Extended Data Fig. 3. Cortical neuron and lymphocyte subtype analysis in MS lesions.

(a) tSNE plots for neuron subtype specific expression of RORB, THY1, NRGN, SST, SV2C and PVALB (left). LaST (ctrl, n= 5) showing layer-specific expression of neuronal RORB in intermediate cortical layer 4 and widespread expression of pyramidal neuron marker THY1 with enrichment in layer 5; note that SST-expressing interneurons preferentially map to deep cortical layers. Co-expression studies (ctrl, n= 5) with SYT1 confirm neuronal expression of RORB, THY1 and SST (black arrowheads). (b) Heatmap with hierarchical clustering of lymphocyte-associated transcripts allowing sub clustering of lymphocytes in T cells, B cells and plasma cells based on marker gene expression (upper left). tSNE plots for typical B/plasma cell and T cell marker genes enriched in lymphocyte clusters (upper right). IHC for T cell marker SKAP1 (black arrowheads mark SKAP1⁺ T cells) together with spatial transcriptomics for B cell-associated IGHG1 encoding immunoglobulin G1 (IgG1) (magenta-colored arrowheads; lower left); note preferential clustering of plasma cell-associated MZB1⁺ and IGHG1-expressing B cells (white arrowheads, lower right) in inflamed meningeal tissue versus mixed T and B cell infiltration in perivascular cuffs of subcortical lesions (lower panels). One caveat to these findings is the relatively small number of MS cases samples, which limited our ability to cluster T cell populations. For tSNE plots (a, b) and hierarchical clustering (b), data shown from 9 control and 12 MS samples. For tSNE plots, data shown for all 48,919 nuclei; for hierarchical clustering, data shown from 53 nuclei in the B cell cluster. For ISH and IHC experiments in b, representative images shown from individual tissue sections (ctrl, n= 4; MS, n= 7).