Gene expression analysis of CD45RB/CD69 subsets of spleen (SPL) and gut. Single-cell suspensions of SPL and ileum (ILE) were stained for RNA extraction after BD FACS Aria sorting of naive and MBC subsets defined by CD45RB and CD69 expression. (A) PC analysis of MBC subsets from SPL and ILE with genes that are differentially expressed between splenic DP and DN MBCs. The dotted ellipses represent the 95% confidence interval boundary for cell clusters. (B) PC analysis of MBC subsets from SPL and ILE with genes that are differentially expressed between CD69⁺ and CD69^– MBCs. (C) Heatmap of top 40 DEGs between CD69⁺ and CD69^– MBCs ranked by adjusted P value for all splenic MBC subsets. (D) Heatmap of differentially expressed TFs between splenic DP and DN MBCs. (E) Analysis of tissue-specific genes in DP and DN MBCs. Scatterplots display log2 fold-change of SPL DP vs DN samples on the x-axis and ILE DP samples vs SPL DN samples on the y-axis. Gray dots represent genes with significant (P adjusted <.1 and absolute value of log2 fold-change >1) differential expression in at least one of the 2 DP vs DN comparisons. Magenta dots denote “ILE-specific” and purple dots denote “SPL-specific” genes. Light blue and blue dots represent genes that are upregulated or downregulated, respectively, in both comparisons. (F) GSEA for ILE DP vs SPL DN on the TRM gene signature of CD8⁺ T-cell lineage (left panel) and CD4⁺ T-cell lineage (right panel) in lung tissue. In each plot, the x-axis shows the genes ranked by log fold-change between ILE DP vs SPL DN, and the y-axis shows the running enrichment score on the signature gene set with nominal P value indicated for 1000 permutations.