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.

Conflict of interest statement

Competing interests

H. Chi is a consultant for Kumquat Biosciences, Inc.

Figures

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

(a) Diagram of de novo lipid synthesis biochemical pathways. (b) Enrichment plots of SREBP gene targets in intratumoural versus PLN-derived Treg cells from B16 melanoma-bearing Foxp3Cre mice. (c) Top 15 upstream transcriptional regulators enriched in intratumoural Treg cells as compared to PLN Treg cells from B16 melanoma-bearing Foxp3Cre mice, analyzed by Ingenuity pathway analysis. (d, e) Enrichment of SREBP gene targets in intratumoural and PLN Treg cells from public mouse B16 melanoma scRNA-seq datasets,. (f, g) Enrichment of SREBP gene targets in intratumoural Treg cells from human patients with breast cancer (f) and HNSCC (g). (h) Enrichment plots of SREBP gene targets from the public dataset of in vivo activated Treg (aTreg) compared to 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 scRNA-seq analysis. (i) Unsupervised clustering of cells in the spleen and CNS was analyzed by UMAP, with the splenic and CNS cells annotated with different colors (left), and the expression of SREBP gene targets visualized on the UMAP plot (right). (j) Comparison of SREBP gene targets between splenic and CNS Treg cells on the UMAP plot. (k) Relative (values in splenic samples were set to 1) uptake of a fluorescent glucose analog, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), in splenic and intratumoural Treg cells from MC38 tumour-bearing mice on day 21 after tumour inoculation (n = 17). (l) Relative (values in splenic samples were set to 1) 2-NBDG expression (n = 14) in Treg cells from the spleen and spinal cord of MOG-induced EAE mice on day 16 after MOG immunization. (m) Scap mRNA expression was examined in Treg, naïve CD4+, and CD8+ T cells from Foxp3CreScap+/fl and Foxp3CreScapfl/fl mice under steady state (n = 5 samples per genotype). (n) Heat map of SREBP gene target expression normalized by row (z-score) in the tumour-infiltrating Treg cells from B16 melanoma-bearing mice on day 19 after tumour inoculation (n = 4, Foxp3CreScap+/+ or +/fl 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 (k–m) or two-way ANOVA (o). Data are mean ± s.e.m. in d–g, j–m, o. Data are representative of two (o), or compiled from two (k, l) independent experiments.

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

(a–d) Analyses of Foxp3CreScap+/+ or +/fl (Foxp3CreScap+/+ or Foxp3CreScap+/fl mice that were phenotypically indistinguishable) and Foxp3CreScapfl/fl mice (12–18 weeks) under steady state (n = 4 mice per genotype). (a) Cellularity of Foxp3–CD4+ and CD8+ T cells in spleen and PLNs. (b) Flow cytometry analysis of the expression of CD62L and CD44 (left) and quantification of the frequency of CD44hiCD62Llo cells (right) in Foxp3–CD4+ (upper) or CD8+ T cells (lower) from spleen and PLNs. (c) Flow cytometry analysis of the expression of IFN-γ, IL-4 and IL-17 (left), and quantification of frequencies of IFN-γ+, IL-4+ and IL-17+ cells (right) in Foxp3–CD4+ T cells from spleen and PLNs. (d) Flow cytometry analysis of the expression of IFN-γ (left), and quantification of frequency of IFN-γ+ cells (right) in CD8+ T cells from spleen and PLNs. (e) Hematoxylin and eosin staining of spleen, PLNs, colon, pancreas, lung, liver, and skin from age-matched Foxp3CreScap+/+ or +/fl 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 (> 3 months old) (n = 23, Foxp3CreScap+/fl mice; n = 20, Foxp3CreScapfl/fl mice). (h) Survival curve of Foxp3CreScap+/+ or +/fl (n = 17) and Foxp3CreScapfl/fl mice (n = 27). **P < 0.01, NS, not significant. Two-tailed unpaired Student’s t-test (a–d, f, g). Data are mean ± s.e.m. Data are compiled from two (a–d) independent experiments.

Extended Data Figure 3.. SCAP is dispensable for Treg cell function in autoimmune and acute inflammation.

(a) Foxp3CreScap+/+ or +/fl (n = 13) and Foxp3CreScapfl/fl (n = 14) mice were immunized with MOG and EAE disease score was measured every day for 20 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 sacrificed in the recovery phase (day 22–25) of EAE for histological analysis of clinical disease. Hematoxylin and eosin staining of the spinal cord (left) and the histological scores of the indicated regions of the brain and spinal cord (right) (n = 9, Foxp3CreScap+/+ or +/fl mice; n = 10, Foxp3CreScapfl/fl mice). (c, d) Foxp3CreScap+/+ or +/fl and Foxp3CreScapfl/fl mice were immunized with MOG to induce EAE (n = 5 mice per genotype) and sacrificed on day 16 for analysis. (c) Cellularity of Foxp3–CD4+ (upper) and CD8+ (lower) T cells in PLNs, spleen and spinal cord. (d) Quantification of percentages (left) and numbers (right) of IFN-γ+ (upper) and IL-17+ (lower) Foxp3–CD4+ T cells from PLNs, spleen and spinal cord. (e–h) Foxp3Cre/DTRScap+/+ or +/fl and Foxp3Cre/DTRScapfl/fl mosaic mice were treated with diphtheria toxin (DT) (e, upper, for experimental design; n = 6 mice per genotype) and sacrificed 3 days after the final DT injection. (e) Quantification of cellularity of Foxp3–CD4+ (lower left) and CD8+ T cells (lower right). (f) Quantification of the frequency of CD44hiCD62Llo cells in Foxp3–CD4+ (upper) and CD8+ (lower) T cells from spleen and PLNs. (g) Quantification of frequencies of IFN-γ+ (left), IL-4+ (middle) and IL-17+ (right) cells in Foxp3–CD4+ T cells from spleen and PLNs. (h) Quantification of frequency of IFN-γ+ cells in CD8+ T cells from spleen and PLNs. NS, not significant. Two-tailed unpaired Student’s t-test (b–h) or two-way ANOVA (a). Data are mean ± s.e.m. in a–h. Data are compiled from three (a) or two (b, e–h) independent experiments.

Extended Data Figure 4.. SCAP/SREBP signalling maintains Treg cell functional state in the TME.

(a–d) Foxp3CreScap+/+ or +/fl (n = 11) and Foxp3CreScapfl/fl (n = 14) mice were inoculated with B16 cells. Cellularity of CD8+ T cells (gated as CD8α+TCRβ+) (a) and Foxp3–CD4+ T cells (b) in PLNs (left) and tumours (right; normalized to tumour weight to account for differences in tumour size between the genetic models) on day 19. (c, d) Quantification of numbers of CD44hiCD62Llo (Tem) cells (c) and CD44hiCD62Lhi (Tcm) cells (d) in CD8+ T cells from tumours. (e) Foxp3CreScap+/+ or +/fl (n = 12) and Foxp3CreScapfl/fl (n = 14) mice were inoculated with B16 cells. Quantification of number of Tim3+PD-1+ (Tex) cells in CD8+ T cells from tumours. (f, g) Foxp3CreScap+/+ or +/fl (n = 11) and Foxp3CreScapfl/fl (n = 14) mice were inoculated with B16 cells. Flow cytometry analysis of the expression of TNF-α and IFN-γ (left), and relative frequency of TNF-α+ cells (middle) and IFN-γ+ cells (right) in CD8+ T cells (f) and Foxp3–CD4+ T cells (g) from PLNs and tumours. (h) Foxp3CreScap+/+ or +/fl and Foxp3CreScapfl/fl mice were inoculated with MC38 cells on day 0 and treated with anti-CD8 on days 1, 2, 5, 8 and 11. Tumour growth was measured (n = 6 mice per genotype). *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant. Two-tailed unpaired Student’s t-test (a–g) or two-way ANOVA (h). Data are mean ± s.e.m. in a–h. Data are representative of two (h), or compiled from two (a–g) independent experiments.

Extended Data Figure 5.. Scap-deficient Treg cellular state in tumours and homeostasis.

(a) Foxp3CreScap+/+ or +/fl and Foxp3CreScapfl/fl mice were inoculated with B16 melanoma cells and sacrificed on day 19. Flow cytometry analysis (left panel) and quantification of frequency (second panel) and number (third panel shows raw values of cell number; right panel shows cell number normalized to tumour weight) of Foxp3+CD4+ T cells in PLNs and tumours (n = 11, Foxp3CreScap+/+ or +/fl mice; n = 14, Foxp3CreScapfl/fl mice). (b) Quantification of number of CD45.2+ cells from B16 melanoma-bearing Foxp3CreScap+/+ or +/fl control (n = 11) and Foxp3CreScapfl/fl (n = 14) mice. (c) Quantification of frequency (left) and number (right) of Foxp3+CD4+ Treg cells in spleen, lung, fat tissue and skin in Foxp3CreScap+/+ or +/fl control and Foxp3CreScapfl/fl mice under steady state (n = 6 mice per genotype). (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. Treg cells were pre-treated with cholesterol for 1 h or vehicle before activation (n = 6 per group). Relative frequency of CTVlo cells. (e) Foxp3CreScap+/+ or +/fl control (n = 11) and Foxp3CreScapfl/fl (n = 13) mice were inoculated with B16 cells. Relative frequency of BrdU+ cells among Foxp3+CD4+ Treg cells from PLNs and tumours. (f) Treg cells from Foxp3CreScap+/+ or +/fl control and Foxp3CreScapfl/fl mice were labelled with CTV and transferred into Rag1−/− mice. Mice were sacrificed on day 10 for analysis. Quantification of cellularity of Foxp3+CD4+ Treg cells in spleen and PLNs (n = 7 mice per genotype). (g) Foxp3CreScap+/+ or +/fl control and Foxp3CreScapfl/fl mice received i.p. injection of IL-2/anti-IL-2 complex daily on days 0–2 to induce Treg cell expansion, and analyzed on day 5. Quantification of number of Foxp3+CD4+ Treg cells in spleen and PLNs (n = 4 mice per genotype). (h) Treg cells from Foxp3CreScap+/+ or +/fl control and Foxp3CreScapfl/fl mice were transferred into Rag1−/− recipients and analyzed as in (f). Quantification of mean fluorescence intensity (MFI) of CTLA4 in Treg cells (n = 7 mice per genotype) from spleen and PLNs. (i) Relative MFI of CTLA4 in Treg cells from IL-2/anti-IL-2 complex-treated Foxp3CreScap+/+ or +/fl control and Foxp3CreScapfl/fl mice (n = 4 mice per genotype) as in (g). (j) Foxp3CreScap+/+ or +/fl and Foxp3CreScapfl/fl mice were inoculated with B16 melanoma cells and sacrificed on day 19. Flow cytometry analysis (left) and relative frequency (right) of active caspase-3+ Treg cells from PLNs and tumours (n = 11, Foxp3CreScap+/+ or +/fl control mice; n = 13, Foxp3CreScapfl/fl mice). (k–m) Female Foxp3Cre/+Scap+/fl control and Foxp3Cre/+Scapfl/fl mosaic mice were challenged with B16 cells and sacrificed on day 14 (n = 15 mice per genotype). (k) Relative frequency of 7AAD+ cells in 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. (n–p) Foxp3CreScap+/+ or +/fl control and Foxp3CreScapfl/fl mice were inoculated with B16 cells and sacrificed on day 19. CD36 expression (n = 8, Foxp3CreScap+/+ or +/fl mice; n = 10, Foxp3CreScapfl/fl mice) (n), uptake of BODIPY FL C12 (n = 5 mice per genotype) (o), MitoTracker (n = 3, Foxp3CreScap+/+ or +/fl mice; n = 6, Foxp3CreScapfl/fl mice) (p, left), TMRM expression (n = 4, Foxp3CreScap+/+ or +/fl mice; n = 6, Foxp3CreScapfl/fl mice) (p, middle) and MitoSOX expression (n = 11, Foxp3CreScap+/+ or +/fl mice; n = 14, Foxp3CreScapfl/fl mice) (p, right) in Treg cells. (q) Quantification of the frequency (left) and number (right) of CD44hiCD62Llo aTreg cells among Foxp3+CD4+ Treg cells from spleen and PLNs under steady state (n = 4 mice per genotype). (r) Quantification of frequency (left) and number of aTreg cells in tumours (right; normalized to tumour weight) from Foxp3CreScap+/+ or +/fl control (n = 11) and Foxp3CreScapfl/fl (n = 14) mice challenged with B16 cells. * P < 0.05, **P < 0.01, *** P < 0.001, NS, not significant. Two-tailed unpaired Student’s t-test (a–c, e–r) and one-way ANOVA (d). Data are mean ± s.e.m. in a–r. Data are representative of two (f; h; o; p, left, middle), or compiled from three (d, e, j) or two (a–c; g; i; k–n; p, right; q; r) independent experiments. Values in control samples were set to 1 (d; e; i; j; k–m; p, right).

Extended Data Figure 6.. Scap-deficient Treg functional integrity in tumours and homeostasis.

(a) Flow cytometry analysis (left) and quantification of the mean fluorescence intensity (MFI) of Foxp3-YFP (right) in intratumoural Treg cells from B16 melanoma-bearing Foxp3CreScap+/+ or +/fl control (n = 5) and Foxp3CreScapfl/fl (n = 6) mice. (b) Flow cytometry analysis (left) and quantification of the MFI (right) of Foxp3 protein in intratumoural Treg cells from B16 melanoma-bearing Foxp3CreScap+/+ or +/fl control (n = 5) and Foxp3CreScapfl/fl (n = 6) mice. (c) Flow cytometry analysis (left) and quantification of the MFI (right) of Foxp3 protein in Treg cells (from Foxp3CreScap+/fl control and Foxp3CreScapfl/fl mice) transferred into Rag1−/− recipients (n = 7 mice per genotype). (d) Quantification of frequency of IFN-γ+ cells in Foxp3+CD4+ Treg cells in spleen and PLNs from Foxp3CreScap+/+ or +/fl control and Foxp3CreScapfl/fl mice under steady state (n = 4 mice per genotype). (e) Violin plots of gene expression in macrophages from scRNA-seq analysis of tumour-infiltrating CD45+ cells in mice challenged with B16 melanoma cells (n = 2 mice per genotype; see also Fig. 2a). (f–h) Foxp3GFP-Cre-ERT2Scap+/flRosa26YFP and Foxp3GFP-Cre-ERT2Scapfl/flRosa26YFP mice were injected with MC38 colon adenocarcinoma cells on day 0, treated with tamoxifen daily on days 7–11 (n = 6 mice per genotype), and analyzed on day 21 after tumour injection. (f) Quantification of the frequencies of IFN-γ+ cells among GFP+YFP– and GFP+YFP+ Treg cells from PLNs (left) and tumours (right). (g) Quantification of frequencies of IFN-γ+ (left) and TNF-α+ (right) cells among CD8+ T cells in PLNs and tumours. (h) Quantification of frequencies of IFN-γ+ (left) and TNF-α+ (right) cells among Foxp3–CD4+ T cells in PLNs and tumours. *P < 0.05, NS, not significant. Two-tailed unpaired Student’s t-test (a–d, f–h). Data are mean ± s.e.m. in a–h. Data are representative of two (a–c), or compiled from two (d) independent experiments.

Extended Data Figure 7.. FASN is dispensable for Treg cell function in steady state and autoimmunity.

(a) Hmgcr mRNA expression was examined in Treg cells from Foxp3CreScap+/fl and Foxp3CreScapfl/fl mice under steady state (n = 5 samples per genotype). (b) Hmgcr mRNA expression was examined in Treg cells from Foxp3CreHmgcr+/+ (n = 4), Foxp3CreHmgcr+/fl (n = 6), and Foxp3CreHmgcrfl/fl (n = 4) mice under steady state. (c) Analyses of T cell homeostasis in Foxp3CreHmgcr+/+ and Foxp3CreHmgcr+/fl mice (6–10 weeks; n = 3 mice per genotype). Quantification of the frequency of CD44hiCD62Llo cells in Foxp3–CD4+ (left) and CD8+ T cells (right) from spleen and PLNs. (d) Fasn mRNA expression was examined in Treg, naïve CD4+, and CD8+ T cells from Foxp3CreFasn+/fl and Foxp3CreFasnfl/fl mice under steady state (n = 3 samples per genotype). (e–i) Analysis of spleen and PLNs from Foxp3CreFasn+/+ or +/fl control and Foxp3CreFasnfl/fl mice (8–12 weeks) under steady state (n = 4 mice per genotype). Quantification of numbers of Foxp3+CD4+ Treg cells (e) and Foxp3–CD4+ (f, left) and CD8+ (f, right) T cells. (g) Quantification of frequencies of CD44hiCD62Llo cells in Foxp3–CD4+ (left) and CD8+ (right) T cells. (h) Quantification of frequencies of IFN-γ+ (left), IL-4+ (middle) and IL-17+ (right) cells among Foxp3–CD4+ T cells. (i) Quantification of frequency of IFN-γ+ cells among CD8+ T cells. (j) Foxp3CreFasn+/+ or +/fl (n = 5) and Foxp3CreFasnfl/fl (n = 6) mice were immunized with MOG and EAE disease score was measured every day until day 24. (k) Mice as in (j) were sacrificed in the recovery phase (day 24) of EAE for histological analysis of clinical disease. Hematoxylin and eosin staining of the spinal cord (left) and the histological scores of the indicated regions of the brain and spinal cord (right) (n = 5, Foxp3CreFasn+/+ or +/fl mice; n = 6, Foxp3CreFasnfl/fl mice). ***P < 0.001, NS, not significant. Two-tailed unpaired Student’s t-test (a, c–i, k), one-way ANOVA (b), or two-way ANOVA (j). Data are mean ± s.e.m. in a–k. Data are compiled from two (e–i) independent experiments.

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

(a, b) Foxp3CreFasn+/+ or +/fl control (a, n = 9; b, n = 13) and Foxp3CreFasnfl/fl (a, n = 10; b, n = 16) bone marrow chimeras were inoculated with B16 cells and sacrificed on day 17 (a) or days 17–19 (b). Relative frequencies of TNF-α+ cells (left) and IFN-γ+ cells (right) among CD8+ T cells (a) and Foxp3–CD4+ T cells (b). (c–h) Foxp3CreFasn+/+ or +/fl control (n = 9) and Foxp3CreFasnfl/fl (n = 10) bone marrow chimeras were inoculated with B16 cells and sacrificed on day 17. (c, d) Quantification of numbers of CD8+ T cells (c) and Foxp3–CD4+ T cells (d) in PLNs (left) and tumours (right; normalized to tumour weight). (e, f) Quantification of frequency of CD44hiCD62Llo cells in CD8+ T cells (e) and Foxp3–CD4+ T cells (f). (g) Quantification of frequency (left) and number of Foxp3+CD4+ T cells in PLNs (middle) and tumours (right; normalized to tumour weight). (h) Quantification of CD8/Treg cell ratio. (i) Foxp3CreFasn+/+ or +/fl control (n = 13) and Foxp3CreFasnfl/fl (n = 16) bone marrow chimeras were inoculated with B16 cells and sacrificed on days 17–19. Relative frequency of IFN-γ+ cells among Foxp3+CD4+ Treg cells in PLNs and tumours. (j, k) GSEA enrichment plots of fatty acid metabolism (j) and Treg cell-specific TCR gene signature (genes downregulated in TCRα-deficient aTreg cells compared to TCRα-sufficient aTreg cells) (k) in intratumoural Treg cells from B16 melanoma-bearing Foxp3CreFasn+/+ or +/fl and Foxp3CreFasnfl/fl mice (n = 3 samples per genotype). (l) Treg cells from Foxp3CreFasn+/fl control and Foxp3CreFasnfl/fl mice were used for in vitro suppression assays at multiple Treg versus TN (naïve CD4+ T cell) ratios. (m, n) Treg cells from control 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. Flow cytometry analysis for frequency of CTVlo cells (m) and relative expression of GITR (n, left) and CD44 (n, right) (n = 6 per genotype). (o) Relative expression of GITR (n = 16 per genotype) (o, left) and CD44 (n = 15 per genotype) (o, right) on Treg cells in PLNs and tumours of B16 melanoma-bearing mice. *P < 0.05, **P < 0.01, NS, not significant. Two-tailed unpaired Student’s t-test (a–i, l, n, o). Data are mean ± s.e.m. in a–i, l, n, o. Data are representative of four (m) or two (l), or compiled from three (b, i) or two (a, c–h, n, o) independent experiments. Values in control samples were set to 1 (a, b, i, n, o).

Extended Data Figure 9.. SCAP/SREBP signalling promotes Treg cell PD-1 expression in the TME and upon TCR stimulation.

(a) Violin plot of Pdcd1 expression in Treg cells from scRNA-seq analysis of tumour-infiltrating CD45+ cells in mice challenged with B16 cells (n = 2 samples per genotype; see also Fig. 2a). (b) Quantification of mean fluorescence intensity (MFI) of CTLA4, CD69, CD25, Nrp-1, CD39 and CD73 on Foxp3+CD4+ Treg cells in PLNs and tumours from Foxp3CreScap+/+ or +/fl control (n = 5) and Foxp3CreScapfl/fl (n = 6) mice challenged with B16 melanoma cells. (c) Quantification of PD-1 MFI in Foxp3+CD4+ Treg cells in spleen, lung, fat tissue and skin from Foxp3CreScap+/+ or +/fl and Foxp3CreScapfl/fl mice under steady state (n = 6 mice per genotype). (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 sacrificed on day 14, and frequency of IFN-γ+ cells in Foxp3+CD4+ T cells in PLNs and tumours were analyzed by flow cytometry and quantified. (e–g) Violin enrichment plot of Pdcd1 expression from public scRNA-seq datasets from the mouse model of B16 melanoma tumours, (e, f) and human patients with breast cancer (g). (h, i) Quantification of CTLA4 (h) and ICOS (i) MFIs in Treg cells from spleen and tumours in MC38 colon adenocarcinoma tumour-bearing mice (left panels; n = 7) or from spleen and spinal cord in MOG-induced EAE mice (right panels; n = 5). (j) Enrichment plot of Treg cell-specific TCR gene signature in transcriptome analysis of intratumoural PD-1hi and PD-1lo Treg cells from MC38 tumour-bearing mice. (k) Flow cytometry analysis of PD-1 expression on Treg cells cultured in the presence of IL-2 with or without anti-CD3 or anti-CD3/28 antibody stimulation for 3 days. Numbers in graphs indicate MFI. (l) Heat map of SREBP gene target expression 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 of 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 +/fl control and Foxp3CreScapfl/fl mice (n = 3 samples per genotype) with or without anti-CD3/28 stimulation for 72 h. *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant. Two-tailed unpaired Student’s t-test (b–d, h, i, n). Data are mean ± s.e.m. in a–j, m, n. Data are representative of two (d, h, i), or compiled from two (c) independent experiments.

Extended Data Figure 10.. Metabolic dependence of PD-1 expression on intratumoural Treg cells and model of metabolic adaptation.

(a) Flow cytometry analysis (left) and quantification of relative mean fluorescence intensity (MFI) (right) of PD-1 on intratumoural Treg cells from B16 melanoma-bearing Foxp3CreFasn+/+ or +/fl (n = 9) and Foxp3CreFasnfl/fl (n = 10) mice on day 17. (b) Resting Treg cells were sorted from Foxp3CreHmgcr+/fl control and Foxp3CreHmgcrfl/fl mice under steady state (n = 4 samples per genotype), labelled with CellTrace Violet (CTV), and cultured with anti-CD3/28 antibodies plus IL-2 for 3 days. Relative MFI of PD-1 on CTVlo cells. (c, d) Foxp3-YFP+CD4+ cells were sorted from Foxp3CreScap+/fl control and Foxp3CreScapfl/fl mice, labelled with CTV, and cultured with anti-CD3/28 antibodies plus IL-2 for 3 days. Treg cells were pre-treated with cholesterol or vehicle for 1 h before activation (n = 6 samples per group). Flow cytometry analysis (left) and quantification of 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). (f) Treg cells from Foxp3Cre mice were labelled with CTV and pre-treated 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. 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 control 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. Flow cytometry analysis (left) and relative MFI (right) of PD-1 on CTVlo cells. (h) Resting Treg cells were sorted from young (1–3 weeks) Foxp3CreFntb+/+ control (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. Flow cytometry analysis (left) and relative MFI (right) 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. Flow cytometry analysis (left) and relative MFI (right) 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 mM), GGTI (5 mM), or two different AP-1 inhibitors [Sr11302 (30 mM) or T-5224 (120 mM)]. Flow cytometry analysis (upper) and relative MFI of PD-1 (lower). (k) Schematic of SREBP-driven metabolic and functional adaptation of Treg cells in the TME. SREBPs are activated in intratumoural Treg cells as a key driver of Treg cell functional specialization 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 Treg cell suppressive function 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 IFN-γ production by Treg cells in response to TCR activation in the TME, suggesting that SREBP signalling supports intratumoural Treg cell functional fitness, in part, by preventing Treg cell fragility. Therefore, SREBP-dependent metabolic reprogramming enforces Treg cell functional specialization in tumours by coordinating lipid synthesis and inhibitory receptor signalling pathways. *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant. Two-tailed unpaired Student’s t-test (a, b, g–i) or one-way ANOVA (c–f, j). Data are mean ± s.e.m. in a–j. Data are compiled from three (b–d, f, g) or two (a, e, h–j) independent experiments. Values in control samples were set to 1 (a–d, f–j).

Figure 1.. Treg cells upregulate SREBP signalling and require SCAP/SREBP activity for their functional adaptation in tumours.

(a) List of the enriched cell metabolism-related pathways in intratumoural Treg cells compared to those from PLNs (n = 4 per group) from B16 melanoma tumour-bearing mice. (b, c) Foxp3CreScap+/+ or +/fl (n = 6) and Foxp3CreScapfl/fl (n = 5) mice were inoculated with MC38 cells (b) or B16 cells (c), and tumour growth was measured. (d, e) Foxp3GFP-Cre-ERT2Scap+/flRosa26YFP (d, n = 6; e, n = 6) and Foxp3GFP-Cre-ERT2Scapfl/flRosa26YFP (d, n = 6; e, n = 7) mice were injected with MC38 cells on day 0 and treated with tamoxifen on days 7–11 (d, left) or days 21–25 (e, left). Tumour growth (right) was measured. (f) Foxp3CreScap+/fl and Foxp3CreScapfl/fl mice were inoculated with B16 cells on day 0 and treated with either anti-PD-1 or isotype control antibody on days 7, 10, 13, and 16 (left). Tumour growth (right) was measured (n = 7, Foxp3CreScap+/fl + isotype; n = 6, Foxp3CreScap+/fl + anti-PD-1; n = 5, Foxp3CreScapflfl + isotype; n = 5, Foxp3CreScapfl/fl + anti-PD-1). *P < 0.05, ***P < 0.001. Two-way ANOVA (b–f). Data are mean ± s.e.m. in b–f. Data are representative of five (c) or two (b, d, e) independent experiments.

Figure 2.. SREBPs maintain Treg cell functional fitness in the TME.

(a, b) Foxp3CreScap+/+ or +/fl and Foxp3CreScapfl/fl mice (n = 2 mice per genotype) were inoculated with B16 cells and CD45+ tumour-infiltrating lymphocytes (TILs) were isolated on day 19, followed by scRNA-seq analysis. (a) UMAP embeddings of merged scRNA-seq showing distribution of CD45+ TILs (left) and immune cell subsets (right). (b) Proportions of immune cell subsets of scRNA-seq. (c) Foxp3CreScap+/+ or +/fl (n = 18) and Foxp3CreScapfl/fl (n = 23) mice were inoculated with B16 cells and sacrificed on day 19 for the quantification of CD8/Treg cell ratio in PLNs and tumours. (d) Foxp3CreScap+/+ or +/fl control (n = 11) and Foxp3CreScapfl/fl (n = 14) mice were inoculated with B16 cells, and flow cytometry analysis of IFN-γ expression in Foxp3+CD4+ T cells (left panel) and relative frequency (right panel; values in control samples were set to 1) of IFN-γ in Foxp3+CD4+ T cells from PLNs and tumours. (e) B16 cells were inoculated into Foxp3Cre/+Scap+/+ or +/fl and Foxp3Cre/+Scapfl/fl bone marrow chimeras (n = 4 mice per genotype). Tumour growth was measured. (f) MC38 cells were inoculated into Foxp3Cre/+Scap+/+ or +/fl and Foxp3Cre/+Scapfl/fl mosaic mice (n = 7 mice per genotype). Tumour growth was measured. **P < 0.01, ***P < 0.001, NS, not significant. Two-tailed unpaired Student’s t-test (c, d) or two-way ANOVA (e, f). Data are mean ± s.e.m. in c–f. Data are representative of two (e, f), or compiled from four (c) or two (d) independent experiments.

Figure 3.. SREBPs orchestrate intratumoural Treg cell function through coordinating de novo lipid synthesis and PD-1 expression.

(a, b) Enrichment plots showing downregulated cholesterol homeostasis (a) and fatty acid metabolism (b) in intratumoural Treg cells from Foxp3CreScap+/+ or +/fl (n = 4) mice versus Foxp3CreScapfl/fl (n = 3) mice. (c, d) Foxp3CreFasn+/+ or +/fl (c, n = 6; d, n = 5) and Foxp3CreFasnfl/fl (c, n = 7; d, n = 5) mice were inoculated with MC38 (c) or B16 cells (d). Tumour growth was measured. (e) Control and FASN-deficient Treg cells were activated in the presence or absence of palmitate, followed by in vitro suppression assay. TN, naïve CD4+ T cells. (f) PD-1 (left) and ICOS (right) levels on Treg cells in PLNs and tumours from B16 melanoma-bearing Foxp3CreScap+/+ or +/fl (n = 5) and Foxp3CreScapfl/fl (n = 6) mice on day 19. (g, h) MC38 tumour-bearing mice were treated with anti-PD-1 or isotype control antibody on days 7, 10, and 13 (n = 20 per group). Quantified p-AKT (g) and p-S6 (h) MFIs in Foxp3+CD4+ T cells in PLNs and tumours on day 14. (i, j) Foxp3CreScap+/+ or +/fl control and Foxp3CreScapfl/fl mice were inoculated with B16 cells. Quantified p-AKT (i) and p-S6 (j) MFIs in intratumoural Treg cells in PLNs (n = 17 per genotype) and tumours (n = 14, Foxp3CreScap+/+ or +/fl mice; n = 15, Foxp3CreScapfl/fl mice) on day 14. *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant. Two-tailed unpaired Student’s t-test (f–j) or one-way ANOVA (e) or two-way ANOVA (c, d). Data are mean ± s.e.m. in c–i. Data are representative of three (d) or two (c, e, f), or compiled from two (g–j) independent experiments. Values in control samples were set to 1 (g–j).

Figure 4.. Mevalonate metabolism-driven protein geranylgeranylation enforces Treg cell PD-1 upregulation.

(a) PD-1 expression on Treg cells from MC38 tumour-bearing mice (n = 7; day 21) or MOG-induced EAE mice (n = 5; day 16). (b) Violin plot of Pdcd1 expression in Treg cells from scRNA-seq dataset of human patients with HNSCC. (c) Real-time PCR analysis of relative Pdcd1 and Icos mRNA expression in Treg cells from B16 melanoma-bearing Foxp3CreScap+/+ or +/fl and Foxp3CreScapfl/fl mice (n = 4 samples per genotype). (d) Flow cytometry analysis (left) and quantification (right) of Nur77-GFP expression in PD-1hi and PD-1lo Treg cells in B16 melanoma (n = 4). (e) Relative PD-1 expression on Foxp3CreScap+/+ or +/fl or Foxp3CreScapfl/fl Treg cells (n = 6 samples per genotype) that were stimulated by anti-CD3/28 antibodies plus IL-2 for 3 days. (f) Incorporation of 13C into cholesterol in control and SCAP-deficient Treg cells (n = 3 samples per genotype) that were stimulated with anti-CD3/28 antibodies plus IL-2 in the presence of [13C2]-sodium acetate for 2 days. (g) Relative PD-1 expression on vehicle- or simvastatin-treated Treg cells that were stimulated with anti-CD3/28 antibodies plus IL-2 for 3 days (n = 8). (h) Flow cytometry analysis (left) and quantification (right) of relative PD-1 expression on vehicle- or simvastatin-treated CellTrace Violet (CTV)lo Treg cells that were stimulated with anti-CD3/28 antibodies plus IL-2 for 3 days in the presence of mevalonate, FPP, GGPP, or DMSO (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant. Two-tailed unpaired Student’s t-test (a, c–g) or one-way ANOVA (h). Data are mean ± s.e.m. in a–h. Data are representative of two (a), or compiled from three (g) or two (e, h) independent experiments.