Abstract
CD8+ effector T (TE) cell proliferation and cytokine production depends on enhanced glucose metabolism. However, circulating T cells continuously adapt to glucose fluctuations caused by diet and inter-organ metabolite exchange. Here we show that transient glucose restriction (TGR) in activated CD8+ TE cells metabolically primes effector functions and enhances tumour clearance in mice. Tumour-specific TGR CD8+ TE cells co-cultured with tumour spheroids in replete conditions display enhanced effector molecule expression, and adoptive transfer of these cells in a murine lymphoma model leads to greater numbers of immunologically functional circulating donor cells and complete tumour clearance. Mechanistically, TE cells treated with TGR undergo metabolic remodelling that, after glucose re-exposure, supports enhanced glucose uptake, increased carbon allocation to the pentose phosphate pathway (PPP) and a cellular redox shift towards a more reduced state—all indicators of a more anabolic programme to support their enhanced functionality. Thus, metabolic conditioning could be used to promote efficiency of T-cell products for adoptive cellular therapy.
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Data availability
The data that support the findings of this study, as well as further information and requests for resources and reagents, will be made available on reasonable request by the corresponding author. scRNA-seq data have been deposited in the Gene Ontology Omnibus (accession no. GSE152018). Source data are provided with this paper.
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Acknowledgements
We thank members of the Pearce laboratories for support and helpful discussions and A. Quintana and J. Sutherland mouse colony management. This work was funded by the National Institutes of Health (NIH; CA181125 to E.L.P. and AI110481 to E.J.P.) and the Max Planck Society. R.Z. was supported by the Deutsche Forschungsgemeinschaft (DFG; SFB1160, B09; TRR167, B06) and the European Research Council (ERC; GvHDCure no. 681012).
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R.I.K.G., J.E.-H., P.A., D.O., D.E.S., D.J.P., J.M.B., K.M.G., A.M.K., M.S., F.M.U., M.F., R.Z., E.J.P. and E.L.P. designed the research, analysed data and provided conceptual input. A.E.P., N.A.M.L., J.D.C. and F.H. analysed data and performed the experiments. R.I.K.G., J.E.H. and E.L.P. wrote the manuscript.
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E.L.P. is a SAB member of ImmunoMet Therapeutics, and E.L.P. and E.J.P. are founders of Rheos Medicines. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 TGR CD8+ TE do not sustain mitochondrial metabolism by incorporating more glutamine-derived carbons into the TCA cycle.
WT CD8+ T cells isolated from spleens of C7Bl/6 mice were activated with anti-CD3 (5 μg/mL), anti-CD28 (0.5 μg/mL), IL-2 (100 U/mL), expanded for a total of 72 h, and exposed to 10 mM, 3 mM, or 1 mM glucose as indicated in cultures set to 1 million per ml. a, Polar metabolites were extracted from 500 µl media supernatant from 20 h cultures of 10 mM and 1 mM cells. Bar graphs represent glutamine concentration in the indicated conditions compared to complete culture media for n = 3 biological replicates. Significance was calculated using 2-tailed Student’s t tests, no significant changes (ns) were observed. b, TE were generated as above, but during the final 20 h all groups were cultured in 4 mM heavy labelled (U-13C) glutamine (100% U-13C glutamine). Polar metabolites were extracted and isotopologue distribution assessed by targeted mass spectrometry. TCA intermediates are plotted as percent label from newly metabolized U-13C (open bars) or remaining U-12C (black bars) glutamine carbons. Data are from n = 3 biological replicates, representative of 2 independent experiments. Significance was calculated using 2-tailed Student’s t tests, no significant changes were observed. c, TE were generated as above, but during the final 6 h 2 mM U-13C-lactate was added to the culture. Polar metabolites were extracted and isotopologue distribution assessed by targeted mass spectrometry. Significance was calculated using 2-tailed Student’s t tests comparing the predominant m + 2 isotopologue group in both conditions. Data shown for n = 3 biological replicates. Significance was calculated using 2-tailed Student’s t tests, No significant changes were observed. All error bars show SEM.
Extended Data Fig. 2 Limited transcriptional reprogramming during TGR.
WT CD8+ T cells isolated from spleens of C57BL/6 mice were activated with anti-CD3 (5 μg/mL), anti-CD28 (0.5 μg/mL), IL-2 (100 U/mL), expanded for a total of 72 h, and exposed to 10 mM (black) or 1 mM (orange) glucose in cultures set to 1 million per ml. RNA isolated from single cells was sequenced using the 10X genomics platform and analysed to explore transcriptional changes in each population of cells. a, A Uniform Manifold Approximation and Projection (UMAP) of the overlapped cells from each treatment (control, black; TGR, orange), clustered on the basis of transcriptional similarity is shown highlighting clusters and treatment distribution. Bar graphs depicting cluster distribution for each condition are also shown. b, Clusters 0 and 1, where least overlap between treatments was observed, were contrasted to look for differentially expressed genes (> 1.2-fold change and < 0.1 adjusted p-value); a heatmap of top up and down regulated genes is shown, along with UMAPs of example genes. c, The top 5 most enriched pathways in differentially regulated genes between cluster 0 and 1. d, Module scores based on average expression levels of gene programs were calculated for 18 differentially expressed OXPHOS genes (Atp5g1, Atp5g3, Cox5a, Cox6a1, Cyc1, Ndufa11, Ndufa12, Ndufa4, Ndufa8, Ndufab1, Ndufb7, Ndufb8, Ndufc1, Ndufc2, Ndufs6, Sdhb, Uqcr10, Uqcr11). Violin plots depict the global OXPHOS module score per condition (left) or per cluster and condition (right). e, Module scores based on average expression levels of gene programs were also calculated for enzyme-coding genes involved in glycolysis (mmu00010) or the pentose phosphate pathway (PPP, mmu00030) based on Kegg annotation. UMAP and Violin plots illustrate the OXPHOS, glycolysis and PPP modules, with the violin plots divided per cluster.
Extended Data Fig. 3 Signalling timecourse during TGR.
Protein isolates were prepared as described in (Fig. 3f), taking samples every 2 h over the 20 h exposure to limiting glucose concentration. Immunoblot analysis of protein extracts from equal cell numbers probed for phosphorylated acetyl-coA carboxylase at Ser79 (p-ACC1Thr79), total ACC1, phosphorylated AMPK at Thr172 (p-AMPkThr172), total AMPK, phosphorylated ribosomal protein S6 at Ser235/236 (p-S6Ser235/236), total S6, phosphorylated 4E-binding protein 1 at Thr37/46 (p-4E-BP1Thr37/46), total 4E-BP1, and Glut1. Tubulin was used as a loading control. Representative of 3 biological independent samples. Biological replicate data is shown in Fig. 3f.
Extended Data Fig. 4 TGR TE have altered glucose reallocation upon re-exposure to drive anabolic metabolism.
WT CD8+ T cells isolated from spleens of C57BL/6 mice were activated with anti-CD3 (5 μg/mL), anti-CD28 (0.5 μg/mL), IL-2 (100 U/mL), expanded for a total of 72 h, and exposed to 10 mM (control) or 1 mM (TGR) glucose during the final 5 min cells were re-exposed to normal (U-12C) glucose or heavy labelled (U-13C) glucose. 20 h cultures were started at 1 × 106 (control) and 1.5 × 106 (TGR) cells per/ml to generate similar end concentrations for glucose pulse experiments. a, Table showing the results of a Kegg-pathway analysis of the significantly increased and decreased metabolite pools in TGR TE after normal (U-12C) glucose re-feeding when compared to re-fed control TE. b, Table shows the results of a Kegg pathway analysis of the metabolites that exhibited increased glucose-derived U-13C assimilation (as determined by X13 CMS software) after re-feeding TGR TE with heavy labelled (U-13C) glucose compared to re-fed control TE. Data are from 3 biological replicates, representative of 3 independent experiments. Kegg Pathways were ranked according to the number of metabolite hits in that pathway (from highest to lowest). Metabolites in the highest ranked pathways were further validated by targeted analysis. c, Polar metabolites were extracted and untargeted metabolomic analysis was performed using XCMS on cells re-exposed to normal (U-12C) glucose. Volcano plot depicts relative metabolite pools compared between control and TGR cells. Orange circle represents a pentose phosphate pathway metabolite Pentose-phosphate, significantly increased in glucose re-fed TGR TE. Statistical significance was calculated using a Welch’s t-test, comparing relative intensities of each isotopologue in labelled samples of control TE versus those of TGR TE.
Extended Data Fig. 5 6h U-13C-glucose re-exposure following TGR.
WT CD8+ T cells isolated from spleens of C57BL/6 mice were activated with anti-CD3 (5 μg/mL), anti-CD28 (0.5 μg/mL), IL-2 (100 U/mL), expanded for a total of 72 h, and exposed to 10 mM (black) or 1 mM (orange) glucose. Cells were re-exposed to 10 mM U-13C-glucose for 6 h prior to polar metabolite extraction and analysis by LC-MS. 20 h cultures were started at 1 × 106 (control) and 1.5 × 106 (TGR) cells per/ml to generate similar end concentrations for glucose pulse experiments. a, Percent 13C label incorporation into nucleotide monophosphates was analysed. Data are from n = 3 biological replicates. Significance was calculated using 2-tailed Student’s t tests. * p < 0.05. b, Relative 13C label incorporation into serine was analysed. Data are from n = 3 biological replicates. Significance was calculated using 2way ANOVA. Significance is indicated for the 13C portion (open bars). *** p < 0.001. The 12C portion (filled bars) was also significant. ** p < 0.01. c, The GSH/GSSG ratio was calculated from summed isotopologues for each metabolite. Data are from n = 3 biological replicates. Significance was calculated using 2-tailed Student’s t tests, no significant difference was found. d, Relative 13C label incorporation into lactate was analysed. Data are from n = 3 biological replicates. Significance was calculated using 2way ANOVA. Significance is indicated for the 13C portion (open bars), which was not significant. The 12C portion (filled bars) was significant. *** p < 0.001. All error bars show SEM.
Extended Data Fig. 6 TGR enhances CD8+ tumour-specific antitumour function in vivo.
As depicted in Fig. 6f, congenically marked (CD45.1) C57BL/6 female recipient mice were injected subcutaneously with 1×106 B16-OVA melanoma cells and tumours established for 5 d. 5 ×106 control (black) or TGR (orange) OT-I+ TE were injected intravenously per tumour bearing mouse. Mice receiving no TE were used as controls. 3, 6 and 9 d after tumour inoculation, mice received 200 µg anti-PD-1 antibody or IgG2a isotype control in 100 µl PBS intraperitoneally. a, 21 d after tumour inoculation mice were humanely euthanized, blood was collected, red cells were lysed, and the white blood cell fraction stained for congenic markers. Data are presented as % of donor-derived (CD45.2+) CD8+ T cells as a fraction to total circulating CD8+ T cells and each dot represents an individual mouse (n = 5). Statistical significance was calculated by 2-tailed Student’s t test. * p < 0.05; ** p < 0.01. b, 21 d after tumour inoculation mice were humanely euthanized and the tumour mass was excised. Single cell suspensions were analysed by flow cytometry. The number of host (CD45.1) CD8+ T cells per mg tissue was quantified and each dot represents an individual mouse (n = 5). Statistical significance was calculated by 2-tailed Student’s t test. ns not significant. All error bars show SEM.
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Klein Geltink, R.I., Edwards-Hicks, J., Apostolova, P. et al. Metabolic conditioning of CD8+ effector T cells for adoptive cell therapy. Nat Metab 2, 703–716 (2020). https://doi.org/10.1038/s42255-020-0256-z
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DOI: https://doi.org/10.1038/s42255-020-0256-z
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