Background
Early exhaustion of tumor infiltrating CD8 T cells is a major obstacle that restricts the effectiveness of immunotherapy in a broader range of cancer patients.1 Mitochondrial health is a crucial factor determining the performance and endurance of CD8 T cells within the immunosuppressive tumor microenvironment (TME).2 Apart from encountering immunosuppressive signals, CD8 T cells also face metabolic challenges such as hypoxia and nutrient deprivation in the TME.3 4 These metabolic stresses disrupt mitochondrial function, leading to diminished CD8 T cell activity and T cell exhaustion.3 4 Recent studies have shown promising results by employing strategies that enhance mitochondrial function to rejuvenate exhausted tumor-infiltrating lymphocytes (TILs).1 2
Mitophagy, an intrinsic process for removing dysfunctional mitochondria, plays a critical role in maintaining mitochondrial quality and metabolic homeostasis.5 Inhibiting USP30, a mitochondrial deubiquitinase, has been demonstrated to enhance mitophagy and improve mitochondrial function.6 7 We propose that the inhibition of USP30 in CD8 T cells could enhance their performance and endurance within the TME by preserving mitochondrial function.
Methods
In this study, we utilized mitophagy reporter (mt-Keima) mice to investigate the mitophagy activity of tumor-infiltrating CD8 T cells based on their exhaustion statuses. We genetically and pharmacologically inhibited USP30 in CD8 T cells and assessed their mitophagy activity, mitochondrial functions, T cell functions, and exhaustion levels within the TME. Additionally, we treated tumor xenograft mice with a USP30 inhibitor and adoptive CD8 T cells with USP30 deletion to evaluate the potential benefits of targeting USP30 in immunotherapies.
Results
Our findings revealed that mitophagy activity, indicated by the mt-Keima fluorescence, is intrinsically reduced in activated CD8 T cells and further compromised in exhausted CD8 T cells expressing PD1 and TIM3 markers (figure 1). Inhibiting USP30 effectively increased mitophagy activity in exhausted CD8 T cells, thereby enhancing mitochondrial efficiency (figure 2). Furthermore, treating mice with a USP30 inhibitor or transferring USP30 knockout adoptive CD8 T cells resulted in reduced T cell exhaustion and improved CD8 T cell functionality, ultimately enhancing anti-tumor immunity (figure 3).
Conclusions
Mitophagy plays a critical role in maintaining mitochondrial health in CD8 T cells within the TME. However, the exhaustion of CD8 T cells can result in the suppression of mitophagy and impaired mitochondrial function. By promoting mitophagy, exhausted CD8 T cells can be rejuvenated, leading to improved function and endurance within the TME. Targeting USP30 is a promising approach to promote mitophagy activity in CD8 T cells and develop more effective immunotherapy strategies.
Acknowledgements
I am offering my sincere appreciation to all the lab members in Dr. Nuo Sun and Dr. Gang Xin’s lab. This work couldn’t be finished without the help from Dr. Sun in the field of mitochondrial metabolism and Dr. Xin in the field of cancer immunology. Former lab manager, now Ph.D. student, Jianying Li, offered me many helps in using FACS machines.
References
Huang Y, et al. Rewiring mitochondrial metabolism to counteract exhaustion of CAR-T cells. J Hematol Oncol, 2022;15(1):38.
Scharping NE, et al. The Tumor Microenvironment Represses T Cell Mitochondrial Biogenesis to Drive Intratumoral T Cell Metabolic Insufficiency and Dysfunction. Immunity, 2016;45(3):701–703.
Scharping NE, et al. Mitochondrial stress induced by continuous stimulation under hypoxia rapidly drives T cell exhaustion. Nat Immunol, 2021;22(2):205–215.
Yu YR, et al. Disturbed mitochondrial dynamics in CD8(+) TILs reinforce T cell exhaustion. Nat Immunol, 2020;21(12):1540–1551.
Palikaras K, E Lionaki, N Tavernarakis. Mechanisms of mitophagy in cellular homeostasis, physiology and pathology. Nat Cell Biol, 2018;20(9):1013–1022.
Luo H, et al. Pharmacological inhibition of USP30 activates tissue-specific mitophagy. Acta Physiol (Oxf), 2021;232(3):e13666.
Tsefou E, et al. Investigation of USP30 inhibition to enhance Parkin-mediated mitophagy: tools and approaches. Biochem J, 2021;478(23):4099–4118.
Abstract 402 Figure 1
(A, B) Mitophagyactivity (Keima Red) in tumor infiltrated CD8 T cells based on exhaustion degree. (C, D) Mitochondrial membrane potential (TMRM) and mass (Mitotracker Green) in tumor infiltrated CD8 T cells based on exhaustion degree.
Abstract 402 Figure 2
(A, B) Mitophagyactivity (Keima Red) in exhausted CD8 T cells treated with vehicle or USP30 inhibitor in vitro. (C, D) Mitochondrial membrane potential (TMRM) and mass (Mitotracker Green) in exhausted CD8 T cells treated with vehicle or USP30 inhibitor in vitro.
Abstract 402 Figure 3
(A) tumor growth curve of xenograft mice treated with vehicle or USP30 inhibitor (ST-539). (B) tumor growth curve of xenograft mice with or without USP30 knockout on T cells (LCK CRE). (C,D,E) Exhaustion degree (PD1 & TIM3) of tumor infiltrated CD8 T cell with or without USP30 knockout.