Fig. 1: An integrative computational analysis-guided CRISPR screen identifies post-translational regulators of both exhaustion and mitochondrial fitness in T cells.
From: The ubiquitin ligase KLHL6 drives resistance to CD8+ T cell dysfunction
a, Schematic illustration of workflow for computational analysis of RNA-seq atlases of T cell exhaustion. b,c, Samples from two published datasets (GSE89307, GSE86881) were projected onto a two-dimensional map defined by computationally derived gene modules. b, Antigen-specific TCR-transgenic T cells collected across acute Listeria infection and tumour progression at matched time points following adoptive transfer. c, Antigen-specific naive and chronically exhausted T cells isolated during late-stage LCMV infection. d, GSEA enrichment of proteostasis-associated pathways in modules 1 and 2. e, Experimental schematic of CRISPR screen for E3 ligases that regulate T cell exhaustion and mitochondrial function. f,g, Rank plots (left) of gene-level enrichment scores in exhausted versus non-exhausted T cells (f) and in T cell populations with dysfunctional versus functional mitochondria (g). The CRISPR enrichment scores (log2 fold change of PD-1+TIM-3+ versus PD-1−TIM-3− or (MTDR/MTG)lo versus (MTDR/MTG)hi) were determined by comparing the indicated subsets for target genes from Cas9+sgRNA (mCherry)+ cells isolated from tumours on day 7 after ACT. The x axis shows targeted genes; the y axis shows the CRISPR enrichment score of each targeted gene; the dot colour represents false discovery rate (FDR). Distribution of several top-hit sgRNAs (right). Axis represents log2 fold change (FC). The histogram shows distribution of all sgRNAs. Red bars represent targeted sgRNAs, grey bars represent all other sgRNAs. h,i, Representative plots (left) and quantification (right) of the proportions of PD-1+TIM-3+ (h, n = 7 mice) and (MTDR/MTG)lo (i, n = 6 mice) TILs in sgCtrl-transduced or sgKlhl6-transduced Cas9+ OT-I T cells from B16-OVA tumours obtained on day 7 after ACT. Diagram in a created in Biorender. Li, G. (2025) https://BioRender.com/d5c767f. Diagram in e created in BioRender. Li, G. (2025) https://BioRender.com/dw0yfsp. Data are presented as mean ± s.e.m. Statistical analyses were determined by two-way ANOVA with Tukey’s multiple-comparisons test (h,i). *P < 0.05, ***P < 0.001 and ****P < 0.0001.
