Extended Data Fig. 5: Shared microglial response drives Aβ clearance after immunization.

a, UMAP showing cortical Aβ-rich ST spots based on gene and protein expression, colored by brain region and donor. b, UMAP density plots for each group. c, Top two marker genes for each cortical Aβ-rich cluster. d, Bar plots showing C2L predictions of scRNA-seq cell types proportionally in the gray matter per group. e, Volcano plot showing DEGs distinguishing cortical Aβ-rich cluster 6 from all other cortical Aβ-rich clusters. f, Bar plots showing C2L predictions of scRNA-seq microglia and macrophage subtypes proportionally in Aβ-rich cluster 6 per group. g, Bar graphs showing pseudobulked TREM2, TSPO, S100A4, APOE, A2M, TMSB4X, RAB13, FCGBP, CTSB, CHI3L1, and FAM107A expression in Aβ-rich cluster 6. Error bars indicate SEM. P-values are from DESeq2. h, Bar plots showing log2 fold-change in predicted abundance of deconvoluted scRNA-seq microglia types in Aβ-rich ST spots versus the rest in AN1792, nAD, and the lecanemab case. i, Bar plots showing log2 fold-change in pseudobulked expression of A2M, APOE, FAM107A, LIPA, SPP1, and TREM2 in Aβ-associated Mg2-enriched and Mg4-enriched ST spots compared to the nAD control group for AN1792 and the lecanemab case. j, Chord plots showing inferred CellChat cell-cell communication of APOE, complement, and SPP1 signaling pathways among different scRNA-seq cell types. The width of the chords reflects the strength of interaction or communication probability, with thicker chords indicating stronger signaling. k, Visium HD ST method. Created using BioRender.com. l, UMAP showing annotated binned nuclei from the high-definition ST assay, colored by donor. m, Number of features (genes) per binned nuclei in high-definition ST data per donor. n, Percentage of mitochondrial genes per binned nuclei in high-definition ST data per donor. o, Top three marker genes for overarching cell types annotated in the high-definition ST data. p-q, Top 10 upregulated response DEGs in microglia or Aβ DE ranked by their average percentile in Aβ (Y-axis) and microglia (X-axis) DE: o, in AN1792; r, in lecanemab. g, h-i, Bar plots display means ± SEM. m-n, Violin plots showing the data range and median. Points represent individual cells. nAD3 and nAD4 refer to separate samples. a-c, e-f, nAD-AN1792 = 4, iAD-lim = 6; iAD-ext = 4; nAD-LCMB = 3; LCMB = 1. d, nAD-AN1792 = 6, iAD-lim = 6; iAD-ext = 7; nAD-LCMB = 3; LCMB = 1. g, nAD-AN1792 = 4; iAD = 10; nAD-LCMB = 3; LCMB = 1. h, nAD-AN1792 = 4; iAD-lim = 6; iAD-ext = 6; nAD-LCMB = 3; LCMB = 1. i, iAD = 9 (Mg-2), 10 (Mg-4); LCMB = 1. j, iAD = 5; nAD = 3; LCMB = 1. l-o, nAD = 2; LCMB = 1. g, DESeq2 was used to compare expression levels. Covariates included sex, age, average genes detected, gDNA percentage (AN1792 vs. nAD); average genes detected, brain region, gDNA percentage (LCMB vs. nAD). h. Statistical tests, guided by Shapiro–Wilk and F tests, included t-tests, Mann–Whitney, ANOVA with Tukey’s test, Welch ANOVA with Dunnett’s T3 test, and Kruskal–Wallis with Dunn’s test. All P-values are FDR-adjusted using the Benjamini-Hochberg correction. A2M, alpha-2-macroglobulin; Aβ, amyloid-beta; AD, Alzheimer’s disease; APOC1, apolipoprotein C1; APOE, apolipoprotein E; CD68, cluster of differentiation 68; Cort., cortical; Ctx, cortex; CTSB, cathepsin B; DEGs, differentially expressed genes; DESeq2, Differential Expression Analysis for Sequence Count Data (version 2); FDR, False Discovery Rate; FCGBP, Fc fragment of IgG binding protein; GM, gray matter; HD, high-definition; IBA1, ionized calcium-binding adapter molecule 1; ITGAX, integrin subunit alpha X; L, layer; LCMB, lecanemab; LOESS, locally estimated scatterplot smoothing; MSigDB, Molecular Signatures Database; MT, mitochondrial; nAD, non-immunized Alzheimer’s disease; nFeatures, number of features; P-adj, P-value adjusted; SPP1, secreted phosphoprotein 1; ST, spatial transcriptomics.