Comparative kinetic analyses of gene profiles of naïve CD4+ and CD8+ T cells from young and old animals reveal novel age-related alterations

Abstract

It is well established that immune responses are diminished in the old. However, we still do not have a clear understanding of what dictates the dysfunction of old T cells at the molecular level. Although microarray analysis has been used to compare young and old T cells, identifying hundreds of genes that are differentially expressed among these populations, it has been difficult to utilize this information to pinpoint which biological pathways truly affect the function of aged T cells. To better define differences between young and old naïve CD4+ and CD8+ T cells, microarray analysis was performed pre- and post-TCR stimulation for 4, 12, 24 and 72 h. Our data indicate that many genes are differentially expressed in the old compared to the young at all five time points. These genes encode proteins involved in multiple cellular functions such as cell growth, cell cycle, cell death, inflammatory response, cell trafficking, etc. Additionally, the information from this microarray analysis allowed us to underline both intrinsic deficiencies and defects in signaling only seen after activation, such as pathways involving T-cell signaling, cytokine production, and Th2 differentiation in old T cells. With the knowledge gained, we can proceed to design strategies to restore the function of old T cells. Therefore, this microarray analysis approach is a powerful and sensitive tool that reveals the extensive changes seen between young and old CD4+ and CD8+ naïve T cells. Evaluation of these differences provides in-depth insight into potential functional and phenotypical differences among these populations.

Fig. 1

Gene selection algorithm of a microarray-based kinetic analysis of naïve CD4⁺ and CD8⁺ young and old T cells. (A) Schematic representation of experimental design. Venn diagrams overlaying genes that are differentially regulated between old and young naïve T cells. (B) Pre- and post-stimulation in CD8⁺ naïve T cells. (C) Post-stimulation only in CD8⁺ naïve T cells. (D) Pre- and post-stimulation in CD4⁺ naïve T cells. (E) Post-stimulation only in CD4⁺ naïve T cells.

Fig. 2

Cluster analysis of differentially regulated genes between young and old CD4⁺ and CD8⁺ T cells. Gene lists created in Fig 1 containing genes that are differentially regulated at time point 0 and at least 2 of the 4 other time points. (A) Four prominent and biologically relevant expression pattern profile plots with ranges of expression values within each pattern. (B) Gene lists for the patterns in A.

Fig. 3

Heat maps of expression levels of genes contained in the immune processes gene ontology. Differentially regulated genes by 2-fold or more for each time point. Shown in hierarchical clustered heat maps for CD8⁺ (A) and CD4⁺ (B) T cells. Color bar depicting the range of differential gene expression, in log 2. Asterisks denote mentioned in the text.

Fig. 4

Expression profile plots of differentially regulated genes. The gene expression profile of selected gene families pre- and post-stimulation was analyzed in old and young CD8⁺ and CD4⁺ T cells. Axis labels are the same as in Fig. 2.

Fig. 5

Kinetic changes in gene expression of selected components of the Wnt/β-catenin signaling pathway in aging CD8⁺ T cells. Wnt/β-catenin pathway was generated by Ingenuity Pathway analysis. Adjacent to each step of the pathway are the gene profile plots of old and young CD8⁺ T cells. Axis labels are the same as in Fig. 2. Red indicates up-regulated genes, blue down-regulated genes.

Fig. 6

Kinetic changes in gene expression of selected components of Th2 responses. Pathways were generated by Ingenuity Pathway analysis. (A) Pathway analysis of Th2 differentiation. (B) IL-10 Signaling and IL-4Rα expression, and (C) IL-4 signaling and production. Adjacent to each step of the pathway are the gene profile plots of old and young CD4⁺ T cells. Axis labels the same as in Fig. 2.

Fig. 7

Validation of gene expression. (A) expression of IL-4, IL-10, IL-10R, GATA-3, and GATA-1 for CD4 T cells pre- and post-stimulation was evaluated by QRT-PCR. Data indicate the relative gene expression from old CD4⁺ T cells in relation to gene expression from young T cells. (B) Secretion of IL-4, IL-6, IL-10, and INF-γ was evaluated in young and old CD4⁺ T cells post-stimulation by ELISA. Each value represents the mean of triplicate wells ±SE. (C) Analysis of protein profiler for the detection of cytokines and chemokines productions. Supernatants of 72-h stimulated young and old CD4 and CD8 T cells were used for this analysis. Each value represents the mean of duplicate wells. Values indicate relative integrated intensity.