Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Oxidative stress controls regulatory T cell apoptosis and suppressor activity and PD-L1-blockade resistance in tumor

Abstract

Live regulatory T cells (Treg cells) suppress antitumor immunity, but how Treg cells behave in the metabolically abnormal tumor microenvironment remains unknown. Here we show that tumor Treg cells undergo apoptosis, and such apoptotic Treg cells abolish spontaneous and PD-L1-blockade-mediated antitumor T cell immunity. Biochemical and functional analyses show that adenosine, but not typical suppressive factors such as PD-L1, CTLA-4, TGF-β, IL-35, and IL-10, contributes to apoptotic Treg-cell-mediated immunosuppression. Mechanistically, apoptotic Treg cells release and convert a large amount of ATP to adenosine via CD39 and CD73, and mediate immunosuppression via the adenosine and A2A pathways. Apoptosis in Treg cells is attributed to their weak NRF2-associated antioxidant system and high vulnerability to free oxygen species in the tumor microenvironment. Thus, the data support a model wherein tumor Treg cells sustain and amplify their suppressor capacity through inadvertent death via oxidative stress. This work highlights the oxidative pathway as a metabolic checkpoint that controls Treg cell behavior and affects the efficacy of therapeutics targeting cancer checkpoints.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Treg cells are highly apoptotic in the tumor microenvironment.
Figure 2: Apoptotic Treg cells are immunosuppressive.
Figure 3: Apoptotic Treg cells mediate immunosuppression via soluble factor(s).
Figure 4: Apoptotic Treg cells mediate immunosuppression via adenosine.
Figure 5: Apoptotic Treg cells release ATP and generate high levels of adenosine.
Figure 6: Oxidative stress induces Treg cell apoptosis in the tumor environment.

Similar content being viewed by others

Accession codes

Accessions

Gene Expression Omnibus

References

  1. Hori, S., Nomura, T. & Sakaguchi, S. Control of regulatory T cell development by the transcription factor Foxp3. Science 299, 1057–1061 (2003).

    CAS  PubMed  Google Scholar 

  2. Fontenot, J.D., Gavin, M.A. & Rudensky, A.Y. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 4, 330–336 (2003).

    CAS  PubMed  Google Scholar 

  3. Chen, W. et al. Conversion of peripheral CD4+CD25 naive T cells to CD4+CD25+ regulatory T cells by TGF-β induction of transcription factor Foxp3. J. Exp. Med. 198, 1875–1886 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Curiel, T.J. et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat. Med. 10, 942–949 (2004).

    CAS  PubMed  Google Scholar 

  5. Sakaguchi, S. Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self. Nat. Immunol. 6, 345–352 (2005).

    CAS  PubMed  Google Scholar 

  6. Zou, W. Regulatory T cells, tumour immunity and immunotherapy. Nat. Rev. Immunol. 6, 295–307 (2006).

    CAS  PubMed  Google Scholar 

  7. Li, M.O., Sanjabi, S. & Flavell, R.A. Transforming growth factor-β controls development, homeostasis, and tolerance of T cells by regulatory T cell-dependent and -independent mechanisms. Immunity 25, 455–471 (2006).

    CAS  PubMed  Google Scholar 

  8. Liu, Y. et al. A critical function for TGF-β signaling in the development of natural CD4+CD25+Foxp3+ regulatory T cells. Nat. Immunol. 9, 632–640 (2008).

    CAS  PubMed  Google Scholar 

  9. Maruyama, T. et al. Control of the differentiation of regulatory T cells and TH17 cells by the DNA-binding inhibitor Id3. Nat. Immunol. 12, 86–95 (2011).

    CAS  PubMed  Google Scholar 

  10. Ouyang, W. et al. Novel Foxo1-dependent transcriptional programs control Treg cell function. Nature 491, 554–559 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Vander Heiden, M.G., Cantley, L.C. & Thompson, C.B. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324, 1029–1033 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Gubser, P.M. et al. Rapid effector function of memory CD8+ T cells requires an immediate-early glycolytic switch. Nat. Immunol. 14, 1064–1072 (2013).

    CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Chang, C.-H. et al. Metabolic competition in the tumor microenvironment is a driver of cancer progression. Cell 162, 1229–1241 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 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 

  16. Zhao, E. et al. Cancer mediates effector T cell dysfunction by targeting microRNAs and EZH2 via glycolysis restriction. Nat. Immunol. 17, 95–103 (2016).

    CAS  PubMed  Google Scholar 

  17. Clever, D. et al. Oxygen sensing by T cells establishes an immunologically tolerant metastatic niche. Cell 166, 1117–1131 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Eil, R. et al. Ionic immune suppression within the tumour microenvironment limits T cell effector function. Nature 537, 539–543 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Singer, M. et al. A distinct gene module for dysfunction uncoupled from activation in tumor-infiltrating T cells. Cell 166, 1500–1511 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Delgoffe, G.M. et al. The mTOR kinase differentially regulates effector and regulatory T cell lineage commitment. Immunity 30, 832–844 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Zeng, H. et al. mTORC1 couples immune signals and metabolic programming to establish Treg-cell function. Nature 499, 485–490 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Shi, L.Z. et al. HIF1α-dependent glycolytic pathway orchestrates a metabolic checkpoint for the differentiation of TH17 and Treg cells. J. Exp. Med. 208, 1367–1376 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Dang, E.V. et al. Control of TH17/Treg balance by hypoxia-inducible factor 1. Cell 146, 772–784 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Saito, T. et al. Two FOXP3+CD4+ T cell subpopulations distinctly control the prognosis of colorectal cancers. Nat. Med. 22, 679–684 (2016).

    CAS  PubMed  Google Scholar 

  25. Cubillos-Ruiz, J.R. et al. ER stress sensor XBP1 controls anti-tumor immunity by disrupting dendritic cell homeostasis. Cell 161, 1527–1538 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Arvey, A. et al. Inflammation-induced repression of chromatin bound by the transcription factor Foxp3 in regulatory T cells. Nat. Immunol. 15, 580–587 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Ko, K. et al. Treatment of advanced tumors with agonistic anti-GITR mAb and its effects on tumor-infiltrating Foxp3+CD25+CD4+ regulatory T cells. J. Exp. Med. 202, 885–891 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Cui, T.X. et al. Myeloid-derived suppressor cells enhance stemness of cancer cells by inducing microRNA101 and suppressing the corepressor CtBP2. Immunity 39, 611–621 (2013).

    CAS  PubMed  Google Scholar 

  29. Wan, S. et al. Tumor-associated macrophages produce interleukin 6 and signal via STAT3 to promote expansion of human hepatocellular carcinoma stem cells. Gastroenterology 147, 1393–1404 (2014).

    CAS  PubMed  Google Scholar 

  30. Shevach, E.M. CD4+CD25+ suppressor T cells: more questions than answers. Nat. Rev. Immunol. 2, 389–400 (2002).

    CAS  PubMed  Google Scholar 

  31. Vignali, D.A.A., Collison, L.W. & Workman, C.J. How regulatory T cells work. Nat. Rev. Immunol. 8, 523–532 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Deaglio, S. et al. Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression. J. Exp. Med. 204, 1257–1265 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Kryczek, I. et al. Phenotype, distribution, generation, and functional and clinical relevance of Th17 cells in the human tumor environments. Blood 114, 1141–1149 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Huang, S., Apasov, S., Koshiba, M. & Sitkovsky, M. Role of A2a extracellular adenosine receptor-mediated signaling in adenosine-mediated inhibition of T-cell activation and expansion. Blood 90, 1600–1610 (1997).

    CAS  PubMed  Google Scholar 

  35. Erdmann, A.A. et al. Activation of Th1 and Tc1 cell adenosine A2A receptors directly inhibits IL-2 secretion in vitro and IL-2-driven expansion in vivo. Blood 105, 4707–4714 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Chekeni, F.B. et al. Pannexin 1 channels mediate 'find-me' signal release and membrane permeability during apoptosis. Nature 467, 863–867 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Sporn, M.B. & Liby, K.T. NRF2 and cancer: the good, the bad and the importance of context. Nat. Rev. Cancer 12, 564–571 (2012).

    CAS  PubMed  Google Scholar 

  38. DeBerardinis, R.J. & Chandel, N.S. Fundamentals of cancer metabolism. Sci. Adv. 2, e1600200 (2016).

    PubMed  PubMed Central  Google Scholar 

  39. Zou, W., Wolchok, J.D. & Chen, L. PD-L1 (B7-H1) and PD-1 pathway blockade for cancer therapy: mechanisms, response biomarkers, and combinations. Sci. Transl. Med. 8, 328rv4 (2016).

    PubMed  PubMed Central  Google Scholar 

  40. Barnett, B., Kryczek, I., Cheng, P., Zou, W. & Curiel, T.J. Regulatory T cells in ovarian cancer: biology and therapeutic potential. Am. J. Reprod. Immunol. 54, 369–377 (2005).

    CAS  PubMed  Google Scholar 

  41. Dannull, J. et al. Enhancement of vaccine-mediated antitumor immunity in cancer patients after depletion of regulatory T cells. J. Clin. Invest. 115, 3623–3633 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Attia, P., Maker, A.V., Haworth, L.R., Rogers-Freezer, L. & Rosenberg, S.A. Inability of a fusion protein of IL-2 and diphtheria toxin (denileukin diftitox, DAB389IL-2, Ontak) to eliminate regulatory T lymphocytes in patients with melanoma. J. Immunother. 28, 582–592 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Kurose, K. et al. Phase Ia study of FoxP3+ CD4 Treg depletion by infusion of a humanized anti-CCR4 antibody, KW-0761, in cancer patients. Clin. Cancer Res. 21, 4327–4336 (2015).

    CAS  PubMed  Google Scholar 

  44. Tai, X. et al. Foxp3 transcription factor is proapoptotic and lethal to developing regulatory T cells unless counterbalanced by cytokine survival signals. Immunity 38, 1116–1128 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Yang, J. et al. Kupfer-type immunological synapse characteristics do not predict anti-brain tumor cytolytic T-cell function in vivo. Proc. Natl. Acad. Sci. USA 107, 4716–4721 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Wang, W. et al. Effector T cells abrogate stroma-mediated chemoresistance in ovarian cancer. Cell 165, 1092–1105 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Peng, D. et al. Epigenetic silencing of TH1-type chemokines shapes tumour immunity and immunotherapy. Nature 527, 249–253 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Curiel, T.J. et al. Blockade of B7-H1 improves myeloid dendritic cell–mediated antitumor immunity. Nat. Med. 9, 562–567 (2003).

    CAS  PubMed  Google Scholar 

  49. Zou, W. et al. Stromal-derived factor-1 in human tumors recruits and alters the function of plasmacytoid precursor dendritic cells. Nat. Med. 7, 1339–1346 (2001).

    CAS  PubMed  Google Scholar 

  50. Subramanian, A. et al. Gene Set Enrichment Analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 102, 15545–15550 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported (in part) by the US National Institutes of Health (grants CA217540, CA123088, CA099985, CA156685, CA171306, CA190176, CA193136, CA211016, and 5P30CA46592 to W.Z.), the Ovarian Cancer Research Fund, and the Marsha Rivkin Center for Ovarian Cancer Research (W.Z.; I.K.). We are grateful to L. Carter and X. Hu for critical discussions about the A2A pathway. We thank D. Postiff, M. Vinco, R. Craig, and J. Barikdar at the Tissue and Molecular Pathology Core for their assistance. We thank C. Ruan and S. Bridges at the Metabolomics Core for their support. Cd274−/− mice and Pdcd1−/− mice were provided by L. Chen (Yale University, New Haven, Connecticut, USA) and T. Honjo (Kyoto University, Kyoto, Japan), respectively.

Author information

Authors and Affiliations

Authors

Contributions

T.M., Wei Wang, I.K., and W.Z. designed the experiments. T.M., I.K., and W.Z. wrote the paper. Wei Wang, T.M., J.C., S.W., L.V., and I.K. performed the in vivo tumor experiments. L.V., W.S., I.K., and J.R.L. provided and processed clinical specimens and performed immunohistochemical and pathological analysis. T.M., Wei Wang, H.Z., I.S., and Weimin Wang performed the immunological and biochemical assays. I.K., L.Z., T.M., C.L., Wei Wang and W.Z. analyzed data.

Corresponding author

Correspondence to Weiping Zou.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Expression of apoptosis-related genes in tumor Treg cells.

(a) Identification of FOXP3 Treg cells by FACS. CD45+ cells were gated as enriched lymphoid cell populations with low-granularity. Singlet cells were gated on the basis of forward and side scatter W and H parameters. Next, T cell subsets were identified on the basis of CD3, CD4, and CD8 staining. Treg cells were identified as FOXP3+CD4+ T cells. FOXP3-CD4+ T cells were conventional T cells. (b) Ki67 expression in tumor infiltrating T cell subsets. Ki67 expression was detected in human ovarian cancer infiltrating FOXP3+ and FOXP3-CD45+CD3+CD4+ cells. Ki67 expression was shown in CD4+ T cells from two representative ovarian cancer specimens (left panel) and in FOXP3- and FOXP33+CD4+ T cell subsets (right panel). mean ± s.d., n = 10, Student's t-test, * P < 0.05. (c) Split Manders' coefficient plot depicts the colocalization of FOXP3 (red) and cleaved CASP3 (green) in human ovarian cancer section. One representative of 10 is shown. (d,e) Effect of mouse tumor medium on Treg cell gene expression. Normal mouse GFP+ Treg cells and GFP- conventional T cells were cultured with MC38-medium for 24 hours. Expression of pro-apoptotic (d) and anti-apoptotic (e) genes was quantified by real-time PCR. The level of each gene in Treg cells was normalized to that in conventional T cells. Data are shown as mean ± s.d., n = 5; Student's t-test, *P < 0.05.

Supplementary Figure 2 Suppressive activity of mouse live and apoptotic Treg cells.

(a) Representative dot plots show Treg and Tconv apoptosis induced by anti-FAS mAb Jo-1. Annexin V expression was analyzed by FACS at 30 minutes and 4 hours. (b,c) Mouse Treg apoptosis was induced by different conditions. T cell suppressive assay was performed with these apoptotic Treg cells. T cell TNF (b) and IL-2 (c) were measured on day 3 by ELISA, n = 5, Student's t-test, *P < 0.05. (d-f) Effect of live and apoptotic Treg cells on ID8-OVA tumor immunity. ID8-OVA-bearing mice were treated with live and apoptotic Treg cells. Tumor growth is shown as final bioluminescent signal quantification (d). Effector T cell cytokine expression (e, f) was detected in cancer ascites fluid. Data presented as mean ± s.d., n = 10 animals per group; ANOVA with Dunett post-hoc test, *P < 0.05. (g) Scheme of pmel-specific B16-F10 model. B16-F10 tumor bearing RAG2−/− mice received Pmel-specific T cells and intratumoral apoptotic Treg cell administration as indicated.

Supplementary Figure 3 Apoptotic Treg cells mediated immunosuppression via small and non-protein molecules.

(a-c) Effect of CTLA-4 blockade on apoptotic Treg-mediated immunosuppression. T cell immunosuppressive assay was performed with apoptotic Treg cells in the presence of anti-CTLA4 mAb. TNF (a) and IFN-γ (b) were analyzed by FACS on day 3 and IL-2 (c) was detected by ELISA on day 5 n = 5, ANOVA with Dunett's post-hoc test, *P < 0.05. (d) Effect of apoptotic Treg supernatants on T cell IL-2 production. Apoptotic Treg supernatants were collected at 6 hour time point and were added into T cell culture. T cell IL-2 was measured by ELISA. One of 3 experiments is shown. (e-m) Effect of the indicated cytokine blockade on apoptotic Treg cell-mediated immunosuppression. T cell immunosuppressive assay was performed with apoptotic Treg cells in the presence of anti-TGF-β (e-g), anti-EBI3 (h-j), and anti-IL-10 (k-l) mAbs. TNF (e, h, k) and IFN-γ (f, I, l) were analyzed by FACS on day 3. IL-2 (g, j, m) was detected by ELISA on day 5. n = 5, ANOVA with Dunett's post-hoc test, *P < 0.05.

Supplementary Figure 4 Adenosine production by apoptotic Treg cells.

Treg cell apoptosis was induced with anti-Fas mAb. Adenosine was measured by mass spectrometry in supernatants collected at different time points. Based on the standard curve (a) and the extracted ion changed chromatogram (b), adenosine was detected at 0.5 and 6 hours after induction of apoptosis (c). One of 3 independent experiments is shown.

Supplementary Figure 5 The metabolic profile of Treg cells.

(a,b) Purine (a) and pyrimidine (b) associated metabolism pathway in tumor associated Treg cells. GSEA analysis was performed in tumor associated Treg cells compared to conventional T cells at GSE55705 data set from GEO database. (c) Intracellular content of ATP in Treg cells and Tconv. ATP level was measured in cell lysates with comparable amount of protein by colorimetric assay. Data shown as mean ± s.d., Student's t-test, n = 5, *P < 0.05. (d) Effect of the pannexin-1 channel inhibitors on apoptotic Treg ATP release. Apoptosis was induced by anti-FAS treatment in the presence or absence of inhibitors probenecid and carbenoxolone. ATP in the supernatants was measured by colorimetric assay. Data presented as mean ± s.d., Student's t-test, n = 5, *P < 0.05 in comparison with control. (e,f) Intracellular (e) and released (f) ATP in live (e) and apoptotic (f) wild-type or Nt5e−/− mouse Treg cells. ATP level in whole cells was normalized to total protein expression (e). ATP in apoptotic Treg cell supernatants was shown at 30 minutes (f). n = 5, paired Student's t-test, *P > 0.05. (g) Adenosine production by wild-type and Nt5e−/− apoptotic Treg cells. Treg cell apoptosis was induced with anti-FAS and the supernatants were collected at 30 minutes. After deproteinization, adenosine was measured by colorimetric assay. Data shown as mean ± s.d., n = 5, Student's t-test, *P < 0.05.

Supplementary Figure 6 The effect of tumor oxidative stress on Treg cells.

(a,b) Effect of glucose restriction and 2-DG on conventional T cell (a) and Treg (b) apoptosis. Human T cell subsets were cultured with or without glucose or 2-DG for 24 hours. Annexin V+ T cells were measured by flow cytometry. One-way ANOVA with Dunnet's post-hoc test, *P < 0.05. (c) Effect of human ovarian cancer ascites on Treg apoptosis. Mouse Treg cells and conventional T cells (Tconv) were co-cultured with 50% ascites from intraperitoneal ID8 ovarian cancer bearing animals or hydrogen peroxide for 24 hours. Additional cultures were treated with NAC as a free radical scavenger. Annexin V+ Treg cells and Tconv were analyzed by flow cytometry. Data presented as mean ± s.d., n = 6, *P < 0.05. (d) Superoxide level in human ascites. The concentration of superoxide was measured with colorimetric test. Water contains 2 μM H2O2 as a positive control. Data are shown as mean ± s.d., n = 3. (e) Mitochondrial load of mouse Treg cells. The cells were treated with fluorescent mitochondrial activity dye (Mitotracker) and analyzed by flow cytometry. One of 3 assays is shown. (f) Level of reactive oxygen species (ROS) in ovarian cancer infiltrating conventional T cells and Treg cells. The level of ROS was tested by CellROX Green and ROS content was shown as mean fluorescence intensity. Data shown as mean ± s.d., n = 5, Student's t-test, *P < 0.05. (g,h) Expression of human Nrf2 and NRF2-associated genes and protiens in Treg cells. Nfe2l2 and NRF2-associated gene transcripts (g) and proteins (h) were determined in T cell subsets by real-time PCR and immunoblotting, respectively. Data presented as mean ± s.d., n = 5, paired Student's t-test, *P < 0.05

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Tables 1–3 (PDF 1596 kb)

Life Sciences Reporting Summary (PDF 171 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Maj, T., Wang, W., Crespo, J. et al. Oxidative stress controls regulatory T cell apoptosis and suppressor activity and PD-L1-blockade resistance in tumor. Nat Immunol 18, 1332–1341 (2017). https://doi.org/10.1038/ni.3868

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni.3868

This article is cited by

Search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer