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Metabolic reprogramming of terminally exhausted CD8+ T cells by IL-10 enhances anti-tumor immunity

Nature Immunology volume 22, pages 746–756 (2021)Cite this article

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Abstract

T cell exhaustion presents one of the major hurdles to cancer immunotherapy. Among exhausted CD8+ tumor-infiltrating lymphocytes, the terminally exhausted subset contributes directly to tumor cell killing owing to its cytotoxic effector function. However, this subset does not respond to immune checkpoint blockades and is difficult to be reinvigorated with restored proliferative capacity. Here, we show that a half-life-extended interleukin-10–Fc fusion protein directly and potently enhanced expansion and effector function of terminally exhausted CD8+ tumor-infiltrating lymphocytes by promoting oxidative phosphorylation, a process that was independent of the progenitor exhausted T cells. Interleukin-10–Fc was a safe and highly efficient metabolic intervention that synergized with adoptive T cell transfer immunotherapy, leading to eradication of established solid tumors and durable cures in the majority of treated mice. These findings show that metabolic reprogramming by upregulating mitochondrial pyruvate carrier-dependent oxidative phosphorylation can revitalize terminally exhausted T cells and enhance the response to cancer immunotherapy.

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Fig. 1: IL-10–Fc expands terminally exhausted CD8+ TILs.
Fig. 2: IL-10–Fc expands CD8+ T cells through IL-10R and enhances their effector function.
Fig. 3: IL-10–Fc expands antigen-specific terminally exhausted CD8+ T cells in a progenitor exhausted cell–independent manner.
Fig. 4: IL-10–Fc reprograms CD8+ T cell metabolism by promoting OXPHOS.
Fig. 5: IL-10–Fc potentiates ACT therapies to eradicate established tumors in multiple mouse models with durable protection.
Fig. 6: IL-10–Fc upregulates OXPHOS and expression of genes encoding effector function of terminally exhausted CD8+ TILs.
Fig. 7: IL-10–Fc promotes T cell OXPHOS and anti-tumor immunity in an MPC-dependent manner.

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Data availability

Gene sets in the MSigDB database (C2 and C7) were used for gene set enrichment analysis. All data generated and supporting the findings of this study are available within the paper. The RNA-seq data for tumor-infiltrating lymphocytes are available in the Gene Expression Omnibus database under accession code GSE168990. Source data are provided with this paper. Additional information and materials will be made available upon reasonable request.

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Acknowledgements

We thank D. Trono and B.E. Correia for providing plasmids of Delta 8.9 and pVSV-G; J.-C. Martinou (University of Geneva) for providing Mpc1fl/fl mice; and A. Donda (University of Lausanne) for the technical support on human CAR T cells. We acknowledge the EPFL Center of PhenoGenomics, Flow Cytometry Core Facility and Protein Expression Core Facility for technical assistance. This work was supported in part by the Swiss National Science Foundation (SNSF project grant no. 315230_173243), the ISREC Foundation with a donation from the Biltema Foundation, the Swiss Cancer League (grant no. KFS-4600-08-2018), the European Research Council under the ERC grant agreement MechanoIMM (grant no. 805337), the Kristian Gerhard Jebsen Foundation, Fondation Pierre Mercier pour la science, an Anna Fuller Fund grant and the EPFL (L.T.). P.-C.H. was supported in part by the Swiss Institute for Experimental Cancer Research (ISREC grant no. 26075483), SNSF project grants (grant nos. 31003A_163204 and 31003A_182470), the Cancer Research Institute Lloyd J. Old STAR award and the European Research Council Starting Grant (grant no. 802773-MitoGuide). P.R. was supported in part by grants from the SNSF (grant nos. 310030_182735 and 310030E-164187). W.H. was supported in part by the Swiss Cancer League (grant no. KFS-4407-02-2018) and the SNSF (grant no. 310030B_179570). W.X. was supported in part by the Strategic Priority Research Program of the Chinese Academy of Sciences (grant no. XDB29030000) and the Ministry of Science and Technology of China (grant no. 2016YFC1303503). M.G. was supported by the Chinese Scholarship Council (grant no. 201808320453).

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Author notes

  1. These authors contributed equally: Yugang Guo, Yu-Qing Xie.

Authors and Affiliations

  1. Institute of Bioengineering, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

    Yugang Guo, Yu-Qing Xie, Min Gao, Yang Zhao, Bing Feng & Li Tang

  2. Institute of Materials Science & Engineering, EPFL, Lausanne, Switzerland

    Yugang Guo, Bing Feng & Li Tang

  3. Department of Oncology, University of Lausanne, Epalinges, Switzerland

    Fabien Franco, Mathias Wenes, Imran Siddiqui, Alessio Bevilacqua, Haiping Wang, Catherine M. Sabatel, Benjamin Tschumi, Werner Held, Pedro Romero & Ping-Chih Ho

  4. Ludwig Institute for Cancer Research, University of Lausanne, Epalinges, Switzerland

    Fabien Franco, Alessio Bevilacqua, Haiping Wang & Ping-Chih Ho

  5. State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center of Biotherapy, Chengdu, China

    Hanshuo Yang

  6. Center for Genomics and Systems Biology, New York University Abu Dhabi, Abu Dhabi, United Arab Emirates

    Xin Xie

  7. Siriraj Center of Research Excellence for Systems Pharmacology, Department of Pharmacology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand

    Amphun Chaiboonchoe

  8. Department of Respiratory and Critical Care Medicine, West China Medical School/West China Hospital, Sichuan University, Chengdu, China

    Yuxi Wang & Weimin Li

  9. Department of Oncology of the First Affiliated Hospital, Division of Life Sciences and Medicine, The CAS Key Laboratory of Innate Immunity and Chronic Disease, Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, China

    Weihua Xiao

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  1. Yugang Guo
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Contributions

Y.G., Y.-Q.X. and L.T. conceived the study. Y.G., Y.-Q.X., L.T. and P.-C.H. designed the experiments. Y.G., Y.-Q.X., M.G., Y.Z., F.F., M.W., I.S., A.B., H.W., H.Y., B.F., X.X., C.M.S., B.T., A.C., Y.W., W.L., W.X., W.H. and P.R. performed the experiments. Y.G., Y.-Q.X., L.T. and P.-C.H. analyzed the data. Y.G., Y.-Q.X. and L.T. wrote the manuscript. All authors edited the manuscript.

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Correspondence to Ping-Chih Ho or Li Tang.

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Competing interests

Y.G., L.T. and Y.-Q.X. are inventors on the patent (International Publication Number WO 2021053134) related to the technology described in this manuscript. The remaining authors declare no competing interests.

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Extended data

Extended Data Fig. 1 IL-10–Fc promotes tumor infiltration of T cells but shows less effects on other immune cells.

a, Schematic diagram of the production and purification of IL-10–Fc. b, Representative size-exclusion chromatographic traces. Peak 3 (P3) was collected and analyzed. c, Representative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified IL-10–Fc. DTT, dithiothreitol. Results are one representative of three independent experiments. d, Splenocytes from PMEL mice were cultured with human gp10025–33 (hgp100) peptide (1 μM) and IL-2 (10 ng ml−1) for 3 d in the presence of IL-10–Fc or recombinant IL-10 at a series of concentrations. Shown are fold changes of CD8+ T cell counts (normalized by that in PBS control) at each concentration of cytokine. e-i, The experimental setting was the same as described in Fig. 1. Data are one representative of three or four independent experiments. e, Counts of CD45.2+ tumor infiltrating lymphocytes (TILs) and CD3+ TILs in B16F10 tumors. Shown are pooled data of two independent experiments (n = 10 independent animals). f, Representative immunofluorescence images of samples from each group. Green, CD3; blue, DAPI. Scale bar: 100 μm. Results are one representative of two independent experiments. g, Counts of various immune cell subsets in B16F10 tumors. For endogenous CD8+ T cells, PMEL CD8+ T cells, and total CD4+ T cells, shown are pooled data of two independent experiments (n = 10 independent animals). For other immune cells, data are representative of three independent experiments (n = 5 independent animals). NK: natural killer; DCs: dendritic cells; TAMs: tumor associated macrophages; MDSCs: myeloid-derived suppressive cells. h, Mean fluorescence intensity (MFI) of maturation markers on CD11b+CD11c+ tumor infiltrating DCs (n = 5 independent animals). i, Frequencies of M1 phenotype (CD206–MHC-II+) among CD11b+F4/80+ TAMs (n = 5 independent animals). All data represent the mean ± s.e.m. and are analyzed by two-sided Student’s t-test or one-way ANOVA and Tukey’s test; NS, not significant (P > 0.05).

Source data

Extended Data Fig. 2 Tumor infiltrating PD-1+TIM-3+CD8+ T cells are PD-1+TCF-1–TIM-3+ terminally exhausted CD8+ T cells, which respond to IL-10–Fc through IL-10 receptor.

a-d, The experimental setting was the same as described in Fig. 1. a-c, Data are representative of two independent experiments (n = 4 independent animals). a, Representative flow cytometry plots showing the frequencies of progenitor exhausted (TCF-1+TIM-3–) and terminally exhausted (TCF-1–TIM-3+) CD8+ TILs among total CD44+PD-1+CD8+ TILs. b,c, Frequencies and counts of three subpopulations of CD8+ TILs based on TCF-1/TIM-3 four-quadrant gating as shown in a. d, Frequencies of IFNγ+TNFα+ polyfunctional and IL-2+ T cells among PD-1+TIM-3–CD8+ TILs or PD-1+TIM-3+CD8+ TILs from untreated tumors (n = 5 independent animals). Memory PMEL CD8+ T cells (n = 5 independent animals) in spleens from cured mice (schedule shown in Fig. 5c) were stimulated in the same way as positive controls. Data are one representative of two independent experiments. e-g, Activated CD8+ T cells from CRISPR-Cas9 KI P14 T cell receptor (TCR) transgenic mice were transfected with control gRNA or IL-10 receptor-α (IL-10Rα) allele specific gRNA to generate wild type control P14 T cells (Ctrl) or IL-10Rα-KO P14 T cells (IL-10Rα-KO), respectively. e, MFI of IL-10Rα expression. Data are one representative of two independent experiments (n = 6 independent samples). f, Ctrl P14 or IL-10Rα-KO P14 CD8+ T cells were restimulated by dimerized anti-CD3 (α-CD3) antibody in the presence or absence of IL-10–Fc and cultured for 2 d. Cell counts were analyzed by flow cytometry. Shown are relative T cell counts treated with IL-10–Fc versus that with PBS control. Data are one representative of two independent experiments (n = 3 independent samples). g, Experimental timeline of Fig. 2b. All data represent the mean ± s.e.m. and are analyzed by two-sided Student’s t-test or one-way ANOVA and Tukey’s test; NS, not significant (P > 0.05).

Source data

Extended Data Fig. 3 IL-10–Fc expands terminally exhausted CD8+ T cells in a progenitor exhausted cell-independent manner.

a,b, Thy1.2+ C57BL/6 mice were sublethally lymphodepleted and received adoptive transfer of Thy1.1+ naïve PMEL CD8+ T cells. Mice were inoculated with B16F10 tumor cells and were treated with adoptive transfer of Thy1.2+ activated PMEL CD8+ T cells. Subset [1] (PD-1+TIM-3–) and subset [2] (PD-1+TIM-3+) were sorted from pooled Thy1.1+CD8+ TILs (n = 10 independent animals). Sorted subsets (5 × 104 for each) were transferred separately into Thy1.2+ C57BL/6 recipient mice bearing B16F10 tumors followed by administration of IL-10–Fc or PBS control. On day 13, mice were killed (n = 5 independent animals for groups receiving subset [1]; n = 3 independent animals for groups receiving subset [2]). a, Experimental timeline. b, Counts of tumor infiltrating Thy1.1+ donor T cells. c-e, Thy1.2+ C57BL/6 mice bearing B16F10 tumors received adoptive transfer of activated PMEL Thy1.1+CD8+ T cells and were killed. TILs were pooled from 3 ~ 6 mice and two subsets of Thy1.2+ endogenous CD8+ T cells were sorted. Sorted subsets were cultured separately ex vivo in the presence of dimerized α-CD3 and IL-2 with or without IL-10–Fc for 3 d. Data are one representative of two independent experiments (n = 3 independent samples). c, Experimental timeline. d, Fold change of T cell counts after 3-d ex vivo culture (n = 3 independent samples). e, Frequencies of PD-1+TIM-3+ terminally exhausted CD8+ T cells after 3-d ex vivo culture (n = 3 independent samples). f, Stacked bar graphs show the putative origins of PD-1+TIM-3+ cells in IL-10–Fc-treated tumors based on experimental results in d and e. g, The experimental setting was the same as that described in Fig. 1. MFI of active caspase-3 among three subpopulations (n = 4 independent animals). Data are one representative of two independent experiments. h,i, The experimental setting was the same as that shown in Fig. 3e. (n = 4 independent animals). h, Frequencies of Tcf7DTR-GFP+ P14 CD8+ T cells among all CD8+ TILs before (day 15) and after (day 17) the DT treatment. i, Representative flow cytometry plots. All data represent the mean ± s.e.m. and are analyzed by two-sided Student’s t-test; NS, not significant (P > 0.05).

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Extended Data Fig. 4 IL-10–Fc shows minimal effects on T cell glycolysis.

Primed PMEL CD8+ T cells in resting phase (day 7) were co-cultured with B16F10 tumor cells at a ratio of 1 to 1 in the presence or absence of IL-10–Fc. After a 2-d co-culture, CD8+ T cells were isolated for Seahorse assay and flow cytometry analyses, respectively. Data are representative of three independent experiments (n = 3 independent samples). a, Real-time analysis of extracellular acidification rate (ECAR) of PMEL CD8+ T cells from the co-culture assay. b, Basal and maximal ECAR levels of PMEL CD8+ T cells. All data represent the mean ± s.e.m. and are analyzed by one-way ANOVA and Tukey’s test; NS, not significant (P > 0.05).

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Extended Data Fig. 5 Ex vivo generated PD-1+TIM-3+CD8+ T cells by restimulation exhibit exhaustion phenotypes similarly as terminally exhausted CD8+ TILs.

a, Dimerized α-CD3 antibody was produced by mixing rat α-CD3 antibody with goat α-rat IgG Fc antibody at the mole ratio of 2:1. Primed PMEL CD8+ T cells in resting phase (day 7) were restimulated by dimerized α-CD3 at indicated concentrations and cultured in complete RPMI 1640 medium supplemented with IL-2 (10 ng ml−1) for 2 d. Shown are representative flow cytometry plots and frequencies of PD-1+TIM-3+CD8+ T cells among total PMEL CD8+ T cells. Data are representative of three independent experiments (n = 3 independent samples). b, Activated PMEL CD8+ T cells in resting phase were restimulated by dimerized α-CD3 antibody and IL-2 (10 ng ml−1) for 2 d. Freshly isolated naïve PMEL CD8+ T cells from splenocytes and restimulated PMEL CD8+ T cells were compared for the expression of PD-1, TIM-3 surface markers and TCF-1 transcription factor.

Extended Data Fig. 6 IL-10–Fc enhances OXPHOS and proliferation of human CD8+ T cells as well as their killing efficiency of target cells.

a-c, Human HER2 CAR-T cells were co-cultured with SKOV3-HER2 (a) or ME275-HER2 (b,c) tumor cells in vitro at a ratio of 1 to 1 in the presence or absence of IL-10–Fc. After a 2-d co-culture, counts of tumor cells and CD8+ T cells were measured by flow cytometry. CD8+ T cells from the co-culture were further isolated for Seahorse assay (a). Data are one representative of two independent experiments (n = 3 independent samples). a, Average basal OCR of the CD8+ HER2 CAR-T cells. b, Relative counts of PD-1+LAG-3+CD8+ CAR-T cells treated with IL-10–Fc versus that with PBS. c, Percentage of target cell killing by CAR-T cells. d-f, Activated human CD8+ T cells in resting phase were restimulated by coated α-CD3 antibody (0.05 μg mL−1) for 2 d. PD-1+LAG-3+CD8+ T cells were sorted for a Seahorse assay. d, Average basal OCR of PD-1+LAG-3+CD8+ T cells. e, Relative counts of PD-1+LAG-3+CD8+ T cells treated with IL-10–Fc versus that with PBS. f, MFI of IL-10Rα expression on three different subpopulations. Data are one representative of two independent experiments (n = 3 independent samples). All data represent the mean ± s.e.m. and are analyzed by one-way ANOVA and Tukey’s test or two-sided Student’s t-test; NS, not significant (P > 0.05).

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Extended Data Fig. 7 IL-10–Fc potentiates adoptive T cell transfer therapies to eradicate established solid tumors without overt toxicity.

a, Shown are individual tumor growth curves of the therapy experiments in Fig. 5a. Indicated are numbers of long-term surviving mice among the total number of mice in the group. b-c, The experimental setting was the same as that described in Fig. 1. b, Ratio of counts of CD8+ T cells to regulatory T cells (Tregs) in tumors in different treatment groups. c, Frequencies of granzyme B+IFNγ+TNFα+ polyfunctional CD8+ T cells among total CD8+ TILs. Data are one representative of three independent experiments (n = 5 independent animals). d-h, Thy1.2+ C57BL/6 mice were inoculated s.c. with B16F10 melanoma cells (5 × 105) on day 0, and received i.v. adoptive transfer of activated Thy1.1+ PMEL CD8+ T cells (5 × 106) on day 6, followed by p.t. administration of IL-10–Fc (20 µg) or PBS control every 4 days until day 18. d, Experimental timeline. e-h, Shown are average tumor growth curves (e), survival curves (f), and individual tumor growth curves (g, h) of each treatment group. Indicated are numbers of long-term surviving mice out of the total numbers in the group. Data are representative of two independent experiments (n = 10 independent animals). i-k, The experimental setting was the same as that described in Fig. 1. On day 14, mice were killed and serum samples were collected for analysis. Shown are relative body weight (i), serum alanine transaminase (ALT, j) and aspartate transaminase (AST, k) levels of the treated mice. Shown are pooled data of two independent experiments (n = 10 independent animals). All data represent the mean ± s.e.m. and are analyzed by one-way ANOVA and Tukey’s test; NS, not significant (P > 0.05).

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Extended Data Fig. 8 IL-10–Fc synergizes with immune checkpoint blockade therapy to eradicate established tumors.

BALB/c mice were inoculated subcutaneously with CT26 colon adenocarcinoma cells (3 × 105) and received p.t. administration of IL-10–Fc (20 µg) or PBS control every other day until day 14, together with p.t. administration of α-PD-1 (RMP1-14, 100 µg) every 3 days until day 12. Data are one representative of three independent experiments (n = 5 or 10 independent animals). a, Experimental timeline. b, Shown are survival curves of each treatment group. Indicated are numbers of long-term surviving mice among the total number of mice in the group. c, Shown are individual tumor growth curves. d, Cured mice from treatment group of combination of α-PD-1 and IL-10–Fc were re-challenged subcutaneously with CT26 (3 × 105) cells at day 90 post primary inoculation. Naïve wild type mice were inoculated with the same number of tumor cells as controls. Shown are survival curves and numbers of long-term surviving mice against the re-challenges. All data represent the mean ± s.e.m. and are analyzed by Log-rank test for survival curves; NS, not significant (P > 0.05).

Extended Data Fig. 9 IL-10–Fc upregulates OXPHOS and effector function related pathways in terminally exhausted CD8+ TILs in vivo.

The experimental setting for RNA-sequencing (RNA-seq) was shown in Fig. 6a. a, Heatmap of top 100 differentially expressed genes (IL-10–Fc versus PBS) were generated using z-scores derived from log2 (fold change). RNA-seq datasets were generated from two independent animals from PBS treatment group and four independent animals from IL-10–Fc treatment group. b, Volcano plot of differentially expressed genes (IL-10–Fc versus PBS). Transcripts with a false discovery rate (FDR) value < 0.05 and log2 (fold change) > 1 are highlighted in red; transcripts with an FDR value < 0.05 and log2 (fold change) < -1 are highlighted in blue. c, Canonical pathway analysis was performed by using QIAGEN Ingenuity Pathway Analysis (IPA) based on differentially expressed genes (DEGs) from RNA-seq dataset. Enrichment Z-score is generated based on hypergeometric distribution, where the negative logarithm of the significance level (P value) is obtained by Fisher’s exact test at the right tail. Shown are top 15 pathways that were significantly upregulated by IL-10–Fc treatment (-log (P value) > 6). Numbers on the right of each bar indicates the significance -log (P value) of each pathway. d, Bean plot of log2 (fold change) of genes involved in oxidative phosphorylation pathway shown in c.

Extended Data Fig. 10 IL-10–Fc promotes mitochondrial fitness and function of terminally exhausted CD8+ TILs in vivo in an MPC dependent manner.

a-c, CD45.1+CD45.2+ mice were inoculated subcutaneously with B16F10-OVA tumor cells (5 × 105) and received i.v. adoptive co-transfer of activated CD45.1+ WT OT-I CD8+ T cells and CD45.2+ MPC1-KO OT-I CD8+ T cells (1:1, 5 × 106 for each) on day 6 followed by p.t. administration of IL-10–Fc (20 µg) or PBS control every other day until day 12. On day 13, mice were killed and tumors were processed and analyzed by flow cytometry. Data are one representative of two independent experiments (n = 7 independent animals). a, b, Relative MFI of MitoTracker Green FM (MitoGreen) (a) and MitoTracker Deep Red FM (MitoDeepRed) (b) of WT or MPC1-KO PD-1+TIM-3+CD8+ OT-I TILs treated with IL-10–Fc versus that with PBS control. c, Frequencies of MitoSOX+CD8+ T cells among total WT or MPC1-KO PD-1+TIM-3+CD8+ OT-I TILs. d, f, Activated PMEL CD8+ T cells were starved overnight and restimulated by dimerized α-CD3 antibody in the presence or absence of IL-10–Fc for overnight (d) or indicated time (f). Proteins from total cell lysates were separated by SDS-PAGE and MPC1 (d) or pSTAT3 (Tyr705) (f) were detected by Western blot. Results are one representative of three independent experiments. e, Transcript expression of Mpc1, Mpc1-ps, and Mpc2. Data are extracted from analyses of RNA-seq. All data represent the mean ± s.e.m. and are analyzed by two-sided Student’s t-test; NS, not significant (P > 0.05).

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Guo, Y., Xie, YQ., Gao, M. et al. Metabolic reprogramming of terminally exhausted CD8+ T cells by IL-10 enhances anti-tumor immunity. Nat Immunol 22, 746–756 (2021). https://doi.org/10.1038/s41590-021-00940-2

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