Deficiency of metabolic regulator PKM2 activates the pentose phosphate pathway and generates TCF1+ progenitor CD8+ T cells to improve immunotherapy

Restifo, N. P., Dudley, M. E. & Rosenberg, S. A. Adoptive immunotherapy for cancer: harnessing the T cell response. Nat. Rev. Immunol. 12, 269–281 (2012).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Rosenberg, S. A. & Restifo, N. P. Adoptive cell transfer as personalized immunotherapy for human cancer. Science 348, 62–68 (2015).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Klebanoff, C. A., Rosenberg, S. A. & Restifo, N. P. Prospects for gene-engineered T cell immunotherapy for solid cancers. Nat. Med. 22, 26–36 (2016).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Rosenberg, S. A., Parkhurst, M. R. & Robbins, P. F. Adoptive cell transfer immunotherapy for patients with solid epithelial cancers. Cancer Cell 41, 646–648 (2023).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Pardoll, D. M. The blockade of immune checkpoints in cancer immunotherapy. Nat. Rev. Cancer 12, 252–264 (2012).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Sharma, P. & Allison, J. P. Dissecting the mechanisms of immune checkpoint therapy. Nat. Rev. Immunol. 20, 75–76 (2020).

CAS 
PubMed 

Google Scholar 

Ribas, A. & Wolchok, J. D. Cancer immunotherapy using checkpoint blockade. Science 359, 1350–1355 (2018).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Markowitz, G. J. et al. Immune reprogramming via PD-1 inhibition enhances early-stage lung cancer survival. JCI Insight https://doi.org/10.1172/jci.insight.96836 (2018).

Philip, M. & Schietinger, A. CD8(+) T cell differentiation and dysfunction in cancer. Nat. Rev. Immunol. 22, 209–223 (2022).

CAS 
PubMed 

Google Scholar 

Blank, C. U. et al. Defining ‘T cell exhaustion’. Nat. Rev. Immunol. 19, 665–674 (2019).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Siddiqui, I. et al. Intratumoral Tcf1(+)PD-1(+)CD8(+) T cells with stem-like properties promote tumor control in response to vaccination and checkpoint blockade immunotherapy. Immunity 50, 195–211 e110 (2019).

CAS 
PubMed 

Google Scholar 

Miller, B. C. et al. Subsets of exhausted CD8(+) T cells differentially mediate tumor control and respond to checkpoint blockade. Nat. Immunol. 20, 326–336 (2019).

CAS 
PubMed 
PubMed Central 

Google Scholar 

van Loosdregt, J. & Coffer, P. J. The role of WNT signaling in mature T cells: T cell factor is coming home. J. Immunol. 201, 2193–2200 (2018).

PubMed 

Google Scholar 

Gattinoni, L. et al. Wnt signaling arrests effector T cell differentiation and generates CD8⁺ memory stem cells. Nat. Med. 15, 808–813 (2009).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Escobar, G., Mangani, D. & Anderson, A. C. T cell factor 1: a master regulator of the T cell response in disease. Sci. Immunol. https://doi.org/10.1126/sciimmunol.abb9726 (2020).

Gebhardt, T., Park, S. L. & Parish, I. A. Stem-like exhausted and memory CD8(+) T cells in cancer. Nat. Rev. Cancer 23, 780–798 (2023).

CAS 
PubMed 

Google Scholar 

Kurtulus, S. et al. Checkpoint blockade immunotherapy induces dynamic changes in PD-1(-)CD8(+) tumor-infiltrating T cells. Immunity 50, 181–194 e186 (2019).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Kishton, R. J., Sukumar, M. & Restifo, N. P. Metabolic regulation of T cell longevity and function in tumor immunotherapy. Cell Metab. 26, 94–109 (2017).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Reina-Campos, M., Scharping, N. E. & Goldrath, A. W. CD8(+) T cell metabolism in infection and cancer. Nat. Rev. Immunol. 21, 718–738 (2021).

CAS 
PubMed 
PubMed Central 

Google Scholar 

van der Windt, G. J. & Pearce, E. L. Metabolic switching and fuel choice during T-cell differentiation and memory development. Immunol. Rev. 249, 27–42 (2012).

PubMed 
PubMed Central 

Google Scholar 

Wang, R. & Green, D. R. Metabolic reprogramming and metabolic dependency in T cells. Immunol. Rev. 249, 14–26 (2012).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Hermans, D. et al. Lactate dehydrogenase inhibition synergizes with IL-21 to promote CD8(+) T cell stemness and antitumor immunity. Proc. Natl Acad. Sci. USA 117, 6047–6055 (2020).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Pearce, E. L. et al. Enhancing CD8 T-cell memory by modulating fatty acid metabolism. Nature 460, 103–107 (2009).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Sukumar, M. et al. Inhibiting glycolytic metabolism enhances CD8⁺ T cell memory and antitumor function. J. Clin. Invest. 123, 4479–4488 (2013).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Choi, H. et al. Transcriptome analysis of individual stromal cell populations identifies stroma-tumor crosstalk in mouse lung cancer model. Cell Rep. 10, 1187–1201 (2015).

CAS 
PubMed 

Google Scholar 

Kanehisa, M. & Goto, S. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28, 27–30 (2000).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Vardhana, S. A. et al. Impaired mitochondrial oxidative phosphorylation limits the self-renewal of T cells exposed to persistent antigen. Nat. Immunol. 21, 1022–1033 (2020).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Horton, B. L. et al. Lack of CD8(+) T cell effector differentiation during priming mediates checkpoint blockade resistance in non-small cell lung cancer. Sci. Immunol. 6, eabi8800 (2021).

CAS 
PubMed 
PubMed Central 

Google Scholar 

DuPage, M. et al. Endogenous T cell responses to antigens expressed in lung adenocarcinomas delay malignant tumor progression. Cancer Cell 19, 72–85 (2011).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Hogquist, K. A. et al. T cell receptor antagonist peptides induce positive selection. Cell 76, 17–27 (1994).

CAS 
PubMed 

Google Scholar 

Jurica, M. S. et al. The allosteric regulation of pyruvate kinase by fructose-1,6-bisphosphate. Structure 6, 195–210 (1998).

CAS 
PubMed 

Google Scholar 

Gattinoni, L., Powell, D. J. Jr., Rosenberg, S. A. & Restifo, N. P. Adoptive immunotherapy for cancer: building on success. Nat. Rev. Immunol. 6, 383–393 (2006).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Ban, Y. et al. Radiation-activated secretory proteins of Scgb1a1 (+) club cells increase the efficacy of immune checkpoint blockade in lung cancer. Nat. Cancer 2, 919–931 (2021).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Luckey, C. J. et al. Memory T and memory B cells share a transcriptional program of self-renewal with long-term hematopoietic stem cells. Proc. Natl Acad. Sci. USA 103, 3304–3309 (2006).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Liberzon, A. et al. The Molecular Signatures Database (MSigDB) hallmark gene set collection. Cell Syst. 1, 417–425 (2015).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Ecker, C. et al. Differential reliance on lipid metabolism as a salvage pathway underlies functional differences of T cell subsets in poor nutrient environments. Cell Rep. 23, 741–755 (2018).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Pucino, V. et al. Lactate buildup at the site of chronic inflammation promotes disease by inducing CD4(+) T cell metabolic rewiring. Cell Metab. 30, 1055–1074 e1058 (2019).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Seki, S. M. et al. Modulation of PKM activity affects the differentiation of T(H)17 cells. Sci. Signal https://doi.org/10.1126/scisignal.aay9217 (2020).

Kono, M. et al. Pyruvate kinase M2 is requisite for Th1 and Th17 differentiation. JCI Insight https://doi.org/10.1172/jci.insight.127395 (2019).

Angiari, S. et al. Pharmacological activation of pyruvate kinase M2 inhibits CD4(+) T cell pathogenicity and suppresses autoimmunity. Cell Metab. 31, 391–405 e398 (2020).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Stincone, A. et al. The return of metabolism: biochemistry and physiology of the pentose phosphate pathway. Biol. Rev. Camb. Philos. Soc. 90, 927–963 (2015).

PubMed 

Google Scholar 

Bouzier-Sore, A. K. & Bolanos, J. P. Uncertainties in pentose-phosphate pathway flux assessment underestimate its contribution to neuronal glucose consumption: relevance for neurodegeneration and aging. Front. Aging Neurosci. 7, 89 (2015).

PubMed 
PubMed Central 

Google Scholar 

Jang, C., Chen, L. & Rabinowitz, J. D. Metabolomics and isotope tracing. Cell 173, 822–837 (2018).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Xia, J., Psychogios, N., Young, N. & Wishart, D. S. MetaboAnalyst: a web server for metabolomic data analysis and interpretation. Nucleic Acids Res. 37, W652–W660 (2009).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Pang, Z. et al. MetaboAnalyst 5.0: narrowing the gap between raw spectra and functional insights. Nucleic Acids Res. 49, W388–W396 (2021).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Daneshmandi, S. et al. Blockade of 6-phosphogluconate dehydrogenase generates CD8(+) effector T cells with enhanced anti-tumor function. Cell Rep. 34, 108831 (2021).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Ghergurovich, J. M. et al. A small molecule G6PD inhibitor reveals immune dependence on pentose phosphate pathway. Nat. Chem. Biol. 16, 731–739 (2020).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Lu, C. et al. G6PD functions as a metabolic checkpoint to regulate granzyme B expression in tumor-specific cytotoxic T lymphocytes. J. Immunother. Cancer https://doi.org/10.1136/jitc-2021-003543 (2022).

Wang, R. et al. The transcription factor Myc controls metabolic reprogramming upon T lymphocyte activation. Immunity 35, 871–882 (2011).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Hwang, S. et al. Correcting glucose-6-phosphate dehydrogenase deficiency with a small-molecule activator. Nat. Commun. 9, 4045 (2018).

PubMed 
PubMed Central 

Google Scholar 

Raub, A. G. et al. Small-molecule activators of glucose-6-phosphate dehydrogenase (G6PD) bridging the dimer interface. ChemMedChem 14, 1321–1324 (2019).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Horikoshi, N. et al. Long-range structural defects by pathogenic mutations in most severe glucose-6-phosphate dehydrogenase deficiency. Proc. Natl Acad. Sci. USA https://doi.org/10.1073/pnas.2022790118 (2021).

Garcia, A. A. et al. Stabilization of glucose-6-phosphate dehydrogenase oligomers enhances catalytic activity and stability of clinical variants. J. Biol. Chem. 298, 101610 (2022).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Yang, Q. et al. TCF-1 upregulation identifies early innate lymphoid progenitors in the bone marrow. Nat. Immunol. 16, 1044–1050 (2015).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Yao, C. et al. BACH2 enforces the transcriptional and epigenetic programs of stem-like CD8(+) T cells. Nat. Immunol. 22, 370–380 (2021).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Roychoudhuri, R. et al. BACH2 regulates CD8(+) T cell differentiation by controlling access of AP-1 factors to enhancers. Nat. Immunol. 17, 851–860 (2016).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Rao, R. R., Li, Q., Gubbels Bupp, M. R. & Shrikant, P. A. Transcription factor Foxo1 represses T-bet-mediated effector functions and promotes memory CD8(+) T cell differentiation. Immunity 36, 374–387 (2012).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Kim, M. V., Ouyang, W., Liao, W., Zhang, M. Q. & Li, M. O. The transcription factor Foxo1 controls central-memory CD8+ T cell responses to infection. Immunity 39, 286–297 (2013).

CAS 
PubMed 

Google Scholar 

Delpoux, A., Lai, C. Y., Hedrick, S. M. & Doedens, A. L. FOXO1 opposition of CD8(+) T cell effector programming confers early memory properties and phenotypic diversity. Proc. Natl Acad. Sci. USA 114, E8865–E8874 (2017).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Podaza, E. et al. Novel co-culture strategies of tumor organoids with autologous T-cells reveal clinically relevant combinations of immune-checkpoint and targeted therapies. Preprint at bioRxiv https://doi.org/10.1101/2023.07.05.546622 (2023).

Dijkstra, K. K. et al. Generation of tumor-reactive T cells by co-culture of peripheral blood lymphocytes and tumor organoids. Cell 174, 1586–1598 e1512 (2018).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Jin, J. et al. Simplified method of the growth of human tumor infiltrating lymphocytes in gas-permeable flasks to numbers needed for patient treatment. J. Immunother. 35, 283–292 (2012).

PubMed 
PubMed Central 

Google Scholar 

Toriyama, K. et al. T cell-specific deletion of Pgam1 reveals a critical role for glycolysis in T cell responses. Commun. Biol. 3, 394 (2020).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Siska, P. J. et al. Suppression of Glut1 and glucose metabolism by decreased Akt/mTORC1 signaling drives T cell impairment in B Cell leukemia. J. Immunol. 197, 2532–2540 (2016).

CAS 
PubMed 

Google Scholar 

Ho, P. C. et al. Phosphoenolpyruvate is a metabolic checkpoint of anti-tumor T cell responses. Cell 162, 1217–1228 (2015).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Gemta, L. F. et al. Impaired enolase 1 glycolytic activity restrains effector functions of tumor-infiltrating CD8(+) T cells. Sci. Immunol. https://doi.org/10.1126/sciimmunol.aap9520 (2019).

Chang, C. H. et al. Posttranscriptional control of T cell effector function by aerobic glycolysis. Cell 153, 1239–1251 (2013).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Telang, S. et al. Small molecule inhibition of 6-phosphofructo-2-kinase suppresses T cell activation. J. Transl. Med. 10, 95 (2012).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Quinn, W. J. et al. Lactate limits T cell proliferation via the NAD(H) redox state. Cell Rep. 33, 108500 (2020).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Mehta, M. M. et al. Hexokinase 2 is dispensable for T cell-dependent immunity. Cancer Metab. 6, 10 (2018).

PubMed 
PubMed Central 

Google Scholar 

Gu, M. et al. NF-κB-inducing kinase maintains T cell metabolic fitness in antitumor immunity. Nat. Immunol. 22, 193–204 (2021).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Ma, R. et al. A Pck1-directed glycogen metabolic program regulates formation and maintenance of memory CD8(+) T cells. Nat. Cell Biol. 20, 21–27 (2018).

CAS 
PubMed 

Google Scholar 

Utzschneider, D. T. et al. T cell factor 1-expressing memory-like CD8(+) T cells sustain the immune response to chronic viral infections. Immunity 45, 415–427 (2016).

CAS 
PubMed 

Google Scholar 

Shan, Q. et al. Tcf1 preprograms the mobilization of glycolysis in central memory CD8(+) T cells during recall responses. Nat. Immunol. 23, 386–398 (2022).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Im, S. J. et al. Defining CD8⁺ T cells that provide the proliferative burst after PD-1 therapy. Nature 537, 417–421 (2016).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Lu, S. et al. PKM2-dependent metabolic reprogramming in CD4(+) T cells is crucial for hyperhomocysteinemia-accelerated atherosclerosis. J. Mol. Med. 96, 585–600 (2018).

CAS 
PubMed 

Google Scholar 

Walls, J. F. et al. Metabolic but not transcriptional regulation by PKM2 is important for natural killer cell responses. eLife https://doi.org/10.7554/eLife.59166 (2020).

Kahan, S. M. et al. Intrinsic IL-2 production by effector CD8 T cells affects IL-2 signaling and promotes fate decisions, stemness, and protection. Sci. Immunol. 7, eabl6322 (2022).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Ji, Y. et al. Identification of the genomic insertion site of Pmel-1 TCR α and β transgenes by next-generation sequencing. PLoS ONE 9, e96650 (2014).

PubMed 
PubMed Central 

Google Scholar 

Overwijk, W. W. et al. Tumor regression and autoimmunity after reversal of a functionally tolerant state of self-reactive CD8⁺ T cells. J. Exp. Med. 198, 569–580 (2003).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Chang, K., Marran, K., Valentine, A. & Hannon, G. J. Packaging shRNA retroviruses. Cold Spring Harb. Protoc. 2013, 734–737 (2013).

PubMed 

Google Scholar 

Jacobi, A. M. et al. Simplified CRISPR tools for efficient genome editing and streamlined protocols for their delivery into mammalian cells and mouse zygotes. Methods 121, 16–28 (2017).

PubMed 

Google Scholar 

Kloss, C. C. et al. Dominant-negative TGF-β receptor enhances PSMA-targeted human CAR T cell proliferation and augments prostate cancer eradication. Mol. Ther. 26, 1855–1866 (2018).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013).

PubMed 
PubMed Central 

Google Scholar 

Trapnell, C. et al. Differential analysis of gene regulation at transcript resolution with RNA-seq. Nat. Biotechnol. 31, 46–53 (2013).

CAS 
PubMed 

Google Scholar 

Trapnell, C. et al. Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010).

CAS 
PubMed 
PubMed Central 

Google Scholar 

R Core Team. R: A Language and environment for statistical computing (R Foundation for Statistical Computing, 2022).

Wickham, H. ggplot2: Elegant Graphics for Data Analysis. (Springer, 2016).

Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).

PubMed 
PubMed Central 

Google Scholar 

Durinck, S., Spellman, P. T., Birney, E. & Huber, W. Mapping identifiers for the integration of genomic datasets with the R/Bioconductor package biomaRt. Nat. Protoc. 4, 1184–1191 (2009).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Durinck, S. et al. BioMart and Bioconductor: a powerful link between biological databases and microarray data analysis. Bioinformatics 21, 3439–3440 (2005).

CAS 
PubMed 

Google Scholar 

Mootha, V. K. et al. PGC-1α-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat. Genet. 34, 267–273 (2003).

CAS 
PubMed 

Google Scholar 

Korotkevic, G., Sukhov, V. & Sergushichev, A. Fast gene set enrichment analysis. Preprint at bioRxiv https://doi.org/10.1101/060012 (2019).

Anders, S., Pyl, P. T. & Huber, W. HTSeq-a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015).

CAS 
PubMed 

Google Scholar 

Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

PubMed 
PubMed Central 

Google Scholar 

Andrews, S. FastQC: a quality control tool for high throughput sequence data. Babraham Bioinformatics http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ (2010).

Blighe, K., Rana, S. & Lewis, M. EnhancedVolcano: publication-ready volcano plots with enhanced colouring and labeling. GitHub https://github.com/kevinblighe/EnhancedVolcano (2018).

Chen, W. W., Freinkman, E., Wang, T., Birsoy, K. & Sabatini, D. M. Absolute quantification of matrix metabolites reveals the dynamics of mitochondrial metabolism. Cell 166, 1324–1337 e1311 (2016).

CAS 
PubMed 
PubMed Central 

Google Scholar 

Pauli, C. et al. Personalized in vitro and in vivo cancer models to guide precision medicine. Cancer Discov. 7, 462–477 (2017).

PubMed 
PubMed Central 

Google Scholar