Proteasome-guided haem signalling axis contributes to T cell exhaustion: Preclinical validation powered by Cyagen’s NKG mice
In a recent landmark study published in Nature (2026), Xu Y, Shangguan Y, Chuang Y-M, and colleagues mapped the proteasome-guided haem signalling axis to T cell exhaustion (https://doi.org/10.1038/s41586-026-10250-y). Their work addresses a major barrier to durable responses in cancer immunotherapy, particularly for chimeric antigen receptor (CAR)-T cell therapies.
T cell exhaustion remains a major barrier to durable responses in cancer immunotherapy, particularly for chimeric antigen receptor (CAR)-T cell therapies. While mitochondrial dysfunction and metabolic stress in the tumour microenvironment are known contributors, the precise molecular link between depolarised mitochondria and terminal exhaustion has been unclear. This study provides a coherent mechanistic framework that connects mitochondrial integrity, proteasome activity, and transcriptional reprogramming through regulatory haem and the transcription factor BACH2.
Molecular Mechanism: Proteasome-Mediated Mitochondrial Breakdown Drives BACH2/BLIMP1 Axis in T Cell Terminal Exhaustion
The authors demonstrate that terminally exhausted CD8⁺ T cells accumulate depolarised mitochondria due to impaired mitophagy. These cells engage the proteasome as a compensatory homeostatic mechanism, with the E3 ligase CBLB directing selective ubiquitination and degradation of mitochondrial proteins. Proteasome-mediated breakdown of mitochondrial haemoproteins releases regulatory haem (RH). For this RH to exert transcriptional effects, it must translocate into the nucleus via the haem chaperone PGRMC2.
Once in the nucleus, RH binds BACH2, inducing conformational changes that impair its DNA-binding activity and promote its degradation. This relieves BACH2-mediated transcriptional repression of Prdm1 (encoding BLIMP1), thereby shifting the balance from progenitor-exhausted / stem-like programs toward terminal differentiation and exhaustion.
This axis is robustly supported by integrated multi-omics (proteomics, RNA-seq, ATAC-seq) and functional assays in both human and murine systems. Cells harbouring depolarised mitochondria (MDR/MGlow) exhibit elevated proteasome activity, preferential mitochondrial protein turnover, and higher RH levels. Pharmacological elevation of haem (haemin) recapitulates key exhaustion features, whereas a haem-binding-deficient BACH2 mutant (Bach2MUT) resists degradation and preserves progenitor-exhausted and stem-like populations even under mitochondrial stress. Genetic ablation of PGRMC2 similarly restores BACH2, suppresses BLIMP1, improves mitochondrial fitness, enhances cytokine polyfunctionality, and significantly augments anti-tumour efficacy of antigen-specific T cells and HER2 CAR-T cells in vivo, establishing nuclear haem import as a critical and druggable node in the exhaustion cascade.
Actionable Therapeutic Strategy: Transient Proteasome Inhibition for Enhanced CAR-T Cell Manufacturing
A key strength of the work lies in its direct translation from mechanism to intervention. The authors show that transient, low-dose proteasome inhibition with bortezomib during CAR-T cell manufacturing interrupts the exhaustion cascade. This brief pretreatment reduces terminal exhaustion markers after repeated antigen challenge and induces durable epigenetic changes favouring memory-like transcriptional programs. These molecular improvements translate into enhanced functional persistence without compromising initial expansion.
Preclinical Validation of CAR-T Efficacy in the NKG Humanized Leukemia Xenograft Model
To test therapeutic relevance, the team evaluated bortezomib-pretreated human CD19 CAR-T cells in leukaemia-bearing NKG (NOD.Cg-PrkdcscidIl2rgem1cya/Cya) mice purchased from Cyagen Biosciences. In this established immunodeficient model, animals received Nalm6 leukaemia cells followed by adoptive transfer of 1 × 106 CAR-T cells. Mice treated with the proteasome-modulated CAR-T cells maintained body weight for longer periods and demonstrated significantly prolonged survival compared with those receiving standard CAR-T cells. The benefit was attributed to improved resistance to exhaustion rather than quantitative differences in cell numbers, providing clean evidence that manufacturing-stage intervention can enhance in vivo performance.
NKG mice are severe immunodeficient mice generated by Cyagen by deleting the Il2rg gene from a NOD-Scid genetic background. They exhibit deficiency of mature T cells, B cells, and functional NK cells, reduced complement activity, and weak phagocytosis of human-derived cells by macrophages, making them well-suited for human hematopoietic cell and tissue xenotransplantation. The NKG strain proved well-suited for this study, supporting robust human leukemic engraftment and allowing sensitive discrimination of functional outcomes between CAR-T manufacturing conditions.
Broader Impact and Considerations
This work elegantly links mitochondrial quality control to transcriptional fate decisions in CD8⁺ T cells and offers a clinically feasible strategy to mitigate exhaustion during CAR-T production. The use of a short, low-dose bortezomib pulse is particularly attractive because it is compatible with existing GMP processes and appears to induce lasting epigenetic reprogramming.
One interpretive note is that the benefit of proteasome modulation may depend on precise dosing and timing; excessive or prolonged inhibition could impair memory formation, as suggested by prior literature [1]. The clear separation in survival curves observed here, however, indicates that carefully optimised, transient inhibition can be net beneficial.
j, Body weight curves of NKG mice bearing Nalm6 leukaemia after intravenous infusion of 1 × 10⁶ human CD19 CAR-T cells that were pretreated with 0.1 nM bortezomib or vehicle control during manufacturing (n = 6 mice per group). Body weight was monitored as a surrogate indicator of disease burden and therapeutic response. Data are mean ± s.e.m. Statistical analysis was performed using two-way ANOVA.
k, Kaplan–Meier survival curves of the same cohorts of leukaemia-bearing NKG mice shown in j. Mice receiving bortezomib-pretreated CAR-T cells exhibited significantly prolonged survival compared with those receiving vehicle-pretreated CAR-T cells. n = 6 mice per group. Statistical significance was determined by log-rank test.
Conclusion: The Proteasome–Haem–BACH2 Axis and NKG Mice as a Platform for Improving Adoptive Cellular Immunotherapy
Xu, Ho and colleagues deliver both fundamental mechanistic insight and actionable translational data. By identifying the proteasome–haem–BACH2 axis as a driver of terminal exhaustion, they provide a rational basis for manufacturing interventions that enhance CAR-T durability. The robust performance of the NKG model in validating these improvements underscores its utility as a reliable platform for evaluating human cell therapies in immuno-oncology. This study represents a meaningful step toward overcoming exhaustion and improving the long-term efficacy of adoptive cellular immunotherapies.
Accelerate Your Immuno-Oncology Research with Cyagen's Advanced NKG Mouse Model for CAR-T Programs
Reliable in vivo models are the cornerstone of translating novel cellular immunotherapies from bench to clinic. Discover how our NKG models can be integrated into your next preclinical study. [Access our NKG validation data repository] to discuss tailored in vivo strategies for your CAR-T pipeline.
Reference
[1] Widjaja CE, Olvera JG, Metz PJ, Phan AT, Savas JN, de Bruin G, Leestemaker Y, Berkers CR, de Jong A, Florea BI, Fisch K, Lopez J, Kim SH, Garcia DA, Searles S, Bui JD, Chang AN, Yates JR 3rd, Goldrath AW, Overkleeft HS, Ovaa H, Chang JT. Proteasome activity regulates CD8+ T lymphocyte metabolism and fate specification. J Clin Invest. 2017 Oct 2;127(10):3609-3623. doi: 10.1172/JCI90895. Epub 2017 Aug 28. PMID: 28846070; PMCID: PMC5617668.
