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ECSIT facilitates memory CD8+ T cell development by mediating fumarate synthesis during viral infection and tumorigenesis

Nature Cell Biology volume 26, pages 450–463 (2024)Cite this article

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Abstract

Memory CD8+ T cells play a crucial role in infection and cancer and mount rapid responses to repeat antigen exposure. Although memory cell transcriptional programmes have been previously identified, the regulatory mechanisms that control the formation of CD8+ T cells have not been resolved. Here we report ECSIT as an essential mediator of memory CD8+ T cell differentiation. Ablation of ECSIT in T cells resulted in loss of fumarate synthesis and abrogated TCF-1 expression via demethylation of the TCF-1 promoter by the histone demethylase KDM5, thereby impairing memory CD8+ T cell development in a cell-intrinsic manner. In addition, ECSIT expression correlated positively with stem-like memory progenitor exhausted CD8+ T cells and the survival of patients with cancer. Our study demonstrates that ECSIT-mediated fumarate synthesis stimulates TCF-1 activity and memory CD8+ T cell development during viral infection and tumorigenesis and highlights the utility of therapeutic fumarate analogues and PD-L1 inhibition for tumour immunotherapy.

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Fig. 1: ECSIT is upregulated in memory CD8+ T cells and promotes memory CD8+ T cell generation in a cell-intrinsic manner under homeostasis.
Fig. 2: ECSIT deficiency impairs memory CD8+ T cell differentiation in acute viral infection.
Fig. 3: ECSIT deficiency impairs memory CD8+ T cell differentiation by inhibiting the transcription level and regulation of TCF-1 in acute viral infection.
Fig. 4: ECSIT deficiency inhibits memory CD8+ T cell differentiation by reducing fumarate production.
Fig. 5: ECSIT deficiency impairs the formation of stem cell-like memory T cell progenitors and the antitumour function of CD8+ T cells.
Fig. 6: ECSIT deficiency impairs the differentiation of stem-like memory CD8+ T cells by inhibiting the transcriptional level and regulation of TCF-1 in chronic tumour stimulation.
Fig. 7: ECSIT deficiency inhibits the differentiation of stem cell-like memory CD8+ T cells by reducing fumarate production in chronic tumour stimulation.
Fig. 8: ECSIT expression is positively correlated with stem cell-like memory progenitors of CD8+ T cells, antitumour immunity and the survival of patients with cancer.

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Data availability

RNA-seq, ATAC-seq and ChIP–seq data that support the findings of this study have been deposited in the NCBI Sequence Read Archive database under the NCBI Bioproject accession codes PRJNA905206, PRJNA902096, PRJNA905582, PRJNA904827 and PRJNA1016360. Source data are provided with this paper.

References

  1. Kaech, S. M. & Ahmed, R. Memory CD8+ T cell differentiation: initial antigen encounter triggers a developmental program in naive cells. Nat. Immunol. 2, 415–422 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Jameson, S. C. & Masopust, D. Diversity in T cell memory: an embarrassment of riches. Immunity 31, 859–871 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Chen, Y., Zander, R., Khatun, A., Schauder, D. M. & Cui, W. Transcriptional and epigenetic regulation of effector and memory CD8 T cell differentiation. Front. Immunol. 9, 2826 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  4. McLane, L. M., Abdel-Hakeem, M. S. & Wherry, E. J. CD8 T cell exhaustion during chronic viral infection and cancer. Annu. Rev. Immunol. 37, 457–495 (2019).

    Article  CAS  PubMed  Google Scholar 

  5. Joshi, N. S. et al. Inflammation directs memory precursor and short-lived effector CD8+ T cell fates via the graded expression of T-bet transcription factor. Immunity 27, 281–295 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Martin, M. D. & Badovinac, V. P. Defining memory CD8 T cell. Front. Immunol. 9, 2692 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Hashimoto, M. et al. CD8 T cell exhaustion in chronic infection and cancer: opportunities for interventions. Annu. Rev. Med. 69, 301–318 (2018).

    Article  CAS  PubMed  Google Scholar 

  8. Reading, J. L. et al. The function and dysfunction of memory CD8+ T cells in tumor immunity. Immunol. Rev. 283, 194–212 (2018).

    Article  CAS  PubMed  Google Scholar 

  9. Siddiqui, I. et al. Intratumoral Tcf1+ PD-1+ CD8+ T cells with stem-like properties promote tumor control in response to vaccination and checkpoint blockade immunotherapy. Immunity 50, 195–211 (2019).

    Article  CAS  PubMed  Google Scholar 

  10. Gattinoni, L., Speiser, D. E., Lichterfeld, M. & Bonini, C. T memory stem cells in health and disease. Nat. Med. 23, 18–27 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Zhou, X. et al. Differentiation and persistence of memory CD8+ T cells depend on T cell factor 1. Immunity 33, 229–240 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Ichii, H. et al. Role for Bcl-6 in the generation and maintenance of memory CD8+ T cells. Nat. Immunol. 3, 558–563 (2002).

    Article  CAS  PubMed  Google Scholar 

  13. Yang, C. Y. et al. The transcriptional regulators Id2 and Id3 control the formation of distinct memory CD8+ T cell subsets. Nat. Immunol. 12, 1221–1229 (2011).

    Article  CAS  PubMed  Google Scholar 

  14. Rutishauser, R. L. et al. Transcriptional repressor Blimp-1 promotes CD8+ T cell terminal differentiation and represses the acquisition of central memory T cell properties. Immunity 31, 296–308 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Xin, A. et al. A molecular threshold for effector CD8+ T cell differentiation controlled by transcription factors Blimp-1 and T-bet. Nat. Immunol. 17, 422–432 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Chen, J. et al. NR4A transcription factors limit CAR T cell function in solid tumours. Nature 567, 530–534 (2019).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  17. Khan, O. et al. TOX transcriptionally and epigenetically programs CD8+ T cell exhaustion. Nature 571, 211–218 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Escobar, G., Mangani, D. & Anderson, A. C. T cell factor 1: a master regulator of the T cell response in disease. Sci. Immunol. 5, eabb9726 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Xiao, C. et al. Ecsit is required for Bmp signaling and mesoderm formation during mouse embryogenesis. Genes Dev. 17, 2933–2949 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Wi, S. M. et al. TAK1–ECSIT–TRAF6 complex plays a key role in the TLR4 signal to activate NF-κB. J. Biol. Chem. 289, 35205–35214 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Wen, H. et al. Recurrent ECSIT mutation encoding V140A triggers hyperinflammation and promotes hemophagocytic syndrome in extranodal NK/T cell lymphoma. Nat. Med. 24, 154–164 (2018).

    Article  CAS  PubMed  Google Scholar 

  22. West, A. P. et al. TLR signalling augments macrophage bactericidal activity through mitochondrial ROS. Nature 472, 476–480 (2011).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  23. Carneiro, F. R., Lepelley, A., Seeley, J. J., Hayden, M. S. & Ghosh, S. An essential role for ECSIT in mitochondrial complex I assembly and mitophagy in macrophages. Cell Rep. 22, 2654–2666 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Xu, L. et al. ECSIT is a critical limiting factor for cardiac function. JCI Insight 6, e142801 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Yang, S. et al. ECSIT is a critical factor for controlling intestinal homeostasis and tumorigenesis through regulating the translation of YAP protein. Adv. Sci. 10, e2205180 (2023).

    Article  Google Scholar 

  26. Zhang, N. & Bevan, M. J. TGF-β signaling to T cells inhibits autoimmunity during lymphopenia-driven proliferation. Nat. Immunol. 13, 667–673 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Hogquist, K. A. et al. T cell receptor antagonist peptides induce positive selection. Cell 76, 17–27 (1994).

    Article  CAS  PubMed  Google Scholar 

  28. Delpoux, A., Lai, C.-Y., Hedrick, S. M. & Doedens, A. L. FOXO1 opposition of CD8+ T cell effector programming confers early memory properties and phenotypic diversity. Proc. Natl Acad. Sci. USA 114, E8865–E8874 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Arts, R. J. et al. Glutaminolysis and fumarate accumulation integrate immunometabolic and epigenetic programs in trained immunity. Cell Metab. 24, 807–819 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Kornberg, M. D. et al. Dimethyl fumarate targets GAPDH and aerobic glycolysis to modulate immunity. Science 360, 449–453 (2018).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  31. Gattinoni, L. et al. A human memory T cell subset with stem cell-like properties. Nat. Med. 17, 1290–1297 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Thommen, D. S. & Schumacher, T. N. T cell dysfunction in cancer. Cancer Cell 33, 547–562 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Zhu, L. et al. Dapl1 controls NFATc2 activation to regulate CD8+ T cell exhaustion and responses in chronic infection and cancer. Nat. Cell Biol. 24, 1165–1176 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Kaech, S. M. & Cui, W. Transcriptional control of effector and memory CD8+ T cell differentiation. Nat. Rev. Immunol. 12, 749–761 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Ryan, D. G. et al. Coupling Krebs cycle metabolites to signalling in immunity and cancer. Nat. Metab. 1, 16–33 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Levicán, G., Ugalde, J. A., Ehrenfeld, N., Maass, A. & Parada, P. Comparative genomic analysis of carbon and nitrogen assimilation mechanisms in three indigenous bioleaching bacteria: predictions and validations. BMC Genomics 9, 581 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  37. de Castro Fonseca, M., Aguiar, C. J., da Rocha Franco, J. A., Gingold, R. N. & Leite, M. F. GPR91: expanding the frontiers of Krebs cycle intermediates. Cell Commun. Signal. 14, 3 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Keshet, R., Szlosarek, P., Carracedo, A. & Erez, A. Rewiring urea cycle metabolism in cancer to support anabolism. Nat. Rev. Cancer 18, 634–645 (2018).

    Article  CAS  PubMed  Google Scholar 

  39. Nguyen, B. D. et al. Import of aspartate and malate by DcuABC drives H2/fumarate respiration to promote initial Salmonella gut-lumen colonization in mice. Cell Host Microbe 27, 922–936 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Kaufman, S. A model of human phenylalanine metabolism in normal subjects and in phenylketonuric patients. Proc. Natl Acad. Sci. USA 96, 3160–3164 (1999).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  41. King, A., Selak, M. & Gottlieb, E. Succinate dehydrogenase and fumarate hydratase: linking mitochondrial dysfunction and cancer. Oncogene 25, 4675–4682 (2006).

    Article  CAS  PubMed  Google Scholar 

  42. Cohen, N. S. & Kuda, A. Argininosuccinate synthetase and argininosuccinate lyase are localized around mitochondria: an immunocytochemical study. J. Cell. Biochem. 60, 334–340 (1996).

    Article  CAS  PubMed  Google Scholar 

  43. Bergeron, A., D’Astous, M., Timm, D. E. & Tanguay, R. M. Structural and functional analysis of missense mutations in fumarylacetoacetate hydrolase, the gene deficient in hereditary tyrosinemia type 1. J. Biol. Chem. 276, 15225–15231 (2001).

    Article  CAS  PubMed  Google Scholar 

  44. Taha-Mehlitz, S. et al. Adenylosuccinate lyase is oncogenic in colorectal cancer by causing mitochondrial dysfunction and independent activation of NRF2 and mTOR-MYC-axis. Theranostics 11, 4011–4029 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Shcherbakova, V., Fechina, V. & Iakovleva, V. Isolation of spheroplasts from Escherichia coli 85 for aspartate-ammonia-lyase localization (in Russian). Prikladnaia Biokhimiia i Mikrobiologiia 24, 400–404 (1988).

    CAS  PubMed  Google Scholar 

  46. He, B. et al. CD8+ T cells utilize highly dynamic enhancer repertoires and regulatory circuitry in response to infections. Immunity 45, 1341–1354 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Zhai, X. et al. Mitochondrial C1qbp promotes differentiation of effector CD8+ T cells via metabolic-epigenetic reprogramming. Sci. Adv. 7, eabk0490 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  48. Spencer, C. M., Crabtree-Hartman, E. C., Lehmann-Horn, K., Cree, B. A. & Zamvil, S. S. Reduction of CD8+ T lymphocytes in multiple sclerosis patients treated with dimethyl fumarate. Neurol. Neuroimmunol. Neuroinflammation 2, e76 (2015).

    Article  Google Scholar 

  49. Garcia, J. et al. Progressive multifocal leukoencephalopathy on dimethyl fumarate with preserved lymphocyte count but deep T-cells exhaustion. Mult. Scler. 27, 640–644 (2021).

    Article  CAS  PubMed  Google Scholar 

  50. Jiang, Y. et al. Gasdermin D restricts anti-tumor immunity during PD-L1 checkpoint blockade. Cell Rep. 41, 111553 (2022).

    Article  CAS  PubMed  Google Scholar 

  51. Milner, J. J. et al. Delineation of a molecularly distinct terminally differentiated memory CD8 T cell population. Proc. Natl Acad. Sci. USA 117, 25667–25678 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  52. Im, S. J. et al. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Nature 537, 417–421 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  53. Miller, B. C. et al. Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade. Nat. Immunol. 20, 326–336 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Sade-Feldman, M. et al. Defining T cell states associated with response to checkpoint immunotherapy in melanoma. Cell 175, 998–1013 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. De Biasi, S. et al. Circulating mucosal-associated invariant T cells identify patients responding to anti-PD-1 therapy. Nat. Commun. 12, 1669 (2021).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  56. Shan, Q. et al. Tcf1 preprograms the mobilization of glycolysis in central memory CD8+ T cells during recall responses. Nat. Immunol. 23, 386–398 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Shan, Q. et al. Ectopic Tcf1 expression instills a stem-like program in exhausted CD8+ T cells to enhance viral and tumor immunity. Cell. Mol. Immunol. 18, 1262–1277 (2021).

    Article  CAS  PubMed  Google Scholar 

  58. Pritykin, Y. et al. A unified atlas of CD8 T cell dysfunctional states in cancer and infection. Mol. Cell 81, 2477–2493 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Kurtulus, S. et al. Checkpoint blockade immunotherapy induces dynamic changes in PD-1− CD8+ tumor-infiltrating T cells. Immunity 50, 181–194 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Wherry, E. J. et al. Molecular signature of CD8+ T cell exhaustion during chronic viral infection. Immunity 27, 670–684 (2007).

    Article  CAS  PubMed  Google Scholar 

  61. Jolma, A. et al. Multiplexed massively parallel SELEX for characterization of human transcription factor binding specificities. Genome Res. 20, 861–873 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank L. Lu (Zhejiang University, Hangzhou, China), X. Zhang (Huazhong University of Science and Technology, Wuhan, China), H. Wang (ShanghaiTech University, Shanghai, China), X. Wang (Nanjing Medical University, Nanjing, China) and Y. Xiao (Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences) for providing dLck-Cre, Rosa26CreERT2, OT-1, CD45.1 and Rag1−/− mice respectively. We thank C. Xu (Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences) for providing B16F10 and B16F10-OVA cells. We also thank C. Y. Yang (Sun Yat-sen University, Zhongshan School of Medicine, Guangzhou, China) for providing VSV-OVA and LM-OVA. This work was supported by the National Key Research and Development Program of China (grant number 2022YFA1303900 to S.Y.), National Natural Science Foundation of China (grant numbers 32270921 and 82070567 to S.Y., 32170742 to X.W. and 82204354 to Y.H.), the Start Fund for Specially Appointed Professor of Jiangsu Province (S.Y. and X.W.), the Open Project of State Key Laboratory of Reproductive Medicine of Nanjing Medical University (grant number SKLRM-2021B3 to S.Y.), the talent cultivation project of ‘Organized scientific research’ of Nanjing Medical University (grant number NJMURC20220014 to S.Y.), the Start Fund for High-level Talents of Nanjing Medical University (grant number NMUR2020009 to X.W.), the Open Project of Chinese Materia Medica First-Class Discipline of Nanjing University of Chinese Medicine (grant number 2020YLXK017 to B.W.), the Priority Academic Program Development of Jiangsu Higher Education Institutions (to B.W.), the Natural Science Foundation of Jiangsu Province (grant number BK20221352 to B.W.), the Jiangsu Provincial Outstanding Postdoctoral Program (grant number 2022ZB419 to Y.H.) and the Postdoctoral Research Funding Project of Gusu School (grant number GSBSHKY202104 to Y.H.). F.H. is supported by a Charles Hood Child Health Grant.

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Author notes

  1. These authors contributed equally: Yongbing Yang, Yanan Wang.

Authors and Affiliations

  1. Department of Immunology, State Key Laboratory of Reproductive Medicine, Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Collaborative Innovation Center for Personalized Cancer Medicine, Gusu School, the Affiliated Wuxi People’s Hospital of Nanjing Medical University, Wuxi People’s Hospital, Wuxi Medical Center, National Vaccine Innovation Platform, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, China

    Yongbing Yang, Yanan Wang, Yi Gong, Yingchao Hu, Yuying Jiang, Shuang Wen & Shuo Yang

  2. Department of Medical Laboratory, Affiliated Children’s Hospital of Jiangnan University, Wuxi, China

    Yongbing Yang

  3. School of Pharmacy, Nanjing Medical University, Nanjing, China

    Zhongcheng Wang, Feifei Xu & Yun Chen

  4. State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, China

    Huanyu Yan & Xi Wang

  5. Department of Pharmacology, Nanjing University of Chinese Medicine, Nanjing, China

    Bingwei Wang

  6. Division of Innate Immunity, Department of Medicine, UMass Chan Medical School, Worcester, MA, USA

    Fiachra Humphries

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Contributions

Y.Y., Y.W., Z.W., H.Y., Y.G., Y.H., Y.J., S.W. and F.X. designed and performed the experiments, analysed the data and prepared the figures. F.H., Y.C. and X.W. provided the key technique mentoring, data analysis and research resources. B.W. and F.H. contributed to the experimental design and edited the manuscript. S.Y. supervised the project. Y.Y., Y.W. and S.Y. wrote the manuscript.

Corresponding authors

Correspondence to Fiachra Humphries, Yun Chen, Xi Wang or Shuo Yang.

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Extended data

Extended Data Fig. 1 ECSIT is upregulated in memory CD8+ T cells, and promotes memory CD8+ T cells generation in a cell-intrinsic manner under the homeostasis.

(a) Representative flow cytometry plots of Ecsit–EGFP expression in immune cells in Fig. 1d. (b) Representative flow cytometry plots of Ecsit–EGFP expression in TN, TCM, and TEM of CD8+ T cells in Fig. 1e. (c) Representative flow cytometry plots of Ecsit–EGFP expression in MPEC and SLEC of CD8+ T cells in Fig. 1f. (d) Representative flow cytometry plots of Ecsit–EGFP expression in TEM and TCM of CD8+ T cells in Fig. 1g. (e) Representative flow cytometry plots of Ecsit–EGFP expression in TSCMP and TEXH of CD8+ T cells in Fig. 1h. (f) CD45.1 mice bone marrows were mixed equally with Ecsitfl/fl or Ecsitfl/fl dLck-Cre (CD45.2) mice bone marrows then injected intravenously into irradiated Rag1−/− mice. 8 weeks later, draining lymph nodes were collected and analysed by flow cytometry for CD4+ T cells, CD8+ T cells, and CD8+ TN, TM cells. Data are pooled from three independent experiments and n = 6 for (f). Error bars show mean ± sem. Two-tailed unpaired student’s t-test for (f).

Source data

Extended Data Fig. 2 ECSIT promotes the recall response of memory CD8+ T cells.

(a) Schematic of memory CD8+ T cells recall response. Naive CD8+ T cells obtained from Ecsitfl/fl OT-1 or Ecsitfl/fl dLck-Cre OT-1 mice were transferred into WT recipient mice. Subsequently, the recipient mice were infected with VSV-OVA 1 day later. After 35 days, memory OT-1 T cells were sorted and transferred into naive WT recipient mice, followed by LM-OVA challenge. Spleen and liver were collected for analyses 5 days later. (b, e) Representative flow cytometry plots (top) and the analysis of percentage and number (bottom) of Kb-OVA+ CD44+ CD8+ cells in the spleen (b) and liver (e). (c, d, f, g) Representative flow cytometry plots (top) and the analysis of percentage and number (bottom) of IFN-γ+ (c) and TNF-α+ (d) T cells gated on Kb-OVA+ CD44+ CD8+ cells in the spleen, and IFN-γ+ (f) and TNF-α+ (g) T cells gated on Kb-OVA+ CD44+ CD8+ cells in the liver. (h, i) Bacterial load of LM-OVA in the spleen (h) and liver (i). (j) Schematic of memory T cells formation in acute infection. Naive CD8+ T cells from Ecsitfl/fl OT-1 or Ecsitfl/fl Rosa26CreERT2 OT-1 mice were transferred into WT recipient mice, followed by VSV-OVA infection 1 day later. Tamoxifen was intraperitoneally injected from day21 to day25. OT-1 memory cells were analysed on day35. (k) Schematic of memory T cells formation in acute infection with adaptive co-transplantation model. Naive CD8+ T cells from Ecsitfl/+ CD45.1.2 OT-1 and Ecsitfl/fl Rosa26CreERT2 CD45.2 OT-1 mice were mixed equally and transferred into CD45.1 recipient mice, and then the recipient mice were infected with VSV-OVA 1 day later. Tamoxifen was intraperitoneally injected from day21 to day25. OT-1 memory cells were analysed on day35. Data are pooled from three independent experiments and n = 5 for (b-i). Error bars show mean ± sem. Two-tailed unpaired student’s t-test for (b-i).

Source data

Extended Data Fig. 3 ECSIT deficiency impairs memory CD8+ T cells formation and function without affecting proliferation and apoptosis during acute viral infection.

(a) Immunoblot analysis of ECSIT expression in Ecsitfl/fl OT-1 and Ecsitfl/fl Rosa26CreERT2 OT-1 cells 35 days after VSV-OVA infection. (b) Representative flow cytometry plots (top) and the analysis of percentage and number (bottom) of Kb-OVA+ CD44+ CD8+ cells in the liver. (c, d) Representative flow cytometry plots and the analysis of percentage and number of IFN-γ+ (c) and TNF-α+ (d) T cells gated on Kb-OVA+ CD44+ CD8+ cells in the liver. (e, f) Representative flow cytometry plots (left) and the analysis of percentage and number (right) of Annexin V+ (e) and Ki-67+ (f) in Kb-OVA+ CD44+ CD8+ cells in the spleen. (g, h) Flow cytometry analysis of CD44 (g) and T-BET (h) expression in Kb-OVA+ CD44+ CD8+ cells in the liver. (i) Representative flow cytometry plots (top) and the analysis of percentage and number (bottom) of Ecsitfl/+ CD45.1.2 OT-1 and Ecsitfl/fl Rosa26CreERT2 CD45.2 OT-1 cells gated on Kb-OVA+ CD44+ CD8+ cells in the spleen. (j, k) Representative flow cytometry plots (top) and the analysis of percentage and number (bottom) of Ecsitfl/+ CD45.1.2 OT-1 and Ecsitfl/fl Rosa26CreERT2 CD45.2 OT-1 cells gated on Kb-OVA+ CD44+ CD8+ IFN-γ+ (j) and TNF-α+ (k) T cells in the liver. Data are pooled from three independent experiments for (a-k), and n = 5 for (b-k). Error bars show mean ± sem. Two-tailed unpaired student’s t-test for (b-k).

Source data

Extended Data Fig. 4 ECSIT affects chromatin accessibility of memory CD8+ T cells and promotes transcription regulation of Tcf7 during acute viral infection.

(a) Flow cytometry gating strategy of sorting Ecsitfl/+ CD45.1.2 OT-1 and Ecsitfl/fl Rosa26CreERT2 CD45.2 OT-1 cells (memory) from CD45.1 recipient mice 35 days after VSV-OVA infection for RNA-seq and ATAC-seq. (b) Venn diagrams of differentially and commonly expressed genes between Ecsitfl/+ CD45.1.2 OT-1 and Ecsitfl/fl Rosa26CreERT2 CD45.2 OT-1 cells. (c) Pie charts of the genomic distribution of differentially and commonly accessible chromatin regions between Ecsitfl/+ CD45.1.2 OT-1 and Ecsitfl/fl Rosa26CreERT2 CD45.2 OT-1 cells. (d) GO and KEGG analysis of differentially accessible chromatin region genes. (e) Genome track view of the Ccr7 and Slamf6 locus showing ATAC-seq and Tcf7 ChIP–seq peaks (GSE177064). Tcf7 predictive binding sites (red shadows) are listed. One-side Fisher’s Exact test for (b, d).

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Extended Data Fig. 5 TCF-1 overexpression promotes memory T cells formation in ECSIT deficiency CD8+ T cells in vitro.

(a, b) Ecsitfl/fl OT-1 (WT) and Ecsitfl/fl Rosa26CreERT2 OT-1 (iKO) splenocytes were activated with OVA and IL-2 for 2 days, followed by sorting of CD8+ T cells, then CD8+ T cells were transduced with vector (EV) or TCF-1 (TCF-1 OE) by the retroviral system and cultured in IL-15 for 3 days to induce IL-15-mediated differentiation of memory T cells in the presence of 4−OHT. (a) Flow cytometry gating strategy of TCF-1 overexpression CD8+ T cells. (b) Immunoblot analysis of ECSIT expression in WT + EV, WT + TCF-1 OE, iKO+EV and iKO+TCF-1 OE cells. (c) Schematic of the experiments. Ecsitfl/+ CD45.1.2 OT-1(WT) and Ecsitfl/fl Rosa26CreERT2 CD45.2 OT-1(iKO) mice splenocytes were activated with OVA and IL-2 for 2 days, followed by sorting of CD8+T cells, then CD8+T cells were transduced with vector (EV) or TCF-1 (TCF-1 OE) by the retroviral system and cultured in IL-15 for 3 days to induce IL-15-mediated differentiation of memory T cells in the presence of 4-OHT. WT and iKO memory CD8+T (GFP+) were mixed at a 1:1 ratio and transferred into CD45.1 recipient mice, and then the recipient mice were infected with VSV-OVA 1 day later. OT-1 cells were analysed on day 5 after infection in the spleen. (d, e) Representative flow cytometry plots (left) and the analysis of percentage (right) of OT-1 cells (GFP+ Kb-OVA+ CD44+ CD8+ T) (d) and IFN-γ+ cells (e) gated on GFP+ Kb-OVA+ CD44+ CD8+ T cells in the spleen. Data are pooled from two independent experiments and n = 4 for (d-e). Error bars show mean ± sem. ***P ≤ 0.001, ns, not significant. Two-tailed unpaired student’s t-test for (d-e).

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Extended Data Fig. 6 ECSIT deficiency dysregulates mitochondrial metabolism and inhibits fumarate production.

(a-d) Ecsitfl/fl OT-1 (WT) and Ecsitfl/fl Rosa26CreERT2 OT-1 (iKO) splenocytes were activated with OVA and IL-2 for 2 days, followed by sorting of CD8+ T cells, then cultured in IL-15 for 3 days to induce IL-15-mediated differentiation of memory T cells in the presence of 4-OHT. Memory T cells were collected for metabolite detection. (a) Schematic of memory CD8+ T cells induced in vitro. (b) PCA analysis of metabolomics data. (c) Pathway enrichment analysis of changed metabolites. (d) Heat map of relative abundance of significantly changed metabolites. (e) GSEA analysis of oxidative Phosphorylation genes (Hallmark) with RNA-seq data. (f,g,k,l) Ecsitfl/fl OT-1 and Ecsitfl/fl dLck-Cre OT-1 (CKO) splenocytes were activated with OVA and IL-2 for 2 days, followed by sorting of CD8+ T cells, then CD8+ T cells were cultured in IL-15 for 3 days to induce IL-15-mediated differentiation of memory T cells. (f, g) Seahorse extracellular flux analysis of OCR; n = 4 biological replicates per group. (k) Transmission electron microscope images of mitochondria. (l) Confocal microscopy images of mitochondria. Mitochondria are red (MitoTracker Red) and nuclei are blue (Hoechst). (h) GSEA analysis of MOOTHA GLYCOLYSIS Pathway (KEGG) with RNA-seq data. (i-j) Ecsitfl/fl OT-1 and Ecsitfl/fl dLck-Cre OT-1 (CKO) splenocytes were activated with OVA and IL-2 for 2 days, followed by sorting of CD8+ T cells, then CD8+ T cells were cultured in IL-15 for 3 days to induce IL-15-mediated differentiation of memory T cells. Memory T cells were collected for Seahorse extracellular flux analysis of ECAR. Data are pooled from three independent experiments for (f, g, i-l), and n = 4 for (f, g) or 5 for (I, j) Error bars show mean ± sem. One-side Fisher’s Exact test for (c). Two-tailed unpaired student’s t-test for (g, j).

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Extended Data Fig. 7 DMF supplement significantly rescue H3K4me3 modification and H3K4me3 level at the Tcf7 promoters of ECSIT-deficient memory CD8+ T cells.

(a) Immunoblot analysis of H3K4me3 expression in WT + NT, WT + DMF, iKO+NT and iKO + DMF memory T cells induced in vitro. (b) Comparison of entire H3K4me3 ChIP–seq peaks in WT + NT, WT + DMF, iKO + NT and iKO + DMF memory T cells induced in vitro. (c) Genome track view of the Tcf7 locus showing H3K4me3 ChIP–seq peaks in WT + NT, WT + DMF, iKO + NT and iKO + DMF memory T cells induced in vitro. H3K4me3 predictive binding sites (red shadows) are listed. (d) ChIP–qPCR analysis of H3K4me3 levels at the promoters of Tcf7 in WT + NT, WT + DMF, iKO + NT and iKO + DMF memory T cells induced in vitro; n = 4 biological replicates per group. (e) Schematic of memory T cells formation with DMF supplement in acute infection. The equal number of OT-1 naive CD8+ T cells from Ecsitfl/+ CD45.1.2 OT-1 and Ecsitfl/fl Rosa26CreERT2 CD45.2 OT-1 mice were mixed and transferred into CD45.1 recipient mice, and then the recipient mice were infected with VSV-OVA 1 day later. Tamoxifen was administered every day for five treatments starting at day 21 to day 25, and DMF was also injected every other day from day 21 to day 35. OT-1 memory cells from the spleen were analysed on day 35. Data are pooled from three independent experiments for (a, d), and n = 4 for (d). Error bars show mean ± sem. Two-tailed unpaired student’s t-test for (d).

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Extended Data Fig. 8 ECSIT deficiency impairs CD8+ T cells antitumour function through cell-intrinsic mechanisms.

(a) Schematic of tumour model. WT recipient mice were implanted subcutaneously with B16F10-OVA cells, and then Ecsitfl/fl OT-1 or Ecsitfl/fl Rosa26CreERT2 OT-1 naive CD8+ T cells were transferred into recipient mice 1 day later. Tamoxifen was injected intraperitoneally 5 times from day10 to day15. The growth of B16F10-OVA melanomas and the survival of mice were assessed. And transferred OT-1 cells were analysed on day 20. (b) Schematic of tumour model with adaptive co-transplantation. CD45.1 recipient mice were implanted subcutaneously with B16F10-OVA cells, and then Ecsitfl/+ CD45.1.2 OT-1 and Ecsitfl/fl Rosa26CreERT2 CD45.2 OT-1 naive CD8+ T cells were mixed and transferred into recipient mice 1 day later. Tamoxifen was injected intraperitoneally 5 times from day10 to day15. Transferred OT-1 cells were analysed on day 20.

Extended Data Fig. 9 ECSIT affects chromatin accessibility of TME CD8+ T cells and promotes transcription regulation of Tcf7 in chronic tumour stimulation.

(a) Flow cytometry gating strategy of sorting Ecsitfl/+ CD45.1.2 OT-1 and Ecsitfl/fl Rosa26CreERT2 CD45.2 OT-1 cells (memory) from B16F10-OVA tumour-bearing CD45.1 recipient mice for RNA-seq and ATAC-seq. (b) GO and KEGG analysis of differentially accessible chromatin region genes. (c) Genome track view of the Ccr7 and Slamf6 locus showing ATAC-seq and Tcf7 CUT&RUN peaks (GSE139056). Tcf7 predictive binding sites (red shadows) are listed. One-side Fisher’s exact test for (b).

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Extended Data Fig. 10 ECSIT deficiency inhibits fumarate production of CD8+ memory T cells in chronic stimulation, and DMF supplementation improves the antitumour function of ECSIT-deficient memory CD8+ T cells in vivo.

(a-d) Ecsitfl/fl OT-1 (WT) and Ecsitfl/fl Rosa26CreERT2 OT-1 (iKO) splenocytes were activated with OVA and IL-2 for 2 days, followed by sorting of CD8+ T cells, and then sorted CD8+ T cells were cultured in IL-15 and OVA for 3 days to mimic tumour antigen chronic stimulation in the presence of 4-OHT. CD8+ T cells were collected for metabolite detection. (a) Schematic of CD8+ T cells stimulated chronically in vitro. (b) PCA analysis of metabolomics data. (c) Pathway enrichment analysis of changed metabolites. (d) Heat map of relative abundance of significantly changed metabolites. (e) Schematic of tumour model with DMF supplement. An equal number of OT-1 naive CD8+ T cells from Ecsitfl/+ CD45.1.2 OT-1 and Ecsitfl/fl Rosa26CreERT2 CD45.2 OT-1 mice were mixed and transferred into CD45.1 recipient mice that had been engrafted with B16F10-OVA melanoma cells. Tamoxifen was administered every day for five treatments starting at day 11 to day 15 and DMF was also injected every other day from day 11 to day 20. OT-1 cells from the tumour were analysed on day 20. One-side Fisher’s exact test for (c).

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Supplementary information

Supplementary Information

Supplementary Figs. 1–11 and source unprocessed images for Supplementary Figs. 1,8.

Reporting Summary

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Supplementary Table 1

Sheet 1. Metabolisms for WT and iKO memory T cells. Sheet 2. Metabolisms for WT and iKO exhausted T cells. Sheet 3. Gene set of Tcf1 target gene.

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Yang, Y., Wang, Y., Wang, Z. et al. ECSIT facilitates memory CD8+ T cell development by mediating fumarate synthesis during viral infection and tumorigenesis. Nat Cell Biol 26, 450–463 (2024). https://doi.org/10.1038/s41556-024-01351-9

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