS/MS analysis was performed for m + 2, m + 3 citrulline, m + 2 ornithine and m + 2 urea. In a-d, n = 3 independent experiments. Two-tailed unpaired Student’s t-tests followed by Bonferroni’s test (a-d). The data represent mean ± s.d.

Source data

Extended Data Fig. 6 Cps1 is crucial for nitrogen disposal and memory development.

a, The gating strategy of flow cytometry analysis on GFP+CD8+ T cells. b,.The same as Fig. 6a, except that on day 7, CD8+GFP+ T cells were analyzed. c, The same as Fig. 6a, except that On day 30, Annexin V, Ki-67 and BrdU in spleen CD8+GFP+ Tm cells were analyzed. d, C57BL/6 mice were co-transferred with 1×104 mCherry-NC and 1×104 GFP-OE-Cps1 transduced CD8+ OT-I T cells, and then infected with Lm-OVA. On day 30, the mCherry+CD8+(NC) and GFP+CD8+(OE-Cps1) T cells in the LNs, PBMCs and spleen were analyzed by flow cytometry. e, 1×105 GFP-shNC or GFP-shCps1 CD45.1+ OT-I Tm cells were adoptively transferred into CD45.2+ mice (left), 1×105 GFP-NC or GFP-OE-Cps1 CD45.1+ OT-I Tm cells were adoptively transferred into CD45.2+ mice (right), On day 30, the CD8+CD45.1+GFP+ T cells in the spleen were analyzed by flow cytometry. f, The gating strategy of flow cytometry analysis on CD45.1+CD8+ T cells. g, Mice were transferred with 1×105 OT-l T cells transduced with shCps1, 30 days later; the percentage of cytokine-producing CD8+ OT-l cells were analyzed 4 h after Lm-OVA reinfection. h, The same as g, except that mice were transferred with 1×105 OT-l T cells transduced with OE-Cps1. i, 1×105 GFP-shNC or GFP-shCps1 CD45.1+ OT-I Tm cells were adoptively transferred into naïve mice followed by infection with Lm-OVA, 5 days after infection, the percentage and cell numbers of CD8+ OT-I T cells are analyzed. j, The same as i, except that 1×105 NC or OE-Cps1 CD45.1+ OT-I Tm cells were adoptively transferred into naive mice followed by infection with Lm-OVA. k, 1×105 shNC or shCps1 CD45.1+ OT-I Tm cells were adoptively transferred into naïve mice(left), or 1×105 NC or OE-Cps1 CD45.1+ OT-I Tm cells were adoptively transferred into naive C57BL/6 mice (right), followed by infection with Lm-OVA, 5 days after infection, the percentage of cytokine-producing CD8+ OT-I T cells were analyzed. In a-k, n = 5 mice. Two-tailed unpaired Student’s t-tests followed by Bonferroni’s test (b,e and g-k). The data represent mean ± s.d.

Source data

Extended Data Fig. 7 β-hydroxybutyrylation of Cps1 triggers CP metabolic cascade.

a, The knockdown efficiency of shBdh1 in IL-15-derived Tm cells. b, IL-15-derived Tm cells transduced with shBdh1, the expression of Cps1 was analyzed by qPCR. In a and b, n = 3 independent experiments. Two-tailed unpaired Student’s t-tests followed by Bonferroni’s test (a and b). The data represent mean ± s.d.

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Supplementary information

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Supplementary Tables 1–4

Reagent information, sequences of shRNA information, antibody information and sequences of qPCR primer information.

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Tang, K., Zhang, H., Deng, J. et al. Ammonia detoxification promotes CD8+ T cell memory development by urea and citrulline cycles. Nat Immunol 24, 162–173 (2023). https://doi.org/10.1038/s41590-022-01365-1

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