Mitochondrial translation drives activated CD8+ T cell dysfunction

Enhancing CD8+ T cell function is crucial for improving cancer immunotherapy. Through CRISPR screening and validation, we identified that mitochondrial translation is upregulated in activated and exhausted CD8+ T cells, leading to the accumulation of misfolded proteins that induce mitochondrial stress and contribute to T cell dysfunction. Inhibition of this over-translation using low doses of chloramphenicol (CAP) significantly enhanced the antitumor activity of activated CD8+ T cells by promoting their expansion and reducing apoptosis and exhaustion. Low-dose CAP treatment preserved oxidative phosphorylation (OXPHOS) and cytokine production while restoring mitochondrial membrane potential. Furthermore, CAP facilitated the import of mitochondrial protein precursors, shifted HSP70 pools binding toward NFATc1, and limited its nuclear translocation, thereby reducing exhaustion markers of TIM-3 and PD1. Notably, a short course of low-dose Linezolid, a commonly used antibiotic and mitochondrial translation inhibitor, improved the CD8+ T cell ratio in the peripheral blood of patients with diffuse large B cell lymphoma. These findings highlight mitochondrial translation inhibition as a promising strategy to enhance CD8+ T cell function and potentially improve the effectiveness of cancer immunotherapy. Overall design: For the pooled CRISPR screen, we used CRISPR-FOCUS (http://cistrome.org/crispr-focus/) to design two libraries: a mini-library targeting 171 mitochondrial tRNA-binding proteins, and a broader library targeting 1,331 translation-related proteins, with 10 sgRNAs per gene. Naïve OT1 CD8+ T cells expressing Cas9 were first activated with anti-CD3/CD28 antibodies, followed by retroviral transduction with the sgRNA library, achieving a transduction efficiency of approximately 30%. After a two-day culture to allow for sgRNA editing and mCherry expression, 3 × 106 mCherry-positive cells were sorted as the Input sample using a FACSAria Fusion Cell Sorter (BD). Subsequently, 2 × 107 cells were co-cultured with 1 × 106 purified CD11c+ cells in the presence of 10 ng/ml IL-2 and 5 µg/ml OVA peptide for three days. After expansion, 3 × 106 mCherry-positive cells were sorted as the expanded sample. Genomic DNA was then extracted using the Blood and Cell Culture DNA Maxi Kit (Qiagen), and two rounds of PCR amplification were performed to amplify the sgRNAs and add Illumina adaptors, using NEBNext High-Fidelity 2× PCR Master Mix (New England BioLabs).