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The central nervous system (CNS) is under close surveillance by immune cells, which mediate tissue homeostasis, protection, and repair. Conversely, in neuroinflammation, dysregulated leukocyte invasion into the CNS leads to immunopathology and neurological disability. To invade the brain parenchyma, autoimmune encephalitogenic T helper (T H ) cells must encounter their cognate antigens (Ags) presented via local Ag-presenting cells (APCs). The precise identity of the APC that samples, processes, and presents CNS-derived Ags to autoaggressive T cells is unknown. Here, we used a combination of high-dimensional single-cell mapping and conditional MHC class II ablation across all CNS APCs to systematically interrogate each population for its ability to reactivate encephalitogenic T H cells in vivo. We found a population of conventional dendritic cells, but not border-associated macrophages or microglia, to be essential for licensing T cells to initiate neuroinflammation., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)