AbstractMultiple sclerosis (MS) is a chronic demyelinating disease characterised by immune cell infiltration resulting in lesions that preferentially affect periventricular areas of the brain. Despite research efforts to define the role of various immune cells in MS pathogenesis, the focus has been on a few immune cell populations while full-spectrum analysis, encompassing others such as natural killer (NK) cells, has not been performed. Here, we used single-cell mass cytometry (CyTOF) to profile the immune landscape of brain periventricular areas – septum and choroid plexus – and blood from MS donors and controls with and without other neurological diseases. Using a 37-marker panel, we revealed the infiltration of T cells and antibody-secreting cells in periventricular brain regions and identified a novel NK cell signature specific to MS. CD56bright NK cells accumulated in the septum of MS donors, displaying an activated and migratory phenotype that was similar to that of CD56bright NK cells in their circulation. We validated this signature by multiplex immunohistochemistry and found that NK cells with high expression of granzyme K, which is typical of the CD56bright subset, accumulated in both periventricular lesions and the choroid plexus. Together, our multi-tissue single-cell data suggests that CD56bright NK cells infiltrate the periventricular brain regions in MS patients via both the blood-brain and blood-CSF barriers and brings NK cells to the spotlight of MS pathology.