Extended Data Fig. 5: Enforced expression of KLHL6 restricts T cell exhaustion and promotes mitochondrial function.
From: The ubiquitin ligase KLHL6 drives resistance to CD8+ T cell dysfunction
a, Experimental design, related to Fig. 3a–e. b, Percentages of transferred Control and KLHL6-OE CD45.1+ OT-I T cells detected in the spleen and dLN on day 14 post-transfer (n = 7 mice). c, CellTrace Violet (CTV)-labeled CD45.1/2+ Control and CD45.1+ KLHL6-OE OT-I T cells were cotransferred into B16-OVA tumour-bearing recipient mice. TILs were analyzed on day 4 post-ACT (n = 5 mice). d-f, Proliferation (Ki-67hi) (d), apoptosis (Annexin V+) (e), and expression of apoptotic molecules (Bcl-2, Bcl-XL, and Bim) (f) in transferred Control and KLHL6-OE TILs at day 14 post-ACT (n = 6 mice). g,h, Percentages (g) and cell numbers (h) of PD-1−TIM-3−, PD-1+TIM-3− and PD-1+TIM-3+ populations in transferred CD45.1+CD8+ TILs (n = 7 mice). i,j, TOX (i) and TCF-1 (j) expression in CD8+PD-1+ TILs (n = 7 mice). k, Percentages of CD44+CD62L+ cells in spleen and dLN (n = 7 mice). l,m, Frequencies (l) and numbers (m) of Tpex (Ly108+TIM-3−) and Texterm (Ly108−TIM-3+) subsets in WT and KO TILs (n = 14 mice), related to Extended Data Fig. 2p. n,o, TOX (n) and PD-1 (o) expression in the Tpex and Texterm subsets at day 14 post-ACT (n = 7 mice). p-t, CD45.1/2+ KLHL6-OE and CD45.1+ Control OT-I T cells (2 × 106 each) were cotransferred into CD45.2+ mice bearing B16-OVA tumour. At day 14 post-ACT, TILs were isolated for metabolic analysis, including OCR (p), SRC (q), mitochondrial ATP production (r), glycoPER (s), and the percentage of (MTDR/MTG)lo cells (t) (Seahorse: n = 6 independent tests, 20 mice; (MTDR/MTG)lo subset: n = 8 mice). u-y, CD8+ OT-I T cells transduced with KLHL6-OE or Control retrovirus were analyzed on day 6 for MTG and TMRE staining (u, n = 3 independent samples), as well as OCR (v,w), SRC (x), and mitochondrial ATP production (y) (n = 10 tests). Diagram in a created in BioRender. Li, G. (2025) https://BioRender.com/aicmpdn. Data are presented as mean ± s.e.m. Statistical analyses were performed by unpaired two-tailed Student’s t-test (b-f,i-k,q,r,t,u,x,y) or two-way ANOVA with Sidak’s multiple-comparisons (g,h,l-p,s,w). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant.
