Gene expression profiles of naive CD4⁺T cells and CD19⁺B cells isolated from HSC-transplanted RAG1^−/−mice. (A) Schematic representation of the experimental setup: HSCs were isolated from young and old B6.SJL (CD45.1) mice and cultured for 16 hours ± 5 µM CASIN. 600 HSCs were transplanted into sublethally irradiated young RAG1^−/− (CD45.2) recipients. Twelve weeks after transplantation, naive CD4⁺ T and CD19⁺ B cells were isolated and RNA sequencing was performed (B) Venn diagram of significantly differentially expressed genes in naive CD4⁺ T cells between DY vs DO (blue circle) and DO+C vs DO (green circle). The heat map shows unsupervised clustering of the genes expressed differentially in the same direction between the 2 groups. (C) Venn diagram of significantly differentially expressed genes in CD19⁺ B cells between DY vs DO (orange circle) and DO+C vs DO (violet circle). Genes, differentially expressed in the same direction between the 2 groups, are depicted as heat map (unsupervised clustering). (D) Venn diagram shows GSEA of significantly enriched GO gene sets differentially expressed in naive CD4⁺ T cells between DY vs DO (blue circle) and DO+C vs DO (green circle). Normalized enrichment scores (NES) of GO categories differentially expressed in the same direction between the 2 groups are depicted. (E-F) Venn diagrams show GSEA of significantly enriched GO and REACTOME gene sets, respectively, differentially expressed in CD19⁺ B cells between DY vs DO (orange circle) and DO+C vs DO (violet circle). NES of GO and REACTOM categories differentially expressed in the same direction between the 2 groups are depicted. (G) GSEA enrichment plots of correlation between gene sets previously identified by Mirza et al.³³  to be differentially expressed in naive CD4⁺ T cells from nontransplanted young and aged mice and differentially expressed genes between DY and DO HSC recipients as well as DO+C and DO recipients. DY, DO and DO+C, n = 3. (D-F) FDR q-value < 0.05; (G) FDR q-value < 0.25.