319 Comprehensive analysis of proteome turnover dynamics during T cell exhaustion

Background Adoptive T-cell therapies are widely considered to be the next frontier in cancer immunotherapy treatment but are currently only effective at treating cancers that do not form solid tumors. Tumor-infiltrating lymphocytes (TILs) become exhausted in the hostile tumor environment where they lose effector function, hindering their ability to control tumor progression. We seek to understand the proteins T-cells depend on to ensure adequate plasticity when adapting to stress in the tumor microenvironment. Maintaining proper proteostasis is essential for a cell to balance proteome flexibility and energy saving proteome stability. We have employed proteomic approaches exploring global proteome turnover changes and the differential expression of E3 ubiquitin ligases associated with T-cell exhaustion. Collectively, these findings offer valuable layer of insight into the molecular mechanisms governing T-cell exhaustion, ultimately constraining T-cell effector function and plasticity. Methods For T-cell exhaustion, CD8+ T-cells were stimulated using αCD3/αCD28 either on day 0 (acute stimulation), or every 2 days for 8 days (chronic stimulation). Cells were harvested at day 8 for proteomic analysis to look for differential E3 ubiquitin ligase expression and to assess global proteome turnover changes (>6000 half-lives). The exhaustion phenotype was validated by quantification of inhibitory receptor expression using flow cytometry. Differential E3 ubiquitin ligase expression and protein half lives were validated via western blot analysis. Results Proteomic analysis identified 33 differentially expressed E3 ubiquitin ligases and 364 changes in protein half-lives associated with T-cell exhaustion. We found destabilization of the TCR complex results in decreased protein abundance, whereas stabilization of T-cell exhaustion markers results in increased protein abundance. Surprisingly, many glycolytic enzymes are destabilized without a loss of abundance, indicating a rapid renewal and possible compensation for protein damage and misfolding. In support of this interpretation, we found an accumulation K48-linked ubiquitination, which is a well-known signal for protein degradation. Conclusions Our overarching hypothesis is that the ability of a T-cell to persist and function in the hostile tumor microenvironment can be enhanced by dynamic control of its proteome through advantageous E3 ubiquitin ligases. Here we show dramatic changes in proteome turnover in exhausted T cells which is in part due to changes in E3 ubiquitin ligase expression and the accumulation of misfolded proteins. It remains to be determined if the manipulation of E3 ubiquitin ligases can protect T-cell proteostasis and enhance T-cell function in solid tumors.