Tim-4 and CD163 expression identify small intestinal macrophages in different sub-tissular locations. (A) Uniform manifold approximation and projection (UMAP) plot of scRNA-seq data from 3,864 live CD45⁺Lin⁻CD11b⁺MHCII⁺CD64⁺ cells from the small intestine of two WT C57BL/6 and four Ccr2^−/− mice. Colors denote individual cells assigned to the same cluster. (B) UMAP plot showing the composition of clusters as derived from WT (blue) or Ccr2^−/− (red) mice. (C) UMAP plot showing expression level of Ccr2. (D) UMAP plot showing expression level of Timd4. (E) UMAP plot showing expression level of Cd63.(F) Violin plots showing the similarity score of each cluster against ImmGen datasets for left, LP macrophages; right, serosal macrophages. (G) UMAP visualization of combined WT and Ccr2^−/−-derived small intestinal macrophages showing their differentiation trajectory from Ccr2⁺ macrophages, colored by pseudotime as computed in Monocle 3. (H) Representative immunofluorescence image of small intestine section from WT mice. DAPI (blue), IBA1 (red), and Tim-4 (green). Dashed line demarcates the boundary between the LP (above the line) and S/M (below the line). (I) Heatmap showing the top DEGs for all small intestinal macrophage populations identified in Fig. 1 A, with Cd163 highlighted. (J) UMAP plot showing expression level of Cd163.(K) Representative immunofluorescence image of small intestine section from WT mice. DAPI (blue), IBA1 (red), and CD163 (green). Scale bar = 100 μm (H and K). Dashed line demarcates the boundary between the LP (above the line) and S/M (below the line). (L) Expression of CD163 and Tim-4 on small intestinal live CD45⁺Lin^–CD11b⁺MHCII⁺CD64⁺ macrophages assessed by flow cytometry. Numbers denote the percentages of cells within the gate. (M) Frequency of macrophages expressing Tim-4 and/or CD163 in the small intestine. Error bars show mean ± SD. Data (n = 8 per group) are pooled from eight independent experiments.