Fig. 1: An in vivo CRISPR screen identifies metabolic genes regulating CD8+ T cell fitness in the tumour and metastatic organs.
a–c, Experimental design of anti-PD-1 treatment resistance on KPC_OVA bearing mice (a); representative haematoxylin and eosin colouration of untreated KPC_OVA primary tumour, 5 days post-injection, scale bar 100 μM (b); KPC_OVA tumour weight at 12 days post-injection (c) (IgG-treated n = 4 versus anti-PD-1 n = 4). d, KPC_OVA tumour weights at 12 days with (n = 7) or without activated OT-I adoptive transfer (n = 3) at day 5 (ACT) (Extended Data Fig. 1a). e,f, Percentage of OVA-specific TCRvα2+ TCRvβ5+ T cells (left) and PD-1 expression (right) in lung (e) and liver (f) from naive or KPC_OVA-bearing mice receiving ACT (naive + ACT n = 3, KPC_OVA + ACT n = 7). g, Workflow of in vivo metabolic CD8+ T cells CRISPR/ Cas9 screening design from KPC_OVA bearing mice. Isolation and activation of OT-I T cells from OT-I:Rosa26-Cas9 mice; transduction of OT-I T cells with the lentiviral sgRNA metabolic library; Enrichment of transduced CD90.1+ OT-I; adoptive transfer of CD90.1+ OT-I T cells into recipient KPC_OVA-bearing mice and treatment with anti-PD-1 blocking antibody; 7 days post-ACT, sort of CD90.1+ OT-I cells from primary tumour, metastatic niches and lymphoid organs. NGS and bioinformatic identification of the candidate metabolic targets (IgG n = 41, anti-PD-1 n = 40, three independent sequencing experiments). Figure created in BioRender (Mazzone (2025) p04f031). h–j, Representative volcano plot generated with MAGeCK of enriched and depleted genes in CD90.1+ OT-I T cells sorted from primary tumour (h), lungs (i) and liver (j) of KPC_OVA tumour-bearing mice treated with anti-PD-1. k, Venn diagram representing the genes corresponding to the significantly enriched sgRNAs, at least in one organ and one treatment, using STAR (P ≤ 0.05) and MAGeCK (false discovery rate (FDR) ≤ 0.05) algorithms. l, Tissue distrubution of the 83 genes enriched in STAR and MAGeCK. m, Top five GO terms for the 83 genes enriched in STAR and MAGeCK. Data are presented as mean ± s.e.m. Statistical significance was assessed by two-tailed unpaired Student’s t-test.
