Extended Data Fig. 11: KLHL6 modulates mitochondrial fitness and anti-tumour T cell responses via ubiquitinating PGAM5.

a, Immunoblot of mitochondrial fusion/fission proteins in WT or KLHL6 KO OT-I T cells activated by anti-CD3/CD28 for indicated times (n = 3 independent samples). b, Immunoblot of p62 and LC3 (upper band: LC3-I; lower band: LC3-II) in WT and KO OT-I T cells at day 5 post-activation. c, CD45.2⁺ B16-OVA-bearing mice received 4×10⁶ WT or KO CD45.1⁺ OT-I cells pretreated with DMSO or M1 (20 μM) + Mdivi-1 (10 μM) (MM) for 3 days. Survival of mice was monitored (n = 8 mice). d, Interaction between introduced KLHL6-Myc and endogenous PGAM5 in Jurkat cells. e, Ubiquitination of endogenous PGAM5 in Jurkat cells transduced with KLHL6-Myc. f,g, Immunoblot analysis of endogenous PGAM5 in Jurkat cells expressing an empty vector or KLHL6-Myc with or without MG132 treatment (f) and in WT and KO T cells collected at different time points after activation (g). h,i, WT and KLHL6 KO OT-I T cells transduced with shPgam5 (shP5) or shCtrl retrovirus were cultured in vitro for 5 days. TMRE/MTG ratio (h, n = 5 independent samples) and mitochondrial morphology and area (i; scale bar, 1 μm; n = 60 cells) were analyzed. j-m, CD45.1/2⁺ WT and CD45.1⁺ KLHL6 KO OT-I T cells transduced with GFP-shCtrl or Thy1.1-shPgam5 (shP5) were mixed at a 1:1:1:1 ratio and cotransferred into CD45.2⁺ B16-OVA tumour-bearing mice. Mice were sacrificed for analysis on day 14 after ACT. Experimental design (j), CD8⁺ TIL numbers (k), PD-1 and TIM-3 expression in TILs (l), and numbers of CD44⁺CD62L⁺ cells in dLN and spleen from the four groups (m) (n = 7 mice). n-r, WT and KO OT-I T cells were activated and cultured for 3 days in vitro, then treated with DMSO or the PGAM5 inhibitor LFHP-1c (P5i, 2 μM) for another 3 days before analysis. Immunoblotting of Mfn2, Opa1, Drp1, p-Drp1^S637, PGAM5, and Actin (n). OCR, SRC, mitochondrial ATP production (o-q, n = 10 tests), and TMRE/MTG ratio (r, n = 3 independent samples) were assessed. s-y, CD45.2⁺ mice bearing B16-OVA tumours received 4×10⁶ activated WT or KO CD45.1⁺ OT-I cells pretreated with DMSO or P5i. The mice were sacrificed for analysis at day 14 after ACT. Schematic of the experiment (s), tumour weights (t), total numbers (u), cytokine production (v), PD-1 and TIM-3 levels (w), and cell numbers of indicated subsets (x) of OT-I TILs were evaluated (n = 6 mice); percentages of T_CM populations in dLN and spleen (y, n = 6 mice). Diagram in j created in BioRender. Li, G. (2025) https://BioRender.com/md3c1bz. Diagram in s created in BioRender. Li, G. (2025) https://BioRender.com/ap5vq6h. Data in (b,d-g,n) are representative of three 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,h,i,k-m,o-r,t-y) and Log-rank (Mantel-Cox) test (c). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant.

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