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 (T_RM) cell development. T_RM cells depended on mitochondrial translation and respiration. Conversely, three nutrient-dependent lysosomal signaling nodes—Flcn, Ragulator, and Rag GTPases—inhibited intestinal T_RM cell formation. Depleting these molecules or amino acids activated the transcription factor Tfeb, thereby linking nutrient stress to T_RM programming. Further, Flcn deficiency promoted protective T_RM 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 T_RM cell formation, while Acss1 controlled T_RM cell development under Flcn deficiency-associated lysosomal dysregulation. Thus, the interplay between nutrients, organelle signaling, and metabolic adaptation dictates tissue immunity.