Single-cell transcriptomic analysis of B220 ^low cells reveals unique developing B cell signatures. RNA sequencing was performed on single cells from young and aged SJL/J A/T EAE dura at homeostasis (young n = 2, aged n = 1), acute phase of disease (young n = 3, aged n = 2), and postacute phase (young n = 1, aged n = 1). Results are data from three experimental replicates. Unique B cell and PC clusters were identified after QC and data integration. (A) Annotation of B cell/PC clusters based on expression of canonical genes. (B) Bc-1 versus FOBc gene expression in B220^high versus B220^low cells. (C) Volcano plot showing differentially expressed genes between B220^high and B220^low clusters discovered based on Wilcoxon rank-sum test. (D) DEGs identified in B220-low cells (logFC > 2, P < 0.05) reveal upregulation of pathways associated with B cell development. (E) Chord diagrams showing CellChat interaction inferencing at homeostasis, acute phase, and postacute phase. Networks are shown with B cells as senders. (F) Total number and weight of inferred interactions at each time point from B220^high versus B220^low subsets. (G) Bubble plot showing predicted receptor–ligand interactions originating from B cells to fibroblast subsets, P-values calculated using CellChat’s prediction algorithm.