Memory CD8 T cells, unlike their naive precursors, are capable of rapidly producing high levels of cytokines, killing target cells, and proliferating into numerous secondary effectors immediately upon Ag encounter. This ready-to-respond state contributes to their superior ability to confer protective immunity, yet the underlying molecular basis remains unknown. In this study, we show that memory CD8 T cells have increased histone acetylation compared with naive CD8 T cells; however, those activated without CD4 T cell help (“unhelped”) remain hypoacetylated and fail to develop into functional, protective memory. Treatment with a histone deacetylase inhibitor during activation results in increased histone acetylation in unhelped CD8 T cells and restores their ability to differentiate into functional memory cells capable of immediate cytokine production and providing protective immunity. These results demonstrate that CD4 T help-dependent chromatin remodeling provides a molecular basis for the enhanced responsiveness of memory CD8 T cells.

The CD8 T cell plays a critical role in immune responses against intracellular pathogens. Following an acute infection, Ag-specific naive CD8 T cells expand and differentiate into a large population of effector CD8 T cells that contribute to the control of infection (1). After clearance of the pathogen, the majority of effector CD8 T cells die (2); however, a small percentage survive and further differentiate into mature memory CD8 T cells (CD8 TM)3 (3, 4, 5). Unlike naive CD8 T cells, which require days to differentiate into cytokine-producing effectors, CD8 TM are immediately able to produce high levels of effector cytokines and kill target cells upon Ag recognition, even before rapid proliferation into large numbers of secondary effectors (6, 7, 8, 9). The molecular basis that confers this poised, ready-to-respond state to CD8 TM remains largely unknown.

Under certain conditions, such as the absence of adequate CD4 T cell help (CD4 TH), a poor immune response is induced that often leads to persistent viral infection and/or generation of CD8 TM that are defective and unable to provide protective immunity (10, 11, 12, 13). Although CD4 T cells are needed during priming for programming the differentiation of CD8 TM (14), they have also been shown to play a role in the maintenance of CD8 TM (15). The precise nature of CD4 TH during the priming and maintenance of CD8 TM is still unclear. Furthermore, the molecular basis that underlies the defective phenotype of “unhelped” CD8 TM remains to be elucidated. Our recent results show that defects in unhelped CD8 TM are not due to aberrations in the TCR repertoire nor to impairment in functional avidity maturation (16). Instead, our results suggest a correlation between diminished histone acetylation and poor functionality in CD8 TM generated in the absence of CD4 TH (16). In this report, we demonstrate a direct role for CD4 TH-dependent histone modifications in contributing to the enhanced functionality of memory CD8 T cells.

C57BL/6J (B6) and B6.129S2-Cd4tm1Mak/J (B6-CD4−/−) mice were purchased from The Jackson Laboratory. B6-P14 Thy1.1 mice were obtained from in-house breeding colonies crossing B6;D2-Tg(TcrLCMV)327Sdz/JDvsJ with B6.PL-Thy1a/CyJ. All experiments were performed with female age-matched (6–10 wk) mice in accordance with Institutional Animal Care and Use Committee-approved protocols at the University of Pennsylvania School of Medicine Animal Facility (Philadelphia, PA).

P14 CD8 T cells (≈20% of total) in coculture with B6 or B6-CD4−/− splenocytes were activated with soluble anti-CD3ε (1.0 μg/ml) and anti-CD28 (0.5 μg/ml) Abs for 3 days. For TSA treatment, a final concentration of 5 ng/ml (17 nM) TSA was added to the above in vitro cocultures on day 2. This concentration was based on pilot experiments in which we determined the optimal concentration and duration of TSA treatment that resulted in increased histone acetylation with minimal toxicity.

Chromatin immunoprecipitation (ChIP) with an anti-acetyl histone-H3 Ab was performed as described previously, and the quantity of DNA was determined by quantitative PCR (16). Values for specific enrichment of immunoprecipitated DNA from each sample are normalized to the mean value for the standards (naive P14 CD8 T cell samples taken from stock −80°C frozen aliquots) set at 1 to facilitate comparison of ChIP results from different experiments.

In vitro activated P14 cells were transferred into B6 or B6-CD4−/− recipient mice (1–2 × 106 per mouse). At different time points, splenocytes were harvested for FACS analysis and intracellular cytokine staining. To assess protective immunity, recipient mice were infected i.p. with 1.5 × 107 CFU of a recombinant strain of Listeria monocytogenes expressing glycoprotein (GP)-derived peptide GP33–41 of the lymphocyte choriomeningitis virus for which P14 CD8 T cells are specific. Bacterial loads were determined on day 3 postinfection.

To further define the role of CD4 T cells and the molecular basis of the defective phenotypes in unhelped CD8 TM, we used an experimental system of in vitro activation followed by adoptive transfer into naive hosts (4, 17). A population of homogenous, monoclonal CD8 T cells from P14 TCR transgenic mice were purified and stimulated with anti-CD3 and anti-CD28 Abs in the presence of splenocytes from either B6 or B6-CD4−/− mice. Three days after in vitro activation, P14 CD8 T cells were adoptively transferred into congenic naive hosts to examine whether the presence of CD4 T cells during activation influences the development of CD8 TM.

Before transfer, levels of histone H3 acetylation (AcH3) at the Ifng promoter and enhancer were analyzed by ChIP. P14 cells from coculture with B6 splenocytes (“helped”) were observed to have a significant increase in AcH3 at the Ifng locus relative to unstimulated naive CD8 T cells (Fig. 1, A and B). In contrast, P14 cells cocultured with splenocytes from B6-CD4−/− mice (“unhelped”) failed to substantially increase AcH3 at the Ifng enhancer (Fig. 1, A and B). Similar results were observed when splenocytes from B6 mice depleted of CD4 T cells by MACS beads (<1% CD4 T cells; Miltenyi Biotec) were used in the cocultures (data not shown). Irrespective of the presence of CD4 T cells, activated P14 cells restimulated with PMA and ionomycin (P+I) produced equal amounts of IFN-γ (Fig. 1,C) and exhibited similar activation phenotypes (Fig. 1 D). Thus, the difference in AcH3 levels is not merely due to suboptimal activation of P14 cells in the absence of CD4 TH.

FIGURE 1.

Diminished histone H3 acetylation at the Ifng enhancer of CD8 T cells activated without CD4 T cells. A, Specific enrichment of DNA from the Ifng enhancer of P14 cells from helped (⊕) or unhelped (⊖) cocultures. Individual replicates from B6 (♦) and B6-CD4−/− () cocultures are normalized to values from naive P14 cells (X), and a horizontal bar indicates the mean for each group. The dashed line shows the average background signal from control ChIP assays with polyclonal rabbit IgG (0.006). B, A 250-bp region of the Ifng enhancer was amplified by conventional PCR from total cell extract (left band) or ChIP fraction (right band) from control Ab (∅), naive (N), helped (H), and unhelped (U) CD8 T cells. C, IFN-γ production by restimulated P14 cells. Dot plots are gated on CD8 T cells and the percentage of IFN-γ+ is shown in the upper right corner (mean ± SD). D, Expression of activation markers CD69 and CD44 (mean fluorescence intensity of each at the respective axes). ChIP data are from three experiments with 6–9 repeat samples per group. Flow cytometric data are representative of five independent experiments. D, Day.

FIGURE 1.

Diminished histone H3 acetylation at the Ifng enhancer of CD8 T cells activated without CD4 T cells. A, Specific enrichment of DNA from the Ifng enhancer of P14 cells from helped (⊕) or unhelped (⊖) cocultures. Individual replicates from B6 (♦) and B6-CD4−/− () cocultures are normalized to values from naive P14 cells (X), and a horizontal bar indicates the mean for each group. The dashed line shows the average background signal from control ChIP assays with polyclonal rabbit IgG (0.006). B, A 250-bp region of the Ifng enhancer was amplified by conventional PCR from total cell extract (left band) or ChIP fraction (right band) from control Ab (∅), naive (N), helped (H), and unhelped (U) CD8 T cells. C, IFN-γ production by restimulated P14 cells. Dot plots are gated on CD8 T cells and the percentage of IFN-γ+ is shown in the upper right corner (mean ± SD). D, Expression of activation markers CD69 and CD44 (mean fluorescence intensity of each at the respective axes). ChIP data are from three experiments with 6–9 repeat samples per group. Flow cytometric data are representative of five independent experiments. D, Day.

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Following transfer into B6 hosts, the in vitro activated, helped P14 cells contracted within 1 wk and survived at stable levels to at least 45 days posttransfer. However, only low numbers of unhelped P14 cells transferred into B6-CD4−/− mice could be detected 1 wk posttransfer (Fig. 2,A), and these cells expressed lower levels of the high affinity IL-7Rα (CD127) compared with the helped P14 cells (Fig. 2,B). Furthermore, significantly less IFN-γ was produced by fewer unhelped P14 cells following GP33–41 peptide restimulation or with P+I (Fig. 2, C and D). The failure of P+I to even partially restore IFN-γ production by the unhelped CD8 TM further indicates that the IFN-γ defect in these cells lies downstream of TCR signaling. Together, these results from a well-defined in vitro experimental system clearly show that CD4 TH influences epigenetic modification in CD8 T cells and the development of functional CD8 TM, consistent with our in vivo results observed in the context of an infection (16).

FIGURE 2.

CD8 T cells activated in coculture with B6-CD4−/− splenocytes develop into defective memory cells in vivo. Helped (⊕) or unhelped (⊖) in vitro activated P14 cells were transferred into congenic B6 (+/+) or B6-CD4−/− (−/−) host mice, respectively, and analyzed 14 days later. A, Numbers in the dot plots indicate P14 cells as a percentage of total CD8 T cells (mean ± SD; p = 0.001). Total P14 cells recovered per spleen are as follows: ⊕, 704 ± 221 × 103; and ⊖, 15 ± 8 × 103 (p = 0.0001). B, CD127 expression for helped (black line, mean fluorescence intensity = 26 ± 5) vs unhelped donor cells (gray tinting, 17 ± 1; p = 0.003). C, Percentage of IFN-γ+ donors (mean ± SD) following ex vivo restimulation with GP33–41 (gp33) (p = 0.002) and P+I (p = 0.0003). D, Mean fluorescence intensity (MFI) of IFN-γ+ donors, indicating the amount of IFN-γ produced per cell in response to restimulation with gp33 (p = 0.02) or P+I (p = 0.0007). Data are from 3–6 mice per group and are representative of four independent experiments.

FIGURE 2.

CD8 T cells activated in coculture with B6-CD4−/− splenocytes develop into defective memory cells in vivo. Helped (⊕) or unhelped (⊖) in vitro activated P14 cells were transferred into congenic B6 (+/+) or B6-CD4−/− (−/−) host mice, respectively, and analyzed 14 days later. A, Numbers in the dot plots indicate P14 cells as a percentage of total CD8 T cells (mean ± SD; p = 0.001). Total P14 cells recovered per spleen are as follows: ⊕, 704 ± 221 × 103; and ⊖, 15 ± 8 × 103 (p = 0.0001). B, CD127 expression for helped (black line, mean fluorescence intensity = 26 ± 5) vs unhelped donor cells (gray tinting, 17 ± 1; p = 0.003). C, Percentage of IFN-γ+ donors (mean ± SD) following ex vivo restimulation with GP33–41 (gp33) (p = 0.002) and P+I (p = 0.0003). D, Mean fluorescence intensity (MFI) of IFN-γ+ donors, indicating the amount of IFN-γ produced per cell in response to restimulation with gp33 (p = 0.02) or P+I (p = 0.0007). Data are from 3–6 mice per group and are representative of four independent experiments.

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Importantly, the in vitro system allowed us to use chemical inhibitors to alter AcH3 levels and ask whether the AcH3 level at the Ifng locus is simply correlative or plays a direct role in regulating IFN-γ production. TSA is a chemical inhibitor of histone deacetylase (HDAC) activity that increases the total cellular levels of hyperacetylated histones (18, 19, 20). P14 CD8 T cells were cultured with anti-CD3 and anti-CD28 and splenocytes from either B6 or B6-CD4−/− mice. On day 2 of the culture, 5 ng/ml TSA was added and the cells were cultured for an additional 24 h. P14 CD8 T cells were purified and analyzed by ChIP for AcH3 at the Ifng locus (Fig. 3 A). TSA treatment resulted in increases in AcH3 in the unhelped P14 cells to levels similar to those in the helped P14 cells.

FIGURE 3.

TSA treatment increases AcH3 of in vitro activated CD8 T cells and the survival and IFN-γ production of these cells rested in vivo. A, Specific enrichment for AcH3 at the Ifng enhancer in P14 cells from B6 (♦), B6-CD4−/− (), and TSA-treated B6-CD4−/− (△) cocultures compared with naive (X) controls. B, In vitro activated P14 cells were rested in congenic B6 hosts for 1 wk and IFN-γ production was measured by intracellular cytokine staining upon restimulation with GP33–41 peptide. Numbers in the dot plots indicate a percentage of P14 cells producing IFN-γ, and the bar graphs show mean fluorescence intensity of IFN-γ (e3, ×1,000). There are significant differences in percentage (p = 0.001) and mean fluorescence intensity (MFI) (∗∗, p = 0.0006) between TSA-treated unhelped and untreated, unhelped P14 cells. C, Survival of P14 cells in vivo. Numbers in dot plots indicate a percentage of total CD8 T cells that are P14 cells (⊖ vs ⊖+TSA; p = 0.00006), and bar graph show the total numbers of donor P14 cells recovered per spleen (#s per spl; e4, ×10,000) (⊖ vs ⊖+TSA; 7.0-fold (7.0×) increase; ∗∗, p = 0.0006). ChIP data are compiled from 4–9 repeat samples from two independent TSA experiments. Transfer data (mean ± SD) are from 4–5 individual host mice representative of four independent experiments.

FIGURE 3.

TSA treatment increases AcH3 of in vitro activated CD8 T cells and the survival and IFN-γ production of these cells rested in vivo. A, Specific enrichment for AcH3 at the Ifng enhancer in P14 cells from B6 (♦), B6-CD4−/− (), and TSA-treated B6-CD4−/− (△) cocultures compared with naive (X) controls. B, In vitro activated P14 cells were rested in congenic B6 hosts for 1 wk and IFN-γ production was measured by intracellular cytokine staining upon restimulation with GP33–41 peptide. Numbers in the dot plots indicate a percentage of P14 cells producing IFN-γ, and the bar graphs show mean fluorescence intensity of IFN-γ (e3, ×1,000). There are significant differences in percentage (p = 0.001) and mean fluorescence intensity (MFI) (∗∗, p = 0.0006) between TSA-treated unhelped and untreated, unhelped P14 cells. C, Survival of P14 cells in vivo. Numbers in dot plots indicate a percentage of total CD8 T cells that are P14 cells (⊖ vs ⊖+TSA; p = 0.00006), and bar graph show the total numbers of donor P14 cells recovered per spleen (#s per spl; e4, ×10,000) (⊖ vs ⊖+TSA; 7.0-fold (7.0×) increase; ∗∗, p = 0.0006). ChIP data are compiled from 4–9 repeat samples from two independent TSA experiments. Transfer data (mean ± SD) are from 4–5 individual host mice representative of four independent experiments.

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Because TSA treatment increased histone acetylation levels in CD8 T cells primed in the absence of CD4 TH, we next tested whether this impacted positively on the functionality of unhelped memory cells. P14 cells activated in vitro in the presence or absence of TSA were transferred into naive hosts. On day 7 posttransfer, splenocytes were harvested from hosts, restimulated for 4 h with GP33–41 peptide and monensin, and stained for intracellular IFN-γ. Compared with helped P14 cells, less IFN-γ was produced by fewer unhelped P14 cells following GP33–41-peptide restimulation (Fig. 3,B). However, when unhelped P14 cells were treated with TSA during priming, more P14 cells produced IFN-γ compared with untreated P14 cells, and the level of IFN-γ production was similar to those in helped P14 cells. Interestingly, TSA treatment had no significant effect on IFN-γ production by helped P14 cells (Fig. 3 B), indicating that helped CD8 T cells already achieve optimal histone acetylation during priming. The positive effect of TSA treatment in unhelped P14 cells was not limited to IFN-γ, as TNF-α production upon restimulation was also increased in P14 cells from the TSA-treated, unhelped group (data not shown). Thus, TSA treatment during priming had a positive impact on the ability of unhelped CD8 TM to produce effector cytokines.

Additionally, TSA treatment had a positive effect on the ability of unhelped P14 cells to survive in the adoptive host (Fig. 3,C). On day 7 after transfer, significantly fewer numbers of P14 cells were present in mice that received unhelped P14 than those that received helped P14 cells (Fig. 3,C). TSA treatment of unhelped P14 cells resulted in a significant increase in the number of P14 cells surviving in vivo, with frequencies and cell numbers similar to those of helped P14 cells at 7 (Fig. 3 C) and 30 days posttransfer (data not shown). Thus, TSA treatment of unhelped CD8 T cells had a positive effect on the subsequent numbers of cells recovered from host mice after 1 wk of resting. These results suggest that defective survival of unhelped CD8 T cells may also be due to impaired histone acetylation, possibly at loci involved in prosurvival, anti-apoptosis, and/or homeostatic proliferation.

Because TSA treatment rescued the ability of unhelped P14 cells to survive and form functional memory fully capable of producing immediate effector cytokines upon restimulation, we next tested whether TSA treatment could also restore the ability of unhelped CD8 T cells to provide protective immunity. Equal numbers of in vitro activated P14 CD8 T cells and naive P14 cells were transferred into B6 recipients. One week following transfer, mice were challenged with a high dose of recombinant L. monocytogenes expressing the GP33–41 epitope. Three days following challenge, bacterial loads in the spleen were assessed. The transfer of helped CD8 T cells afforded increased protection compared with the transfer of naive P14 cells (41-fold fewer CFU; Fig. 4). Unhelped CD8 T cells offered significantly less protection compared with mice that received helped CD8 T cells (36-fold difference in CFU; Fig. 4). Importantly, transfer of TSA-treated, unhelped CD8 T cells provided substantially greater protection than the transfer of untreated, unhelped CD8 T cells (62-fold decrease in CFU, p = 0.004; Fig. 4), and the level of protection was similar to that seen in mice receiving helped CD8 T cells (Fig. 4).

FIGURE 4.

TSA treatment of unhelped CD8 T cells during priming enhances the immune protection of subsequent CD8 TM to infectious challenge. In vitro activated P14 cells were transferred into congenic B6 hosts. One week posttransfer, mice were challenged with recombinant L. monocytogenes expressing the GP33–41 epitope, and bacterial numbers in the spleen were enumerated on day 3 after challenge. Bacterial load is shown on a log scale bar graph of CFU per gram of spleen. The lower limit of detection (LOD) is 230 CFU per gram of spleen. A group of mice that received naive P14 cells 1 day before challenge was included as a control. Mice receiving no P14 cells had ∼8 × 106 CFU per gram of spleen (data not shown). Protection data are from 6–9 individual mice and representative of four independent experiments.

FIGURE 4.

TSA treatment of unhelped CD8 T cells during priming enhances the immune protection of subsequent CD8 TM to infectious challenge. In vitro activated P14 cells were transferred into congenic B6 hosts. One week posttransfer, mice were challenged with recombinant L. monocytogenes expressing the GP33–41 epitope, and bacterial numbers in the spleen were enumerated on day 3 after challenge. Bacterial load is shown on a log scale bar graph of CFU per gram of spleen. The lower limit of detection (LOD) is 230 CFU per gram of spleen. A group of mice that received naive P14 cells 1 day before challenge was included as a control. Mice receiving no P14 cells had ∼8 × 106 CFU per gram of spleen (data not shown). Protection data are from 6–9 individual mice and representative of four independent experiments.

Close modal

Our previous in vivo studies have shown that the absence of CD4 TH results in defective CD8 TM that have diminished histone acetylation (16). Although these in vivo results established biological significance in the context of infection, a direct role for CD4 T cells was difficult to establish because of potential confounding factors, such as slower viral clearance in CD4 T cell-deficient mice. Results from this study, using a well-defined ex vivo system, clearly demonstrate that CD8 T cells primed in the absence of CD4 T cells fail to undergo chromatin remodeling at the Ifng locus and develop into CD8 TM with an impaired ability to produce IFN-γ. TSA treatment at priming rescues key functional defects of the unhelped CD8 TM and restores long-term persistence in vivo, immediate effector cytokine production, and, most importantly, protective immunity. HDAC inhibition was effective in overcoming defects normally imprinted on unhelped CD8 TM. These results establish chromatin remodeling as a molecular basis for enhanced functionality of CD8 TM and provide direct evidence for a role of CD4 T cells in this process.

Heritable epigenetic modifications play an essential role in regulating gene expression for a broad range of biological processes (21, 22, 23, 24). In CD4 T cells, chromatin remodeling at the Ifng and Il4 loci occurs during differentiation into TH1 or TH2 polarized effectors (25, 26, 27, 28). In addition to histone acetylation, other epigenetic modifications such as DNA methylation likely play a role in rapid expression of effector functions by CD8 TM upon restimulation (16, 29). Although our analyses have focused on IFN-γ as a representative effector cytokine, our results show the rescue of multiple functions by TSA treatment and thus suggest a model in which the transition of naive CD8 T cells into quality memory involves epigenetic modifications of numerous genes involved in T cell survival, proliferation, and effector functions. Consistent with this model, increased histone acetylation has been observed in other loci in CD8 TM (30). Thus, histone acetylation likely plays a role in maintaining a broad range of genes in an accessible state, which represents an important aspect of CD8 TM physiology critical to its ability to mediate immune protection.

We thank the members of the Shen and Wells laboratories, especially Joanna DiSpirito, Rajan Thomas, Matthew Cohen, and Steven Saenz for technical assistance and helpful discussions.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by National Institutes of Health Grant AI045025.

3

Abbreviations used in this paper: CD8 TM, memory CD8 T cell; AcH3, histone H3 acetylation; CD4 TH, CD4 T cell help; ChIP, chromatin immunoprecipitation; GP, glycoprotein; HDAC, histone deacetylase; P+I, PMA and ionomycin; TSA, trichostatin A.

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