Immunotherapy has underscored a revolution in cancer treatment. Yet, many patients fail to respond due to T cell exhaustion. Here, an intervention that restores mitochondrial function reversed the exhausted T cell phenotype to promote cytotoxicity and durable anti-tumour responses in vivo

Frezza, Christian

Ryan, Dylan

English

Apollo (Cambridge)

 ij.hapres.com Immunometabolism. 2021;3(4):e210030. https://doi.org/10.20900/immunometab20210030 Commentary IL-10-Mediated Refueling of Exhausted T Cell Mitochondria Boosts Anti-Tumour Immunity Dylan Ryan, Christian Frezza * MRC Cancer Unit, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0XZ, UK * Correspondence: Christian Frezza, Email: cf366@cam.ac.uk. ABSTRACT Immunotherapy has underscored a revolution in cancer treatment. Yet, many patients fail to respond due to T cell exhaustion. Here, an intervention that restores mitochondrial function reversed the exhausted T cell phenotype to promote cytotoxicity and durable anti-tumour responses in vivo. KEYWORDS: immunotherapy; mitochondria; metabolism; T cell; exhaustion; IL-10 Immunotherapy is a type of biological therapy that uses the bodys own immune system to treat cancer. It is often achieved by adoptive T cell transfer (ACT) and immune checkpoint blockades (ICBs), which preferentially target tumour-infiltrating lymphocytes (TILs), but fails to work in most patients. This failure is often attributed to the adoption of an “exhausted” T cell phenotype. Defining T cell exhaustion is complex, but the term primarily describes CD8+ effector T cells with a reduced capacity to secrete cytokines and elevated expression levels of inhibitory receptors [1]. In tumours, exhaustion of TILs can impair cytotoxic killing, which subsequently enables tumour progression. Two major subsets of exhausted CD8+ T cells include an ICB-responsive “progenitor exhausted” subset that can proliferate and differentiate into an ICB-unresponsive “terminally exhausted”. Nutrient restriction in the tumour microenvironment (TME) and impaired mitochondrial bioenergetics of TILs is a contributing factor to exhaustion and impaired anti-tumour killing. Writing in Nature Immunology, Guo et al. explored the possibility of reinvigorating terminally exhausted CD8+ T cells by combining a “metabolic intervention”, comprised of an IL-10-Fc fusion protein to restore mitochondrial fitness, in combination with ACT or ICB immunotherapy [2]. Their findings provide strong preclinical evidence that metabolic interventions can promote proliferation and cytotoxic activity of terminally exhausted effector T cells to enhance cancer immunotherapy. Interleukin-10 (IL-10) is a cytokine that can promote anti-tumor immunity. In this work, the authors first synthesized a recombinant fusion protein between human IL-10 and IgG1 (IL-10-Fc), which would cross-react  Open Access Received: 19 August 2021 Accepted: 20 September 2021 Published: 24 September 2021 Copyright © 2021 by the author(s). Licensee Hapres, London, United Kingdom. This is an open access article distributed under the terms and conditions of Creative Commons Attribution 4.0 International License.  Immunometabolism 2 of 5 with the mouse IL-10 receptor and extend the half-life of the protein. To test the ability of IL-10-Fc to regulate T cell responses in vivo, the authors employed the poorly immunogenic B16F10 melanoma mouse model. They found that ACT of tumour antigen-specific PMEL CD8+ T cells with IL-10-Fc enhanced tumour infiltration of TILs and increased the number of CD8+ T cells with minimal impact on other lymphocytes or myeloid cells. Importantly, subset analysis revealed a specific expansion and improved effector functioning of terminally exhausted CD8+ T cells (PD1+TIM-3+ CD8+ T cells) in an IL-10 receptor-dependent manner. To confirm a direct effect of IL-10-Fc on terminally exhausted CD8+ T cells, independent of progenitor exhausted T cells, the authors first demonstrated an antigen-specific increase in PD1+TIM-3+-double positive populations when they were co-administered with OT-I CD8+ T cells. Likewise, comparing the transfer of PD1+TIM-3−CD8+ (progenitor exhausted) and PD1+TIM-3+CD8+ (terminally exhausted) T cells confirmed a specific expansion of terminally exhausted T cells even in the absence of progenitor exhausted T cells. Finally, in vivo depletion of progenitors using diphtheria toxin (DT) and Tcf7DTR-GFP transgenic P14 T cells had no impact on the expansion and tumour-killing capacity of terminally exhausted T cells in B16-gp33 tumours. These results show that IL-10-Fc can directly reinvigorate terminally exhausted CD8+ T cells and promote tumour killing via a progenitor-independent mechanism. IL-10 signaling is reported to oppose pro-inflammatory macrophage activation by antagonizing mTOR and promoting mitophagy [3]. Given the profound effect of IL-10 on macrophage metabolic reprogramming, the authors examined whether IL-10-Fc could elicit effects on the respiratory activity of CD8+ T cells. Using in vitro co-cultures of activated PMEL CD8+ T cells and B16F10 cancer cells, and ex vivo production of PD1+TIM-3+CD8+ T cells, respirometry analysis revealed an increase in oxygen consumption rates (OCR) upon treatment with IL-10-Fc. The increase in OCR was also accompanied by increased PMEL CD8+ T cell proliferation and cytotoxicity against B16F10 cells and was dependent on the IL-10 receptor. To determine whether the metabolic response was conserved between mouse and human T cells, the authors validated the ability of IL-10-Fc to increase respiration, proliferation and the cytotoxic activity of CAR-T cells targeting HER2-expressing cancer cells. These results clearly demonstrated that IL-10-Fc could promote oxidative phosphorylation (OXPHOS) of mouse and human T cells and suggested that IL-10-Fc-induced metabolic reprogramming may underlie the enhanced proliferation and killing capacity in vitro.  To explore the utility of using IL-10-Fc in vivo as an adjunct to cancer immunotherapy, Guo et al. utilized four different solid tumour models to assess safety and efficacy. Firstly, IL-10-Fc was shown to reduce B16F10 tumour burden on its own and to synergize with ACT of PMEL CD8+ T cells. Secondly, ACT of OT-I CD8+ T cells with IL-10-Fc led to potent tumour regression in a large and established YUMM1.7-OVA melanoma model. Immunometabolism. 2021;3(4):e210030. https://doi.org/10.20900/immunometab20210030  Immunometabolism 3 of 5 Thirdly, a combination of HER2-targeting CAR-T cells led to a 90% reduction in tumour size using the HER2-expressing MC38 model of colon adenocarcinoma. This finding was particularly notable as no pre-conditioning was required for the robust anti-tumour activity of CAR-T cells. Overall, combination treatment of IL-10-Fc with ACT led to greater survival rates, and remarkably, sustained anti-tumour immune memory with many IL-10-Fc-treated mice surviving tumour re-challenge. Lastly, IL-10-Fc was also found to synergize with anti-PD1 ICB immunotherapy in a mouse CT26 colorectal tumour model leading to tumour eradication and durable immune responses. These preclinical studies suggest that IL-10-Fc may successfully potentiate ACT and ICB immunotherapies with no overt toxicity. To mechanistically dissect how IL-10-Fc reinvigorated terminally exhausted CD8+ T cells in vivo, RNA sequencing (RNA-seq) analysis of PD1+TIM-3+CD8+ TILs from B16F10 tumours treated with either PBS or IL-10-Fc was performed. Enrichment analysis revealed increases in genes relating to OXPHOS, effector T cell responses and a decrease in inhibitory receptor expression. PD1+TIM-3+CD8+ TILs isolated from tumours were also found to have elevated basal OCR and mitochondrial reactive oxygen species (mtROS) levels. To uncover a causal relationship between metabolic reprogramming and T cell reinvigoration, the authors took a pharmacological approach targeting key bioenergetic pathways, including glycolysis, glutaminolysis, and fatty acid oxidation (FAO). Here, the authors found an important role for glycolysis-derived pyruvate uptake into mitochondria via the mitochondrial pyruvate carrier (MPC) for the increase in IL-10-Fc-driven respiration and validated this using MPC-KO T cells. Furthermore, feeding OT-I CD8+ T cells with sodium pyruvate circumvented the need for IL-10-Fc and promoted PD1+TIM-3+CD8+ T cell proliferation. IL-10-Fc failed to promote expansion and anti-tumour cytotoxic activity of terminally exhausted CD8+ T cells in MPC-KO OT-I CD8+ T cells in both B16F10-OVA tumours and YUMM1.7-OVA tumours. These findings demonstrate that IL-10-Fc promotes OXPHOS and anti-tumour killing via a pyruvate and MPC-dependent refueling of mitochondria (Figure 1).  While the involvement of mitochondrial pyruvate uptake and OXPHOS in reinvigorating terminally exhausted T cells is evident, precisely how IL-10-Fc regulates MPC-dependent OXPHOS remains to be established. Given the known role of IL10-Fc in promoting mitophagy of damaged mitochondria [3], perhaps a similar mechanism is at play in terminally exhausted CD8+ T cells. In addition, improved mitochondrial fitness achieved by enforced peroxisome proliferator-activated receptor-gamma coactivator (PGC)--1α overexpression (and subsequently increased mitochondrial biogenesis) in CD8+ TILs improved anti-tumour efficacy in a mouse model of melanoma [4]. This work highlights the need to develop more therapeutic approaches that enhance mitochondrial fitness to improve T cell function and patient responses to immunotherapy. Since Immunometabolism. 2021;3(4):e210030. https://doi.org/10.20900/immunometab20210030  Immunometabolism 4 of 5 pyruvate alone was sufficient to re-activate mitochondrial functions in exhausted CD8+ T cells, it is tempting to speculate that other dietary interventions could have similar effects, paving the way for diet-based immunomodulation. Whilst this work presents a very exciting development and compelling case for the combination of metabolic interventions and immunotherapies, it remains to be seen if these results will be applicable to a broad range of cancer types. The relevance of these findings to the clinic is also unclear as these experiment use highly immunogenic tumour antigens and involved a peritumoral mode of delivery for IL-10-Fc. Finally, a large gap still remains in the translation of preclinical mouse model therapies to the effective treatment of human disease.  Figure 1. IL-10-Mediated Refueling of Exhausted T Cell Mitochondria Boosts Anti-Tumour Immunity. Figure created with Biorender.com. CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest. ACKNOWLEDGEMENTS This work is funded by the MRC Core award grant MRC_MC_UU_12022/6, the ERC (ERC819920), and the CRUK Programme Foundation award C51061/A27453. REFERENCES 1. Blank CU, Haining WN, Held W, Hogan PG, Kallie A, et al. Defining ‘T cell exhaustion’. Nat Rev Immunol. 2019;19:665-74.  2. Guo Y, Xie YQ, Gao M, Zhao Y, Franco F, Wenes M, et al. Metabolic reprogramming of terminally exhausted CD8+ T cells by IL-10 enhances anti-tumor immunity. Nat Immunol. 2021 Jun;22(6):746-56. Immunometabolism. 2021;3(4):e210030. https://doi.org/10.20900/immunometab20210030  Immunometabolism 5 of 5 3. Ip EWK, Hoshi N, Shouval DS, Snapper S, Medzhitov R. Anti-inflammatory effect of IL-10 mediated by metabolic reprogramming of macrophages. Science. 2017;356:513-9. 4. Dumauthioz N, Tschumi B, Wenes M, Marti B, Wang H, Franco F, et al. Enforced PGC-1α expression promotes CD8 T cell fitness, memory formation and antitumor immunity. Cell Mol Immunol. 2021 Jul;18(7):1761-771.    How to cite this article: Ryan D, Frezza C. IL-10-Mediated Refueling of Exhausted T Cell Mitochondria Boosts Anti-Tumour Immunity. Immunometabolism. 2021;3(4):e210030. https://doi.org/10.20900/immunometab20210030   Immunometabolism. 2021;3(4):e210030. https://doi.org/10.20900/immunometab20210030 

IL-10-Mediated Refueling of Exhausted T Cell Mitochondria Boosts Anti-Tumour Immunity.

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IL-10-Mediated Refueling of Exhausted T Cell Mitochondria Boosts Anti-Tumour Immunity.

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