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Lipid signalling enforces functional specialization of Treg cells in tumours

Abstract

Regulatory T cells (Treg cells) are essential for immune tolerance1, but also drive immunosuppression in the tumour microenvironment2. Therapeutic targeting of Treg cells in cancer will therefore require the identification of context-specific mechanisms that affect their function. Here we show that inhibiting lipid synthesis and metabolic signalling that are dependent on sterol-regulatory-element-binding proteins (SREBPs) in Treg cells unleashes effective antitumour immune responses without autoimmune toxicity. We find that the activity of SREBPs is upregulated in intratumoral Treg cells. Moreover, deletion of SREBP-cleavage-activating protein (SCAP)—a factor required for SREBP activity—in these cells inhibits tumour growth and boosts immunotherapy that is triggered by targeting the immune-checkpoint protein PD-1. These effects of SCAP deletion are associated with uncontrolled production of interferon-γ and impaired function of intratumoral Treg cells. Mechanistically, signalling through SCAP and SREBPs coordinates cellular programs for lipid synthesis and inhibitory receptor signalling in these cells. First, de novo fatty-acid synthesis mediated by fatty-acid synthase (FASN) contributes to functional maturation of Treg cells, and loss of FASN from Treg cells inhibits tumour growth. Second, Treg cells in tumours show enhanced expression of the PD-1 gene, through a process that depends on SREBP activity and signals via mevalonate metabolism to protein geranylgeranylation. Blocking PD-1 or SREBP signalling results in dysregulated activation of phosphatidylinositol-3-kinase in intratumoral Treg cells. Our findings show that metabolic reprogramming enforces the functional specialization of Treg cells in tumours, pointing to new ways of targeting these cells for cancer therapy.

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Fig. 1: Treg cells upregulate SREBP signalling and require SCAP/SREBP activity for their functional adaptation in tumours.
Fig. 2: SREBPs maintain the functional fitness of Treg cells in the TME.
Fig. 3: SREBPs orchestrate the function of intratumoral Treg cells by coordinating de novo lipid synthesis and PD-1 expression.
Fig. 4: Protein geranylgeranylation driven by mevalonate metabolism enforces the upregulation of PD-1 by Treg cells.

Data availability

Data generated here are included within the paper (and its Supplementary Information) or are available from the corresponding author upon reasonable request. Microarray and scRNAseq data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE165259Source data are provided with this paper.

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Acknowledgements

We thank M. Hendren, A. KC and S. Rankin for management of animal colonies; Y. Wang and S. Brown for help with immunological experiments; J. Easton and St Jude Genomics Lab of Computational Biology for scRNAseq; and the St Jude Immunology fluorescence-activated cell sorting (FACS) core facility for cell sorting. This work was supported by National Institutes of Health (NIH) grants CA221290, CA250533, AI105887, AI131703, AI140761, AI150241 and AI150514. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Author information

Authors and Affiliations

Authors

Contributions

S.A.L. and J.W. designed and performed in vitro and in vivo experiments, analysed data and wrote the manuscript; T.-L.M.N. designed, performed and analysed cellular experiments; H.S. and Y.D. performed bioinformatic analyses; W.S. and N.M.C. carried out experiments relating to protein prenylation; G.P. performed metabolomic tracing experiments; L.L. and J.S. helped with molecular experiments; P.V. analysed immunohistochemistry and provided histopathology scoring; and H.C. helped to design experiments, co-wrote the manuscript, and provided overall direction.

Corresponding author

Correspondence to Hongbo Chi.

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Competing interests

H.C. is a consultant for Kumquat Biosciences.

Additional information

Peer review information Nature thanks Ulf Beier, Axel Kallies and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Different metabolic states of Treg cells in tumours and inflammatory contexts.

a, Biochemical pathways for de novo lipid synthesis. b, Enrichment plots for SREBP gene targets in intratumoral versus PLN Treg cells from Foxp3Cre mice bearing B16 melanoma (n = 4 per group). c, Top 15 upstream transcriptional regulators enriched in intratumoral Treg cells as compared with PLN Treg cells from Foxp3Cre mice bearing B16 melanoma (n = 4 per group), analysed by Ingenuity pathway analysis. d, e, Enrichment of SREBP gene targets in intratumoral and PLN Treg cells from public scRNAseq datasets for mouse B16 melanoma7,8. f, g, Enrichment of SREBP gene targets in intratumoral Treg cells from individuals with breast cancer9 (f) or HNSCC10 (g). h, Enrichment plots for SREBP gene targets from the public dataset11 of in vivo activated Treg (aTreg) compared with resting Treg (rTreg) cells in the acute inflammation model. i, j, Treg cells were isolated from spleen and CNS of MOG-induced Foxp3RFP EAE mice for scRNAseq analysis. i, Unsupervised clustering of cells in the spleen and CNS was analysed by UMAP: left, splenic and CNS cells are annotated with different colours; right, the expression of SREBP gene targets is visualized. j, Comparison of SREBP gene targets between splenic and CNS Treg cells on the violin plot. k, Relative (values in splenic samples were set to 1) uptake of a fluorescent glucose analogue, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), in splenic and intratumoral Treg cells from mice bearing MC38 tumours on day 21 after tumour inoculation (n = 17). l, Relative (values in splenic samples were set to 1) uptake of 2-NBDG (n = 14) in Treg cells from the spleen and spinal cord of MOG-induced EAE mice on day 16 after MOG immunization. m, Expression of Scap mRNA was examined in Treg, naive CD4+ and CD8+ T cells from Foxp3CreScap+/fl and Foxp3CreScapfl/fl mice under steady state (n = 5 samples per genotype). n, Heat map showing expression of SREBP gene targets, normalized by row (z-score), in tumour-infiltrating Treg cells from mice bearing B16 melanoma on day 19 after tumour inoculation (n = 4, Foxp3CreScap+/+ or Foxp3CreScap+/fl control mice; n = 3, Foxp3CreScapfl/fl mice). o, Foxp3GFP–Cre–ERT2Scap+/flRosa26YFP and Foxp3GFP–Cre–ERT2Scapfl/flRosa26YFP mice were injected with MC38 cells without tamoxifen treatment (n = 6 mice per genotype). Tumour growth was measured. ***P < 0.001; NS, not significant; two-tailed unpaired Student’s t-test (km) or two-way ANOVA (o). Data are means ± s.e.m. in dg, jm, o. Data are representative of two (o) or compiled from two (k, l) independent experiments.

Source data

Extended Data Fig. 2 SCAP is dispensable for Treg cells to maintain immune homeostasis under steady state.

ad, Analyses of Foxp3CreScap+/+ or Foxp3CreScap+/fl (these mice are phenotypically indistinguishable) control and Foxp3CreScapfl/fl mice (12–18 weeks) under steady state (n = 4 mice in each group). a, Cellularity of conventional Foxp3CD4+ and CD8+ T cells in spleen and PLNs. b, Left, flow-cytometry analysis of the expression of CD62L and CD44, and right, quantification of the frequency of CD44hiCD62Llo cells, in conventional Foxp3CD4+ (top panels) or CD8+ T cells (bottom panels) from spleen and PLNs. c, Left panels, flow-cytometry analysis of the expression of IFNγ, IL-4 and IL-17, and right panels, quantification of frequencies of IFNγ+, IL-4+ and IL-17+ cells amongst conventional Foxp3CD4+ T cells from spleen and PLNs. d, Left, flow-cytometry analysis of the expression of IFNγ, and right, quantification of the frequency of IFNγ+ cells amongst CD8+ T cells from spleen and PLNs. e, Haematoxylin-and-eosin staining of spleen, PLNs, colon, pancreas, lung, liver and skin from age-matched Foxp3CreScap+/+ or Foxp3CreScap+/fl control and Foxp3CreScapfl/fl mice (28–35 weeks). f, g, Quantification of anti-nuclear antibodies (ANAs) (f) and anti-dsDNA antibodies (g) in the serum of Foxp3CreScap+/fl and Foxp3CreScapfl/fl mice (older than 3 months) (n = 23, Foxp3CreScap+/fl mice; n = 20, Foxp3CreScapfl/fl mice). h, Survival curve of Foxp3CreScap+/+ or Foxp3CreScap+/fl control (n = 17) and Foxp3CreScapfl/fl (n = 27) mice. **P < 0.01; two-tailed unpaired Student’s t-test (ad, f, g). Data are means ± s.e.m. Data are compiled from two (ae) independent experiments.

Source data

Extended Data Fig. 3 SCAP is dispensable for the function of Treg cells in autoimmune and acute inflammation.

a, Foxp3CreScap+/+ or Foxp3CreScap+/fl control (n = 13) and Foxp3CreScapfl/fl (n = 14) mice were immunized with MOG, and their EAE disease score was measured every day for 21 days. 0, no overt signs of disease; 1, limp tail; 2, limp tail plus hindlimb weakness; 3, total hindlimb paralysis; 4, hindlimb paralysis plus 75% of body paralysis (forelimb paralysis/weakness); 5, moribund. b, Mice as in a were euthanized in the recovery phase (days 22–25) of EAE for histological analysis of clinical disease. Left, haematoxylin-and-eosin staining of the spinal cord, and right, histological scores of the indicated regions of the brain and spinal cord (n = 9, Foxp3CreScap+/+ or Foxp3CreScap+/fl control mice; n = 10, Foxp3CreScapfl/fl mice). c, d, Foxp3CreScap+/+ or Foxp3CreScap+/fl control and Foxp3CreScapfl/fl mice were immunized with MOG to induce EAE (n = 5 mice in each group) and euthanized on day 16 for analysis. c, Cellularity of Foxp3 CD4+ (top) and CD8+ (bottom) T cells in PLNs, spleen and spinal cord. d, Percentages (left) and numbers (right) of IFNγ+ (top) and IL-17+ (bottom) Foxp3 CD4+ T cells from PLNs, spleen and spinal cord. eh, Foxp3Cre/DTRScap+/+ or Foxp3Cre/DTRScap+/fl control and Foxp3Cre/DTRScapfl/fl mosaic mice were treated with diphtheria toxin (DT) (e, top, experimental design; n = 6 mice in each group) and euthanized 3 days after the final DT injection. e, Quantification of cellularity of Foxp3 CD4+ (lower left) and CD8+ (lower right) T cells. f, Quantification of the frequency of CD44hi CD62Llo cells in Foxp3 CD4+ (top) and CD8+ (bottom) T cells from spleen and PLNs. g, Quantification of frequencies of IFNγ+ (left), IL-4+ (middle) and IL-17+ (right) cells amongst Foxp3 CD4+ T cells from spleen and PLNs. h, Frequency of IFNγ+ cells amongst CD8+ T cells from spleen and PLNs. Two-tailed unpaired Student’s t-test (bh) or two-way ANOVA (a). Data are means ± s.e.m. in ah. Data are compiled from three (a) or two (b, eh) independent experiments.

Source data

Extended Data Fig. 4 SCAP/SREBP signalling maintains the functional state of Treg cells in the TME.

ad, Foxp3CreScap+/+ or Foxp3CreScap+/fl control (n = 11) and Foxp3CreScapfl/fl (n = 14) mice were inoculated with B16 cells. a, b, Cellularity of CD8+ T cells (gated as CD8α+TCRβ+; a) and Foxp3CD4+ T cells (b) in PLNs (left panels) and tumours (right panels; normalized to tumour weight to account for differences in tumour size between the genetic models) on day 19. c, d, Numbers of CD44hiCD62Llo (Tem) CD8+ cells (c) and CD44hiCD62Lhi (Tcm) CD8+ cells (d) from tumours. e, Foxp3CreScap+/+ or Foxp3CreScap+/fl control (n = 12) and Foxp3CreScapfl/fl (n = 14) mice were inoculated with B16 cells, and the number of Tim3+PD-1+ (Tex) CD8+ T cells from tumours was quantified. f, g, Foxp3CreScap+/+ or Foxp3CreScap+/fl control (n = 11) and Foxp3CreScapfl/fl (n = 14) mice were inoculated with B16 cells. Shown are flow-cytometry analysis of the expression of TNFα and IFNγ (left), and the relative frequencies of TNFα+ cells (middle) and IFNγ+ cells (right) amongst CD8+ T cells (f) and Foxp3CD4+ T cells (g) from PLNs and tumours. h, Foxp3CreScap+/+ or Foxp3CreScap+/fl control and Foxp3CreScapfl/fl mice were inoculated with MC38 cells on day 0 and treated with anti-CD8 antibody on days –1, 2, 5, 8 and 11. Tumour growth was measured (n = 6 mice in each group). *P < 0.05; **P < 0.01; ***P < 0.001; two-tailed unpaired Student’s t-test (ag) or two-way ANOVA (h). Data are means ± s.e.m. in ah. Data are representative of two (h) or compiled from two (ag) independent experiments.

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Extended Data Fig. 5 Cellular state of Scap-deficient Treg cells in tumours and homeostasis.

a, Foxp3CreScap+/+ or Foxp3CreScap+/fl control and Foxp3CreScapfl/fl mice were inoculated with B16 cells and euthanized on day 19. Shown are flow-cytometry analysis (left) and quantification of frequency (second left) and number (second right, raw values of cell number; right, cell number normalized to tumour weight) of Foxp3+CD4+ T cells in PLNs and tumours (n = 11, Foxp3CreScap+/+ or Foxp3CreScap+/fl control mice; n = 14, Foxp3CreScapfl/fl mice). b, Number of CD45.2+ cells from Foxp3CreScap+/+ or Foxp3CreScap+/fl control (n = 11) and Foxp3CreScapfl/fl (n = 14) mice bearing B16 melanoma. c, Frequency (left) and number (right) of Foxp3+CD4+ Treg cells in spleen, lung, fat tissue and skin from Foxp3CreScap+/+ or Foxp3CreScap+/fl control and Foxp3CreScapfl/fl mice under steady state (n = 6 mice in each group). d, Foxp3–YFP+CD4+ cells were sorted from Foxp3CreScap+/fl and Foxp3CreScapfl/fl mice, labelled with CellTrace Violet (CTV), and cultured with anti-CD3/28 antibodies plus IL-2 for 3 days in the presence of cholesterol or vehicle (Treg cells were pretreated with cholesterol for 1 h or vehicle before activation; n = 6 per group). The relative frequency of CTVlo cells is shown. e, Foxp3CreScap+/+ or Foxp3CreScap+/fl control (n = 11) and Foxp3CreScapfl/fl (n = 13) mice were inoculated with B16 cells. Shown is the relative frequency of BrdU+ cells amongst Foxp3+CD4+ Treg cells from PLNs and tumours. f, Treg cells from Foxp3CreScap+/+ or Foxp3CreScap+/fl control and Foxp3CreScapfl/fl mice were labelled with CTV and transferred into Rag1−/− mice. Mice were euthanized on day 10 for analysis, and the cellularity of Foxp3+CD4+ Treg cells in spleen and PLNs was quantified (n = 7 mice in each group). g, Foxp3CreScap+/+ or Foxp3CreScap+/fl control and Foxp3CreScapfl/fl mice received an intraperitoneal injection of IL-2/anti-IL-2 complex daily on days 0–2 to induce the expansion of Treg cells19, and were analysed on day 5. Shown is the number of Foxp3+CD4+ Treg cells in spleen and PLNs (n = 4 mice in each group). h, Treg cells from Foxp3CreScap+/+ or Foxp3CreScap+/fl control and Foxp3CreScapfl/fl mice were transferred into Rag1−/− recipients and analysed as in f. The mean fluorescence intensity (MFI) of CTLA4 in Treg cells (n = 7 mice in each group) from spleen and PLNs was quantified. i, Relative MFI of CTLA4 in Treg cells from Foxp3CreScap+/+ or Foxp3CreScap+/fl control and Foxp3CreScapfl/fl mice (n = 4 mice in each group) treated with IL-2/anti-IL-2 complex as in g. j, Foxp3CreScap+/+ or Foxp3CreScap+/fl control and Foxp3CreScapfl/fl mice were inoculated with B16 cells and euthanized on day 19. Left, flow-cytometry analysis and right, relative frequency of Treg cells with active caspase-3 in PLNs and tumours (n = 11, Foxp3CreScap+/+ or Foxp3CreScap+/fl control mice; n = 13, Foxp3CreScapfl/fl mice). km, Female Foxp3Cre/+Scap+/fl control and Foxp3Cre/+Scapfl/fl mosaic mice were challenged with B16 cells and euthanized on day 14 (n = 15 mice per genotype). k, Relative frequency of 7AAD+ cells amongst Foxp3+CD4+ Treg cells from tumours. l, Relative frequency of Foxp3+CD4+ Treg cells from tumours. m, Relative number of Foxp3+CD4+ Treg cells from tumours (normalized to tumour weight). np, Foxp3CreScap+/+ or Foxp3CreScap+/fl control and Foxp3CreScapfl/fl mice were inoculated with B16 cells and euthanized on day 19. Shown are CD36 expression (n = 8, Foxp3CreScap+/+ or Foxp3CreScap+/fl control mice; n = 10, Foxp3CreScapfl/fl mice) (n), uptake of BODIPY FL C12 (n = 5 mice in each group) (o), MitoTracker expression (n = 3, Foxp3CreScap+/+ or Foxp3CreScap+/fl control mice; n = 6, Foxp3CreScapfl/fl mice) (p, left), TMRM expression (n = 4, Foxp3CreScap+/+ or Foxp3CreScap+/fl control mice; n = 6, Foxp3CreScapfl/fl mice) (p, middle) and MitoSOX expression (n = 11, Foxp3CreScap+/+ or Foxp3CreScap+/fl control mice; n = 14, Foxp3CreScapfl/fl mice) (p, right) in Treg cells. q, Frequency (left) and number (right) of CD44hiCD62Llo aTreg cells amongst Foxp3+CD4+ Treg cells from spleen and PLNs under steady state (n = 4 mice in each group). r, Frequency (left) and number (right; normalized to tumour weight) of aTreg cells in tumours from Foxp3CreScap+/+ or Foxp3CreScap+/fl control (n = 11) and Foxp3CreScapfl/fl (n = 14) mice challenged with B16 cells. *P < 0.05; **P < 0.01; *** P < 0.001; two-tailed unpaired Student’s t-test (ac, er) or one-way ANOVA (d). Data are means ± s.e.m. in ar. Data are representative of two (f; h; o; p, left, middle) or compiled from three (d, e, j) or two (ac; g; i; kn; p, right; q; r) independent experiments. Values in control samples were set to 1 (d; e; im; p, right).

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Extended Data Fig. 6 Scap-deficient Treg cells in tumours and homeostasis.

a, Left, flow-cytometry analysis and right, MFI of Foxp3–YFP in intratumoral Treg cells from Foxp3CreScap+/+ or Foxp3CreScap+/fl control (n = 5) and Foxp3CreScapfl/fl (n = 6) mice bearing B16 melanoma. b, Left, flow-cytometry analysis and right, MFI of Foxp3 protein in intratumoral Treg cells from Foxp3CreScap+/+ or Foxp3CreScap+/fl control (n = 5) and Foxp3CreScapfl/fl (n = 6) mice bearing B16 melanoma. c, Left, flow-cytometry analysis and right, MFI of Foxp3 protein in Treg cells (from Foxp3CreScap+/+ or Foxp3CreScap+/fl control and Foxp3CreScapfl/fl mice) transferred into Rag1−/− recipients (n = 7 mice in each group). d, Frequency of IFNγ+ cells in Foxp3+CD4+ Treg cells in spleen and PLNs from Foxp3CreScap+/+ or Foxp3CreScap+/fl control and Foxp3CreScapfl/fl mice under steady state (n = 4 mice in each group). e, Violin plots showing gene expression in macrophages from scRNAseq analysis of tumour-infiltrating CD45+ cells in mice challenged with B16 cells (n = 2 mice in each group; see also Fig. 2a). fh, Foxp3GFP–Cre–ERT2Scap+/flRosa26YFP and Foxp3GFP–Cre–ERT2Scapfl/flRosa26YFP mice were injected with MC38 cells on day 0, treated with tamoxifen daily on days 7–11, and analysed on day 21 after tumour injection (n = 6 mice per genotype). f, Frequencies of IFNγ+ cells amongst GFP+YFP and GFP+YFP+ Treg cells from PLNs (left) and tumours (right). g, Frequencies of IFNγ+ (left) and TNFα+ (right) cells amongst CD8+ T cells in PLNs and tumours. h, Frequencies of IFNγ+ (left) and TNFα+ (right) cells amongst conventional Foxp3CD4+ T cells in PLNs and tumours. *P < 0.05; two-tailed unpaired Student’s t-test (ad, fh). Data are means ± s.e.m. in ah. Data are representative of two (ac) or compiled from two (d) independent experiments.

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Extended Data Fig. 7 FASN is dispensable for the function of Treg cells in steady state and autoimmunity.

a, Expression of Hmgcr mRNA in Treg cells from Foxp3CreScap+/fl and Foxp3CreScapfl/fl mice under steady state (n = 5 samples per genotype). b, Expression of Hmgcr mRNA in Treg cells from Foxp3CreHmgcr+/+ (n = 4), Foxp3CreHmgcr+/fl (n = 6) and Foxp3CreHmgcrfl/fl (n = 4) mice under steady state. c, Analysis of T cell homeostasis in Foxp3CreHmgcr+/+ and Foxp3CreHmgcr+/fl mice (6–10 weeks; n = 3 mice per genotype): frequency of CD44hiCD62Llo cells amongst conventional Foxp3CD4+ (left) and CD8+ T cells (right) from spleen and PLNs. d, Expression of Fasn mRNA in Treg, naive CD4+ and CD8+ T cells from Foxp3CreFasn+/fl and Foxp3CreFasnfl/fl mice under steady state (n = 3 samples per genotype). ei, Analysis of spleen and PLNs from Foxp3CreFasn+/+ or Foxp3CreFasn+/fl control and Foxp3CreFasnfl/fl mice (8–12 weeks) under steady state (n = 4 mice in each group). e, f, Numbers of Foxp3+CD4+ Treg cells (e) and conventional Foxp3CD4+ (f, left) and CD8+ (f, right) T cells. g, Frequencies of CD44hiCD62Llo cells amongst conventional Foxp3CD4+ (left) and CD8+ (right) T cells. h, Frequencies of IFNγ+ (left), IL-4+ (middle) and IL-17+ (right) cells amongst conventional Foxp3CD4+ T cells. i, Frequency of IFNγ+ cells amongst CD8+ T cells. j, Foxp3CreFasn+/+ or Foxp3CreFasn+/fl control (n = 5) and Foxp3CreFasnfl/fl (n = 6) mice were immunized with MOG, and the EAE disease score was measured every day until day 24. k, Mice as in j were euthanized in the recovery phase (day 24) of EAE for histological analysis of clinical disease. Left, haematoxylin-and-eosin staining of the spinal cord, and right, histological scores of the indicated regions of the brain and spinal cord (n = 5, Foxp3CreFasn+/+ or Foxp3CreFasn+/fl control mice; n = 6, Foxp3CreFasnfl/fl mice). ***P < 0.001; two-tailed unpaired Student’s t-test (a, ci, k), one-way ANOVA (b), or two-way ANOVA (j). Data are means ± s.e.m. in ak. Data are compiled from two (ei) independent experiments.

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Extended Data Fig. 8 Antitumour response of Foxp3CreFasnfl/fl mice.

a, b, Foxp3CreFasn+/+ or Foxp3CreFasn+/fl control (a, n = 9; b, n = 13) and Foxp3CreFasnfl/fl (a, n = 10; b, n = 16) bone-marrow chimaeras were inoculated with B16 cells and euthanized on day 17 (a) or days 17–19 (b). Shown are relative frequencies of TNFα+ cells (left) and IFNγ+ cells (right) amongst conventional CD8+ T cells (a) and Foxp3CD4+ T cells (b). ch, Foxp3CreFasn+/+ or Foxp3CreFasn+/fl control (n = 9) and Foxp3CreFasnfl/fl (n = 10) bone-marrow chimaeras were inoculated with B16 cells and euthanized on day 17. c, d, Numbers of conventional CD8+ T cells (c) and Foxp3CD4+ T cells (d) in PLNs (left) and tumours (right; normalized to tumour weight). e, f, Frequencies of CD44hiCD62Llo cells amongst conventional CD8+ T cells (e) and Foxp3CD4+ T cells (f). g, Frequency (left) and number of Foxp3+CD4+ T cells in PLNs (middle) and tumours (right; normalized to tumour weight). h, Ratio of CD8+ T cells to Treg cells. i, Foxp3CreFasn+/+ or Foxp3CreFasn+/fl control (n = 13) and Foxp3CreFasnfl/fl (n = 16) bone-marrow chimaeras were inoculated with B16 cells and euthanized on days 17–19. Shown is the relative frequency of IFNγ+ cells amongst Foxp3+CD4+ Treg cells in PLNs and tumours. j, k, GSEA enrichment plots showing gene signatures of fatty-acid metabolism (j) and genes downregulated in TCR-α-deficient aTreg cells compared with TCR-α-sufficient aTreg cells (k), in intratumoral Treg cells from Foxp3CreFasn+/+ or Foxp3CreFasn+/fl control and Foxp3CreFasnfl/fl mice (n = 3 samples in each group) bearing B16 melanoma. l, Treg cells from Foxp3CreFasn+/fl and Foxp3CreFasnfl/fl mice were used for in vitro suppression assays (n = 3 technical replicates) at multiple ratios of Treg cells to naive CD4+ T cells (TN cells). m, n, Treg cells from Foxp3CreFasn+/fl and Foxp3CreFasnfl/fl mice were labelled with CellTrace Violet (CTV), and cultured with anti-CD3/28 antibodies plus IL-2 for 3 days. Shown are flow-cytometry analysis of the frequency of CTVlo cells (m) and the relative expression of GITR (n, left) and CD44 (n, right) (n = 6 per genotype). o, Relative expression of GITR (left; n = 16 per genotype) and CD44 (right; n = 15 per genotype) on Treg cells in PLNs and tumours of mice bearing B16 melanoma. *P < 0.05; **P < 0.01; two-tailed unpaired Student’s t-test (ai, l, n, o). Data are means ± s.e.m. in ai, l, n, o. Data are representative of four (m) or two (l) or compiled from three (b, i) or two (a, ch, n, o) independent experiments. Values in control samples were set to 1 (a, b, i, n, o).

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Extended Data Fig. 9 SCAP/SREBP signalling promotes the expression of PD-1 by Treg cells in the TME and upon TCR stimulation.

a, Violin plot of Pdcd1 expression in Treg cells from scRNAseq analysis of tumour-infiltrating CD45+ cells in mice challenged with B16 cells (n = 2 samples in each group; see also Fig. 2a). b, MFI of CTLA4, CD69, CD25, Nrp-1, CD39 and CD73 in Foxp3+CD4+ Treg cells in PLNs and tumours from Foxp3CreScap+/+ or Foxp3CreScap+/fl control (n = 5) and Foxp3CreScapfl/fl (n = 6) mice challenged with B16 cells. c, Quantification of PD-1 MFI on Foxp3+CD4+ Treg cells in spleen, lung, fat tissue and skin from Foxp3CreScap+/+ or Foxp3CreScap+/fl control and Foxp3CreScapfl/fl mice under steady state (n = 6 mice in each group). d, C57BL/6 mice were inoculated with MC38 cells on day 0 and treated with either anti-PD-1 (n = 8) or isotype control (n = 10) antibody on days 7, 10, and 13. Mice were euthanized on day 14, and the frequency of IFNγ+ cells amongst Foxp3+CD4+ T cells in PLNs and tumours was analysed by flow cytometry and quantified. eg, Violin enrichment plots analysing Pdcd1 expression in public scRNAseq datasets from the mouse model of B16 melanoma7,8 (e, f) and in individuals with breast cancer9 (g). h, i, Quantification of CTLA4 (h) and ICOS (i) MFIs in Treg cells from spleen and tumours of mice bearing MC38 tumours (left panels; n = 7) or from spleen and spinal cord of mice with MOG-induced EAE (right panels; n = 5). j, Enrichment plot analysing the Treg-cell-specific TCR-dependent gene signature in transcriptome analysis of intratumoral PD-1hi and PD-1lo Treg cells from mice bearing MC38 tumours. k, Flow-cytometry analysis of PD-1 expression on Treg cells cultured in the presence of IL-2, with or without stimulation by anti-CD3 or anti-CD3/28 antibodies, for 3 days. Numbers within graphs indicate MFI. l, Heat map showing the expression of SREBP genes, normalized by row (z-score), in transcriptome analysis of Treg cells (from Foxp3Cre mice) stimulated with anti-CD3/28 antibodies for 0 h and 8 h (n = 5 per time point). m, Enrichment plot analysing SREBP gene targets in transcriptome analysis of Treg cells (from Foxp3Cre mice) stimulated with anti-CD3/28 antibodies for 0 h and 8 h. n, Real-time PCR analysis of Pdcd1 mRNA expression in Treg cells from Foxp3CreScap+/+ or Foxp3CreScap+/fl control and Foxp3CreScapfl/fl mice (n = 3 samples in each group) with or without anti-CD3/28 stimulation for 3 days. *P < 0.05; **P < 0.01; ***P < 0.001; two-tailed unpaired Student’s t-test (bd, h, i, n). Data are means ± s.e.m. in aj, m, n. Data are representative of two (d, h, i) or compiled from two (c) independent experiments.

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Extended Data Fig. 10 Metabolic dependence of PD-1 expression on intratumoral Treg cells, and model of metabolic adaptation.

a, Left, flow-cytometry analysis, and right, quantification of PD-1 MFI on intratumoral Treg cells from Foxp3CreFasn+/+ or Foxp3CreFasn+/fl control (n = 9) and Foxp3CreFasnfl/fl (n = 10) mice bearing B16 melanoma on day 17. b, Resting Treg cells were sorted from Foxp3CreHmgcr+/fl and Foxp3CreHmgcrfl/fl mice under steady state (n = 4 samples per genotype), labelled with CTV, and cultured with anti-CD3/28 antibodies plus IL-2 for 3 days. Shown is relative PD-1 MFI on CTVlo cells. c, d, Foxp3–YFP+CD4+ cells were sorted from Foxp3CreScap+/fl and Foxp3CreScapfl/fl mice, labelled with CTV, and cultured with anti-CD3/28 antibodies plus IL-2 for 3 days in the presence of cholesterol or vehicle (Treg cells were pretreated with cholesterol or vehicle for 1 h before activation; n = 6 samples per group). Shown are flow-cytometry analysis (left) and relative MFI (right) of CTLA4 (c) and PD-1 (d) in CTVlo cells. e, Foxp3–YFP+CD4+ cells were sorted from Foxp3Cre mice, treated with vehicle or simvastatin (2 μM), and then stimulated with anti-CD3/28 antibodies plus IL-2 for 18 h in the presence of mevalonate (500 μM), FPP (50 μM), GGPP (5 μM) or DMSO for real-time PCR analysis of Pdcd1 mRNA expression (n = 4 per group). f, Treg cells from Foxp3Cre mice were labelled with CTV and pretreated with vehicle, FTI-277 (FTI) (10 μM) or GGTI-2147 (GGTI) (5 μM) for 1 h and then stimulated with anti-CD3/28 antibodies plus IL-2 for 72 h. Shown is relative MFI of PD-1 on CTVlo cells (n = 8 per group). g, Resting Treg cells were sorted from young (1–3 weeks) Foxp3CrePggt1b+/fl and Foxp3CrePggt1bfl/fl mice (n = 4 samples per genotype) under steady state; young mice were used to limit the influence of systemic inflammation. Treg cells were labelled with CTV and cultured with anti-CD3/28 antibodies plus IL-2 for 3 days. Left, flow-cytometry analysis, and right, relative MFI of PD-1 on CTVlo cells. h, Resting Treg cells were sorted from young (1–3 weeks) Foxp3CreFntb+/+ (n = 2) and Foxp3CreFntbfl/fl (n = 3) mice under steady state. Treg cells were labelled with CTV and cultured with anti-CD3/28 antibodies plus IL-2 for 3 days. Left, flow-cytometry analysis, and right, relative MFI of PD-1 on CTVlo cells. i, Resting Treg cells were sorted from Foxp3Cre control and Foxp3CreRac1fl/flRac2–/– mice (n = 4 mice per genotype) under steady state, and cultured with anti-CD3/28 antibodies plus IL-2 for 3 days. Left, flow-cytometry analysis, and right, and relative MFI of PD-1. j, Foxp3–YFP+CD4+ cells were sorted from Foxp3Cre mice (n = 5) and stimulated with anti-CD3/28 antibodies plus IL-2 for 3 days, in the presence of vehicle control, simvastatin (2 μM), GGTI (5 μM) or two different AP-1 inhibitors (Sr11302 (30 μM) or T-5224 (120 μM)). Top, flow-cytometry analysis, and bottom, relative MFI of PD-1. k, SREBP-driven metabolic and functional adaptation of Treg cells in the TME. SREBPs are activated in intratumoral Treg cells as a key driver of the functional specialization of Treg cells in the TME, which leads to suppression of effective antitumour immune responses and the reduced efficacy of immune-checkpoint therapy. Mechanistically, SREBP-dependent de novo fatty-acid synthesis and PD-1 signalling in Treg cells enforce the suppressive function of Treg cells in tumours. Treg cells show enhanced Pdcd1 expression in tumours, and this upregulation is dependent on SREBP-dependent mevalonate metabolism that further signals to Pggt1b-driven protein geranylgeranylation. PD-1 induction is required to repress overt production of IFNγ by Treg cells in response to TCR activation in the TME, suggesting that SREBP signalling supports the functional fitness of intratumoral Treg cells, in part by preventing the fragility of Treg cells. Therefore, SREBP-dependent metabolic reprogramming enforces the functional specialization of Treg cells in tumours by coordinating lipid synthesis and inhibitory receptor signalling pathways. *P < 0.05; **P < 0.01; ***P < 0.001; two-tailed unpaired Student’s t-test (a, b, gi) or one-way ANOVA (cf, j). Data are means ± s.e.m. in aj. Data are compiled from three (bd, f, g) or two (a, e, hj) independent experiments. Values in control samples were set to 1 (a–d, f–j).

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Supplementary Table 1: Cell metabolism-related pathways. This file contains the list of cell metabolism-related pathways curated from the KEGG (Kyoto Encyclopedia of Genes and Genomes) and Reactome databases. The first column lists the pathway name, the second column is original gene size, and the third column lists the gene size after restricting to dataset.

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Lim, S.A., Wei, J., Nguyen, TL.M. et al. Lipid signalling enforces functional specialization of Treg cells in tumours. Nature 591, 306–311 (2021). https://doi.org/10.1038/s41586-021-03235-6

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