(A) Heatmap showing log(fold change) of 6 marker genes (rows) in trained macrophages relative to the average expression in control macrophages (columns). Red and blue colors correspond to upregulation and downregulation, respectively. (B) KEGG enrichment of training response (TR) genes (comparing trained conditions with RPMI controls) in each subgroup of trained cells. (C) Annotation of the subgroups of trained cells in UMAP plots. (D) Comparison of the cell frequency of subgroups between trained tissues. M-MONO, monocytes trained in the absence of lymphocytes; M-PBMC, monocytes trained in the presence of lymphocytes. Dirichlet’s regression model was applied to test the differences in cell frequency between groups; P values are shown on the box-and-whisker plot. (E) UMAP of cellular trajectories inferred by Monocle 3 with trained subgroups or original clusters. (F) UMAP and violin plot of pseudo-time state of trained cells estimated by Monocle 3. P values from Wilcoxon’s rank-sum test are shown on the violin plot. (G) Integrated UMAP of cells from the initial and replicate in vitro experiments showing the distribution of cells sampled at different time points. (H) Violin plots showing AUCell-based scores (R/AUCell package) of trained-immunity signatures from MCI and MC subgroups in trained cells and nontrained controls sampled from the replicate experiment. The lines in the violin plots represent the median of the AUC scores and the 0.25 and 0.75 quantiles, and colors represent the average scores centered on zero. Wilcoxon’s rank-sum test was applied to ascertain whether the AUC scores in trained cells were larger than in nontrained controls. T1, 4 hours after training (or RPMI) stimulation; pre-T2, 5 days after training (or RPMI) stimulation and before LPS restimulation; T2, 4 hours after LPS restimulation. P values are shown at the top in D, F, and H.