Highlights d Live monitoring of guidance receptors reveals specific dynamics in commissural axons d Slit and Semaphorin receptors have different temporal patterns of membrane insertion d A front-rear polarity organizes PlxnA1 and Robo1 receptors at the growth-cone surface d Robo1, but not Robo2, is sorted during midline crossing

The mechanisms underlying the chronicity of autoimmune diseases of the central nervous system (CNS) are largely unknown. In particular, it is unclear whether tissue-resident memory T cells (T RM ) contribute to lesion pathogenesis during chronic CNS autoimmunity. Here, we observed that a high frequency of brain-infiltrating CD8 + T cells exhibit a T RM -like phenotype in human autoimmune encephalitis. Using mouse models of neuronal autoimmunity and a combination of T single-cell transcriptomics, high-dimensional flow cytometry, and histopathology, we found that pathogenic CD8 + T cells behind the blood-brain barrier adopt a characteristic T RM differentiation program, and we revealed their phenotypic and functional heterogeneity. In the diseased CNS, autoreactive tissue-resident CD8 + T cells sustained focal neuroinflammation and progressive loss of neurons, independently of recirculating CD8 + T cells. Consistently, a large fraction of autoreactive tissue-resident CD8 + T cells exhibited proliferative potential as well as proinflammatory and cytotoxic properties. Persistence of tissue-resident CD8 + T cells in the CNS and their functional output, but not their initial differentiation, were crucially dependent on CD4 + T cells. Collectively, our results point to tissue-resident CD8 + T cells as essential drivers of chronic CNS autoimmunity and suggest that therapies targeting this compartmentalized autoreactive T cell subset might be effective for treating CNS autoimmune diseases.