Ketogenesis-generated β-hydroxybutyrate is an epigenetic regulator of CD8+ T-cell memory development

Abstract

Glycogen has long been considered to have a function in energy metabolism. However, our recent study indicated that glycogen metabolism, directed by cytosolic phosphoenolpyruvate carboxykinase Pck1, controls the formation and maintenance of CD8+ memory T (Tmem) cells by regulating redox homeostasis1. This unusual metabolic program raises the question of how Pck1 is upregulated in CD8+ Tmem cells. Here, we show that mitochondrial acetyl coenzyme A is diverted to the ketogenesis pathway, which indirectly regulates Pck1 expression. Mechanistically, ketogenesis-derived β-hydroxybutyrate is present in CD8+ Tmem cells; β-hydroxybutyrate epigenetically modifies Lys 9 of histone H3 (H3K9) of Foxo1 and Ppargc1a (which encodes PGC-1α) with β-hydroxybutyrylation, upregulating the expression of these genes. As a result, FoxO1 and PGC-1α cooperatively upregulate Pck1 expression, therefore directing the carbon flow along the gluconeogenic pathway to glycogen and the pentose phosphate pathway. These results reveal that ketogenesis acts as an unusual metabolic pathway in CD8+ Tmem cells, linking epigenetic modification required for memory development.

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Fig. 1: CD8+ Tmem cells display an active ketogenesis.
Fig. 2: BHB plays an important role in CD8+ Tmem-cell formation.
Fig. 3: BHB upregulates Pck1 expression in CD8+ Tmem cells.
Fig. 4: BHB increases β-hydroxybutyrylation of histone H3.
Fig. 5: BHB upregulates Pck1 by epigenetically modifying FoxO1-bound H3K9.

Data availability

The ChIP–seq data of this study have been deposited in the Gene Expression Omnibus (GEO) repository under accession number GSE129723. Previously published ChIP–seq data18 that were reanalysed here are available under accession code GSE69617. Mass spectrometry data have been deposited in ProteomeXchange with the primary accession code PXD016044. Source data for Figs. 15 and Extended Data Figs. 15 are available online. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank H. Deng (MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University) for the mass spectrometry analysis of histone β-hydroxybutyrylation; Y. Peng (State Key Laboratory of Biogeology and Environmental Geology, China University of Geosciences, Wuhan) for metabolite analysis; T. Jiang, W. Zhu (Suzhou Institute of Systems Medicine, Suzhou, China) and X. He (Huazhong University of Science and Technology) for the ChIP–seq analysis. This work was supported by the National Natural Science Foundation of China (grant numbers 81788101, 81530080 and 81701544), the CAMS Initiative for Innovative Medicine (2016-I2M-1-007) and China Postdoctoral Science Foundation funded project (2018T110773).

Author information

B.H. conceived the project. H.Z., K.T., J.M., L.Zhou, J.Liu, L.Zeng, L.Zhu, P.X., J.C., K.W., X.L., J.Lv and J.X. performed the experiments. H.Z. and B.H. analysed the data. H.Z., Y.L., Y.W. and B.H. contributed to manuscript preparation. H.Z. and B.H. wrote the manuscript.

Correspondence to Bo Huang.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Ketogenesis activity in mouse CD4+ and human CD8+ Tm cells.

a, The levels of BHB in mouse CD4+ Tn, OVA-specific Teff and Tm cells from mice were analyzed (n = 4 independent experiments). b, The levels of BHB in human CD8+ Tn, Teff and Tm cells were analyzed (n = 4 independent experiments). c,d, The expression of ketogenic enzymes were analyzed in human CD8+ Tn, Teff and Tm cells (n = 3 independent experiments). e,f, The expression of ketogenic enzymes were analyzed in mouse CD4+ Tn, Teff and Tm cells (n = 3 independent experiments). g, mouse CD8+ Tn, Teff and Tm cells were incubated with [U6]-13C glucose for 24 h, and LC/MS was performed for m+2-labelled BHB and AcAc (n = 4 independent experiments). h, mouse CD8+ Tn, Teff and Tm cells were incubated with [U3]-13C pyruvate for 24 h, and LC–MS was performed for m+2-labelled BHB and AcAc (n = 4 independent experiments). i, CD8+ Tn, Teff and Tm cells were incubated with 1 mM [U16]-13C-Palmitate/BSA for 24 h, and LC/MS analysis was performed for m+2-labelled BHB and AcAc (n = 4 independent experiments). Error bars are mean ± s.d., P values were calculated by two tailed unpaired Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, the precise P value and statistics source data are in Source Data Extended Data Fig. 1. Scanned images of unprocessed blots are shown in Source Data Extended Data Fig. 1. Source Data

Extended Data Fig. 2 BHB regulates the formation of CD8+ Tm cells.

a,b The effect of AcAc on the survival (a) and memory genes expression (b) of IL-15 CD8+ Tm cells. c, BHB levels of AcAc treated IL-15 CD8+ Tm cells were measured (n = 4 independent experiments). d-f, C57BL/6 mice were treated with BHB and control saline 7 days after OT-I cell adoptive transfer and Lm-OVA infection. On day 30, mice were re-challenged with Lm-OVA. 5 days later, spleens were harvested and CD25/CD69 (d), IFN-γ (e) and T cell expansion (f) of CD45.1+ CD8+ OT-I T cells were analyzed. (n = 5 mice per group). g, mice were fed with KD, HF or normal diet, beginning on day 7 after OT-I cell adoptive transfer and Lm-OVA infection. On day 30, the CD45.1+CD8+ Tm cells in the spleen, LN and PBMC were analyzed (n = 5 mice per group). h,i, mice were treated as (g). On day 30, the plasma (h) and liver (i) BHB levels were measured (n = 6 mice per group pooled from two independent experiments). j,k, The expression of Bdh1 in T cells transduced with shNC or shBdh1 was analyzed. l, AcAc levels of CD8+ Tm cells transduced with shNC or shBdh1 were analyzed. m, CD8+ Tm cells transduced with shNC or shBdh1 were incubated with 1 mM [U16]-13C-Palmitate/BSA for 24h, and LC/MS analysis was performed for m+2-labelled BHB (n =4 independent experiments). n-p, Mice were transferred with shNC or shBdh1 transduced CD8+ OT-I Tm cells, followed by infection of Lm-OVA. 7 days later, mice were injected with BHB and control saline once per day. On day 30, CD8+GFP+ T cells in the spleen were isolated for Bdh1 expression (n). CD8+GFP+ T cells in the lung, liver and bone marrow were analyzed (o). Annexin V, Ki-67 and BrdU in spleen Tm cells were analyzed (p). (n = 6 mice per group). q,r, CD8+ OT-I T cells were activated by OVA257-264 for 48hr in the presence or absence of BHB. The number of CD8+ Teff was counted (q), and the expressions of CD25 and CD69 were analyzed (r). s,t, CD8+ OT-I T cells transduced with shNC or shBdh1 were activated by OVA257-264 for 48hr. The number of CD8+ Teff was counted (s), and the expressions of CD25 and CD69 were analyzed (t). Unless otherwise specified, n = 3 independent experiments. All error bars are mean ± s.d., P values were calculated by two tailed unpaired Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, the precise P value and statistics source data are in Source Data Extended Data Fig. 2. Scanned images of unprocessed blots are shown in Source Data Extended Data Fig. 2. Source Data

Extended Data Fig. 3 Acat1 knockdown impaired the formation of CD8+ Tm cells.

a,b, The expression of Acat1 in T cells transduced with shNC or shAcat1 was analyzed by real-time PCR (a) and western blot (b) (n = 4 independent experiments). c, BHB levels of IL-15-induced Tm cells transduced with shNC or shAcat1 were measured (n =4 independent experiments). d-f, mice were transferred with OT-I CD8+ T cells transduced with shNC or shAcat1, followed by infection of Lm-OVA. 7 days later, mice were i.p. injected with BHB and control saline once per day. On day 30, CD8+GFP+ T cells in the spleen, LN and PBMC were analyzed by flow cytometry (n = 5 mice per group, data shown represent three independent experiments). g, mice were transferred with 1×105 shNC- or shAcat1-transduced OT-I CD8+ T cells, followed by infection of Lm-OVA. On day 6, the proportion and effector markers of GFP+CD8+ T cells in the spleen were analyzed by flow cytometry (n =6 mice pooled from two independent experiments). h-k, CD8+ Tn (IL-7), Teff (IL-2) or Tm (IL-15) cells were cultured with 13C-BHB. 24h later, the incorporation of 13C-BHB into TCA cycle intermediates such as α-KG(h), fumarate(i), malate(j) and citrate(k) was measured by LC/MS respectively (n = 3 independent experiments). l-n, CD8+ Tn, Teff or Tm cells were cultured in the presence or absence of BHB. OCR was measured by Seahorse XF analyzer. o, OCR of CD8+ Tm cells transduced with shNC or shBdh1 was measured (Data shown represent three independent experiments). p, Immunoblot analysis of Pck1 from IL-15-derived Tm cells transduced with shNC or shAcat1 (n = 3 independent experiments). All error bars are mean ± s.d., P values were calculated by two tailed unpaired Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, the precise P value and statistics source data are in Source Data Extended Data Fig. 3. Scanned images of unprocessed blots are shown in Source Data Extended Data Fig. 3. Source Data

Extended Data Fig. 4 BHB related Pck1 upregulation in CD8+ Tm cells is independent of acylation or methylation modification.

a, Immunoblot analysis of histones from IL-15-induced CD8+ Tm cells using anti-H3K9ac antibody. b,c, IL-15-induced CD8+ Tm cells were treated with HDAC inhibitor TSA or Acetate. Pck1 mRNA level was analyzed by real-time PCR (b), and protein level of Pck1 and H3K9ac were analyzed by western blot (c). d, Immunoblot analysis of histones from IL-15-induced CD8+ Tm cells using anti-H3K4me3 and anti-H3K27me3 antibodies. e,f, ChIP-qPCR analyses of H3K4me3 (e) and H3K27me3 (f) enrichment around the promoter of Pck1 in IL-15-induced CD8+ Tm cells. g, Methylation of CpG in the promoter region of Pck1 was determined. h, Immunoblot analysis of Kbhb from human CD8+ T cells and mouse CD4+ T cells using pan anti-Kbhb antibody and anti-H3K9bhb antibody. i, H3K9bhb in IL-15-derived Tm cells transduced with shNC or shAcat1 was measured by western blot. j, Immunoblot analysis of H3K9bhb and Pck1 in IL-15-derived Tm cells treated with AcAc. k, CD8+ Tn, Teff or Tm cells were cultured in the presence of 10 mM 13C-BHB for 48h. Percent of 13C-β-hydroxybutyrylation of H3K9 were measured by LC/MS/MS. n = 3 independent experiments for all panels. Error bars are mean ± s.d., P values were calculated by two tailed unpaired Student’s t-test. **P < 0.01, ***P < 0.001, the precise P value and statistics source data are in Source Data Extended Data Fig. 4. Scanned images of unprocessed blots are shown in Source Data Extended Data Fig. 4. Source Data

Extended Data Fig. 5 BHB upregulates Pck1 by epigenetically modifying FoxO1-bound H3K9.

a, ChIP-qPCR analyses of H3K9bhb enrichment around the promoter of FoxO1 in IL-15 Tm cells transduced with shNC or shAcat1. b, FoxO1 expression in IL-15 Tm cells was measured. c, The expression of FoxO1 in T cells transduced with shNC or shFoxO1 was analyzed. d,e, The effect of FoxO1 on Pck1 expression in CD8+ Tm cells was analyzed in vivo using shFoxO1 retrovirus (d) or Foxo1 inhibitor (AS1842856, 100 mg kg-1) (e). f, ChIP-qPCR analyses of H3K9bhb enrichment around the promoter of PGC1α in IL-15 Tm cells transduced with shNC or shAcat1. g-i, IL-15 Tm cells were treated with BHB, and the expression of PGC1α was analyzed by real-time PCR (g), western blot (h) and Immunofluorescence (i). Scale bar, 10 μm. j, PGC1α expression in IL-15-derived Tm cells was measured. k, the expression of PGC1α in T cells transduced with shNC or shPGC1α was analyzed. l,m, The effect of PGC1α on Pck1 expression in CD8+ Tm cells was analyzed in vivo using shPGC1α retrovirus (l) or PGC1α inhibitor (SR18292, 45 mg kg-1) (m). n, Average read count frequency of H3K9 β-hydroxybutyrylation marks surrounding the TSSs were generated for genes that are expressed in CD8+ Tm cells treated with BHB. o, Gene ontology analysis of H3K9bhb ChIP-seq data in CD8+ Tm cells treated with BHB (p.adjust means FDR(false discovery rate)-adjusted p values). p, Venn diagram of genes marked by H3K9bhb in CD8+ Tm cells, ST-liver cells and AL-liver cells. q, Distribution of H3K9bhb modification at Foxo1 and Pck1 in CD8+ Tm cells treated with BHB. The data represent the mean ± s.d. of n = 3 biologically independent experiments (a-m), P values were calculated by two tailed unpaired Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, the precise P value and statistics source data are in Source Data Extended Data Fig. 5. Scanned images of unprocessed blots are shown in Source Data Extended Data Fig. 5. Source Data

Supplementary information

Supplementary Information

Supplementary Fig. 1: flow cytometry gating strategy.

Reporting Summary

Supplementary Tables

Supplementary Table 1: quantification of Kbhb on histone H3 from CD8+ Tmem cells treated with BHB. Supplementary Table 2: MS/MS spectra of histone H3 peptide from BHB-treated CD8+ Tmem cells. Supplementary Table 3: MS/MS spectra of histone H3 peptide from 13C-labelled BHB-treated CD8+ Tmem cells. Supplementary Table 4: list of primer sequences used for qPCR.

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Zhang, H., Tang, K., Ma, J. et al. Ketogenesis-generated β-hydroxybutyrate is an epigenetic regulator of CD8+ T-cell memory development. Nat Cell Biol 22, 18–25 (2020) doi:10.1038/s41556-019-0440-0

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