Fig. 5

EOMES expression is sufficient to drive T_IM transcriptional program. a Representative flow cytometry plots of total thymocytes from WT and Eomes^Tg mice showing DN, DP, CD4SP, and CD8SP proportions. Histograms indicate the frequency of each population and their EOMES expression (MFI). b Expression of the indicated markers in WT and Eomes^Tg CD3⁺CD8SP thymocytes and in EOMES^lo and EOMES^hi CD3⁺CD8SP thymocytes from Balb/c mice, shown as a reference. SPADE trees indicate cell frequency of CD3⁺CD8SP thymocytes from WT and Eomes^Tg mice using the tree constructed from WT Balb/c CD3⁺CD8SP thymocytes as shown in Fig. 1. c IFNγ expression measured by flow cytometry in CD3⁺CD8SP thymocytes from WT and Eomes^Tg mice cultured with IL-12+IL-18 for 16 h or with phorbol-myristate-acetate and ionomycin (P/I) for 4 h. For P/I, EOMES^lo and EOMES^hi CD3⁺CD8SP thymocytes from Balb/c mice are shown. d Volcano plot of RNA-seq data of CD3⁺CD8SP thymocytes from WT versus Eomes^Tg mice show the adjusted P-value versus fold-change (up in Eomes^Tg, red; up in WT, green). The numbers of differentially expressed genes are indicated. e GSEA plots of innate memory (T_IM)-specific (left) or naïve (N)-specific (right) gene sets in CD3⁺CD8SP thymocytes from Eomes^Tg mice. NES normalized enrichment score. f Expression heatmaps of RNA-seq data comparing mean fold-change (FC) of T_IM and Eomes^Tg CD3⁺CD8SP thymocytes in comparison to their respective naïve and WT controls, respectively. CPM counts per million. In a–c horizontal bars indicate median ± interquartile range and are representative of more than three experiments. Each point represents an individual mouse. Statistics were calculated using Mann–Whitney test. ns not significant, *P < 0.05; **P < 0.01; and ***P < 0.001