Limiting metabolic competition in the tumour microenvironment may increase the effectiveness of immunotherapy. Owing to its crucial role in the glucose metabolism of activated T cells, CD28 signalling has been proposed as a metabolic biosensor of T cells1. By contrast, the engagement of CTLA-4 has been shown to downregulate T cell glycolysis1. Here we investigate the effect of CTLA-4 blockade on the metabolic fitness of intra-tumour T cells in relation to the glycolytic capacity of tumour cells. We found that CTLA-4 blockade promotes metabolic fitness and the infiltration of immune cells, especially in glycolysis-low tumours. Accordingly, treatment with anti-CTLA-4 antibodies improved the therapeutic outcomes of mice bearing glycolysis-defective tumours. Notably, tumour-specific CD8+ T cell responses correlated with phenotypic and functional destabilization of tumour-infiltrating regulatory T (Treg) cells towards IFNγ- and TNF-producing cells in glycolysis-defective tumours. By mimicking the highly and poorly glycolytic tumour microenvironments in vitro, we show that the effect of CTLA-4 blockade on the destabilization of Treg cells is dependent on Treg cell glycolysis and CD28 signalling. These findings indicate that decreasing tumour competition for glucose may facilitate the therapeutic activity of CTLA-4 blockade, thus supporting its combination with inhibitors of tumour glycolysis. Moreover, these results reveal a mechanism by which anti-CTLA-4 treatment interferes with Treg cell function in the presence of glucose.
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Human melanoma RNA-seq datasets investigated in this study were previously reported9,10 and have been deposited to the Gene Expression Omnibus (GEO) repository with accession number GSE165278. The 4T1 RNA-seq datasets generated for this study have been submitted to the GEO repository with accession number GSE164051. Source data are provided with this paper.
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We thank the Flow Cytometry and Integrated Genomics Operation Cores at MSK for technical assistance. We thank A. Rudensky for the constructive discussions for the revisions of this manuscript. This research was funded in part through the NIH/NCI R01 CA215136-01A1 and Cancer Center Support Grant P30 CA008748, the Swim Across America, Ludwig Institute for Cancer Research, Parker Institute for Cancer Immunotherapy and Breast Cancer Research Foundation. R.Z. was supported by the Parker Institute for Cancer Immunotherapy scholar and bridge scholar awards. I.S. was supported by R50 CA221810 (NIH/NCI) grant. M.J.W. was supported by T32CA082084 and F31AI149971. P.-C.H. was supported by the SNSF project grants (31003A_182470) and European Research Council Staring Grant (802773-MitoGuide). G.M.D. was supported by DP2AI136598 and R21AI135367.
R.Z. is inventor on patent applications related to work on GITR, PD-1 and CTLA-4. R.Z. is consultant for Leap Therapeutics and iTEOS Belgium SA. Y.S. is currently employed by Genentech and holds equity in Roche. P.-C.H. received research support from Roche-pRED and honorarium from Chungai and Pfizer. P.-C.H. is also a scientific advisory board member of Elixiron Immunotherapeutics and Acepodia. G.M.D. consults for and/or is on the scientific advisory board of BlueSphere Bio, Century Therapeutics, Novasenta, Pieris Pharmaceuticals, and Western Oncolytics/Kalivir; has grants from bluebird bio, Novasenta, Pfizer, Pieris Pharmaceuticals, TCR2, and Kalivir; G.M.D. owns equity in BlueSphere Bio and Novasenta. T.M. is a cofounder and holds an equity in IMVAQ Therapeutics. T.M. is a consultant of Immunos Therapeutics, Pfizer and Immunogenesis. T.M. has research support from Bristol-Myers Squibb; Surface Oncology; Kyn Therapeutics; Infinity Pharmaceuticals, Inc.; Peregrine Pharmaceuticals, Inc.; Adaptive Biotechnologies; Leap Therapeutics, Inc.; and Aprea. T.M. has patents on applications related to work on oncolytic viral therapy, alpha virus-based vaccines, neo-antigen modelling, CD40, GITR, OX40, PD-1 and CTLA-4. J.D.W. is consultant for Adaptive Biotech; Amgen; Apricity; Ascentage Pharma; Astellas; AstraZeneca; Bayer; Beigene; Boehringer Ingelheim; Bristol Myers Squibb; Celgene; Chugai; Eli Lilly; Elucida; F Star; Georgiamune; Imvaq; Kyowa Hakko Kirin; Linneaus; Merck; Neon Therapeutics; Polynoma; Psioxus; Recepta; Takara Bio; Trieza; Truvax; Sellas; Serametrix; Surface Oncology; Syndax; Syntalogic, Werewolf Therapeutics. J.D.W. reports grants from Bristol Myers Squibb and Sephora. J.D.W. has equity in Tizona Pharmaceuticals; Adaptive Biotechnologies; Imvaq; Beigene; Linneaus; Apricity; Arsenal IO; Georgiamune. J.D.W. is inventor on patent applications related to work on DNA vaccines in companion animals with cancer, assays for suppressive myeloid cells in blood, oncolytic viral therapy, alphavirus-based vaccines, neo-antigen modelling, CD40, GITR, OX40, PD-1 and CTLA-4. The other authors declare no competing interests.
Peer review information Nature thanks the anonymous reviewers for their contribution to the peer review of this work.
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Extended data figures and tables
a, Quantification of glucose and lactate by 1H-NMR in supernatants from 72 h cultures of activated T cells (100,000 cells), 4T1 cells (3,000 cells) and the two cell types together. Plots show combined results from two independent experiments (n = 2 per experiment). b, Percentage of proliferating (CFSElow) (left) and dead (right) CD4+ or CD8+ T cells assessed by flow cytometry after 48 h activation in the presence of the indicated concentrations of lactic acid to define the workable lactate dose range (n = 3 per condition, except for 0 μM lactic acid, n = 2). c, d, Flow cytometry analysis of the indicated parameters in CD8+ and CD4+ T cells activated for 48 h in the presence or absence of 4T1 cells (c) or of 10 mM lactate (d) from two independent experiments (n = 3). e, f, Expression of immune cell signatures by CIBERSORT (top) and glycolysis-related genes (bottom) in RNA-seq datasets from human melanoma samples at baseline (e, n = 7) and after ipilimumab treatment (f, n = 15). Each column in the heat maps represents an independent tumour sample. Data are mean ± s.d. P values determined by two-sided unpaired t-test.
a, Expression of LDHA in 4T1-KD and 4T1-Sc whole-cell protein extracts by western blot analysis. Vinculin was used as a loading control. Representative of three independent experiments. b, Glycolytic proton efflux rate (glycoPER) in 4T1-KD and 4T1-Sc cultures (n = 20). 2-DG, 2-deoxy-d-glucose; Rot/AA, rotenone plus antimycin A. c, In vivo growth of 4T1-KD and 4T1-Sc tumours orthotopically implanted in the mammary fat pad of immunocompetent wild-type (WT) and immunodeficient RAG2-knockout (KO) BALB/c mice (n = 10 mice per group; representative of two independent experiments). d, Growth of primary 4T1-Sc and 4T1-KD tumours in mice treated as in Fig. 2a (left) and average tumour diameter on the day of tumour resection (right) (IgG, n = 9; anti-CTLA-4, n = 12). e, LDH activity in 4T1-Sc (n = 4) and 4T1-KD (n = 5) tumour extracts on the day of tumour resection after treatment as in d. f, Tumour growth after a second injection with 4T1-Sc in 4T1-KD- and 4T1-Sc-bearing mice that survived neoadjuvant treatment with CTLA-4 blockade as in Fig. 2a (n = 4 per group, except for naive, n = 5). Data are mean ± s.d. (b) or mean ± s.e.m. (c–f). P values determined by two-way ANOVA with Bonferroni correction (b, c, f) or two-sided unpaired t-test (e).
Extended Data Fig. 3 Neoadjuvant anti-CTLA-4 treatment schedule for same-day 4T1-Sc and 4T1-KD-tumour resection.
a, Top, additional treatment schedule modified to obtain 4T1-Sc and 4T1-KD tumours for flow cytometry analysis on the same day. Separate groups of BALB/c mice were injected with 106 4T1-KD and 4T1-Sc cells 3 days apart and then treated with three cycles of anti-CTLA-4 or the matched isotype control (IgG) every 3 days before surgery and flow cytometry analysis of tumour and tumour draining lymph node (DLN) samples. a, Bottom, primary tumour growth (left) and tumour weight (right) on the day of surgery, showing similar tumour size across groups during treatment and on the day of surgery (n = 5 mice per group). b, Overall survival of mice treated as in a (n = 5 mice per group). c, Frequency of the indicated T cell subsets among total CD45+ leukocytes in tumours and DLNs from the indicated treatment groups (n = 5 mice per group except for 4T1-Sc IgG, n = 4). d, Frequency of CD11b+ myeloid cell subsets among total CD45+ leukocytes, M1 and M2 macrophages according to MHC-II and CD206 staining among total CD11b+F4/80+ macrophages, and Gr1+ granulocyte subsets among total CD11b+ myeloid cells in 4T1-Sc and 4T1-KD tumours as well as DLNs from mice treated as indicated (n = 5 mice per group except for 4T1-Sc IgG, n = 4). Representative plots (left) showing the flow cytometry gating strategy for M1 and M2 macrophages and granulocytes (granulo) are reported. Data are representative of at least two independent experiments. Data are mean ± s.e.m. *P < 0.05. P values determined by log-rank test (b) or two-sided unpaired t-test (c, d).
Extended Data Fig. 4 Selective loss of Treg cell functional stability in LDHA-deficient tumours treated with CTLA-4 blockade.
a, Representative gating strategy for tumour-infiltrating CD8+, CD4+FOXP3− Teff cells and CD4+FOXP3+ Treg cells, in which expression of IFNγ and TNF was assessed. b, Representative flow cytometry plots showing IFNγ and TNF expression in Treg and Teff cells, CD8+ TILs gated as in a from 4T1-Sc- and 4T1-KD-bearing BALB/c mice treated as in Fig. 3a. c, d, Quantification of TNF and IFNγ expression in CD4+FOXP3− Teff cells and CD8+ TILs from 4T1-Sc- and 4T1-KD-bearing BALB/c mice treated as in Fig. 3a (c; n = 5 mice per group) and Fig. 3b (d; n = 5 mice per group except for 4T1-Sc IgG, n = 4). e, Quantification of IFNγ (top) and TNF (bottom) expression in CD8+ T cells, CD4+FOXP3− Teff and Treg cells from DLNs of 4T1-Sc- and 4T1-KD-bearing BALB/c mice treated as in Fig. 3b (n = 5 mice per group except for 4T1-Sc IgG, n = 4). f, Quantification (left) and representative plots (right) of CTLA-4 expression by flow cytometry in CD8+ T cells, CD4+FOXP3− Teff and Treg cells from tumour and DLN samples of 4T1-Sc and 4T1-KD tumour-bearing mice (n = 5 mice per group). Data are mean ± s.e.m. and representative of at least two independent experiments. P values determined by two-sided unpaired (c–e) or paired (f) t-test.
Extended Data Fig. 5 Treg cell destabilization and CD8+ TIL activation in additional LDHA-deficient tumour models treated with CTLA-4 blockade.
a–e, Primary tumour growth and overall survival, reporting the number of tumour-free mice at the end of the experiment, in BALB/c mice implanted in the mammary fat pads with the LDHA-KD 4T1 A3-8KD cell line (106 cells per mouse) treated with neoadjuvant anti-CTLA-4 (n = 9) or IgG control (n = 10), as indicated. CTLA-4 and CD25 in tumour-infiltrating Treg cells (b), quantification of Treg cells and CD8+ TILs as well as expression of the indicated markers by flow cytometry (c), and flow cytometry analysis of IFNγ expression in CTLA-4lo and CTLA-4hi tumour-infiltrating Treg cells (d) from mice treated as in a (CTLA-4lo versus CTLA-4hi Treg cells, two-sided paired t-test; IgG versus anti-CTLA-4 CTLA-4lo Treg cells, two-sided unpaired t-test). e, Pearson correlation analyses of indicated parameters in Treg cells and CD8+ TILs from mice treated as in a (black, IgG; red, anti-CTLA-4). n = 9–10 mice per group; one independent experiment. f–j, 4T1-KD-bearing BALB/c mice were treated with the standard IgG2b 9D9 anti-CTLA-4 antibody (n = 10) or its IgG2a variant (n = 9) or IgG control (n = 10) (f), and overall survival (g), quantification of CTLA-4 and GITR expression in Treg cells (h), and tumour-infiltrating Treg cells and their expression of FOXP3 and IFNγ by flow cytometry (i) are shown. j, Pearson correlation analyses between the indicated parameters in Treg cells and CD8+ TILs from mice treated as in f. n = 1 experiment with 9D9 IgG2a. k–m, LDHA protein expression by western blot (k), LDH activity (l), and glycolytic proton efflux rate (GlycoPER) (m) by Seahorse analysis in B16-KD and B16-Sc cells (n = 3, representative of 2–3 independent experiments). n–p, C57BL/6J mice were implanted with B16-KD and B16-Sc tumours and treated with anti-CTLA-4 or IgG control as indicated in n. Quantification of CTLA-4 and CD25 (o; n = 5 per group except for B16-KD IgG, n = 4) and IFNγ expression (p) in tumour-infiltrating Treg cells by flow cytometry (B16-Sc IgG, n = 4; B16-Sc anti-CTLA-4, n = 6; B16-KD IgG, n = 4; B16-KD anti-CTLA-4, n = 3; representative of two experiments). GzmB, granzyme B; i.d., intradermal; TM, tumour. Data are mean ± s.e.m. (a–d, h–i, o–p) or mean ± s.d. (l–m). Unless stated otherwise, P values were determined by two-sided unpaired t-test (b, c, h, i, l, m, o, p), Pearson correlation coefficient (e, j) or log-rank test (g).
a, b, LDHA protein expression by western blot (representative of three independent experiments) (a) and LDH activity (b) in 4T1-KD and 4T1-EtBr cells in comparison to control 4T1-Sc cells (n = 3). c, Complete cell energetic map with mitochondrial and glycolytic production rates in the indicated 4T1 cell variants using a real-time ATP rate assay by Seahorse (Sc and EtBr, n = 22; KD and A3-8KD, n = 24) (representative of two independent experiments). d–g, BALB/c mice (n = 5 per group) were orthotopically implanted with 106 4T1-KD or 4T1-EtBr cells and tumours were surgically resected 13 days later (d). d, Overall survival and number of surviving mice out of total. Frequency of FOXP3+ Treg cells among tumour-infiltrating CD4+ T cells (e), CD25 and CTLA-4 (f) and IFNγ (g) expression in intra-tumour Treg cells by flow cytometry; representative of two independent experiments. h, Schematic representation of anti-CTLA-4 or control IgG treatment in BALB/c mice implanted with 4T1-Sc and 4T1-KD in opposite mammary fat pads, and tumour weight on day 13 for samples analysed in i and j. i, GlucoseCy3 staining by flow cytometry in CD45− tumour cells gated as indicated to enrich in live CD45− tumour cells by comparing CD45 and DAPI staining between tumour and spleen samples from mice treated as in h. j, GlucoseCy3 staining by flow cytometry in Treg cells gated based on surface staining of CD4, CD25 and GITR in tumour samples as in h. h–j, 4T1-Sc, n = 4 (1 tumour sample in each treatment group was contaminated by DLN and was excluded); 4T1-KD, n = 5; n = 1 experiment. k, In vitro glucose consumption by B16-Sc and B16-KD cells. l, m, Ex vivo glucose uptake potential by flow cytometry analysis of glucoseCy3 staining of CD45− tumour cells (l) and intra-tumour FOXP3-GFP+ Treg cells (m) from B16-Sc- and B16-KD-bearing FOXP3–GFP transgenic C57BL/6J mice treated with anti-CTLA-4 (n = 3; representative of two independent experiments). Data are mean ± s.d. (b, c, k–m) or mean ± s.e.m. (e–g, h–j). P values determined by two-sided unpaired t-test.
Extended Data Fig. 7 Ex vivo and in vitro Treg cell response to tumour glucose metabolism and CTLA-4 blockade.
a, b, FOXP3–GFP transgenic (Tg) mice were implanted with B16-Sc or B16-KD cells and treated with anti-CTLA-4 as indicated in a, and tumour-infiltrating FOXP3–GFP+ Treg cells were FACS-sorted and tested in ex vivo suppression assays with CellTrace Violet (CTV)-labelled CD8+ T cells activated with anti-CD3 in the presence of 0.5 or 10 mM glucose (b). b, Flow cytometry of CD44 and CD25 expression in CD8+ T cells cultured with B16-Sc- and B16-KD-derived Treg cells (top) and quantification of proliferation (CTV dilution by CTV MFI) of dividing CTVlo CD8+ T cells and Treg cell suppression of CD8+ T cell proliferation in the same culture conditions (bottom) (n = 3, representative of two independent experiments). c, Quantification by flow cytometry of IFNγ and TNF expression in Treg cells co-cultured with 4T1-Sc or 4T1-KD cells in 5 mM glucose RPMI1640 for 24 h in the presence of soluble anti-CD3, IL-2 and anti-CTLA-4 (n = 3, n = 1 experiment). d, Glucose consumption and lactate production by NMuMg benign mammary gland cell line in comparison with 4T1 cells (n = 6, n = 1 experiment with NMuMg). e, Quantification by flow cytometry of IFNγ and TNF expression in Treg cells cultured for 48 h with 4T1-Sc, 4T1-KD- or NMuMg-conditioned medium (11 mM glucose complete RPMI1640) in the presence of plate-bound anti-CD3, IL-2 and anti-CTLA-4 or an IgG control (n = 3, n = 1 experiment). Data are mean ± s.d. *P < 0.05; **P < 0.01; ***P < 0.001. P values determined by two-sided unpaired t-test.
Extended Data Fig. 8 Loss of functional stability of Treg cells induced by anti-CTLA-4 depends on Treg cell glycolysis and CD28 signalling.
a, Quantification and representative plots of GlucoseCy3 staining by flow cytometry of Treg cells activated as in Fig. 4c in the presence of 10 mM glucose ± rotenone/antimycin A or oligomycin and treated with anti-CTLA-4 or IgG control (average of two biological replicates per condition; representative of two independent experiments). b, FOXP3 expression by flow cytometry and IL-10 production by Luminex-based bead immunoassay in Treg cells activated in the presence of 10 mM glucose ± rotenone/antimycin A or oligomycin (n = 3, representative of three independent experiments). c, d, Representative plots of in vitro assays reported in Fig. 4f, g. Representative proliferation (CellTraceViolet dilution) by flow cytometry of activated CD8+ T cells cultured alone or in the presence of Treg cells at the indicated glucose concentrations and treated with anti-CTLA-4 or an IgG control (c). d, Representative CD86 staining by flow cytometry on B cells from co-cultures with CD8+ T cells and Treg cells treated as in c. e, In vitro suppression assay with CD25hi Treg cells immunomagnetically purified from spleens of naive wild-type or CD28-knockout mice cultured for 48 h with CellTraceViolet-labelled CD8+ T cells and B cells and activated with anti-CD3 in the presence of anti-CTLA-4 or IgG control and the indicated glucose concentrations (n = 3 per conditions except for ‘+CD28 KO Tregs’ at 1–10 mM glucose, n = 2; representative of two independent experiments). Data are mean ± s.d. P values determined by two-sided unpaired t-test. Oligo, oligomycin.
Extended Data Fig. 9 CD28 agonism and CTLA-4 blockade, but not PD-1 blockade, drive loss of Treg cell functional stability.
a, b, Representative flow cytometry plots of in vitro assays reported in Fig. 4i, j. a, Representative proliferation (CellTraceViolet dilution) by flow cytometry of activated CD8+ T cells cultured alone or in the presence of Treg cells at the indicated glucose concentrations and treated with anti-CD28 (2 μg ml−1) or IgG control. b, Representative CD86 staining by flow cytometry on B cells from co-cultures with CD8+ T cells and Treg cells treated as in a. c, Proliferation of CD8+ T cells cultured alone or with Treg cells in 0.5 mM (grey) or 10 mM glucose (black) and activated with increasing concentrations of anti-CD28 (0–0.2 μg ml−1) (n = 3, representative of two independent experiments). d, Quantification and representative plots showing suppression of CD4+ T cell proliferation (left) and CD86 expression on B cells (right) by flow cytometry in culture with Treg cells treated with anti-CTLA-4, anti-PD-1 or an IgG control in complete RPMI1640 containing 11 mM glucose. Percentage suppression was calculated relative to proliferation of CD4+ T cells cultured alone in the same treatment conditions (n = 3; n = 1 experiment with anti-PD-1). e, Suppression of proliferation of CD8+ T cells cultured at the indicated ratios with FOXP3–GFP+PD-1+ Treg cells (top) or FOXP3–GFP+PD-1− Treg cells (bottom) FACS-sorted from spleens of naive FOXP3–GFP mice and incubated with anti-PD-1 or IgG control for 48 h (representative results from one experiment conducted with CD8+ and CD4+ as target T cells with similar results). f, Quantification and representative plots of GlucoseCy3 staining by flow cytometry in Treg cells activated as in Fig. 4c and treated with anti-PD-1 or IgG control (n = 3, representative of two independent experiments). g, Flow cytometry quantification and phenotypic analysis of Treg cells from 4T1-KD tumours treated with anti-CTLA-4, anti-PD-1 or IgG control as indicated (n = 10 mice per group, representative of two independent experiments). Data are mean ± s.d. (c, d, f) or mean ± s.e.m. (g). **P < 0.01; ***P < 0.001. P values determined by two-sided unpaired t-test.
Extended Data Fig. 10 Limiting Treg cell glucose metabolism prevents anti-CTLA-4-mediated Treg cell destabilization in glycolysis-defective tumours.
a, Foxp3GFP-cre-ERT2;Slc2a1fl/fl (Glut1 cKO) and Foxp3GFP-cre-ERT2 control mice (ctrl) were implanted with B16-KD cells and treated with anti-CTLA-4 or IgG after induction of Slc2a1 deletion with tamoxifen as indicated. Tamoxifen treatment was continued throughout the treatment duration. b, Quantification of Slc2a1 mRNA relative to Actb in FOXP3–GFP− Teff cells and FOXP3–GFP+ Treg cells from the spleens of control and Glut1 cKO mice at the end of treatment as in a (n = 3). c, d, Flow cytometry analysis of CD25 and CTLA-4 (c; n = 3 except for ctrl IgG, n = 1), and IFNγ and TNF expression (d; n = 2) in tumour-infiltrating Treg cells from mice treated as in a (representative of two independent experiments). e, Ex vivo suppression assay with Treg cells sorted from the spleens of control and Glut1 cKO mice treated with anti-CTLA-4 as in a. Treg cell suppression of CD8+ T cell expansion after 48 h co-culture in 10 mM glucose and representative flow cytometry plot showing CTV dilution and generation (G) overlay of CD8+ T cells cultured alone (grey) or in the presence of control (black) or Glut1 cKO (red) Treg cell (n = 3; representative of two independent experiments). f–l, Foxp3YFP-cre;Slc2a1fl/+ (Glut1 HET) or Foxp3YFP-cre;Ldhafl/fl (Ldha cKO) and Foxp3YFP-cre mice (ctrl) were implanted with B16-KD cells and treated with anti-CTLA-4 (f). g, Quantification of Slc2a1 mRNA relative to Actb in FOXP3–GFP+ Treg cells from spleens of control and Glut1 HET mice (n = 2). h, i, Quantification by flow cytometry of intra-tumour Treg cells (h) and their expression of Ki67 (i) in control and Glut1 HET mice treated as in f (control, n = 4; HET, n = 2; representative of two independent experiments with mice carrying Glut1 HET or cKO Treg cells). j, Quantification of Ldha mRNA relative to Actb in FOXP3–GFP+ Treg cells from spleens of control and Ldha cKO mice (n = 3). k, l, Quantification by flow cytometry of intra-tumour Treg cells (k) and their expression of Ki67 (l) in control or Ldha cKO mice treated as in f (ctrl, n = 3; Ldha cKO, n = 2; representative of two independent experiments). m, Schematic representation of the culture conditions used in n, o. CD5+ T cells from Ldha cKO or control mice were co-cultured for 48 h with CD45.1+ congenic antigen-presenting cells (either B cells or T-cell depleted splenocytes) as scaffold for soluble anti-CD3 crosslinking in low (0.5 mM) or higher (10 mM) glucose concentrations as indicated. n, Quantification by flow cytometry of Ldha cKO or control FOXP3+CD4+ Treg cells and their expression of Ki67 after activation as in m. o, FOXP3 and CTLA-4 expression by flow cytometry (MFI) in Ki67-negative Ldha cKO or control Treg cells from cultures as in m. Ctrl 0.5 mM glucose, n = 3 except for anti-CD28, n = 2; ctrl 10 mM glucose, n = 3; cKO, n = 4; representative of two independent experiments. aCTLA-4, anti-CTLA-4; aCD28, anti-CD28. Data are mean ± s.d. (b, e, g, j, n, o) or mean ± s.e.m. (c, d, h, i, k, l). **P < 0.01; ***P < 0.001. P values determined by two-sided unpaired t-test.
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Zappasodi, R., Serganova, I., Cohen, I.J. et al. CTLA-4 blockade drives loss of Treg stability in glycolysis-low tumours. Nature 591, 652–658 (2021). https://doi.org/10.1038/s41586-021-03326-4
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