277 Proteome turnover dynamics analysis uncovers E3 ligases that enhance T-cell persistence during exhaustion

Background Adoptive T-cell therapies (ACT) have revolutionized cancer immunotherapy, achieving substantial success in hematologic cancers. However, their efficacy in solid tumors remains limited, largely due to T-cell exhaustion (TEX), a dysfunctional state induced by chronic antigen exposure and the hostile tumor microenvironment (TME). TEX is characterized by impaired effector functions and increased expression of inhibitory receptors. Overcoming TEX requires novel strategies to engineer T-cells that maintain persistence. E3 ubiquitin ligases (E3s), key regulators of protein stability and stress responses, represent a largely untapped avenue for enhancing T-cell function. Using proteomic turnover analysis and TEX modeling, we identified multiple E3s differentially expressed during exhaustion, along with widespread changes in protein half-lives. These findings reveal proteostasis remodeling as a key feature of TEX and provide insight into the molecular mechanisms that govern T-cell fitness, offering potential targets to enhance ACT.Methods To model TEX, primary human CD8 + T-cells were stimulated with αCD3/αCD28 either once on Day 0 (acute stimulation) or every two days for 8-days (chronic stimulation). Cells underwent proteomic profiling to assess expression of E3s and to measure global proteome turnover, capturing over 6,000 protein half-lives. E3s downregulated during TEX were overexpressed in primary murine tumor-specific CD8+ T-cells, and their impact on T-cell persistence was evaluated through serial in vitro cytotoxicity assays measuring their ability to control tumor growth.Results Proteomic turnover analysis revealed the destabilization and loss of the TCR complex, stabilization of inhibitory receptors, and the destabilization of costimulatory receptors during TEX ( figure 1). Notably, many glycolytic enzymes showed increased turnover without changes in abundance, suggesting renewal mechanisms potentially compensating for protein damage or misfolding. Through the overexpression screen, we identified RNF166 to enhance T-cell persistence, prolonging the cytotoxic function of the T-cells by 8 days (p<0.002). Transcriptomic data further showed that elevated RNF166 expression in tumor-infiltrating lymphocytes (TILs) correlates with improved response to immune checkpoint blockade (ICB) in metastatic melanoma. In primary human CD8+ T-cells, RNF166 overexpression reduced expression of inhibitory receptors under chronic stimulation and enhanced persistence of CAR T-cells in vitro (p<0.0032).Conclusions Our overarching hypothesis is that the ability of a T-cell to persist and function within the hostile TME can be enhanced by dynamically regulating its proteome through advantageous E3s. We demonstrate widespread alterations in proteome turnover in exhausted T-cells, driven in part by changes in E3 expression. Notably, overexpression of RNF166 in T-cells significantly enhances persistence, highlighting a promising strategy for improving the efficacy of ACT.Abstract 277 Figure 1Comprehensive analysis of proteome turnover dynamics during T cell exhaustion. Pulsed stable isotope labeling and high-resolution mass spectrometry reveal extensive proteome remodeling in exhausted T cells, driven by accelerated protein turnover and altered E3 ubiquitin ligase expression