Aging increases cell-to-cell transcriptional variability upon immune stimulation

Abstract

Aging is characterized by progressive loss of physiological and cellular functions, but the molecular basis of this decline remains unclear. We explored how aging affects transcriptional dynamics using single-cell RNA sequencing of unstimulated and stimulated naïve and effector memory CD4⁺ T cells from young and old mice from two divergent species. In young animals, immunological activation drives a conserved transcriptomic switch, resulting in tightly controlled gene expression characterized by a strong up-regulation of a core activation program, coupled with a decrease in cell-to-cell variability. Aging perturbed the activation of this core program and increased expression heterogeneity across populations of cells in both species. These discoveries suggest that increased cell-to-cell transcriptional variability will be a hallmark feature of aging across most, if not all, mammalian tissues.

Fig. 1. Divergence in gene expression levels and cell-to-cell variability among naive CD4⁺ T cells isolated from two species of mice.

(A) Single cells were isolated from spleens of young (~3 month) and old (~21 month) individuals of two mouse sub-species, and sequenced before or after activation (13) ; (B) Species-specific tSNE clustering of naive CD4⁺ T cells from young animals; (C) Representative heatmap of 30 genes and 30 cells randomly selected from young animals from both species shows species-specific variation; (D) Violin plots show distribution of expression levels of selected, species-specifically transcribed genes (grey background).

Fig. 2. Activation of naive CD4⁺ T cells induces a transcriptomic switch.

(A) Activation of CD4⁺ T cells from young B6 mice induces large-scale transcriptional changes (see S4 for CAST CD4⁺ T cells); (B) Genes up-regulated (red) and down-regulated (blue) by immune stimulation in young B6 mice (13); (C) Genes with no overall gene expression differences (black inset, (B)) during activation show decreased cell-to-cell variability in transcription (Mann-Whitney-Wilcoxon test, ***: p< 10 ^-10); (D) Up-regulated genes were expressed in a large fraction of activated CD4⁺ T cells after stimulation (median 36%); down-regulated genes were expressed in a smaller fraction of naive CD4⁺ T cells (median 18%); (E-G) Example genes showing transcriptional changes in unstimulated and activated CD4⁺ T cells: (E) Il2ra (Cd25); (F) Eif1; (G) Sell (Cd62l).

Fig. 3. Interspecies comparison of CD4⁺ T cell activation.

(A) CD4⁺ T cells isolated from B6 (black) and CAST (gold) show similar large-scale transcriptional changes upon immune stimulation; (B) Immune activation of CD4⁺ T cells triggers up-regulation of conserved and species-specific transcriptional programs. For visualization purposes, genes in shared and species-specific categories were proportionately and randomly selected (Material and Methods). Thirty cells were randomly selected for each condition/species (13); (C) Only genes up-regulated in both B6 and CAST highly enrich for known T cell functionality (Bonferroni multiple testing corrected p-values, red line is 0.1); (D) Fractions of cells in which a gene is detected are displayed as histograms; 70 genes were randomly selected from each gene set.

Fig. 4. Aging increases the cell-to-cell variability during early activation of CD4⁺ T cells.

(A) Upon activation, changes in expression of the core immune activation program are greater in younger mice than in older mice; (B) Among the genes from (A) that do not change their averaged gene expression, transcriptional variability increases during aging (13); (C) The fraction of cells in which genes of the shared activation process are expressed is reduced in CD4⁺ T cells from old animals. The distribution of fraction values is plotted on each corresponding axis (medians of fraction values are indicated in red); changes in fraction values were tested using a binomial test (significant changes are shaded in red or blue where multiple testing corrected p-values <0.1).