Neonatal single-positive CD8⁺T cells from the thymus express different genes and proliferate faster than adults. (A-F) RNAseq was performed on neonate and adult gBT-I CD8⁺ thymocytes and splenocytes. (A) Principal component analysis. Mean FPKM values from well-expressed genes were used from adult and neonatal naïve splenic and thymic CD8⁺ T cells. The naïve splenic adult sample consists of 3 pooled biological replicates; the remaining samples consist of 2 pooled biological replicates. The percentage of the overall variation accounted for by principal components 1 (x-axis) and 2 (y-axis) is indicated for each axis. Gene loadings are shown for principal components 1 and 2 in supplemental Figure 4. (B) Color-coded pairwise Spearman rank correlation coefficients comparing FPKM values for genes that are significantly differentially expressed between adults and neonates in at least one sample; P < 10¹⁵ for all comparisons. (C) Gene expression values for adults and neonates in splenic cells. Gray indicates lowly expressed genes, black indicates nondifferentially expressed genes, orange indicates the 118 genes upregulated in neonatal cells, and blue indicates the 153 genes upregulated in adult cells. (D) Gene expression values for adults and neonates in thymic cells, where 264 genes are upregulated in neonatal cells and 199 genes are upregulated in adult cells. Clustering of genes in naïve splenic and thymic CD8⁺ T-cell transcriptomes. Fold-change differences for significantly differentially expressed genes were calculated between adults and neonates. (E) Clustering was performed to identify genes with similar differences in expression in each sample; fold-change for each gene is plotted in each sample, and genes are shown in their clusters. (F) Genes in each cluster were compared with genes that define naïve, effector, or memory cells. Enrichment was calculated as number of genes in each cluster compared with the number expected. See supplemental Table 1 for gene expression values and clustering.