Extended Data Fig. 12: TOX and mitochondrial fitness are key mediators of KLHL6-driven anti-tumour immune responses.

a,b, CD8⁺ WT and KLHL6 KO T cells transduced with either Empty vector (Empty) or PGC1α-overexpression (PGC1α) plasmids were cultured for 6 days. OCR (a, n = 10 tests) and SRC and mitochondrial ATP production (b, n = 10 tests) were measured. c-e, Tumour weights (c), absolute numbers of transferred CD8⁺ TILs (d), and proportion of damaged mitochondria in transferred CD8⁺ TILs (e) were assessed in the indicated groups (n = 6 mice), related to Fig. 5l,m. f-i, Experimental design related to Fig. 5n,o (f), tumour weights (g), absolute numbers of total (h) and Tpex (Ly108⁺TIM-3⁻) and Tex^term (Ly108⁻TIM-3⁺) subsets (i) of transferred CD8⁺ TILs among the five groups at day 14 after ACT (n = 6 mice). j-n, CD8⁺ OT-I T cells transduced with either TOX-overexpressing (TOX-OE) or Empty vector (Control) retrovirus were cultured for 5 days in vitro, and then restimulated with or without CD3 antibody for 24 h before analysis. Representative plots and quantification of mitochondrial depolarization (TMRE/MTG)^lo (j) and MitoSOX level (k) were assessed (n = 3 independent samples). OCR (l), SRC (left) and mitochondrial ATP production (right) (m), and glycoPER (n) were measured (n = 10 tests). o,p, Activated WT and KO CD8⁺ OT-I T cells were transduced with either shCtrl or shTox retrovirus at 24 h post-activation and cultured for an additional 5 days prior to analysis. OCR (o), SRC (left) and mitochondrial ATP production (right) (p) were measured (n = 8 tests). Diagram in f created in BioRender. Li, G. (2025) https://BioRender.com/md3c1bz. Data in (f-i) are representative of two independent experiments. Data are presented as mean ± s.e.m. Statistical analyses were determined by two-way ANOVA with Tukey’s multiple-comparisons test (a-e,g-p). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant.

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