IL-2 regulates tumor-reactive CD8+ T cell exhaustion by activating the aryl hydrocarbon receptor

Abstract

CD8+ T cell exhaustion dampens antitumor immunity. Although several transcription factors have been identified that regulate T cell exhaustion, the molecular mechanisms by which CD8+ T cells are triggered to enter an exhausted state remain unclear. Here, we show that interleukin-2 (IL-2) acts as an environmental cue to induce CD8+ T cell exhaustion within tumor microenvironments. We find that a continuously high level of IL-2 leads to the persistent activation of STAT5 in CD8+ T cells, which in turn induces strong expression of tryptophan hydroxylase 1, thus catalyzing the conversion to tryptophan to 5-hydroxytryptophan (5-HTP). 5-HTP subsequently activates AhR nuclear translocation, causing a coordinated upregulation of inhibitory receptors and downregulation of cytokine and effector-molecule production, thereby rendering T cells dysfunctional in the tumor microenvironment. This molecular pathway is not only present in mouse tumor models but is also observed in people with cancer, identifying IL-2 as a novel inducer of T cell exhaustion.

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Fig. 1: CD8+ T cell exhaustion is induced by activating the IL-2 signaling pathway.
Fig. 2: IL-2 induces CD8+ T cell exhaustion by activating the TPH1–5-HTP pathway.
Fig. 3: CD8+ T cell exhaustion induced by IL-2 is due to the activation of the 5-HTP–AhR pathway.
Fig. 4: IL-2 signaling regulates AhR stability.
Fig. 5: IL-2 induces tumor CD8+ T cell exhaustion by activating the TPH1–5-HTP–AhR pathway in vivo.
Fig. 6: The source of IL-2 switches from CD3+CD8+ T cells to CD3+CD4+ T cells in the tumor microenvironment.
Fig. 7: IL-2 induced CD8+ T cell exhaustion by activating the 5-HTP–AhR pathway in people with cancer.

Data availability

The ChIP–seq data reported in this paper have been deposited in the Genome Sequence Archive in National Genomics Data Center, Beijing Institute of Genomics (China National Center for Bioinformation), Chinese Academy of Sciences, under accession number CRA003170. The TCGA data reported in this paper derive from UCSC xena (https://xenabrowser.net/datapages/). Source data are provided with this paper.

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Acknowledgements

This work was supported by National Natural Science Foundation of China (81788101, 81773062, 91942314) and Chinese Academy of Medical Sciences (CAMS) Initiative for Innovative Medicine (CAMS-I2M) 2016-I2M-1-007.

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Authors

Contributions

B.H. conceived of the project. Y.L., N.Z., L.Z., J.W., Y.Z., T.Z., Y.F., Y.G., X.L., J.L., Z.W., J.X., Huafeng Zhang, J.M., K.T., Y.F., F.C. and Haizeng Zhang performed the experiments. Y.L., N.Z., L.Z., J.W., Y.X., J.D., C.Z., B.D., Y.Z., Q.G. and P.Y. developed methodology. Y.L., B.H., F.X.-F.Q., N.Z. and L.Z. performed data analysis. B.H. and Y.L. wrote the manuscript.

Corresponding author

Correspondence to Bo Huang.

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The authors declare no competing interests.

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Peer review information Nature Immunology thanks Yang-Xin Fu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Zoltan Fehervari was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 IL-2 signaling induces CD8+ T cells exhaustion.

a, TCGA database RNA-Seq analysis of correlation between the expression of IL-2 and exhausted T cell signature genes in breast (n = 195) and lung (n = 61) paracancerous tissues, uterine corpus endometrial carcinoma (n = 176), and colon paracancerous tissues (n = 43); or IL-2R and exhausted T cell signature genes in colon cancer (n = 590), breast cancer (n = 1080), lung squamous cell carcinoma (n = 482) or lung adenocarcinoma (n = 508) patients. GEO database RNA-Seq analysis of correlation between the expression of IL-2 and exhausted T cell signature genes in liver normal tissue from chronic hepatitis B patients (n = 124, GSE84044). b, Activated OVA-CTLs were treated with IL-2, IL-2 + anti-CD25, IL-2 + anti-CD122 or IL-2 + anti-CD25 + anti-CD122 neutralizing antibodies for 72 hr. Expression profiles of IRs and cytokines were determined by flow cytometry. c, Activated CD45.1+ gp-100-CTLs were adoptively transferred into C57BL/6 J mice bearing with 5 × 5 mm B16 melanoma. At the same time, these mice were intraperitoneally injected with PBS, IL-2, anti-IL-2 neutralizing antibody or anti-CD25 + anti-CD122 neutralizing antibody every two days for 3 times. At day 7, CD45.1+ transferred cells were isolated from the tumors for flow cytometric analysis of the expression of IRs (PD-1, LAG3, TIM3) and cytokines. d, The same as (c), except that CD45.1+ OVA-CTLs were transferred into mice. e-g, Activated CTLs were treated with IL-2 for the indicated time periods. Expression profiles of IRs (e), viable cell number (f) and the level of cytokines from the supernatants (g) were determined by flow cytometry or ELISA. h-j, Splenocytes from OVA-OT1 mice were stimulated by OVA peptides for 48 hr, and then treated with IL-2 for indicated times periods. The expression of IRs (h), the level of cytokines (i) and viable cells number (j) were determined by flow cytometry. k,l, CTLs were treated with different doses of IL-2 for 48 hr (k) or IL-2 for indicated time periods (l). The expression of IRs was determined by flow cytometry. In c,d, n = 5 mice; b,e-l, n = 3 independent experiments. Two- tailed Student’s t test (f) or by one-way ANOVA Bonferroni’s test (b-e, g-l), or Pearson’s correlation test (a). The data represent mean ± SD. Source data

Extended Data Fig. 2 The Tph1-5HTP pathway regulates CD8+ T cell exhaustion induced by IL-2.

a, Activated CTLs were treated with STAT5 inhibitor (Stat5in), fludarabine, Stattic, LY294002, SCH772984, SP00125 or SB203580 for 48 hr. The expression of IRs was determined by flow cytometry. b,h The knockdown efficiency of STAT5 (b) or TPH1 (h) was confirmed by western blot. c,d, The resting or activated CTLs were treated with IL-2 for 48 hr (c). Or, activated CTLs were treated with IL-2 or IL-2 + Stat5in for 48 hr (d). Tph1 mRNA was determined by real-time PCR. e, The expression of Tph1 from resting or activated Stat5 shRNAs- CD8+ T cells treated with IL-2 or IL-2 + Stat5in for 48 hr. f, The same as (c), except that activated scramble or Stat5 shRNAs-CTLs were used. g, Activated CTLs were treated with IL-2, Stat5in or IL-2 + Stat5in for 48 hr. The level of 5HTP was measured by HPLC-MS. i-k, Activated CTLs were treated with different doses of 5HTP as indicated for 48 hr (i,j) or 5HTP for the indicated time periods (k). Flow cytometric analysis of the expression of IRs, TNF or IFN-γ. l,m, The same as (i-k), except that resting CD8+ T cells were used. In a-m, n = 3 independent experiments. One-way ANOVA Bonferroni’s test (a, c, d, f, g, i-m). The data represent mean ± SD. Source data

Extended Data Fig. 3 The Kyn-AhR pathway regulates IL-2 induced CD8+ T cells exhaustion.

a,b, Activated CTLs were treated with 5HTP or different doses of carbidopa (a), serotonin or melatonin (b) for 48 hr. The expression of IRs was determined by flow cytometry. c, Resting or activated CTLs were treated with 5HTP for 48 hr. The mRNA expression of Cyp1a1 and Cyp1b1 was determined by real-time PCR. d,e, Activated murine CTLs were treated with StemRegenin 1 (SR1), CH223191 (CH), 5HTP, 5HTP/SR1 or 5HTP/CH for 48 hr. Flow cytometric analysis of the expression of IRs (d), or TNF and IFN-γ (e). f, The same as (d,e), except that CTLs were treated with IL-2, IL-2 + SR1 or IL-2 + CH. g, NIH3T3 cells expressing Pdcd-1, Lag3, Havcr2 or Entpd1 promoter-luciferase reporter PGL3 were co-transfected with AhR-plasmid. Cells were treated with 5HTP for 48 hr and analyzed by luciferase assay. h, The same as g, except that cells expressed Pdcd-1, Havcr2 or Entpd1 promoter mutant-luciferase reporter PGL3. i, The same as f, except that cells were treated with Kyn or tryptophan (Trp) for 48 hr. Flow cytometric analysis of the expression of IRs, TNF and IFN-γ. In a-i, n = 3 independent experiments. One-way ANOVA Bonferroni’s test (a, c, d, e, f, g, h, i). The data represent mean ± SD. Source data

Extended Data Fig. 4 IL-2 treatment stabilizes AhR.

a, Activated human CD8+ T cells were treated with IL-2 or IL-2 antibody for 48 hr. The mRNA expression of AhR was measured by real-time PCR. b, Activated CTLs were treated with IL-2 in the presence of cycloheximide (CHX) (2 μg/mL) for the indicated time periods. The level of AhR was determined by western blot analysis. c,The same as (b), except that stably overexpressing HA-AhR human CD8+ T cells were used and the level of AhR was determined by western blot with anti-HA antibody. d, Activated CTLs were treated with IL- 2, anti-IL-2 antibody or IL-2 + anti-IL-2 antibody for 48 hr. AhR level was determined by western blot. e,I, The same as d, except that cell lysates were immunoprecipitated with anti-AhR antibody and immunoblotted with an anti-ubiquitination I, SUMO1 or SUMO2/3 (i) antibody. f,j, Activated human CD8+ T cells stably overexpressing HA-AhR were transfected with a Flag-UbG76V (f) or Flag-SUMO1 plasmid (j). Then, cells were treated with IL- 2, anti-IL-2 antibody or IL-2 + anti-IL-2 antibody. Cell lysates were immunoprecipitated with anti-HA antibody and immunoblotted with an anti-ubiquitination or Flag antibody. g,h, Anti-CD3 antibody-activated human (g) or mouse (h) CD8+ T cells were treated with IL-2 in the presence of MG132 (10 μM) for the indicated time periods. Cells were lysed to perform western blot analysis with an anti-AhR antibody. k, Activated HA-AhR-human CD8+ T cells were transfected with Flag-SUMO1 plasmid. Cells were stained with anti-Flag and anti-HA antibodies and observed under a confocal microscope. Bar, 10 μm. In a-k, n = 3 independent experiments. The data represent mean ± SD. Source data

Extended Data Fig. 5 The Tph1-5HTP-AhR pathway regulates IL-2-induced CD8+ T cells exhaustion in vivo.

a, The mRNA expression of Tph1 in CD8+ T cells from tumor tissues of C57BL/6 J mice with 3 × 3 mm or 8 × 8 mm MC38 tumors. b, Correlation between Tph1 expression and IL-2 levels in MC38 tumors. c, Activated CD45.1+ OVA-CTLs were transferred into C57BL/6 J mice bearing with OVA-B16 melanoma, and then mice were intratumorally injected with 5HTP. CD45.1+ cells were isolated from the tumors for flow cytometric analysis of the expression of IRs, or TNF and IFN-γ. d, C57BL/6 J mice bearing MC38 tumor were intratumorally injected with Stat5in, fenchlonine. CD8+ T cells were isolated from the tumor for flow-cytometric analysis of the expression of IRs, or TNF and IFN-γ. e, The same as c, except that mice were treated with Stat5in or fenchlonine. f,g, Activated Stat5 shRNAs (f) or Tph1 shRNAs-CD45.1+ gp-100-CTLs (g) were transferred into CD45.2+ C57BL/6 J mice with B16 melanoma. At day 7, CD45.1+ T cells were isolated from tumor tissues and the expression of IRs, TNF and IFN-γ were determined by flow cytometry. h-j, The same as (f,g), except that cells were transferred into mice every five days for 3 times. Some mice were intraperitoneally injected with anti-CD25 + anti-CD122 antibody. Tumor growth and mouse survival were analyzed. k, The same as (d), except that mice were intratumorally injected with CH223191 or SR1. l, The same as c, except that mice were intratumorally injected with CH223191or SR1. m, C57BL/6 J mice with 3 × 3 mm MC38 tumor were injected with IL-2, SR1, Stat5in, IL-2 + SR1 or IL-2 + Stat5in every two days for 5 times. Tumor growth was analyzed. n, The same as (m), except that mice with 8 × 8 mm MC38 tumors were treated with IL-15. o,p, Activated CTLs were treated with IL-2 or IL-15 for 48 hr (o) or 72 hr (p). The expression of Tph1 (o) or IRs (p) was determined by real-time PCR or flow cytometry, respectively. In a, d-g, k, l, n = 5 mice; b, n = 15 mice; c, n = 6 mice; h-j, n = 10 mice; m, n, n = 8 mice; o, p, n = 3 independent experiments. Two-tailed Student’s t test (a, c, n), Pearson’s correlation test (b), one-way ANOVA Bonferroni’s test (d-g, h (left), i (left), j-m, o, p), or Log-rank survival analysis (h (right), i (right)). The data represent mean ± SD (a, c-g, k, l, o, p) or mean ± SEM (h-j, m, n). Source data

Extended Data Fig. 6 IL-2 is released by CD45+CD3+ T cells to induce CD8+ T cells exhaustion in the tumor microenvironment.

a, IL-2 mRNA expression in murine CD8+ T cells (mCD8+ T cells) or the PB of healthy volunteer (hCD8+ T cells), MC38, 4T1, B16, HCT15, HCT116, MCF-7 or MP-1 human primary melanoma cells. b, IL-2 mRNA expression in CD45 and CD45+ cells from 5 × 5 mm MC38 tumor tissues. c, The CD11c+ IL-2+, B220+ IL-2+ or CD11b+ F4/80+ IL-2+ cells from 3 × 3 mm or 8 × 8 mm MC38 tumor tissues were determined by flow cytometry. d,e The CD3+ IL-2+ CD4+ or CD3+ IL-2+ CD8+ cells from 3 × 3 mm or 8 × 8 mm B16 melanoma tumor tissues (d) or H22 ascites (e) were determined by flow cytometry. f,g, C57BL/6 J mice with 3 × 3 mm or 8 × 8 mm MC38 tumors were treated with IL-2 or IL2 + anti-IL-2Ra antibody. The CD3+, CD3+CD8+ (f), or PD1+TCF1TIM3+ (g) T cells in the tumors were analyzed by flow cytometry. h, Activated OVA-CTLs were treated with IL-2 for indicated times periods. The PD1+TCF1TIM3+ T cells were analyzed by flow cytometry. i,j, The same as (f,g), except that mice were i.p. injected with anti-IL-2 (i), anti- anti-CD4 or CTLA-4 (j) neutralizing antibody. Tumor growth was analyzed. k-m, C57BL/6 J mice with 8 × 8 mm B16 melanoma were intraperitoneally injected with anti-CD4 neutralizing antibody. CD8+ T cells were isolated from the tumor for flow-cytometric analysis of the expression of IRs (k), or TNF and IFN-γ (l). Tumor growth and mouse survival was analyzed (m). n, The same as (k,l), except that BALB/c mice with H22 ascites were used. o, Activated murine CD4+ and CD8+ T cells were treated with IL-2 for 72 hr. The expression of IRs was determined by flow cytometry. In b-g, k, l, n, n = 5 mice; i, j, n = 8 mice; m, n = 7 mice; a, h, o, n = 3 independent experiments. Two-tailed Student’s t test (b, d, e, g, h, k, l, m, n), one-way ANOVA Bonferroni’s test (f, i, j, o), or Log-rank survival analysis (m (down)). The data represent mean ± SD (a-h, k, l, n, o) or mean ± SEM (i, j, m). Source data

Extended Data Fig. 7 IL-2 induces CD8+ T cells exhaustion by activating TPH1-5HTP-AhR pathway in patients.

a, IL-2 mRNA expression in CD45+ and CD45 cells from colon or breast cancer tissues of patients (n = 5). b, The CD3+ CD8+ IL-2+ or CD3+ CD4+ IL-2+ cells from the fresh tissues of colon cancer patients (n = 5) was determined by flow cytometry. c, Resting CD8+ T cells from the PB of colon or breast cancer patients (n = 6) were treated with IL-2 for 72 hr. The expression of IRs and cytokines was determined by flow cytometry. d, The same as (c), except that resting or activated T cells from healthy donors were used. e, Resting or activated CD8+ T cells from PB of colon or lung cancer patients (n = 15 scopes from 3 patients) were treated with IL-2 for 48 hr. Cells were stained with anti-TPH1 antibody and observed by confocal microscopy. Bar, 10 μm. f, The same as (c), except that activated TPH1shRNAs- CD8+ T cells from healthy volunteers were used. g, Activated CD8+ T cells from the PB of healthy volunteers or colon cancer patients were treated with IL-2, IL-2 + Stat5in or IL-2 + fenchlonine for 72 hr. The expression of IRs and cytokines were determined by flow cytometry. h, The same as (c), except that STAT5 shRNAs-CD8+ T cells from healthy volunteers or colon cancer patients were used. i, Activated CD8+ T cells from the PB of colon or lung cancer patients were treated with IL-2 for 48 hr, or CD3+CD8+ T cells were sorted from peripheral blood (n = 50 scope from 10 patients) or tumor tissues (n = 30 scope from 6 patients) of colon or lung cancer patients. These cells were stained with anti-AhR antibody and observed by confocal microscopy. Bar, 10 μm. j, The same as (g), except CD8+ T cells from colon cancer patients were treated with IL-2, IL-2 + SR1 or IL-2 + CH223191. k, The same as (g), except that CD8+ T cells were from healthy donors or lung or breast cancer patients. In d, f-h, n = 3 independent experiments; j, k, n = 5. Two- tailed Student’s t test (a, b, c, d, i), one-way ANOVA Bonferroni’s test (e-h, j, k). The data represent mean ± SD. Source data

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Supplementary Tables 1–7

Clinical information of cancer patients, reagent information, antibody information, primer sequences information and CHIP-seq information.

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Liu, Y., Zhou, N., Zhou, L. et al. IL-2 regulates tumor-reactive CD8+ T cell exhaustion by activating the aryl hydrocarbon receptor. Nat Immunol 22, 358–369 (2021). https://doi.org/10.1038/s41590-020-00850-9

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