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Repetitive antigen stimulation induces stepwise transcriptome diversification but preserves a core signature of memory CD8(+) T cell differentiation - PubMed

  • ️Fri Jan 01 2010

Repetitive antigen stimulation induces stepwise transcriptome diversification but preserves a core signature of memory CD8(+) T cell differentiation

Thomas C Wirth et al. Immunity. 2010.

Abstract

Repetitive antigen stimulation by prime-boost vaccination or pathogen reencounter increases memory CD8(+) T cell numbers, but the impact on memory CD8(+) T cell differentiation is unknown. Here we showed that repetitive antigen stimulations induced accumulation of memory CD8(+) T cells with uniform effector memory characteristics. However, genome-wide microarray analyses revealed that each additional antigen challenge resulted in the differential regulation of several hundred new genes in the ensuing memory CD8(+) T cell populations and, therefore, in stepwise diversification of CD8(+) T cell transcriptomes. Thus, primary and repeatedly stimulated (secondary, tertiary, and quaternary) memory CD8(+) T cells differed substantially in their molecular signature while sharing expression of a small group of genes and biological pathways, which may constitute a core signature of memory differentiation. These results reveal the complex regulation of memory CD8(+) T cell differentiation and identify potential new molecular targets to dissect the function of memory cells generated by repeated antigen stimulation.

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Figures

Figure 1
Figure 1. Repeated antigen stimulation impacts memory CD8+ T cell kinetics and homeostatic proliferation

Naïve (5×102) or memory (6×104) Thy1.1 OT-I T cells were adoptively transferred into naïve Thy1.2 C57Bl/6 recipient mice prior to infection with 5×106 att LM-OVA. A) OT-I T cell responses (% of OT-I T cells of all PBL) in peripheral blood. Data are mean +/− SD for 3–10 mice per group per time point. B) Percentages of OT-I T cells in PBL were normalized to the expansion peak at day 7 for each individual group. Data are mean +/− SD for 3–10 mice per group per time point. C) Proliferation of memory cells was determined by BrdU incorporation (day 70–84). Bar graphs represent the percentage of BrdU+ OT-I T cells in spleen (mean +/− SD, n=5).

Figure 2
Figure 2. Repeated antigen stimulation impacts memory CD8+ T cell phenotype, function and tissue distribution

A) Phenotype of OT-I T cells in the spleen 90 days post infection. Numbers inside dot plots represent the percentage of cells positive for the indicated molecules. B) Peptide-stimulated intracellular cytokine staining (ICS) of OT-I T cells from spleen. Numbers represent the percentage of OT-I T cells that stain positive for a given cytokine combination. C) OT-I T cell frequency in total CD8+ T cells in tissues was normalized to the frequency in PBL. Data are mean +/− SD for 3 mice per group.

Figure 3
Figure 3. Dynamic and stable gene expression in repetitively stimulated memory CD8+ T cells

Genome-wide mRNA expression in memory OT-I T cells was analyzed via microarray and normalized to naive OT-I T cells. An FDR q-value <0.01 defined significant changes. Genes with significant mRNA changes in all memory OT-I T cell populations (1° to 4°) were grouped according to gene expression patterns: A) transcriptional upregulation with consistent increase (pattern I), B) transcriptional downregulation with consistent decrease (pattern II), C) transcriptional upregulation with no consistent increase (pattern III), or D) transcriptional downregulation with no consistent decrease (pattern IV) from 1° to 4° memory. E) Biological pathway analysis of genes with significant mRNA changes in all memory CD8+ T cell populations was generated using KEGG pathway tool in DAVID bioinformatics resources. Arrows indicate whether genes are upregulated (↑) or downregulated (↓) in all memory CD8+ T cell populations when compared to naïve cells.

Figure 4
Figure 4. New genes with significant transcriptional regulation appear in memory populations after each antigen encounter

Genes with the following expression patterns were identified: A) transcriptional upregulation or B) downregulation in 2°, 3° and 4° memory, C) transcriptional upregulation or D) downregulation in 3° and 4° memory, E) transcriptional upregulation or F) downregulation in 4° memory, G) transcriptional upregulation in 1° memory followed by decreasing mRNA expression from 1° to 4° memory. H) Protein expression of CD11b and CD11c on memory CD8+ T cells 90 days after infection (see Figure 3A+G for mRNA expression). Representative histograms are shown.

Figure 5
Figure 5. Repetitive antigen stimulation impacts global gene transcription in memory CD8+ T cell populations

A) Genome-wide gene transcription in 1°, 2°, 3°, and 4° memory OT-I T cell (day 85–95 p.i.) populations was analyzed via microarray and compared to naïve OT-I T cells. An FDR q-value <= 0.01 defined statistically significance. Data represent the number of genes with significant regulation inside each group. B) Venn diagram presentation of genes with significant mRNA changes in indicated memory CD8+ T cell populations. Overlapping circles represent number of genes whose expression is different from naïve cells but is either shared or unique to indicated memory CD8+ T cell groups. C) Significantly altered genes were assessed for all four memory groups individually. Each memory group is represented by an individual ellipse, overlapping ellipses indicate genes shared by two or more groups.

Figure 6
Figure 6. Similarities and differences between primary, quaternary memory and exhausted CD8+ T cell populations

A) Genes with significant mRNA changes between 1° and 4° memory CD8 T cells (Table S4) were evaluated using KEGG pathway analysis in DAVID. Arrows indicate whether genes are upregulated (↑) or downregulated (↓) in 4° compared to 1° memory CD8 T cells. B) Fold changes in mRNA of ribosomal genes in 4° compared to 1° memory CD8+ T cells. C) GSEA was performed to compare whether exhaustion-associated genes (down (left panel) or up (right panel)) showed specific enrichment in 4° memory CD8+ T cells. Rectangles with solid lines indicate down- or up-regulated genes common to exhausted and 4° memory CD8+ T cells. Rectangles with dotted lines indicate genes showing opposite expression patterns between exhausted and 4° memory CD8+ T cells. Genes are shown in Table S5. D) Fold changes in mRNA quantity for selected inhibitory receptors (implicated in exhausted CD8+ T cells (Shin et al., 2009)) in 4° compared to 1° memory CD8+ T cells.

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