Fig. 3

Skull bone marrow-derived B cells, particularly ABCs, invade the brain parenchyma of 3-month-old 5 × FAD mice. a, b Schematic diagram depicting dye tracing of bone marrow (a). APC⁺ tracers and FITC⁺ tracers were injected into the cavities of the skulls and femurs of 3-month-old 5 × FAD mice, respectively, and flow cytometry of the brain was performed at 24 h and 96 h. Gating strategy for APC⁺ B cells (CD45⁺ CD19⁺) (left), frequency of APC⁺ B cells in APC⁺ cells in brain tissue (right) (b). n = 6 5 × FAD mice. c Flow cytometry analysis of lymphocytes in the brain tissue of 3-month-old 5 × FAD mice and WT counterparts. Gating strategy for lymphocytes in brain tissue (top). The counts and frequencies of B cells, CD4⁺ T cells and CD8⁺ T cells (bottom). n = 8 mice per group. d Flow cytometry analysis of myeloid cells in the brain tissue of 3-month-old 5 × FAD mice and WT counterparts. Gating strategy for myeloid cells in brain tissue (top). The counts and frequency of neutrophils (CD45^high CD11b⁺ Ly6G⁺), Ly6C^high monocytes (CD45^high CD11b⁺ Ly6G⁻ F4/80⁻ Ly6C ^high) and Ly6C^low monocytes (CD45^high CD11b⁺ Ly6G⁻ F4/80⁻ Ly6C ^low) (bottom). n = 4 mice per group. e Flow cytometry analysis of B-cell subpopulations in the skull BM of 3-month-old 5 × FAD mice and WT counterparts. n = 7–8 mice per group. Counts and frequency of plasma cells (PCs) (CD45⁺ CD3⁻ CD19⁺CD138⁺), B1a (CD45⁺ CD3⁻ CD19⁺ CD11b⁺CD5⁺), B1b (CD45⁺ CD3⁻ CD19⁺ CD11b⁺CD5⁻), B2 (CD45⁺ CD3^- CD19⁺CD11b⁻CD5⁻), ABCs (CD45⁺ CD19⁺ CD11b⁺ CD11c⁺ CD21⁻ CD23^-), CD21^-CD23^-B cells (CD45⁺ CD19⁺ CD21⁻CD23⁻), CD21⁺CD23⁻B cells (CD45⁺ CD19⁺ CD21⁺CD23⁻), and FOB (CD45⁺ CD19⁺ CD21⁺CD23⁺). The gating strategy is depicted in supplementary Fig. 1d. f Dye-tracing of bone marrow analysis of ABC output. Gating strategy for APC⁺ and FITC⁺ ABCs (top). The histogram represents the frequency of APC⁺ and FITC⁺ ABCs in brain tissue (bottom). n = 6 5 × FAD mice. g Flow cytometry analysis of ABCs in the brain tissue of 3-month-old 5 × FAD mice and WT counterparts. n = 8 mice per group. h Bulk RNA sequencing analysis of bone marrow-derived ABCs from 5 × FAD mice and WT counterparts. n = 3 mice per group. Heatmap showing the DEGs (fold change>2.0; adj. FDR value < 0.05) between 5 × FAD mice and WT counterparts; representative genes are listed on the right (left). Plot showing the top pathways enriched with the DEGs according to KEGG analysis (right). The data are expressed as the means ± SEMs. *p < 0.05 and **p < 0.01; two-tailed unpaired Student’s t test was used