Journal: Nature

Article Title: The ubiquitin ligase KLHL6 drives resistance to CD8 + T cell dysfunction

doi: 10.1038/s41586-025-09926-8

Figure Lengend Snippet: 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 4×10 6 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 4×10 6 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.

Article Snippet: For drug treatment experiments, DMSO (Sigma, D2650), 2 μM LFHP-1c (MCE, HY-139598) or 10 μM Mdivi-1 (Selleck, S7162) and 20 μM M1 (Selleck, S3375) were added to cultures daily starting on day 3 after T cell activation.

Techniques: Western Blot, Activation Assay, Ubiquitin Proteomics, Transduction, Expressing, Plasmid Preparation, Cell Culture, In Vitro