Cluster 3 cells possess a monocytic-like transcriptional signature and respond to pathogen infection. (A) Heatmap of correlation matrix of transcriptomes between mouse MK subpopulations and immune cells from published data set, including MKs, monocytes, dendritic cells (DCs), granulocytes, macrophages, CD4⁺ T cells, and CD8⁺ T cells. (B) UMAP plot of MKs from different organs and species by integrated analysis. (C) Violin plots showing the expression levels of genes involved in phagocytosis and antigen presentation in immune MKs derived from fetal mouse lung⁴³ and BM. (D) Representative images of CD53⁺ MKs and monocytes stained with Wright-Giemsa stained. Scale bars, 10 μm. (E) Representative FACS plot of CD53⁺ MKs (CD53⁺CD41⁺) among BM samples from mice at 0, 6, 12, 24, and 30 hours after injection of LPS 4 mg/kg. Numbers in boxes represent the percentage of CD53⁺ MKs (CD53⁺CD41⁺) in total MKs; mean ± standard error of the mean (SEM). (F) Quantification of the frequency of MKs (CD41⁺) in total BM cells and CD53⁺ MKs (CD53⁺ CD41⁺) in total MKs from mice at 0, 6, 12, 24, and 30 hours after injection of LPS 4 mg/kg (n = 3; mean ± SEM; unpaired Student t test). (G) Heatmap displaying relative expression for selected genes involved in indicated biological processes in CD53^– and CD53⁺ MKs isolated from mice at 0, 6, and 12 hours after injection of LPS 4 mg/kg . (H) Representative images of fluorescent Escherichia coli particles (purple) incubated with CD53⁺ or CD53^– MKs stained with CD53 (green) and CD41 (red). Scale bars, 20 μm. BF, bright field. Images represent 3 independent experiments. (I) Quantification of the percentage of cells phagocytizing E coli particles in CD53⁺ and CD53^– MKs: CD53⁺CD41⁺, 219 cells; CD53^–CD41⁺, 267 cells. (J) Representative FACS plot of T-cell proliferation. T cells labeled with carboxyfluorescein succinimidyl ester (CSFE) were cultured with or without 3000 to 5000 CD53⁺/CD53^– MKs for 3 days in the presence of anti-CD3 and anti-CD28 antibody. The upper left quadrant represents the proliferating T cells; numbers in the boxes indicate the percentage of proliferating T cells. Results are representative of 3 independent experiments. (K) Bar graph depicting the percentage of proliferating T cells in total T cells cultured with or without 3000 to 5000 CD53⁺/CD53^– MKs for 3 days in the presence of anti-CD3 or anti-CD28 antibody (n = 3; mean ± SEM; 1-way ANOVA; *P ≤ .05; **P ≤ .01. ****P ≤.0001. n.s., not significant.