Subsets of islet macrophages included both proinflammatory and anti-inflammatory populations. (A) t-SNE plot showing clusters of islet macrophages merged from 4-, 8-, and 15-wk-old NOD mice samples. Dashed lines encompass the general populations containing one, two, or three subclusters (see Fig. S4 B). (B) Heatmap of top (limited to 70) differentially expressed genes driving heterogeneity among populations of macrophages (LFC > 0.3, adjusted P < 0.05). Labels on the left indicate the main features of each group. (C) Abundance of each population relative to the total number of islet cells at each stage. (D) Relative proportion fold change (relative to 4-wk-old) for each population as a function of progression. (E, F, and H) The hypergeometric pathway analysis between two indicated conditions based on MSigDB pathways: canonical c2 (E) and hallmark (F and H). (G and I) Volcano plots showing differentially expressed genes in pairwise comparisons between groups of macrophages. (J) Scatter plot illustrating differential gene expression (absolute log2 fold change value > 0.3, adjusted P < 0.05) in activated macrophages and DCs (red, genes with mutually elevated expression in both macrophages and cDCs; green and blue, only in one condition). Differential expression for B, G, I, and J was performed using nonparametric Wilcoxon rank sum test and corrected for multiple testing with Benjamini–Hochberg method.