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Ionic immune suppression within the tumour microenvironment limits T cell effector function

Nature volume 537, pages 539–543 (2016)Cite this article

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Abstract

Tumours progress despite being infiltrated by tumour-specific effector T cells1. Tumours contain areas of cellular necrosis, which are associated with poor survival in a variety of cancers2. Here, we show that necrosis releases intracellular potassium ions into the extracellular fluid of mouse and human tumours, causing profound suppression of T cell effector function. Elevation of the extracellular potassium concentration ([K+]e) impairs T cell receptor (TCR)-driven Akt–mTOR phosphorylation and effector programmes. Potassium-mediated suppression of Akt–mTOR signalling and T cell function is dependent upon the activity of the serine/threonine phosphatase PP2A3,4. Although the suppressive effect mediated by elevated [K+]e is independent of changes in plasma membrane potential (Vm), it requires an increase in intracellular potassium ([K+]i). Accordingly, augmenting potassium efflux in tumour-specific T cells by overexpressing the potassium channel Kv1.3 lowers [K+]i and improves effector functions in vitro and in vivo and enhances tumour clearance and survival in melanoma-bearing mice. These results uncover an ionic checkpoint that blocks T cell function in tumours and identify potential new strategies for cancer immunotherapy.

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Figure 1: Elevated [K+] within TIF silences the TCR-induced anti-tumour function of mouse and human T cells.
Figure 2: [K+]e inhibits TCR-induced transcripts and function by suppressing Akt–mTOR phosphorylation.
Figure 3: [K+]e-mediated T cell suppression requires intact PP2A activity and is associated with elevations in [K+]i.
Figure 4: Kcna3 overexpression lowers [K+]i, enhances Akt–mTOR signalling and augments anti-tumour effector function to improve tumour clearance and host survival.

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Gene Expression Omnibus

Data deposits

RNA-sequencing raw data files are deposited in the Gene Expression Omnibus database with the accession number GSE84996.

References

  1. Mellman, I., Coukos, G. & Dranoff, G. Cancer immunotherapy comes of age. Nature 480, 480–489 (2011)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  2. Richards, C. H., Mohammed, Z., Qayyum, T., Horgan, P. G. & McMillan, D. C. The prognostic value of histological tumor necrosis in solid organ malignant disease: a systematic review. Future Oncol. 7, 1223–1235 (2011)

    Article  CAS  PubMed  Google Scholar 

  3. Li, G. et al. EphB3 suppresses non-small-cell lung cancer metastasis via a PP2A/RACK1/Akt signalling complex. Nat. Commun. 3, 667 (2012)

    Article  ADS  PubMed  CAS  Google Scholar 

  4. Zhou, P. et al. In vivo discovery of immunotherapy targets in the tumour microenvironment. Nature 506, 52–57 (2014)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  5. Sitkovsky, M. & Lukashev, D. Regulation of immune cells by local-tissue oxygen tension: HIF1 alpha and adenosine receptors. Nat. Rev. Immunol. 5, 712–721 (2005)

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Zimmerli, W. & Gallin, J. I. Pus potassium. Inflammation 12, 37–43 (1988)

    Article  CAS  PubMed  Google Scholar 

  8. Mirrakhimov, A. E., Ali, A. M., Khan, M. & Barbaryan, A. Tumor Lysis Syndrome in solid tumors: an up to date review of the literature. Rare Tumors 6, 68–76 (2014)

    Article  Google Scholar 

  9. Tran, E. et al. Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer. Science 344, 641–645 (2014)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  10. Feske, S. ORAI1 and STIM1 deficiency in human and mice: roles of store-operated Ca2+ entry in the immune system and beyond. Immunol. Rev. 231, 189–209 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Omilusik, K. et al. The Ca(v)1.4 calcium channel is a critical regulator of T cell receptor signaling and naive T cell homeostasis. Immunity 35, 349–360 (2011)

    Article  CAS  PubMed  Google Scholar 

  12. Li, F. Y. et al. Second messenger role for Mg2+ revealed by human T-cell immunodeficiency. Nature 475, 471–476 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Wu, C. et al. Induction of pathogenic TH17 cells by inducible salt-sensing kinase SGK1. Nature 496, 513–517 (2013)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  14. Wiig, H., Tenstad, O., Iversen, P. O., Kalluri, R. & Bjerkvig, R. Interstitial fluid: the overlooked component of the tumor microenvironment? Fibrogenesis Tissue Repair 3, 12 (2010)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Haslene-Hox, H. et al. A new method for isolation of interstitial fluid from human solid tumors applied to proteomic analysis of ovarian carcinoma tissue. PLoS One 6, e19217 (2011)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  16. Le, D. T. et al. PD-1 blockade in tumors with mismatch-repair deficiency. N. Engl. J. Med. 372, 2509–2520 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Robbins, P. F. et al. Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. J. Clin. Oncol. 29, 917–924 (2011)

    Article  PubMed  PubMed Central  Google Scholar 

  18. Goldman, D. E. Potential, impedance, and rectification in membranes. J. Gen. Physiol. 27, 37–60 (1943)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Hodgkin, A. L. & Huxley, A. F. A quantitative description of membrane current and its application to conduction and excitation in nerve. J. Physiol. 117, 500–544 (1952)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Buck, M. D., O’Sullivan, D. & Pearce, E. L. T cell metabolism drives immunity. J. Exp. Med. 212, 1345–1360 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Taffs, R. E., Redegeld, F. A. & Sitkovsky, M. V. Modulation of cytolytic T lymphocyte functions by an inhibitor of serine/threonine phosphatase, okadaic acid. Enhancement of cytolytic T lymphocyte-mediated cytotoxicity. J. Immunol. 147, 722–728 (1991)

    CAS  PubMed  Google Scholar 

  22. Liu, Q. H. et al. Modulation of Kv channel expression and function by TCR and costimulatory signals during peripheral CD4+ lymphocyte differentiation. J. Exp. Med. 196, 897–909 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Wulff, H. et al. The voltage-gated Kv1.3 K+ channel in effector memory T cells as new target for MS. J. Clin. Invest. 111, 1703–1713 (2003)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Cahalan, M. D. & Chandy, K. G. The functional network of ion channels in T lymphocytes. Immunol. Rev. 231, 59–87 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Cidad, P. et al. Kv1.3 channels can modulate cell proliferation during phenotypic switch by an ion-flux independent mechanism. Arterioscler. Thromb. Vasc. Biol. 32, 1299–1307 (2012)

    Article  CAS  PubMed  Google Scholar 

  26. Voronkov, M., Braithwaite, S. P. & Stock, J. B. Phosphoprotein phosphatase 2A: a novel druggable target for Alzheimer’s disease. Future Med. Chem. 3, 821–833 (2011)

    Article  CAS  PubMed  Google Scholar 

  27. Hla, T. & Dannenberg, A. J. Sphingolipid signaling in metabolic disorders. Cell Metab. 16, 420–434 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Muñoz-Planillo, R. et al. K+ efflux is the common trigger of NLRP3 inflammasome activation by bacterial toxins and particulate matter. Immunity 38, 1142–1153 (2013)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Wolfs, J. L. et al. Direct inhibition of phospholipid scrambling activity in erythrocytes by potassium ions. Cell. Mol. Life Sci. 66, 314–323 (2009)

    Article  CAS  PubMed  Google Scholar 

  30. Reeves, E. P. et al. Killing activity of neutrophils is mediated through activation of proteases by K+ flux. Nature 416, 291–297 (2002)

    Article  ADS  CAS  PubMed  Google Scholar 

  31. Acquavella, N. et al. Type I cytokines synergize with oncogene inhibition to induce tumor growth arrest. Cancer Immunol. Res. 3, 37–47 (2015)

    Article  CAS  PubMed  Google Scholar 

  32. Budak, Y. U., Huysal, K. & Polat, M. Use of a blood gas analyzer and a laboratory autoanalyzer in routine practice to measure electrolytes in intensive care unit patients. BMC Anesthesiol. 12, 17 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Wargo, J. A. et al. Recognition of NY-ESO-1+ tumor cells by engineered lymphocytes is enhanced by improved vector design and epigenetic modulation of tumor antigen expression. Cancer Immunol. Immunother. 58, 383–394 (2009)

    Article  CAS  PubMed  Google Scholar 

  34. Goff, S. L. et al. Randomized, prospective evaluation comparing intensity of lymphodepletion before adoptive transfer of tumor-infiltrating lymphocytes for patients with metastatic melanoma. J. Clin. Oncol. 34, 2389–2397 (2016)

    Article  PubMed  PubMed Central  Google Scholar 

  35. Vahedi, G. et al. STATs shape the active enhancer landscape of T cell populations. Cell 151, 981–993 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. 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)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. van der Windt, G. J. et al. Mitochondrial respiratory capacity is a critical regulator of CD8+ T cell memory development. Immunity 36, 68–78 (2012)

    Article  CAS  PubMed  Google Scholar 

  38. Tran, E. et al. Immunogenicity of somatic mutations in human gastrointestinal cancers. Science 350, 1387–1390 (2015)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  39. Lu, Y. C. et al. Efficient identification of mutated cancer antigens recognized by T cells associated with durable tumor regressions. Clin. Cancer Res. 20, 3401–3410 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Jiménez-Pérez, L. et al. Molecular determinants of Kv1.3 potassium channels-induced proliferation. J. Biol. Chem. 291, 3569–3580 (2016)

    Article  PubMed  CAS  Google Scholar 

  41. Turowski, P., Favre, B., Campbell, K. S., Lamb, N. J. & Hemmings, B. A. Modulation of the enzymatic properties of protein phosphatase 2A catalytic subunit by the recombinant 65-kDa regulatory subunit PR65α. Eur. J. Biochem. 248, 200–208 (1997)

    Article  CAS  PubMed  Google Scholar 

  42. Xing, Y. et al. Structure of protein phosphatase 2A core enzyme bound to tumor-inducing toxins. Cell 127, 341–353 (2006)

    Article  CAS  PubMed  Google Scholar 

  43. Hand, T. W. et al. Differential effects of STAT5 and PI3K/AKT signaling on effector and memory CD8 T-cell survival. Proc. Natl Acad. Sci. USA 107, 16601–16606 (2010)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  44. Liu, F. & Whitton, J. L. Cutting edge: re-evaluating the in vivo cytokine responses of CD8+ T cells during primary and secondary viral infections. J. Immunol. 174, 5936–5940 (2005)

    Article  CAS  PubMed  Google Scholar 

  45. Palmer, D. C. et al. Cish actively silences TCR signaling in CD8+ T cells to maintain tumor tolerance. J. Exp. Med. 212, 2095–2113 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Clark, J. et al. Quantification of PtdInsP3 molecular species in cells and tissues by mass spectrometry. Nat. Methods 8, 267–272 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The research was supported by the Intramural Research Program of the NCI, Wellcome Trust/Royal Society grant 105663/Z/14/Z (R.R.) and UK Biotechnology and Biological Sciences Research Council grant BB/N007794/1 (R.R. and K.O.). We thank S. A. Rosenberg, K. Hanada and K. J. Swartz for their valuable discussions and intellectual input, A. Mixon and S. Farid for expertise with cell sorting and G. McMullen for expertise with mouse handling.

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Author notes

  1. Robert Eil & Alena Gros

    Present address: †Present addresses: Department of Surgery, Oregon Health and Sciences University, Portland, Oregon 97239, SA (R.E.); Vall d’Hebron Institute of Oncology VHIO, Vall d’Hebron University Hospital, c/Natzaret, 115-117, Barcelona 08035, Spain (A.G.).,

  2. Robert Eil and Suman K. Vodnala: These authors contributed equally to this work.

Authors and Affiliations

  1. National Cancer Institute, National Institutes of Health (NIH), Bethesda, 20892, Maryland, USA

    Robert Eil, Suman K. Vodnala, David Clever, Christopher A. Klebanoff, Madhusudhanan Sukumar, Jenny H. Pan, Douglas C. Palmer, Alena Gros, Tori N. Yamamoto, Shashank J. Patel, Geoffrey C. Guittard, Zhiya Yu, David S. Schrump, W. Marston Linehan & Nicholas P. Restifo

  2. Center for Cell Engineering and Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, 10065, New York, USA

    Christopher A. Klebanoff

  3. Laboratory of Lymphocyte Signalling and Development, The Babraham Institute, Cambridge, CB22 3AT, UK

    Valentina Carbonaro, Klaus Okkenhaug & Rahul Roychoudhuri

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Contributions

R.E., S.V., R.R., J.H.P., C.A.K., and N.P.R. wrote the manuscript. R.E. designed all experiments and carried them all out except those shown in Extended Data (ED) Figs 1c, d, 9e, f. S.V. designed and carried out experiments shown in Figs 1h, i, 4h, i and ED Figs 1c, d, 2a, b, 3e, 8d, f, g, 9e, f. R.R. designed experiments shown in Figs 1a–d, 2a, b, e, 3a, c, k, 4d, f–i and ED Figs 1b, 2c, d, 3c, e, 4e–g, 5b, e, 7c–f, 8a, e–g; N.P.R. designed all experiments. D.C. designed experiments shown in Figs 3b, 4b, and ED Figs 2c, d, 4c–g and 8e–g. C.A.K. designed experiments shown in Figs 1b, c, j, 2, 3c, 4c, e, h, i and ED Figs 2e–h, 8e–g and 9b. J.H.P. designed experiments shown in Fig. 3h–m and ED Figs 2c, d, 7a, b. Z.Y. designed and carried out experiments shown in Fig. 4h, 4i and ED Fig 8f and g. D.P. designed experiments shown in Figs 2a–c, 4h, i, and ED Fig. 3a, c. T.Y. edited the manuscript, provided reagents and designed experiments shown in Fig. 1j and ED Figs 8f, 8g. K.O. and V.C. provided reagents, designed, and carried out experiments shown in Fig. 2h. A.G. provided reagents and designed experiments shown in Fig. 1j and ED Fig. 2f–h. M.S. designed and carried out experiments shown in ED Fig. 4c, 4d. S.P. designed experiments shown in Figs 3a, 1h, i and ED Figs 1c, d, 2a, b. G.C.G. designed experiments shown in Fig. 2d–g and ED Fig. 2b–d and carried out experiments shown in ED Fig. 3c. D.S.S. and W.M.L. contributed reagents for experiments shown in Fig. 1b and ED Fig. 1b.

Corresponding authors

Correspondence to Robert Eil or Nicholas P. Restifo.

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The authors declare no competing financial interests.

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Reviewer Information

Nature thanks G. Chandy and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Extracellular K+ release from apoptotic and necrotic cells inhibits T cell effector function.

a, [K+] in TIF, RPMI medium and mouse serum. b, Ratio of indicated ions in normal human tissue in comparison to serum measured on the day of tissue collection in cancer patients undergoing resection of nearby cancers originating from the same tissue type. c, Representative flow cytometry plots of B16 melanoma tumour cells following the indicated treatment. d, [K+]e quantification following the indicated treatment. e, f, Representative flow cytometry plots of anti-CD3/CD28-stimulated CD8+ T cells cultured in isotonic or hypertonic RPMI medium in the indicated conditions. g, Quantification of f. h, Quantification of cytokine production by CD8+ T cells following stimulation in the indicated conditions; elevated Ca2+ and Mg2+ are 2 mM, in comparison to 0.4 mM for control conditions. i, Cytokine production by T cells across a titration of anti-CD3 in the indicated conditions. j, Representative flow cytometry plots and quantification following anti-CD3/CD28 titration-based activation of CD8+ T cells in the indicated conditions. Centre values and error bars represent mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 between selected relevant comparisons, two-tailed Student’s t-tests (a–d, h), two-way ANOVA (g, i, j). a, n = at least three biological replicates; b, n = 5 biological replicates; c, d, n = 4 experimental replicates; e–j, n = 3 culture replicates per condition; c–j, representative of at least two independent experiments.

Extended Data Figure 2 Potassium-induced T cell suppression is functionally non-redundant with CTLA-4 and PD-L1 co-inhibitory signals and is present in TIL neoantigen responses.

a, b, IFNγ+ from CD8+cells in the indicated conditions. c, d, Flow cytometry analysis of cytokine production by CD4+ T cells polarized under TH1 (c) or TH17 (d) conditions and subsequently re-activated via immobilized anti-CD3/CD28 in the indicated experimental conditions. e, Annexin V and propidium iodide (PI) staining following activation of primed CD8+ T cells in the indicated conditions. f, Representative flow cytometry plots and quantification of human neo-antigen-selected TILs from three patients (Pt.) stimulated in the indicated conditions with cognate mutated (mut) neo-antigen peptide-pulsed target cells (autologous B cells). g, Relevant somatic mutation-induced neoepitopes for three patients A–C in f. h, Representative flow cytometry and quantification of peripheral blood leukocytes from three patients transduced with an HLA*A201-restricted NY-ESO-1 TCR were assayed in the indicated conditions for IFNγ production. Additional [K+]e = 40 mM for a–e, [K+]e = 50 mM for f, h; n = 3 culture replicates per patient per data point, representative of two independent experiments. Centre values and error bars represent mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-tailed Student’s t-tests (a–h). a–h, n = at least three culture replicates per data point and representative of at least two independent experiments.

Extended Data Figure 3 Elevated [K+]e acts independently of TCR-induced tyrosine phosphorylation and Ca2+ to suppress serine/threonine phosphorylation in the Akt–mTOR axis.

a, Flow cytometry analysis of TCR-induced Ca2+ influx in the indicated conditions (AUC, area under the curve). b, Flow cytometry analysis of TCR-induced phosphorylation of the indicated phospho-residues in primed CD8+ T cells. c, Immunoblot analysis of phospho-tyrosine (4G10) residues from primed CD8+ T cells stimulated as above. For immunoblot source image see Supplementary Fig. 1. d, Flow cytometry analysis of CD8+ T cells stimulated via TCR-crosslinking for the indicated phospho-residues and representative histograms at early time points. Filled grey histograms represent unstimulated cells. e, Flow cytometry analysis of the indicated phospho-proteins in CD8+ T cells stimulated at later time points following immobilized anti-CD3 and anti-CD28 stimulation in the indicated conditions. Elevated [K+]e = 40 mM, isotonic. Centre values and error bars represent mean ± s.e.m.; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 between selected relevant comparisons. Two-tailed Student’s t-tests (a, b), two-way ANOVA (d, e). a, n = 3 biologic replicates per condition; b, d, e, n = 3 technical replicates per data point. Representative of three (a–d) or two (e) independent experiments.

Extended Data Figure 4 Suppression of TCR-induced Akt–mTOR signalling by elevated [K+]e limits activation-induced nutrient consumption and T cell effector lineage commitment.

a, b, Flow cytometry analysis of the indicated phospho-proteins in CD8+ T cells stimulated by anti-CD3 and anti-CD28 cross-linking in the indicated conditions. c, 2-NBDG uptake in primed CD8+ T cells induced by TCR stimulation in the indicated conditions with representative histograms and quantification. d, Seahorse XF Bioflux analysis of CD3/CD28 Dynabead-induced extracellular acidification (ECAR) and oxygen consumption rate (OCR) of CD8+ T cells in the indicated conditions. e–g, Flow cytometry analysis of CD4+ T cells polarized in the indicated experimental condition concurrently with TH1 (e), TH17 (f), or iTreg cytokines (g). Elevated [K+]e = 40 mM. Centre values and error bars represent mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 between selected relevant comparisons; two-way ANOVA (a, b), two-tailed Student’s t-tests (c–g). n = 3 technical (a, b) or culture (c–g) replicates per data point. a–g, Representative of two independent experiments.

Extended Data Figure 5 Pharmacologic inhibition and genetic disruption of PP2A function restores T cell effector function in elevated [K+]e.

a, Flow cytometry analysis of the indicated phospho-proteins in primed CD8+ T cells stimulated via TCR crosslinking in the indicated conditions. b, c, Flow cytometry analysis of CD8+ T cell IFNγ production following immobilized anti-CD3/CD28 and induced stimulation in the indicated conditions (b), or among cells expressing a PP2A_DN isoform (c). d, Ppp2r2d expression in the indicated populations followed by flow cytometry analysis of IFNγ production by the populations. e, Flow cytometry analysis of IFNγ production by CD8+ T cells expressing an Akt1-CA isoform stimulated in the indicated conditions. Centre values and error bars represent mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 between selected relevant comparisons. Two-way ANOVA (a), two-tailed Student’s t-tests (b–e). Where noted in a–e, additional [K+]e = 40 mM. a–e, n = 3 culture replicates per condition and representative of at least two independent experiments.

Extended Data Figure 6 Depletion of intracellular potassium restores T cell cytokine production in the presence of elevated [K+]e.

a, b, [K+]i of CD8+ T cells in the indicated conditions assayed via relative fluorescence of Asante-Green 4. c, Flow cytometry analysis of IFNγ production by primed CD8+ T cells following immobilized anti-CD3/CD28 based activation in the indicated conditions. d, Vm of CD8+ T cells in the indicated conditions assayed with the voltage-sensitive fluorescent indicator DiSBAC4. e, Relative [K+]i of CD8+ T cells in the indicated conditions assayed with Asante-Green 4. f, Pictorial representation of the resultant intracellular changes in Vm and [K+]i in the presence of valinomycin. g, Flow cytometry analysis of CD8+ T cells following immobilized anti-CD3/CD28-based re-activation in the indicated conditions. h, Vm of CD8+ T cells in the indicated conditions assayed with DiSBAC4(3). i, Relative [K+]i of CD8+ T cells in the indicated conditions assayed with Asante-Green 4. j, Pictorial representation of the resultant intracellular changes in Vm and [K+]i in the presence of ouabain. k, Flow cytometry analysis of CD8+ T cells following immobilized anti-CD3/CD28 based re-activation in the indicated conditions. Centre values and error bars represent mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 between selected relevant comparisons; two-tailed Student’s t-tests (c, g, k). n = 3 technical (a, b, d, e, h, i) or culture (c, g, k) replicates per data point. a–k, Representative of at least two independent experiments.

Extended Data Figure 7 Elevated [K+]e does not permanently affect T cell [K+]i, Vm, or subsequent response to K+-induced suppression of effector function.

a, Flow cytometry-based calibration of [K+]i. For all values cells were treated with 50 μM gramicidin in titrated doses of [K+]e to provide a known [K+]i. b, Intra-experimental quantification of [K+]i in control conditions and elevated [K+]e based on calibration from a. c, Flow cytometry analysis of the relative Vm of CD8+ T cells of the indicated origin using DiSBAC4(3). d, Cells of the indicated origin assayed in the indicated conditions as in c. e, Flow cytometry analysis of relative [K+]i of CD8+ T cells of the noted origin washed and assayed in indicated conditions, quantified by relative fluorescence of Asante-Green 4. f, Compiled analysis of relative IFNγ production by CD8+cells of the indicated origin washed and subjected to TCR stimulation in the indicated conditions. Centre values and error bars represent mean ± s.e.m. NS, not significant, between experimental and control conditions as assessed by two-tailed Student’s t-test (c) or two-way ANOVA (d–f). For chronic conditioning, additional [K+]e = 40 mM, n = 3 technical (a–e) or culture (f) replicates per condition. a–f, Representative of two independent experiments.

Extended Data Figure 8 Enforced Kcna3 or Kcnn4 expression in CD8+ T cells augments effector function.

a, Flow cytometry analysis of CD8+ T cells retrovirally engineered with Ctrl-Thy1.1 or Kcna3-Thy1.1 encoding constructs assayed for relative [K+]i quantified by relative fluorescence of Asante-Green 4. b, Flow cytometry analysis of CD8+ T cells following re- activation in the indicated conditions. c, Flow cytometry analysis of CD8+ T cells retrovirally engineered with Ctrl-Thy1.1, Kcna3-Thy1.1, or Kcnn4-Thy1.1 constructs and re-activation in the indicated conditions. d, Flow cytometry analysis of IFNγ+ in CD8+ cells following re-activation in the indicated conditions. e, Flow cytometry analysis of IFNγ production in Pmel-1 CD8+Thy1.1+ TILs 6–8 days after transfer into tumour-bearing hosts following ex vivo re-activation. f, g, Pmel-1 CD8+ T cells retrovirally engineered with Ctrl-Thy1.1 or Kcna3-Thy1.1 constructs and transferred into C57BL/6 hosts in conjunction with human gp10025-33-encoding vaccinia virus and quantified by cell number (f) or surface phenotype (g) found in the blood of recipients. Centre values and error bars represent mean ± s.e.m.; two-tailed Student’s t-tests (a–d), two-way ANOVA (f, g); NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. n = 3 technical (a) or culture (b–d) replicates per condition; e, n = 7 mice per group, f, g, n = 5 mice per group. a–g, Representative of two independent experiments.

Extended Data Figure 9 Elevated [K+]i-induced suppression of human CD8+ TIL effector function requires intact PP2A activity.

a, Flow cytometry analysis of relative [K+]i using Asante-Green 4 on human CD8+ TILs in the indicated conditions. b, Representative flow cytometry of the cells in a following TCR-based activation in the indicated conditions; quantification depicted in Fig. 4e. c, Flow cytometry analysis of CD8+ T cells retrovirally engineered with Ctrl-Thy1.1, Kcna3-Thy1.1 or Kcna3_PD-Thy1.1 constructs. d, Flow cytometry analysis of Thy1.1+ (transduced) Pmel-1 CD45.1+CD8+ TILs re-isolated 6 days after transfer into B16 melanoma-bearing mice and re-stimulated ex-vivo. e, f, Immunoprecipitated PP2A protein complexes isolated and assayed for relative phosphatase activity in titrated concentrations of okadaic acid (e) or the indicated conditions (f). Additional [K+]e = 40 mM for mouse cells and 50 mM for human cells unless otherwise indicated. Centre values and error bars represent mean ± s.e.m. NS, not significant between selected relevant comparisons; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; two-tailed Student’s t-tests (c, f). n = 3 technical (a, e, f) or culture (b, c) replicates per condition; d, n = 5 mice per group. a–f, Representative of two independent experiments.

Extended Data Figure 10 Intratumoural inhibition of T cell effector function via an ionic checkpoint.

a, Healthy tissue contains limited local cellular decay, maintaining the interstitial [K+]e close to serum levels. T cells are robustly activated following TCR stimulation. b, Tumour-intrinsic phenomena produce a high density of cell death within cancers. Cell death leads to release of intracellular K+ into the extracellular space. The resultant elevated [K+]e acts to increase the [K+]i of T cells, limiting their activation and effector function. c, Reduction of [K+]i and increased effector function can be imparted to tumour-specific T cells by overexpression of Kv1.3 (Kcna3).

Supplementary information

Supplementary Information

This file contains Supplementary Information 1, a summary statistical analysis of comparative gene-set enrichment analysis comparing the gene expression relationship of indicated T cell populations, Supplementary Information 2, averaged RPKM values for T cells in the indicated conditions and Supplementary Figure 1, immunoblot source data. (PDF 470 kb)

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Eil, R., Vodnala, S., Clever, D. et al. Ionic immune suppression within the tumour microenvironment limits T cell effector function. Nature 537, 539–543 (2016). https://doi.org/10.1038/nature19364

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