Histone acetylation is associated with differential gene expression in the rapid and robust memory CD8(+) T-cell response

Abstract

To understand the molecular basis for the rapid and robust memory T-cell responses, we examined gene expression and chromatin modification by histone H3 lysine 9 (H3K9) acetylation in resting and activated human naive and memory CD8(+) T cells. We found that, although overall gene expression patterns were similar, a number of genes are differentially expressed in either memory or naive cells in their resting and activated states. To further elucidate the basis for differential gene expression, we assessed the role of histone H3K9 acetylation in differential gene expression. Strikingly, higher H3K9 acetylation levels were detected in resting memory cells, prior to their activation, for those genes that were differentially expressed following activation, indicating that hyperacetylation of histone H3K9 may play a role in selective and rapid gene expression of memory CD8(+) T cells. Consistent with this model, we showed that inducing high levels of H3K9 acetylation resulted in an increased expression in naive cells of those genes that are normally expressed differentially in memory cells. Together, these findings suggest that differential gene expression mediated at least in part by histone H3K9 hyperacetylation may be responsible for the rapid and robust memory CD8(+) T-cell response.

Figure 1.

Differentially expressed genes in memory and naive CD8⁺ T cells. (A) Memory cell differentially expressed genes through resting and activated states. Twelve selected genes are presented. (B) Memory cell differentially expressed genes only in the resting states. Eight selected genes are presented. The microarray results derived from 3 independent experiments and normalized to the resting naive cells in panels A and B are presented as the mean ± SEM in log₁₀ scale. (C) Naive cell differentially expressed genes in resting and activated states. Eight selected genes are presented. The microarray results derived from 3 independent experiments and normalized to the resting memory cells in panel C are presented as the mean ± SEM in log₁₀ scale. The detailed information of these genes can be found in Table S9.

Figure 2.

Enhanced production of cytokines in activated memory CD8⁺ T cells. (A) Higher expression levels of 16 cytokine genes in activated memory than naive CD8⁺ T cells. The mean relative expression levels of 16 cytokines are presented. The detailed information of these genes can be found in Table S8. CCL1 indicates chemokine (C-C motif) ligand 1; CCL3L1, chemokine (C-C motif) ligand 3-like 1; CCL4, chemokine (C-C motif) ligand 4 (MIP-1β); CCL17, chemokine (C-C motif) ligand 17; CCL20, chemokine (C-C motif) ligand 20 (MIP-3α); CCL23, chemokine (C-C motif) ligand 23; CSF2, colony-stimulating factor 2 (GM-CSF); IFNG, interferon gamma; IL2, interleukin 2; IL4, interleukin 4; IL5, interleukin 5; IL13, interleukin 13; IL23A, interleukin 23A; IGF2, insulin-like growth factor 2 (somatomedin A); LIGHT, tumor necrosis factor (ligand) superfamily, member 14 (TNFSF14); TNF, tumor necrosis factor (TNF superfamily, member 2). (B) Higher concentrations of cytokine proteins in the supernatant of activated memory than that of activated naive CD8⁺ T cells. Cytokines were measured from the culture supernatant of activated naive and memory CD8⁺ T cells by Bio-plex methods. One representative of 2 independent experiments is shown.

Figure 3.

Fast acquisition of cytotoxicity in activated memory CD8⁺ T cells. (A) Higher levels of expression of 8 effector function-related genes in activated memory than in activated naive CD8⁺ T cells. The mean relative expression levels of 8 cytokines are presented. CARD8 indicates caspase recruitment domain family, member 8; CFLAR, CASP8 and FADD-like apoptosis regulator; CTSC, cathepsin C; CTSH, cathepsin H; GZMA, granzyme A; GZMK, granzyme K; and KLRB1, killer cell lectin-like receptor subfamily B, member 1. The detailed information of these genes can be found in Table S8. (B) Induced cytotoxicity in naive and memory CD8⁺ T cells after anti-CD3/CD28 stimulation. The cytotoxic activity was measured by a redirected cytotoxicity assay at the indicated time after anti-CD3/CD28 stimulation. (C) Cytotoxic activity was measured as in panel B and calculated as lytic units, with data from 3 independent experiments. The mean and standard error of the mean are shown.

Figure 4.

Association of differential gene expression and higher levels of H3K9 acetylation in memory CD8⁺ T cells. (A-B) Resting memory cell differentially expressed genes are associated with high levels of H3K9 acetylation. (A) The representative gel images of one gene with high H3K9 acetylation level (KLRB1) and one control gene (SMARCC1) are shown. N indicates naive; M, memory. (B) Three memory cell differential expressed and 3 similarly expressed genes as shown by microarray (left, n = 3), the real-time RT-PCR (middle, n = 5), and H3K9 acetylation levels (right, n = 8) between naive and memory CD8⁺ T cells. The significantly increased or similarly expressed genes were selected from the microarray data. The levels of H3K9 acetylation are significantly different between the differentially and similarly expressed genes (P < .05). Data are presented as mean ratio ± SEM (log₁₀) for array and RT-PCR, and data are presented as the mean fraction of input ± SEM for H3K9 acetylation levels of naive and memory cells. (C-D) Activated memory cell differentially expressed genes are associated with high levels of H3K9 acetylation in resting memory CD8⁺ T cells. (C) The representative gel images of 4 genes with high H3K9 acetylation levels (ICOS, IFNG, IL2RA, and KLRB1) and 2 control genes (IL2RG and MYST2) are presented. (D) Nine activated memory cell highly expressed and 9 similarly expressed genes as shown by microarray (left, n = 3), the real-time RT-PCR (middle, n = 5), and H3K9 acetylation levels (right, n = 8) between naive and memory CD8⁺ T cells after 72 hours of stimulation. The significantly increased or similarly expressed genes were selected from the array data. The levels of H3K9 acetylation are significantly different between the differentially and similarly expressed genes (P < .01) in activated memory CD8⁺ T cells. Data are presented as the same as described in panel B.

Figure 5.

Histone H3K9 acetylation prior to rapid and robust gene expression. (A) High levels of H3K9 acetylation precede activation-induced gene expression changes in memory CD8⁺ T cells. The relative gene expression levels of 5 activated memory cell highly expressed genes in resting (left) and activated (middle) memory cells versus naive cells, and the levels of H3K9 acetylation of resting memory cells versus naive cells (right). Gene expression levels are presented as the mean ± SEM in log₁₀ scale (n = 5 for RT-PCR), and the dashed line represents a 2-fold difference. The H3K9 acetylation levels are presented as the mean fraction of input ± SEM of naive and memory cells (n = 8-12, P < .01). (B) Increased levels of H3K9 acetylation and mRNA after TSA treatment in naive CD8⁺ T cells. The relative changes of H3K9 acetylation levels were determined by ChIP and real-time PCR between TSA-treated and untreated naive CD8⁺ T cells (left) and the relative levels of mRNA of the same 4 genes (right) between TSA-treated and untreated naive CD8⁺ T cells after anti-CD3/CD28 stimulation for 24 hours. H3K9 acetylation levels are presented as the mean fraction of input of TSA-treated and untreated naive CD8⁺ T cells (mean ± SEM, n = 4-6, P < .01). mRNA levels are presented as the ratio (mean ± SEM, n = 4-6) of TSA-treated and untreated naive CD8⁺ T cells in log₁₀ scale.