IL-12 Signaling Contributes to the Reprogramming of Neonatal CD8+ T Cells

Abstract

Neonates are highly susceptible to intracellular pathogens, leading to high morbidity and mortality rates. CD8⁺ T lymphocytes are responsible for the elimination of infected cells. Understanding the response of these cells to normal and high stimulatory conditions is important to propose better treatments and vaccine formulations for neonates. We have previously shown that human neonatal CD8⁺ T cells overexpress innate inflammatory genes and have a low expression of cytotoxic and cell signaling genes. To investigate the activation potential of these cells, we evaluated the transcriptome of human neonatal and adult naïve CD8⁺ T cells after TCR/CD28 signals ± IL-12. We found that in neonatal cells, IL-12 signals contribute to the adult-like expression of genes associated with cell-signaling, T-cell cytokines, metabolism, and cell division. Additionally, IL-12 signals contributed to the downregulation of the neutrophil signature transcription factor CEBPE and other immaturity related genes. To validate the transcriptome results, we evaluated the expression of a series of genes by RT-qPCR and the promoter methylation status on independent samples. We found that in agreement with the transcriptome, IL-12 signals contributed to the chromatin closure of neutrophil-like genes and the opening of cytotoxicity genes, suggesting that IL-12 signals contribute to the epigenetic reprogramming of neonatal lymphocytes. Furthermore, high expression of some inflammatory genes was observed in naïve and stimulated neonatal cells, in agreement with the high inflammatory profile of neonates to infections. Altogether our results point to an important contribution of IL-12 signals to the reprogramming of the neonatal CD8⁺ T cells.

Keywords: CD8+ T cells; IL-12; RNA-sequencing; T cell activation; neonatal T cells; neonatal immunity.

Figure 1

TCR or TCR/IL-12 induced response in neonatal and adult CD8⁺ T cells. (A) Number of differentially expressed Genes (adjusted p < 0.05) between non-stimulated and TCR or TCR/IL-12 neonatal and adult CD8⁺ T cells. US, unstimulated cells. (B) Top 20 significantly enriched REACTOME pathways (adjusted p < 0.05) in neonatal and adult naïve CD8⁺ T cells stimulated through the TCR or TCR/IL-12 signals.

Figure 2

Basal levels and response patterns of differentially expressed genes between neonatal and adult CD8⁺ T cells. (A) Venn diagram of differentially expressed genes between naïve neonatal and adult naïve CD8⁺ T (adjusted p < 0.05 and Log₂ fold change ≥ 2). (B) k-means clustering of the pattern expression of genes, based in their base levels and response to TCR or TCR/IL-12 treatment in neonatal and adult CD8⁺ T cells. The percentage of genes belonging to each cluster is shown to the left. (C) Genome browser screenshots of RNA-seq data, showing four genes (TBX21, SLAMF7, JAKMIP1, and FASLG) from Cluster 4 that reached the adult's expression level in neonates stimulated cells.

Figure 3

Comparison between TCR and TCR/IL-12 response genes of neonatal and adult naïve CD8⁺ T cells. (A,D) Venn diagrams (B,E) heatmaps of induced genes after TCR or TCR/IL-12 treatment of neonatal (left) and adult (right) CD8⁺ T cells (adjusted p < 0.05). (C,F) Enriched KEGG pathways returned by the DAVID software for the differentially expressed genes in neonatal (left) and adult (right) CD8⁺ T cells.

Figure 4

Genes induced by TCR signals in adult naïve CD8⁺ T cells, but only induced by TCR/IL-12 in the neonatal cells. (A) Venn Diagram showing the genes induced by TCR in adult cells that are only induced by TRC/IL12 in the neonatal cells, and (B) heatmaps of genes induced by TCR in adults CD8⁺ T cells, that only were induced by TCR/IL-12 in neonatal CD8⁺ T cells (adjusted p < 0.05 and log₂ fold change ≥ 2), bars on the right display manual annotations of the functional categories.

Figure 5

Genes significantly downregulated by TCR/IL-12 signals in neonatal and adult naïve CD8⁺ T cells. (A,C) Venn Diagrams showing the genes overexpressed genes in the neonatal (A) and adult (C) cells but down-regulated by TCR/IL-12. (B,D) heatmaps of genes significantly downregulated by TCR/IL-12 in neonatal (B) and adult (D) CD8⁺ T cells (adjusted p < 0.05 and log₂ fold change ≥ 2), bars on the right display manual annotations of functional categories. (E) RT-PCR evaluations of selected significantly down-regulated genes in independent samples (n = 5) of neonatal cells, after TCR or TCR/IL-12 treatment, normalized to β2-microglobulin. Data presented are means ± standard deviations. Statistical significance was assessed by a Student's t-test (unpaired; *p < 0.05).

Figure 6

Genes Overexpressed in neonatal CD8⁺ T cells and showing an exacerbated response to TCR/IL-12 signals. (A) Venn Diagrams showing the genes analyzed and (B) heatmaps with manual annotation of the genes with an exacerbated response to TCR and TCR/IL-12 signals in the neonatal CD8⁺ T cells (adjusted p < 0.05 and log₂ fold change ≥ 2).

Figure 7

Changes in the promoter methylation induced by TCR or TCR/IL-12 signals. Histograms displaying the changes in levels of DNA methylation in the promoters of selected genes related with innate immunity vs. those of selected genes related to CD8⁺ T cell functions. Methylation was evaluated by MeDIP/qPCR of anti-Methylcytosine chromatin immuno-precipitates against non-treated DNA from the same sample (Input). Data presented are means ± standard deviation, with n = 6 for all assays. Statistical significance was assessed by a Student's t-test (unpaired; *p < 0.05).

Figure 8

Activation of neonatal CD8⁺ T cells in the presence of IL-12 enables their transcriptional maturation.