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The kinase complex mTORC2 promotes the longevity of virus-specific memory CD4+ T cells by preventing ferroptosis

Nature Immunology volume 23, pages 303–317 (2022)Cite this article

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Abstract

Antigen-specific memory CD4+ T cells can persist and confer rapid and efficient protection from microbial reinfection. However, the mechanisms underlying the long-term maintenance of the memory CD4+ T cell pool remain largely unknown. Here, using a mouse model of acute infection with lymphocytic choriomeningitis virus (LCMV), we found that the serine/threonine kinase complex mammalian target of rapamycin complex 2 (mTORC2) is critical for the long-term persistence of virus-specific memory CD4+ T cells. The perturbation of mTORC2 signaling at memory phase led to an enormous loss of virus-specific memory CD4+ T cells by a unique form of regulated cell death (RCD), ferroptosis. Mechanistically, mTORC2 inactivation resulted in the impaired phosphorylation of downstream AKT and GSK3β kinases, which induced aberrant mitochondrial reactive oxygen species (ROS) accumulation and ensuing ferroptosis-causative lipid peroxidation in virus-specific memory CD4+ T cells; furthermore, the disruption of this signaling cascade also inhibited glutathione peroxidase 4 (GPX4), a major scavenger of lipid peroxidation. Thus, the mTORC2–AKT–GSK3β axis functions as a key signaling hub to promote the longevity of virus-specific memory CD4+ T cells by preventing ferroptosis.

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Fig. 1: mTORC2 signaling is required for the formation and maintenance of the memory CD4+ T cell pool.
Fig. 2: mTORC2 deficiency induces non-apoptotic cell death in virus-specific memory CD4+ T cells.
Fig. 3: mTORC2 deficiency induces ferroptosis in memory CD4+ T cells.
Fig. 4: Overexpression of GPX4 restricts ferroptosis in mTORC2-deficient memory CD4+ T cells.
Fig. 5: mTORC2 deficiency induces aberrant mitochondrial ROS production in memory CD4+ T cells.
Fig. 6: mTORC2–p-AKTSer 473–p-GSK3βSer 9 signaling regulates HK2–VDAC interaction to orchestrate mitochondrial ROS generation.
Fig. 7: The mTORC2 kinase supports GPX4 peroxidase activity via p-AKTSer 473–p-GSK3βSer 9-mediated NRF2 nuclear translocation.
Fig. 8: Constitutively inactive GSK3β-S9D restores memory CD4+ T cells from mTORC2 deficiency-induced ferroptosis.

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Data availability

The microarray raw data are deposited and available from the GEO database under the accession number GSE151800. All other data supporting the findings are available in this paper or from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank R. Ahmed (Emory University) for SMARTA transgenic mice and retroviral vectors. We thank CapitalBio Corporation (Beijing, China) for performing microarray experiments and data analysis, and we thank the core facility center of Third Military Medical University for cell sorting and TEM. This study was supported by grants from the National Key Research Development Plan (SQ2021YFC2300059 to L.Y.), the National Natural Science Fund for Distinguished Young Scholars (31825011 to L.Y.), the National Natural Science Foundation of China (32030041 to L.Y, 31800748 to Y.W., 31800742 to Q.T. and 31900642 to Y.H.), the China Postdoctoral Science Foundation (2019M663389 to Y.W.) and Chongqing Special Postdoctoral Science Foundation (XmT2018021 to Q.T.).

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Author notes

  1. These authors contributed equally: Yifei Wang, Qin Tian, Yaxing Hao.

Authors and Affiliations

  1. Guangdong Province Key Laboratory of Immune Regulation and Immunotherapy, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, Guangdong, China

    Yifei Wang, Jinjin Lu, Xiangyu Chen & Qizhao Huang

  2. The First Affiliated Hospital of Jinan University, The Biomedical Translational Research Institute, Jinan University, Guangzhou, Guangdong, China

    Yifei Wang

  3. Institute of Immunology, PLA, Third Military Medical University, Chongqing, China

    Qin Tian, Yaxing Hao, Wei Yao, Jinjin Lu, Cheng Chen, Yao Lin, Lifan Xu, Jianjun Hu, Shun Lei, Zhengping Wei, Yuan Luo, Zhirong Li, Li Hu, Jianfang Tang, Qing Wu, Xinyuan Zhou, Yuzhang Wu & Lilin Ye

  4. Cancer Center, The General Hospital of Western Theater Command, Chengdu, China

    Qizhao Huang

  5. Guangdong Provincial Key Laboratory of Tumor Interventional Diagnosis and Treatment, Zhuhai Institute of Translational Medicine Zhuhai People’s Hospital Affiliated with Jinan University, Jinan University, Zhuhai, Guangdong, China

    Zhinan Yin

  6. The Biomedical Translational Research Institute, Faculty of Medical Science, Jinan University, Guangzhou, Guangdong, China

    Zhinan Yin

  7. Shanghai Key Laboratory of Organ Transplantation, Zhongshan Hospital & Institutes of Biomedical Sciences, Fudan University, Shanghai, China

    Jianqing Xu

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Y.W., Q.T., Y.H., W.Y., J.L., C.C., X.C., Y. Lin, Q.H., L.X., J.H., S.L., Z.W., Y. Luo and Q.W. performed the experiments. Z.L., L.H. and J.T. provided reagents, materials and support. L.Y., Y.W. and Q.T. designed the study, analyzed the data and wrote the paper with X.Z. and Y.W. L.Y., J.X. and Z.Y. supervised the study.

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Correspondence to Zhinan Yin, Jianqing Xu or Lilin Ye.

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Peer review information Nature Immunology thanks the anonymous reviewers for their contribution to the peer review of this work. L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 IL-7 signaling sustains mTORC2 activity in memory CD4+ T cells.

a, Naïve SMARTA cells (CD45.1+) were adoptively transferred into C57BL/6J recipient mice (CD45.2+) and followed by infection with LCMV Arm+ the next day. SMARTA cells were analyzed via flow cytometry at D0 (Naïve, green), D5 (early Effector, blue), and D60 (Memory, red) post-infection. CD127 levels on transferred SMARTA cells in the spleens of recipients, from two independent experiments (n=4 for Naive and D5, n=5 for D90). b, p-AKTSer473 levels on memory transferred SMARTA cells as in a after stimulation with (red) or without (blue) recombinant human IL-7 ex vivo, from two independent experiments (n=4). c Experimental setup of IL-7 signaling blockade with αIL-7-mAb at the maintenance stage of CD4+ T cell memory. d, p-STAT5Tyr694 and p-AKTSer473 levels on memory SMARTA cells in the spleens of recipients as in c after administration with αIL-7-mAb at day 60 post-infection. The presented data are representative of two independent experiments (n=4 for αIL-7-mAb, n=5 for Control). e, f, Validation of the knockout of Rictor. Immunoblot analysis (e) of lysates of sorted memory CD4+ T cells from either CD4Cre-Rictorfl/fl (Rictor−/−) or Rictorfl/fl mice. The presented data are representative of four independent experiments. (f) p-AKTSer473 and p-S6Ser235/236 levels on memory CD4+ T cells in the spleens of recipients as in c. The presented data are representative of four independent experiments (n=4). Grey area in histogram indicates staining with rabbit isotype IgG. Data are presented as mean values ± SD and in a-b, d, f. Box plots show min to max, each data point represents one result from an independent experiment, and horizontal bars denote the median in e. Data are analyzed by unpaired two-tailed t-test in a-b, d-f.

Source data

Extended Data Fig. 2 The mTORC2-deficiency impairs memory CD4+ T cell pool but enhances memory CD8+ T cell pool in acute viral infection.

a, b, Analysis of TH1 (SLAMhighCXCR5− in a, CXCR5− in b) and TFH (SLAMlowCXCR5+ in a, CXCR5+ in b) cells among viral-specific memory CD4+ T (positive for LCMV GP66-77 tetramer) cells in the spleens of either Rictorfl/fl (blue) or CD4Cre-Rictorfl/fl (Rictor−/−, red) mice at day 8 (a) and day 100 (b) post-infection of LCMV Arm+. The presented data are representative of three independent experiments (n=6 for Rictorfl/fl and n=4 for Rictor−/− in a, n=6 for both groups in b). c, Viral-specific memory CD8+ T (positive for LCMV GP33-41 tetramer) cells in the spleens of either Rictorfl/fl (blue) or CD4Cre-Rictorfl/fl (Rictor−/−, red) mice. The presented data are representative of three independent experiments (n=4). d, Ratios of total CD8+ T cells and virus-specific CD8+ T cells (positive for LCMV GP33-41 tetramer) between ERT2Cre-Rictorfl/fl origin (CD45.2+) and wild-type origin (CD45.1+) in the spleens of bone marrow chimeras (BMCs) after administration of Tamoxifen (red) or vehicle (blue) at the memory maintenance stage as described in Fig. 2b, from three independent experiments (n=4). Data are presented as mean values ± SD and analyzed by unpaired two-tailed t-test in a-d.

Source data

Extended Data Fig. 3 Torin-1 induced mTORC2-deficiency impairs the virus-specific memory CD4+ T cell pool in vivo.

a, Experimental setup of in vivo administration of Torin-1, Rapamycin or vehicle on adoptively transferred memory SMARTA cells (CD45.1+) in recipients (CD45.2+). b, p-AKTSer473 levels on transferred SMARTA cells in spleens of recipients as in a. The presented data are representative of three independent experiments. c, p-S6Ser235/236 levels on memory SMARTA cells in the presence or absence of αCD3 plus αCD28 ex vivo in spleens of recipients. The presented data are representative of three independent experiments (n=4). d, Frequency change of memory SMARTA cells in PBMCs after administration of either Torin-1, Rapamycin, or vehicle in recipients as in a. The presented data are representative of three independent experiments (n=5). Grey area in histogram indicates staining with rabbit isotype IgG in b-c. Data are presented as mean values ± SD and analyzed by unpaired two-tailed t-test in c. Data are analyzed by paired two-tailed t-test in d.

Source data

Extended Data Fig. 4 No aberrant metabolic profiles in mTORC2-deficient memory CD4+ T cells.

a, b, Profile of (a) extracellular acidification rate (ECAR) and (b) oxygen consumption rate (OCR) of sorted memory CD4+ T cells (CD25−GITR−CD44highCD4+) from either CD4Cre-Rictorfl/fl (Rictor−/−, red) or Rictorfl/fl (blue) mice at day 60 post infection of LCMV Arm+ (n=3 biological replicates, pooled from at least 20 mice in CD4Cre-Rictorfl/fl group and 10 mice in Rictorfl/fl group). The presented data are representative of two independent experiments. Data are presented as mean values ± SD and analyzed by unpaired two-tailed t-test in a-b.

Source data

Extended Data Fig. 5 mTORC2-deficiency induces ferroptosis in both memory TH1 and TFH CD4+ T cells.

a, Bodipy C11 intensity on either wild-type SMARTA (blue) or Rictor−/−-SMARTA (red) TH1 and TFH cells in spleens of recipients at day 60 post infection of LCMV Arm+. The presented data are representative of three independent experiments (n=5 for WT, n=6 for Rictor−/−). b, Bodipy C11 intensity on bulk memory TH1 (CD25−GITR−CD44highCXCR5−CD4+) and TFH (CD25−GITR−CD44highCXCR5+CD4+) cells in spleens of either Rictorfl/fl (blue) or ERT2Cre-Rictorfl/fl (Rictor−/−, red) mice after administration of Tamoxifen. The data presented are representative of three independent experiments (n=4). Data are presented as mean values ± SD and analyzed by unpaired two-tailed t-test in a-b.

Source data

Extended Data Fig. 6 IL-7 signaling is indispensable to protect memory CD4+ T cells from ferroptosis.

a, Experimental setup of IL-7 signaling blockade with αIL-7-mAb at the maintenance stage of CD4+ T cell memory. Naïve SMARTA cells (CD45.1+) were adoptively transferred into C57BL/6J recipient mice (CD45.2+) and followed by infection with LCMV Arm+ the next day. Recipients were injected intraperitoneally with αIL-7-mAb or PBS (Vehicle) at day 60 post infection every three days for a total four doses before sacrifice. b, Representative contour plots of memory SMARTA cell frequency and statistical analysis of the decreased proportion of memory SMARTA cells in the PMBCs before and after αIL-7-mAb administration and the endpoint cell number of memory SMARTA cells in the spleens of recipients at day 60 post LCMV Arm+ infection in the context of IL-7 signaling blockade as described in a. c, d, Dead cells (c, 7-AAD+) and apoptotic cell death (d, Annexin V+ 7-AAD−) in memory SMARTA cells in spleens of recipients as described in a. e, Bodipy C11 intensity on SMARTA cells in spleens of recipients as described in a. The presented data are representative of two independent experiments (b-e, n=4 for αIL-7-mAb, n=5 for Control). Data are presented as mean values ± SD and analyzed by unpaired two-tailed t-test in b-e.

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Extended Data Fig. 7 The mTORC2 signaling generally inhibits ferroptosis in memory CD4+ T cells.

LM-GP61 or IAV-GP61 is the recombinant Listeria monocytogenes or Influenza A Virus expressing LCMV glycoprotein-specific I-Ab restricted CD4 T cell epitope GP66-77. a, Experimental setup of inducible knockout of Rictor in BMCs generated with BM cells from C57BL/6 (WT, CD45.1+) and ERT2Cre-Rictorfl/fl (Rictor−/−, CD45.2+) mice at day 60 post infection of either LM-GP61 or IAV-GP61. b-e, Bodipy C11 intensity on memory CD4+ T cells in spleens of BMCs after administration of Tamoxifen at day 60 post infection with LM-GP61 (b) or IAV-GP61 (d) as described in a. Ratio between Rictor−/− (CD45.2+) and WT (CD45.1+) viral specific memory CD4+ T cells (GP66-77+) in PBMCs from BMCs before and after administration of Tamoxifen at day 60 post infection with LM-GP61(c) or IAV-GP61 (e) as described in a. The presented data are representative of two independent experiments (n=4 in b-e). f, Experimental setup and gating strategy for flow cytometry. Human naïve (Tnaive), central memory (TCM), and effector memory (TEM) CD4+ T cells isolated from PBMCs of healthy human donors were treated with Torin-1, Rapamycin, or vehicle, then analyzed via flow cytometry. g, h, Bodipy C11 intensity (g) and percentage of dead cells (Live/Dead+) in enumerated CD4+ T cell subsets in PBMCs after treatment of Torin-1, Rapamycin, or vehicle as described in f. The presented data are representative of two independent experiments (n=5 in g-h). Data are analyzed by paired two-tailed t-test in b-e. Data are presented as mean values ± SD and analyzed by unpaired two-tailed t-test in g-h.

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Extended Data Fig. 8 GSEA reveals hyperactive mitochondrial ROS generation in mTORC2-deficienct memory CD4+ T cells.

a, b, c, GSEA plots for GO term about mitochondrial organization and respiratory chain complex assembly (a), cellular regulation of reactive oxygen species metabolic process (b), and cellular oxidative stress (c) in sorted memory TH1 cells (CD25−GITR−CD44highCXCR5−CD4+) and memory TFH cells (CD25−GITR−CD44highCXCR5+CD4+) from either CD4Cre-Rictorfl/fl (Rictor−/−) or Rictorfl/fl mice at day 60 post-infection of LCMV Arm+. NES, normalized enrichment score; FDR, false discovery rates.

Extended Data Fig. 9 Blockade of IL-7 signaling results in mitochondrial dysfunction in memory CD4+ T cells.

a, Experimental setup is described as in Extended Data Fig. 6. TMRE, MitoTracker, and MitoSOX intensity on memory SMARTA cells in spleens of recipients at day 60 post LCMV Arm+ infection in the context of IL-7 signaling blockade (αIL-7-mAb) or not (Control). The presented data are representative of two independent experiments (n=4 for αIL-7-mAb, or 5 for Control). Data are presented as mean values ± SD and analyzed by unpaired two-tailed t-test in a.

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Wang, Y., Tian, Q., Hao, Y. et al. The kinase complex mTORC2 promotes the longevity of virus-specific memory CD4+ T cells by preventing ferroptosis. Nat Immunol 23, 303–317 (2022). https://doi.org/10.1038/s41590-021-01090-1

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