Fig. 3: Heterogeneity of the T_SCM and differential engagement of Wnt signaling.

a Heterogeneity of naive CD4 T cells revealed by high-dimension flow cytometry. Naive CD4 T cells of eight donors (four young and four old donors), defined as CCR7⁺CD45RO⁻CD27⁺, were concatenated and analyzed by t-SNE. Clusters were visualized by phenograph. b Phenotype of naive CD4 T-cell clusters. The intensity of fluorescence for each marker was visualized by a normalized heatmap (gradient of increased expression from blue to red). c Preservation of specific gene signature of CD4 T_SCM cells during aging. Naive, T_SCM, T_CM, and T_EM CD4 T cells (n = 5 for all subsets by age except T_EM, n = 3) were sorted, and analyzed for their gene expression by nanostring. Naive T-cell subsets were sorted as naive, T_RTE, T_MNP, and T_SCM CD4 cells and analyzed by RNA-seq (n = 5). PCA analysis of mRNA expression was performed to evaluate the specificity and preservation of T-cell subsets signature during aging. d Heterogeneity of the CD4 T_SCM and Wnt/β-catenin signaling pathway. Identification of CD4 T_SCM clusters in young and old donors by scRNAseq. Single cells corresponding to CD4 T_SCM in all naive CD4 T cells subsets from young (n = 946) or old donors (n = 993) were analyzed by t-SNE, and clusters were automatically identified. The expression of genes coding for the Wnt/β-catenin pathway was quantified and normalized for each cluster in young and old donors. e Canonical and noncanonical Wnt signaling signatures in CD4 T_SCM clusters from young donors. The enrichment of gene expression detected in CD4 T_SCM clusters for each pathway was calculated in comparison with T_RTE signature. Enrichment plot of the gene set reported by GSEA as most enriched among all canonical and noncanonical signaling pathway gene sets (GO:0016055). The profile shows running enrichment score (green curve) and positions of gene set members (black vertical bars) on the rank ordered list of differential gene expression. f Inflammatory signature in CD4 T_SCM clusters from young and old donors. Enrichment plot of the gene set reported by GSEA as most enriched among all inflammatory gene sets (GO:0006954). The profile was displayed as in (e).