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.

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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.

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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.

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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.

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Correspondence to Weiping Zou.

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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

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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

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