Extended Data Fig. 4: Lymphocytes, monocyte-derived dendritic cells, MIMS and AIMS are identified at the chronic active lesion edge.

Chronic active lesion overview. Multiplex immunostaining of a brain tissue block from a 48-year-old woman with progressive MS (case MS4 in Supplementary Table 1), including a chronic active demyelinated lesion (devoid of myelin proteolipid protein (PLP) staining). Numbers indicate areas magnified in subsequent panels for validation of immune cell and inflamed astrocytes and represent the chronic active lesion edge, lesion centre and periplaque white matter. Lymphocytes and microglia at the lesion edge (panels 1 and 2). At the chronic active lesion edge, there are groups of CD8 T cells within the perivascular space and sparsely within the parenchyma (arrows). CD20 B cells are fewer than CD8 T cells and are located prevalently within the perivascular space (dashed arrows). Transition from myelination to demyelination is shown with staining for CNPase, an oligodendrocyte and myelin marker. Residual myelin fragments can be seen at the edge (arrowheads), presumably not yet removed by phagocytes. IBA1-microglia/macrophages are frequent, and they have an activated morphology (round shape without ramifications). Bar graphs (second row) show the gene expression Z-scores of markers implemented for the identification in tissue of the most relevant glial cell populations at the chronic active lesion edge. Homeostatic microglia vs. MIMS: different spatial locations (insets 3–5). In the periplaque white matter, most microglia are P2RY12+ (a homeostatic marker) with short and thick processes, whereas at the chronic active lesion edge, most are CD68+ (indicating upregulation of antigen and lipid processing) with round, activated morphology. At the lesion core, fewer microglia can be identified, and these show a round morphology consistent with activation. Interestingly, some of them are P2RY12+, potentially suggesting the return of some homeostatic markers. MIMS-iron (inset 6). Accumulation of iron-laden phagocytes (CD68 and ferritin light chain, FTL) is seen at the lesion edge. FTL is within the first 100 top differentially expressed genes in MIMS, especially in MIMS-iron (Fig. 2c). Iron retention in phagocytes can be seen by MRI at the chronic active lesion edge as a paramagnetic rim (MRI biomarker; Fig. 1a). MIMS-foamy (inset 7). Colocalization of PPARG and CD68. PPARG and CD68 double positive (white, arrows) microglia are especially seen at the lesion edge, suggesting their involvement in energy metabolism and modulation of inflammation as well as clearance of myelin debris. Monocytes/monocyte-derived dendritic cells and MIMS (inset 8). CD68 microglia outnumber CD83 monocyte-derived mature dendritic cells (arrows) at the chronic active lesion edge. AIMS and MIMS (insets 9, 10a, and 10b). In addition to IBA1+/CD68+ microglia, the lesion edge is enriched for inflamed astrocytes (positive for VIM and APOE but negative for IBA1 and CD68), sometimes in close proximity (dashed white box). Compared to activated microglia, inflamed astrocytes are bigger and show radial processes. moDC,  monocyte-derived dendritic cells; MIMS, microglia inflamed in MS; AIMS, astrocytes inflamed in MS. Scale bar: 20 μm.