Systems immunology guided screening identifies an E3 ubiquitin ligase, KLHL6, as a negative dual regulator of differentiation to T cell exhaustion and mitochondrial dysfunction: bulkRNA-seq

Elucidating the molecular mechanisms that regulate the differentiation of exhausted CD8+ T cells (Tex) can lead to enhanced immunotherapy of cancer and other diseases. Transcriptomic and epigenomic regulation of Tex differentiation have been extensively studied, but the role of proteostasis remains to be defined. We developed a data-driven systems immunology-guided in vivo CRISPR screen and identified the E3 ubiquitin ligase KLHL6 as a dual-negative regulator of T cell exhaustion by maintaining mitochondrial fitness and suppressing TOX-mediated terminal differentiation. Mechanistically, KLHL6 constrains the excessive mitochondrial fission via post-translational regulation of the PGAM5-Drp1 axis, thereby sustaining mitochondrial fitness during T cell chronic stimulation. Simultaneously, KLHL6 interacts with TOX, promoting its poly-ubiquitination and subsequent proteasomal degradation, thereby attenuating the transition of progenitor Tex (Tpex) cells toward terminal exhaustion. Enforced KLHL6 expression in CD8+ T cells markedly boosted mitochondrial fitness, restrained terminal Tex differentiation, and improved the efficacy and long-term persistence of adoptive T cell therapy (ACT) against multiple tumors in vivo. Our studies uncover KLHL6 as a promising and clinically-actionable target for cancer immunotherapy, highlighting the importance of proteostasis and ubiquitin modification in regulating T cell function for therapeutic interventions.