Extended Data Fig. 10: Tim-3 expression is inversely correlated with P2 marker expressions in MGnD cluster.

a, Boxplot of pairwise spearman correlation coefficient (Y-axis) of whole gene expression pseudobulk microglial profiles between conditions (X-axis). Each point represents the spearman correlation coefficient between profiles of two biological replicates. Havcr2^icKO:5xFAD;P1 vs 5xFAD: n = 9 spearman correlations derived from 34 cells from 3 biologically independent Havcr2^icKO:5xFAD samples and 201 cells from 3 biologically independent 5xFAD samples. Havcr2^icKO:5xFAD;P2 vs 5xFAD: n = 9 spearman correlations derived from 23 cells from 3 biologically independent Havcr2^icKO:5xFAD samples and 201 cells from 3 biologically independent 5xFAD samples. Havcr2^icKO:5xFAD;P1 vs Havcr2^icKO:5xFAD;P2: n = 9 spearman correlations derived from 57 cells (34 cells, 23 cells, respectively) from 3 biologically independent Havcr2^icKO:5xFAD samples. b, Twelve human microglial signatures were curated from a previous paper using samples from patients with AD⁵⁸. These signature scores were plotted for cluster 2 in our snRNAseq data. A significant difference was found only in human MG8 signature annotated as inflammatory⁶¹. c, d, Scatterplot of P1(Y-axis, a) or P2 (Y-axis, b), and Hallmark TGFB signature scores among C2 5xFAD and Havcr2^icKO microglia in current study. e-g, Scatterplot of P1 (Y-axis) or P2 (Y-axis), and Hallmark TGFB signature scores among 5xFAD microglia in MGnD clusters in public datasets (GSE98969⁵⁷ cluster 2 (e), GSE98969⁵⁷ cluster 3 (f), GSE140510⁵⁹ (g)). h, i, The cells isolated from the brain were analyzed by flow cytometry, and identified clusters were annotated according to their markers, with cluster 12 annotated as MGnD. The proportion of cluster 12 among all the cells was compared between control, 5xFAD, and Havcr2^icKO:5xFAD mice at the age of 4.5−5 months (n = 3, 3, 3 (female), 2, 4, 3 (male), from the left). j, Phenograph clustering of microglia from 4-month-old control, 5xFAD, and Havcr2^icKO:5xFAD mice using flow cytometry data. Cluster 12 represents Clec7a⁺ MGnD among the microglia. k, Pearson’s correlation between MFI of paired markers of interest within combined Clec7a⁺ MGnD from 4-month-old control, 5xFAD, and Havcr2^icKO:5xFAD was analyzed. The coefficient indicates the strength and direction of the correlation. Data are mean ± s.e.m. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001; Student’s two-tailed t-test for a. One-way ANOVA with Tukey’s HSD correction for b. Spearman correlation test was used for c-g. One-way ANOVA with Holm-Sidak’s multiple comparisons test for i. Pearson’s correlation analysis was adjusted by the false discovery rate (FDR) method for k.

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