CRISPR screens unveil nutrient-dependent lysosomal and mitochondrial nodes impacting intestinal tissue-resident memory CD8+ T cell formation

Nutrient availability and organelle biology direct tissue homeostasis and cell fate, but how these processes orchestrate tissue immunity remains poorly defined. Here, using in vivo CRISPR-Cas9 screens, we uncovered organelle signaling and metabolic processes shaping CD8+ tissue-resident memory T (TRM) cell development. TRM cells depended on mitochondrial translation and respiration. Conversely, three nutrient-dependent lysosomal signaling nodes—Flcn, Ragulator, and Rag GTPases—inhibited intestinal TRM cell formation. Depleting these molecules or amino acids activated the transcription factor Tfeb, thereby linking nutrient stress to TRM programming. Further, Flcn deficiency promoted protective TRM cell responses in the small intestine. Mechanistically, the Flcn-Tfeb axis restrained retinoic acid-induced CCR9 expression for migration and transforming growth factor β (TGF-β)-mediated programming for lineage differentiation. Genetic interaction screening revealed that the mitochondrial protein Mrpl52 enabled early TRM cell formation, while Acss1 controlled TRM cell development under Flcn deficiency-associated lysosomal dysregulation. Thus, the interplay between nutrients, organelle signaling, and metabolic adaptation dictates tissue immunity. [Display omitted] •Systematic discovery of mitochondrial and lysosomal pathways in CD8+ TRM formation•Lysosomal signaling and amino acids shape Tfeb-driven TRM development•Small intestine-specific TRM programming is impeded by the Flcn-Tfeb signaling axis•Acss1 and Mrpl52 empower early siIEL TRM formation in distinct contexts How organelle signaling and metabolic adaptation orchestrate tissue-resident CD8+ T (TRM) cell development remains poorly defined. Here, Raynor et al. establish three nutrient-dependent lysosomal signaling nodes as negative regulators of TRM differentiation in the small intestine. These results uncover mechanisms dictating TRM cell quality and quantity for tissue immunity.