26 apr. 2026
Scharping N.E. et al. (BioRxiv)
Keywords
Proteostasis
CD8+ T cell exhaustion
Cancer Immunotherapy
Chronic Diseases
E3 Ligases
Main Findings
T cell exhaustion is a major barrier to durable anti-tumor immunity, but the molecular programs that preserve more favorable CD8+ T cell states, including progenitor exhausted (TPEX) and tissue-resident-like (TRM) populations, remain incompletely understood. In this preprint, Scharping et al. investigate whether proteostasis-related E3 ubiquitin ligases contribute to T cell differentiation potential and tumor-infiltrating lymphocyte (TIL) function. The authors identify three ligases, NEURL3, RNF149, and WSB1, that are enriched in favorable TPEX/TRM-like states and reduced in terminal exhausted T cell (TEX). In melanoma tumor models, overexpression of these ligases improves TIL accumulation, increases TCF1 expression, enhances pro-inflammatory cytokine production, strengthens tumor control, and improves survival in vivo, whereas ligase deficiency produces the opposite phenotype in tumors. In chronic LCMV infection, ligase overexpression similarly increases TPEX-like cells and improves T cell function, consistent with the tumor model. In acute infection, ligase knockout biases early differentiation toward memory precursors and leads to increased central-memory T cell (TCM) representation at later memory time points.
To define the molecular basis of these effects, the authors compare effector T cell (TEFF), TRM, TPEX, and TEX using both scRNA CITE-seq and low-input mass spectrometry. They show that these T cell states are more clearly separated at the protein level than at the RNA level and identify distinct proteomic programs, representing known cell-type specific pathways, associated with each population. In particular, TEX and TRM are enriched for proteasome-mediated protein catabolism pathways, and TEX additionally shows an accumulation of K48-linked ubiquitinated proteins, short-lived proteins, and unfolded proteins, despite preserved proteasomal activity. Overexpression of NEURL3, RNF149, or WSB1 rescues unfolded protein accumulation in TIL, suggesting that these ligases influence T cell state through protein quality-control pathways. Finally, analysis of human melanoma datasets shows that higher ligase expression is associated with improved checkpoint blockade responses, and in mice, triple-ligase overexpression enhances the efficacy of anti-PD-1 therapy. Together, these findings identify NEURL3, RNF149, and WSB1 as proteostasis-related regulators of CD8+ T cell state and function.
Limitations & Suggestions
Although this study combines tumor models, infection models, proteomics, and human correlative datasets, several limitations remain:
First, the authors identified the ligases NEURL3, RNF149, and WSB1 associated with a tissue-resident signature based on previous transcriptomic profiling, yet the authors describe that about 24% of differential genes were associated with protein degradation and folding. A more systematic reasoning or approach may clarify why these ligases have been chosen for further investigations.
Second, the direct substrates of NEURL3, RNF149, and WSB1 are not identified, making it difficult to determine whether these ligases act through shared or distinct protein quality-control pathways. This would be particularly important in order to understand how these ligases may shape cell identity and expand the TPEX subset. For instance, Cheng et al. (Nature, 2026) showed that poly-ubiquitinlyation of TOX may prevent terminal exhaustion in CD8+ T cells. Moreover, the upstream regulation of these genes is also poorly defined, which limits mechanistic insight into how they are induced and coordinated under proteotoxic stress.
Third, the acute infection data are more difficult to interpret than the tumor and chronic infection data. While ligase deficiency shifts cells toward memory-precursor and TCM-like phenotypes, these cells are less abundant after secondary challenge, indicating a reduced fitness of ligase-deficient memory T cells. The study does not further define how they differ from bona fide functional TCM.
Fourth, the authors could clarify whether impaired protein degradation via autophagy may contribute to the accumulation of ubiquitinylated proteins during CD8+ T cell exhaustion, considering that proteasomal degradation is still intact.
Finally, although human melanoma datasets support the clinical relevance of ligase expression, the core proteomic and mechanistic analyses are performed in mouse systems, and direct proteomic validation in human TIL would strengthen the translational impact of the work.
Significance/Novelty
This preprint is significant because it adds to an important, very recent shift in the T cell exhaustion field toward proteostasis as a biologically and therapeutically relevant axis, rather than viewing exhaustion primarily through transcriptional or epigenetic state maps (see also Cheng et al. 2026 Nature & Wang et al. 2025 & Yu et al. 2026). Its main novelty lies on the identification of three specific E3 ligases, rather than a broad stress signature, as regulators of favorable CD8+ T cell states. By showing that proteomic profiling distinguishes TPEX from TEX more clearly than transcriptomics and by linking ligase expression to both proteotoxic stress control and checkpoint blockade response. As such the authors provide a particularly useful low-input proteomic atlas of CD8+ T cell subsets during chronic diseases that reflect well some of the underlying biological processes driving these subsets. Collectively, the study offers a new framework for thinking about how T cell fate is maintained or lost in chronic disease and may add new therapeutic avenues for the development of CAR T cell therapeutics or prevent CD8+ T cell exhaustion in cancer patients.
Credit
Reviewed by Yen-Tzu Chang (Chester) as part of a cross-institutional journal club between the Icahn School of Medicine at Mount Sinai, the University of Oxford, the Karolinska Institute, the University of Toronto, and the UT MD Anderson James P. Allison Institute.
Reviewed by Felix Richter as part of a cross-institutional journal club between the Max-Delbrück Center Berlin, the Ragon Institute Boston (Mass General, MIT, Harvard), the University of Virginia, the Medical University of Vienna and other life science institutes in Vienna.
The author declares no conflict of interests in relation to their involvement in the review.