(A–D) Bulk RNA sequencing on naive CD8⁺ T cells (sorted on live CD8⁺CD19⁻B220⁻CD44^lo) from 5 WT and 5 MD4 mice. (A) The top 10 most significant gene sets identified by GSEA using the Broad Institute’s Molecular Signatures Database (MSigDB) immunologic signature gene sets (C7) ordered by adjusted P value. (B) ChEA analysis of the 1,027 differentially expressed genes by DESeq2 ranked by combined score. (C) Heatmap of the 242 genes identified by ChEA as being associated with FOXO1 transcriptional activity. (D) Volcano plot where genes known to be differentially expressed in Foxo1-deficient cells (31, 33) are highlighted in black (higher in WT) or maroon (higher in MD4). (E) Flow cytometry staining of CD8⁺ T cells from WT, μMT^−/−, and MD4 mice. Representative histograms are shown for CD44^lo naive cells (top) and for CD44^hi memory and virtual memory cells (middle). A graph plotting the individual gMFI from 5 mice per group for CD44^lo naive cells is shown at bottom. Data shown are means ± SEM, representative of more than 2 experiments. (F) Representative FOXO1 staining in naive CD8⁺ T cells from healthy control versus CAR T cell–treated patients (top) and patients with MS on anti–α₄ integrin versus anti-CD20 therapy (bottom). (G) Summarized results showing the gMFI values (1 × 10³, 10³, 10³, 10², 10³, and 10², respectively) for FOXO1, CD127, IRF4, GZMA, CD49d, and NKG2A protein staining for all 4 groups; n = 19, 8, 20, and 19, respectively; **P < 0.01, ***P < 0.001.