Letter | Published:

CD8+ T cells regulate tumour ferroptosis during cancer immunotherapy

Abstract

Cancer immunotherapy restores or enhances the effector function of CD8+ T cells in the tumour microenvironment1,2. CD8+ T cells activated by cancer immunotherapy clear tumours mainly by inducing cell death through perforin–granzyme and Fas–Fas ligand pathways3,4. Ferroptosis is a form of cell death that differs from apoptosis and results from iron-dependent accumulation of lipid peroxide5,6. Although it has been investigated in vitro7,8, there is emerging evidence that ferroptosis might be implicated in a variety of pathological scenarios9,10. It is unclear whether, and how, ferroptosis is involved in T cell immunity and cancer immunotherapy. Here we show that immunotherapy-activated CD8+ T cells enhance ferroptosis-specific lipid peroxidation in tumour cells, and that increased ferroptosis contributes to the anti-tumour efficacy of immunotherapy. Mechanistically, interferon gamma (IFNγ) released from CD8+ T cells downregulates the expression of SLC3A2 and SLC7A11, two subunits of the glutamate–cystine antiporter system xc, impairs the uptake of cystine by tumour cells, and as a consequence, promotes tumour cell lipid peroxidation and ferroptosis. In mouse models, depletion of cystine or cysteine by cyst(e)inase (an engineered enzyme that degrades both cystine and cysteine) in combination with checkpoint blockade synergistically enhanced T cell-mediated anti-tumour immunity and induced ferroptosis in tumour cells. Expression of system xc was negatively associated, in cancer patients, with CD8+ T cell signature, IFNγ expression, and patient outcome. Analyses of human transcriptomes before and during nivolumab therapy revealed that clinical benefits correlate with reduced expression of SLC3A2 and increased IFNγ and CD8. Thus, T cell-promoted tumour ferroptosis is an anti-tumour mechanism, and targeting this pathway in combination with checkpoint blockade is a potential therapeutic approach.

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Data availability

RNA sequencing data that support the findings of this study have been deposited in NCBI Gene Expression Omnibus (GEO) under accession number GSE128392. All other data that supported the findings of this study are available from the corresponding author upon request.

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Acknowledgements

We thank all members of the Zou laboratory for suggestions. This work was supported in part by NIH/NCI R01 grants (CA217648, CA123088, CA099985, CA193136 and CA152470), and the NIH through the University of Michigan Rogel Cancer Center Support Grant (CA46592) (W.Z.); the NIH/NCI (CA189623) (E.S. and G.G.); Pershing Square Sohn Cancer Research, the PaineWebber Chair, the NIH/NCI (CA205426), the STARR Cancer Consortium, NCI R35 (CA232097), and an NIH/NCI Cancer Center Support Grant (P30 CA008748) (T.A.C.).

Reviewer information

Nature thanks Matthew Albert, Scott Dixon, Valerian Kagan and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

W.W. and W.Z. conceived the project, designed the experiments and wrote the manuscript. W.W. performed most of the experiments with help from A.S., S.W., L.V. and W.S. M. Green performed part of the tumour immunotherapy experiments and data analysis. J.E.C., W.L., J.L. and M.C. performed bioinformatics analysis. M. Gijón, P.D.K. and J.K.J. performed analysis of oxidized phospholipids by LC–MS. P.L., H.X., J.Z., L.V. and H.Z. assisted with tumour xenograft experiments. X.L. performed STAT1 ChIP experiment. G.L. assisted with the generation of knockout cells. I.K. assisted with flow cytometry analysis. C.L., Y.T., E.S. and G.G. contributed reagents. I.K., W.G., R.L., T.S.L., E.S., G.G., T.A.C. and A.C. contributed to discussions and edited the manuscript. W.Z. supervised work and acquired funding.

Competing interests

G.G. and E.S. are inventors on intellectual property related to cyst(e)inase and hold equity interest in Aeglea Biotherapeutics Inc. T.A.C. is a co-founder of and holds equity in Gritstone Oncology. T.A.C. holds equity in An2H. T.A.C. acknowledges grant funding from Bristol-Myers Squibb, AstraZeneca, Illumina, Pfizer, An2H, and Eisai. T.A.C. has served as a paid advisor for Bristol-Myers Squibb, Illumina, Eisai, and An2H. Memorial Sloan Kettering has licensed the use of tumour mutation burden (TMB) for the identification of patients that will benefit from immune checkpoint therapy to PGDx. Memorial Sloan Kettering and T.A.C. receive royalties as part of this licensing agreement. W.Z. has served as a consultant or advisor for Lycera, NGM, Synlogic, and Henlix.

Correspondence to Weiping Zou.

Extended data figures and tables

Extended Data Fig. 1 Immunotherapy increases lipid peroxidation in cancer cells.

a, Flow cytometry analysis of BODIPY fluorescence in CD45 tumour cells isolated from mouse peritoneal cavity. b, ID8 tumour growth in individual mice was monitored by quantifying total flux (photons per second). Animals were treated with either anti-PD-L1 or isotype monoclonal antibodies (Fig. 1b). c, Flow cytometry analysis of oxidized BODIPY fluorescence in CD45OVA-H2Kb+ tumour cells and CD45+ cells isolated from subcutaneous B16 tumour tissue. d, Relative lipid ROS in CD45+ cells isolated from subcutaneous B16 tumour tissue. Control, n = 9; OT-I, n = 10; ns, P = 0.1584 (two-tailed t-test). e, Effect of OT-I cells on MDA concentration in B16 cells in vivo. MDA content in tumour tissue lysate was measured by TBRAS assay and normalized to protein concentration. Control, n = 9; OT-I, n = 9; *P = 0.0285 (two-tailed t-test). f, Subcutaneous B16 tumours from control and OT-I groups were surgically removed and presented. The minimum scale of the rule shows millimetres. Source data

Extended Data Fig. 2 Ferroptosis in cancer cells is regulated by immunotherapy and contributes to the anti-tumour effect of immunotherapy.

a, b, Relative viability of parental or erastin-resistant ID8 cells treated with different concentrations of the ferroptosis inducers RSL3 or erastin (a) or the apoptosis inducers doxrubicin and gemcitabine (b) for 24 h. n = 3 or 4 biological replicates (mean ± s.d.). ****P < 0.0001 (two-way ANOVA). c, Anti-tumour effect of PD-L1 blockade in parental or erastin-resistant ID8 tumour-bearing mice. Mice with luciferase-expressing ID8 tumour cells were treated with anti-PD-L1 or isotype monoclonal antibodies. Tumour growth was monitored by quantifying total flux (photons per second). Parental–isotype, n = 8; parental–anti-PD-L1, n = 8; erastinresis–isotype, n = 9; erastinresis–anti-PD-L1, n = 9; **** P < 0.0001; ns, P = 0.9018 (two-way ANOVA). d, e, Relative viability of parental B16 or RSL3-resistant B16 cells treated with different concentrations of ferroptosis inducers RSL3 or erastin (d) or apoptosis inducers doxrubicin and gemcitabine (e) for 24 h. n = 3 or 4 biological replicates (mean ± s.d.). ****P < 0.0001 (two-way ANOVA). f, g, Effect of anti-PD-L1 therapy on tumour lipid ROS (f) and tumour growth (g) in RSL3-resistant (RSL3resis) B16 tumour bearing mice. Mice with subcutaneous tumours were treated with either anti-PD-L1 or isotype monoclonal antibodies. f, Relative lipid ROS in tumour cells was measured by FACS in gated CD45 cells (isotype, n = 9; anti-PD-L1, n = 10; two-tailed t-test; ns, P = 0.9608). g, Tumour weight was measured on day 17 (isotype, n = 10; anti-PD-L1, n = 10; two-tailed t-test; ns, P = 0.3621). h, Immunoblot of ACSL4 in RSL3resis B16 and erastinresis ID8 cells compared with parental cells. i, j, Relative cell viability of wild-type or ACSL4−/− ID8 cells treated with different concentrations of erastin (i) or RSL3 (j) for 24 h. n = 3 or 4 biological replicates (mean ± s.d.). ****P < 0.0001 (two-way ANOVA). k, l, Anti-tumour effect of PD-L1 blockade in wild-type (k) or ACSL4−/− (l) ID8 tumour-bearing mice. Luciferase-expressing ID8 tumour-bearing mice were treated with either anti-PD-L1 or isotype monoclonal antibodies. Tumour growth was monitored by quantifying total flux (photons per second). WT, isotype, n = 10; WT, anti-PD-L1, n = 10; ACSL4−/−, isotype, n = 9; ACSL4−/−, anti-PD-L1, n = 9; two-way ANOVA, ****P < 0.0001 (k); ns, P = 0.317 (l). m, Percentage of 7-AAD+ ID8-OVA cells in mixed co-cultures with OT-I cells (ID8:OT-I = 1:1) for 24 h followed by treatment with RSL3 (0.1 μM) for 20 h. n = 3 biological replicates. ***P = 0.0004, ****P < 0.0001 (one-way ANOVA). n, Percentage of 7-AAD+ B16-OVA cells in mixed co-cultures with OT-I cells (B16:OT-I = 1:2) in the presence of Fer1 (10 μM) for 40 h. n = 3 biological replicates. ns, P = 0.4640 (one-way ANOVA). o, Relative viability of HT-1080 cells primed with supernatant from anti-CD3 and anti-CD28 activated human CD8+ T cells for 24 h, then treated with RSL3 (0.05 μM) in the presence of Fer1 (10 μM) for another 16 h. n = 4 biological replicates. **P = 0.0015 (one-way ANOVA). Source data

Extended Data Fig. 3 IFNγ sensitizes tumour cells to ferroptosis.

a, Relative lipid ROS in B16 cells treated with supernatant from activated CD8+ T cells in the presence of anti-IFNγ or anti-TNF blocking antibody for 40 h. n = 4 biological replicates. ns, P = 0.1003, ****P < 0.0001 (one-way ANOVA). b, Relative lipid ROS in wild-type or IFNGR1−/− B16 cells treated with supernatant from activated CD8+ T cells for 40 h. n = 4 biological replicates. ns, P = 0.9981, **** P < 0.0001 (one-way ANOVA). c, Lipid ROS in B16 or HT-1080 cells treated with IFNγ for 24 h (representative histogram plot for fluorescence of oxidized BODIPY-C11). d, MFI of LiperFluo in B16 cells treated with IFNγ for 24 h. n = 4 biological replicates. ****P < 0.0001 (two-tailed t-test). e, MFI of LiperFluo in HT-1080 cells primed with IFNγ for 24 h, then treated with RSL3 (0.05 μM) for 6 h in the presence of Fer1 (10 μM). n = 4 biological replicates. **P = 0.0067, ***P = 0.0003, ****P < 0.0001 (two-way ANOVA). f, Relative lipid ROS of HT-1080 cells primed with IFNγ (10 ng ml−1) for 40 h and then treated with erastin (2 μM) for 8 h. n = 3 biological replicates. *P = 0.0426 or 0.0250 (one-way ANOVA). g, h, Relative viability of B16 (g) or HT-1080 (h) cells primed with or without (Ctrl) IFNγ for 40 h in the presence of Fer1 (10 μM), followed by treatment with different concentrations of erastin or RSL3 for 24 h. n = 3 or 4 biological replicates (mean ± s.d.). i, Percentage of 7-AAD+ cells among B16 cells primed with IFNγ (10 ng ml−1) for 40 h and then treated with RSL3 (0.1 μM) for 20 h. Representative images show cell death (left). n = 3 biological replicates. ****P < 0.0001 (one-way ANOVA). j, k, Percentage of 7-AAD+ cells among B16 (j) or HT-1080 (k) cells primed with IFNγ and then treated with RSL3 (0.1 μM, j) or erastin (4 μM, k) in the presence of Fer1(10 μM) or deferoxamine (DFO, 100 μM). n = 2 biological replicates. l, m, Relative content of oxygenated phosphatidylethanolamine (PE) (l) and phosphatidylcholine (PC) (m) species in HT-1080 cells primed with IFNγ (10 ng ml−1) for 48 h. n = 3 biological replicates. ***P = 0.0008, *P = 0.0167 (two-tailed t-test). n, Relative viability of HT-1080 cells primed with IFNγ for 24 h, then treated with ML162 (0.1 μM), ML210 (0.1 μM), or BSO (5 μM) for 24 h in the presence of Fer1 (10 μM). n = 3 (mean ± s.d.), ****P < 0.0001 (two-way ANOVA). o, Percentage of 7-AAD+ cells among HT-1080 cells primed with IFNγ, then treated with SAS (0.5 mM) for 40 h in the presence of Fer1 (10 μM). n = 2 biological replicates. p, Relative viability of B16 cells primed with IFNγ for 24 h, then treated with different concentrations of SAS for an additional 24 h. n = 3 (mean ± s.d.), ****P < 0.0001 (two-way ANOVA). q, Relative viability of HT-1080 cells primed with or without IFNγ, then cultured with medium supplemented with decreased concentrations of cystine in the presence of Fer1 (10 μM) for 20 h. n = 3 or 4 biological replicates (mean ± s.d.). r, Effect of IFNγ and SAS on HT-1080 tumour growth in vivo. HT-1080 cells (2 × 106 cells) were subcutaneously inoculated into NSG mice. Mice were treated either with IFNγ (1.5 × 105 U per mouse), SAS (120 mg/kg) or both. n = 5 animals in each group. *P < 0.05, ****P < 0.0001 (two-way ANOVA). Source data

Extended Data Fig. 4 Tumour cells and T cells are differentially responsive to ferroptosis inducers.

a, b, Percentage of 7-AAD+ cells in naive human (a) and naive mouse (b) CD4+ and CD8+ T cells primed with IFNγ (10 ng ml−1) for 24 h, followed by treatment with Fer1 (10 μM) and different concentrations (μM) of erastin or RSL3 for 24 h. n = 3 biological replicates (mean ± s.d.). c, d, Percentage of IFNγ+ cells in human (c) and mouse (d) CD4+ and CD8+ T cells. T cells were activated with anti-CD3 and anti-CD28 antibodies for 1 day, followed by treatment with Fer1 and different concentrations (μM) of erastin and RSL3 for 2 days. IFNγ expression was determined by FACS. n = 3 biological replicates (mean ± s.d.). Source data

Extended Data Fig. 5 IFNγ targets system xc to regulate tumour cell ferroptosis.

a, Venn diagram showing common genes whose expressions were negatively (z > 5.83) or positively (z < −5.83) associated with cell line sensitivity to erastin and RSL3. b, Box-and-whisker plots show 1st and 99th percentile outlier transcripts (black and coloured dots) whose expression levels are correlated with cell line sensitivity to erastin and RSL3. Plotted values are z-scored Pearson’s correlation coefficients. Line, median; box, 10th–90th percentiles. c, Heat maps of the 16 genes associated with sensitivity to erastin and RSL3 and their expressions in IFNγ-treated HT-1080 cells (bottom). Left twelve genes are negatively associated with drug sensitivity; right four genes are positively associated with drug sensitivity. d, Relative mRNA expression of SLC3A2 and SLC7A11 in HT-1080 cells treated with IFNγ at different time points. n = 3 biological replicates (mean ± s.d.). e, Concentration of glutamate released from HT-1080 cells primed with IFNγ and then treated with DMSO or erastin. n = 3 biological replicates. ***P < 0.001 (one-way ANOVA). f, Intracellular GSH in HT-1080 cells treated with IFNγ (10 ng ml−1) for 24 h and then with erastin (0.5 μM) for 16 h. n = 3 biological replicates. ns, P = 0.8843, ****P < 0.0001 (one-way ANOVA). g, h, Immunoblots of SLC7A11 in HT-1080 cells. HT-1080 cells expressed scramble shRNA, one of three independent shRNAs targeting SLC7A11 (g) or lentivector expressing RFP and SLC7A11 (h). i, Percentage of dead HT-1080 cells bearing RFP or SLC7A11 cDNA, primed with or without IFNγ, then treated with or without erastin (5 μM) for 20 h. n = 2 biological repeats. j, Lipid ROS in HT-1080 cells with empty vector (Empty) or SLC7A11 cDNA primed with IFNγ, then treated with erastin (1 μM) for 20 h (representative histogram plot for fluorescence of oxidized BODIPY-C11). k, Immunoblots of SLC3A2 in HT-1080 cells expressing scramble shRNA or either of two independent shRNAs targeting SLC3A2. l, Relative viability of HT-1080 cells expressing scramble shRNA or shRNA targeting SLC3A2 (shSLC3A2-1, -2), treated with erastin or RSL3 for 24 h. n = 4 biological replicates; ***P < 0.001, ****P < 0.0001 (two-way ANOVA). m, Relative mRNA expression of SLC7A11, SLC3A2, and IRF1 in HT-1080 cells treated for 24 h with supernatant from naive or activated CD8+ T cells. ***P < 0.001 (two-tailed t-test). n, Relative mRNA expression of SLC3A2 and SLC7A11 in human A375 cells treated with IFNγ at different time points. o, Relative mRNA expression of SLC7A11 in B16 cells treated with IFNγ at different time points. p, Immunoblots of mouse SLC7A11 and IRF1 in B16 cells treated with IFNγ (10 ng ml−1) for 24 h. β-actin serves as loading control. Images are representative of two experiments. q, Relative mRNA expression of SLC7A11 in B16 cells expressing shRNA against SLC7A11. r, Relative viability of B16 cells expressing scramble shRNA or shRNA targeting SLC7A11 treated with erastin or RSL3 for 24 h. ****P < 0.0001 (two-way ANOVA). s, Percentage of 7-AAD+ cells in B16 cells expressing scramble shRNA or shRNA targeting SLC7A11 treated with RSL3 for 16 h. ****P < 0.0001 (one-way ANOVA). Source data

Extended Data Fig. 6 IFNγ inhibits SLC7A11 through the JAK–STAT1 pathway.

a, Relative expression of SLC7A11 pre-mRNA in HT-1080 cells treated with IFNγ at different time points. b, c, Relative mRNA expression of SLC7A11 (b) or IRF1 (c) in HT-1080 cells treated with IFNγ and JAK inhibitor I or ruxolitinib (0, 0.5 or 2 µM) for 24 h. d, ChIP of STAT1 in HT-1080 cells treated with or without IFNγ. STAT1 binding to SLC7A11 TSS region was quantified by qPCR. Results are expressed as fold change in site occupancy over control. *P = 0.0156 (two-way ANOVA). e, Immunoblot of STAT1 in wild-type or STAT1−/− HT-1080 cells generated by CRISPR–Cas9. fk, Wild-type or STAT1−/− HT-1080 cells treated with or without IFNγ. SLC7A11 mRNA level (f), SLC7A11 immunoblot (g), IRF1 mRNA level (h), relative lipid ROS (i), erastin-induced cell death (j), and RSL3-induced cell death (k) were analysed. **P = 0.0033, ns, P > 0.9999 (two-way ANOVA) (i, right). l, Percentage of 7-AAD+ cells in wild-type or STAT1−/− B16 cells treated with or without IFNγ, followed by RSL3 treatment for 24 h. n = 3 biological replicates. Source data

Extended Data Fig. 7 Cyst(e)inase and PD-L1 blockade synergistically induce ferroptosis.

a, Relative lipid ROS in B16 cells primed with IFNγ and then treated with 500 nM cyst(e)inase for 12 h. n = 2 biological repeats. b, Percentage of 7-AAD+ cells in B16 cells primed with IFNγ for 24 h and then treated with different concentrations of cyst(e)inase for 40 h. n = 2 biological repeats. c, Percentage of 7-AAD+ cells in B16 cells primed with IFNγ and then treated with 400 nM cyst(e)inase in the presence of 10 µM Fer1 for 24 h. n = 3 biological replicates. ns, P = 0.7290, ****P < 0.0001 (one-way ANOVA). d, Percentage of 7-AAD+ cells in wild-type or STAT1−/− B16 cells treated with or without IFNγ and then treated with 500 nM cyst(e)inase for 40 h. n = 2 or 4 biological replicates. ****P < 0.0001 (one-way ANOVA). e, f, Relative lipid ROS (e) and percentage of 7-AAD+ cells (f) in B16 cells primed with IFNγ or BSA, and then treated with 500 nM heated cyst(e)inase or cyst(e)inase for 24 h (e) or 40 h (f). n = 3 biological replicates. **P = 0.0023, ****P < 0.0001 (two-way ANOVA). g, Effect of cyst(e)inase combined with PD-L1 blockade on IB8 tumour growth. Tumours were monitored over time by quantifying total flux in mouse peritoneal cavity and bioluminescence imaging of representative mice from indicated days is shown. h, Effect of liproxstatin-1 on anti-tumour efficacy of the combination therapy. ID8 tumour-bearing mice treated with a combination of cyst(e)inase and anti-PD-L1 were treated with liproxstatin-1 (10 mg kg−1, n = 9) or DMSO (control, n = 9). Tumour growth was monitored over time by quantifying total flux in peritoneal cavity. Data plotted are mean ± s.e.m. ****P < 0.0001 (two-way ANOVA). Source data

Extended Data Fig. 8 System xc expression correlates with immune signatures and patient outcome.

a, Representative images of dual staining of CD8 and SLC7A11 (top) or CD8 and SLC3A2 (bottom) by immunohistochemistry in human melanoma samples. The levels of SLC7A11 and SLC3A2 expression on tumour cells were assessed by the H-score method. b, c, Kaplan–Meier survival curves for patients with melanoma with low (n = 231) or high (n = 232) CD8A expression (b), and low (n = 231) or high (n = 232) IFNγ signature score (c). P values determined by log-rank test. Source data

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Fig. 1: Immunotherapy-activated CD8+ T cells regulate ferroptosis of cancer cells.
Fig. 2: IFNγ sensitizes tumour cells to ferroptosis by inhibiting system xc.
Fig. 3: Cyst(e)inase and PD-L1 blockade synergistically induce ferroptosis.
Fig. 4: System xc expression correlates with immune signatures and patient outcome.
Extended Data Fig. 1: Immunotherapy increases lipid peroxidation in cancer cells.
Extended Data Fig. 2: Ferroptosis in cancer cells is regulated by immunotherapy and contributes to the anti-tumour effect of immunotherapy.
Extended Data Fig. 3: IFNγ sensitizes tumour cells to ferroptosis.
Extended Data Fig. 4: Tumour cells and T cells are differentially responsive to ferroptosis inducers.
Extended Data Fig. 5: IFNγ targets system xc to regulate tumour cell ferroptosis.
Extended Data Fig. 6: IFNγ inhibits SLC7A11 through the JAK–STAT1 pathway.
Extended Data Fig. 7: Cyst(e)inase and PD-L1 blockade synergistically induce ferroptosis.
Extended Data Fig. 8: System xc expression correlates with immune signatures and patient outcome.

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