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Gene Occurs in Memory but Not Naive CD8 T Cells1
* Emory Vaccine Center and Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322;
Amgen, Seattle, WA 98119; and
Department of Immunology, University of Washington, Seattle, WA 98195
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Abstract |
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locus occur during memory CD8 T cell differentiation in vivo. Expression of the antiviral cytokine IFN- in CD8 T cells is highly developmental stage specific. Most naive cells must divide before they express IFN-, while memory cells vigorously express IFN- before cell division. Ag-specific CD8 T cells were obtained during viral infection of mice and examined directly ex vivo. Naive cells had an IFN- locus with extensive methylation at three specific CpG sites. An inhibitor of methylation increased the amount of IFN- in naive cells, indicating that methylation contributes to the slow and meager production of IFN-. Effectors were unmethylated and produced large amounts of IFN-. Interestingly, while memory cells were also able to produce large amounts of IFN-, the gene was partially methylated at the three CpG sites. Within 5 h of antigenic stimulation, however, the gene was rapidly demethylated in memory cells. This was independent of DNA synthesis and cell division, suggesting a yet unidentified demethylase. Rapid demethylation of the IFN- promoter by an enzymatic factor only in memory cells would be a novel mechanism of differential gene regulation. This differentiation stage-specific mechanism reflects a basic immunologic principle: naive cells need to expand before becoming an effective defense factor, whereas memory cells with already increased precursor frequency can rapidly mount effector functions to eliminate reinfecting pathogens in a strictly Ag-dependent fashion. |
Introduction |
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cytokine, which is critical for the function of CD8 T cells (5, 6), shows strong differential expression in naive and memory CD8 T cells. Naive CD8 T cells require several rounds of proliferation before they can produce large amounts of IFN- (7, 8, 9, 10, 11). Memory CD8 T cells, however, quickly and effectively produce IFN- upon restimulation (12). This functional change is stable and passed on to daughter memory cells during homeostatic proliferation, and is maintained in the absence of Ag (13).
These observations raise the question of what intracellular mechanisms enable the enhanced functional properties of memory CD8 T cells such as increased IFN- expression. More efficient TCR signal transduction (14), increased usage of costimulatory molecules (15), and differential gene expression (16, 17, 18, 19) are all mechanisms that contribute to memory CD8 T cell regulation. Differential gene expression can be achieved through the action of selective transcription factors, and through epigenetic regulatory mechanisms like histone modifications and gene demethylation. In this study, we have examined the differential regulation of gene methylation of the IFN- promoter in naive vs memory CD8 T cells.
DNA methylation negatively affects gene expression (20). In mammals, DNA methylation occurs at cytosines within CpG dinucleotide sequences (20), and often coincides with repressed or inactive chromatin. Cytosine methylation is an important regulatory event as evidenced by the fact that targeted disruption of all known DNA methyltransferases (Dnmts),3 enzymes that add methyl groups to CpGs, causes developmental defects and death (21, 22, 23). Specific abrogation of the maintenance methyl-transferase Dnmt1 in T cells leads to ectopic cytokine expression as well as to impaired proliferative capacity (24, 25). Cell differentiation is often associated with the demethylation of genes (7, 8, 26, 27). It is generally assumed that DNA demethylation is due to a failure of DNA methyltransferases to methylate the newly replicated DNA strand during cell division. However, it has also been postulated that a currently unknown enzymatic factor actively directs the demethylation process independent of cell division (28).
IFN- gene expression is regulated by DNA methylation, although other mechanisms such as specific transcription factors and modulation of chromatin structure also contribute to its transcriptional regulation (9, 29, 30, 31, 32, 33, 34). In naive CD8 T cells, the IFN- gene is methylated at several of the CpG sites in the promoter, and this prevents gene expression (35). Upon activation and proliferation into effector cells, the IFN- gene is demethylated (33, 35, 36), but it is not clear what happens during differentiation into Ag-specific memory cells in vivo.
In this study, we address whether regulated gene methylation is an intracellular mechanism contributing to the differential expression of critical genes in naive and memory CD8 T cells. We assess IFN- gene methylation in vivo during differentiation of naive cells into memory cells, and after re-exposure to Ag. In these studies, freshly isolated, virus-specific memory CD8 T cells with precisely known activation history were examined. IFN- promoter methylation changed from predominantly methylated at three CpG sites to unmethylated, and to partial methylation of the three sites during the transition from naive to effector and then to memory CD8 T cells, respectively. IFN- gene methylation changes were site specific, as the IL-2 promoter was also demethylated in effector cells, but remained demethylated in memory cells. The IFN- promoter was rapidly demethylated upon restimulation of memory cells, while it remained unchanged in naive cells. Thus, IFN- gene methylation was surprisingly dynamic and precisely regulated during all stages of CD8 T cell differentiation and during T cell stimulation. These results suggest a novel mechanism of memory CD8 T cell gene control involving the rapid loss of methylation at a key immune response gene.
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Materials and Methods |
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mRNA determination
IFN- mRNA levels were determined by real-time RT-PCR using an iCycler iQ (Bio-Rad). RNA was prepared using the RNeasy kit (Qiagen). After DNaseI treatment, RNA was reverse-transcribed with oligo dT using the Superscript First-Strand Synthesis System (Invitrogen Life Technologies). For real-time RT-PCR of IFN-, we amplified a 200-bp region spanning exons 2 and 3 using primers CTT CTT GGA TAT CTG GAG GAA CTG GCA AAA and CTC AAA CTT GGC AAT ACT CAT GAA TGC ATC (37). To further ensure specific amplification of mRNA only, we analyzed samples without added reverse transcriptase in every experiment. Real-time RT-PCR was done using the SYBR Green kit with Hot Start Taq (Bio-Rad) to further eliminate nonspecific products. mRNA levels were standardized by determining the expression of the HPRT gene for every sample with primers GAT TCA ACT TGC GCT CAT CTT AGG C and GTT GGA TAC AGG CCA GAC TTT GTT G.
Bisulfite modification, PCR, and sequencing
The methylation status of CpG sequences was determined by "bisulfite sequencing". Bisulfite treatment deaminates all cytosines to uracil unless protected by methylation. Uracils are read as thymidines during PCR, and sequencing then allows identification of methylated cytosines. Genomic DNA was prepared as described (35). DNA was denatured and modified with sodium metabisulfite, purified, desulfonated, and amplified in semi-nested PCR using primers: IFN--1 (GGT GTG AAG TAA AAG TGT TTT TAG AGA ATT TTA T) and IFN--4 (CAA TAA CAA CCA AAA ACA ACC ATA AAA AAA AAC T), then IFN--1 and IFN--3 (CCA TAA AAA AAA ACT ACA AAA CCA AAA TAC AAT A). For the noncoding IFN- strand, primers were IFN--7 (GTT AGA AAT AGT TAT GAG GAA GAG TTG TAA AGT T) and IFN--10 (ACA AAA ACT CCC TAT ACT ATA CTC TAT AAA TAA A), then IFN--7 and IFN--9 (ACA ATT TCC AAC CCC CAC CCC AAA TAA TAT AAA A). For H19, we did nested PCR with primers H19-1 (GAT TAG ATA GTA TTG AGT TTG TTT GGA GT) and H19-4 (CCT AAA ATA CTA AAC TTA AAT AAC CCA CAA), then H19-2 (GAG AAA ATA GTT ATT GTT TAT AGT TTT), and H19-3 (ACC ATT TAT AAA TTC CAA TAC CAA AAA TAA). For IL-2, sites –380 to +64 were examined as described in Ref.28 . Touchdown PCR with decreasing temperatures from 59 to 52°C and a final annealing temperature of 50°C for 30 cycles was used. To prevent contamination, all utensils were UV-irradiated and control reactions showed no amplification of nonspecific reactions. PCR products were separated on agarose gels, excised, and cloned into the pGemT Vector System I (Promega). This allowed the selection of bacterial colonies with a disrupted lacZ reading frame. DNA from individual bacterial colonies was then amplified by PCR using primers T7 and SP6, and sequenced at Agencourt Bioscience by automated sequencing with primers SP6 and T7.
Mice and viral infection
P14 TCR transgenic mice were obtained from The Jackson Laboratory and backcrossed to C57BL/6 (B6) for at least 10 generations. Female B6 mice were from National Cancer Institute. We generated effector and memory P14 cells in normal B6 mice that had adoptively received 2.5 x 105 P14, Thy1.1+ (CD90.1+) splenocytes, followed by infection with 2 x 105 PFU LCMV Armstrong i.p. This was done to reduce the precursor frequency of transgenic P14 cells and to prevent premature elimination of LCMV before all P14 cells were efficiently activated. The technique also allows identification of P14 cells with anti-Thy1.1 Abs, as the recipient B6 mouse is Thy1.2+. All mice were maintained according to Emory University’s Institutional Review Board.
FACS and FACS sorting
For cell purification by cell sorting, T cells from the spleen or lymph node were stained with anti-CD8
and anti-Thy1.1 (clones 53-6.7 and His 51, respectively; BD Pharmingen) to avoid direct stimulation of the TCR, and to anti-CD44 for activation status, and sorted on a FACSVantage to a minimal purity of 96%. For enrichment of memory P14 cells, we depleted splenocytes with anti-Thy1.2-coated columns (Miltenyi Biotec) according to the instructions of the manufacturer.
Cell culture, intracellular cytokine staining
Intracellular staining for IFN- was previously described (38), as was the use of 5-azacytidine (35). For thymidine incorporation, 106 splenocytes were cultured with 1 µM peptide gp33 (KAVYNFATM) and [3H]thymidine was added at 1 µCi/well. Inhibitors were cycloheximide, mizoribine, mitomycin c, actinomycin d, cyclosporin a, and aminopterin (all from Sigma-Aldrich), all used at 50 µg/ml.
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Results |
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mRNA levels in naive and memory P14 CD8 T cells
Several genes are clearly differentially expressed in naive and memory cells (16, 17, 18). The cytokine IFN- is a prime example. Naive peripheral CD8 T cells are slow and inefficient at producing IFN- in response to stimulation. Memory CD8 T cells, however, rapidly produce large amounts of IFN- upon stimulation. This finding is recapitulated in Fig. 1A. To analyze IFN- expression control, the current system used CD8 T cells from P14 TCR-transgenic mice, which are specific for epitope gp33–41 of the glycoprotein of lymphocytic choriomeningitis virus (LCMV) (39), and contained the allelic surface marker Thy1.1 (CD90.1). P14 mice have previously been used to document changes in gene expression during memory CD8 T cell differentiation (16). In naive mice not exposed to LCMV, only 5% of P14 cells can produce intracellular IFN- after peptide stimulation in vitro. When effector cells were harvested on day 7 post-LCMV infection, 95% of P14 cells produced IFN- after stimulation in vitro (Fig. 1A). Importantly, more IFN- was produced, as the mean fluorescence intensity (MFI) had increased 
10-fold compared with naive cells. Memory P14 cells were harvested at least 30 days postinfection, when cellular phenotype was CD127high, CD44high, bcl-2high, and functional, rapid recall responses were observed. Nearly all memory P14 cells (91%) produced large amounts of IFN-. This distinct IFN- production profile exists over a large range of Ag concentration (Fig. 1B).
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protein production were reflected in different IFN- mRNA levels (Fig. 2). IFN- mRNA levels were determined from P14 cells directly ex vivo. Cells were purified by FACS sorting (Fig. 2A), and mRNA levels were measured by comparative real-time RT-PCR (Fig. 2B). Effector cells contained 65 times more IFN- mRNA than naive cells, while memory P14 cells contained 10–20 times more. mRNA levels following activation of naive and memory T cells were assessed in short-term cell cultures for various lengths of time. Production of IFN- mRNA increased rapidly in memory cells, reaching levels of 1,000- to 2,000-fold compared with unstimulated memory cells. This occurred rapidly within the first 2 h (Fig. 2C). Naive cells also increased the amount of IFN- mRNA in this time frame, but more modestly. At 2 h of stimulation, the increase was 130-fold for naive cells compared with resting naive cells, while it was 1,600-fold for memory cells compared with memory cells before stimulation (Fig. 2C). When naive and memory P14 cells were directly compared, and resting naive cells were taken as a point of reference, the difference was 20,000-fold (Fig. 2D). Thus, the IFN- gene is an ideal candidate for the study of differential transcriptional regulation because there is a remarkable difference in IFN- mRNA levels between naive and memory CD8 T cells.
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gene methylation is differentially regulated in naive, effector, and memory cells
There are nine CpG sites within 300 bp of the IFN-transcription start site (Fig. 3A). Methylation of these sites negatively affects transcription (35, 36). For CpG methylation analysis, we used the PCR-based "bisulfite sequencing" method (40). This requires much less biologic material than Southern blotting techniques, a critical factor when studying memory CD8 T cells of limited quantities. We obtained genomic DNA from P14 cells highly purified by FACS sorting to at least 96% purity. Sorts for naive cells were performed to obtain CD8+Thy1.1+CD44low cells to exclude previously activated cells, while sorted effector and memory cells were CD8+Thy1.1+CD44high. DNA was treated with sodium bisulfite, resulting in the deamination of cytosines to uracil, unless protected by methylation. The modified DNA was amplified by PCR, and uracils were replicated as thymidines. PCR products were then subcloned, sequenced, and compared with the wild-type IFN- gene. All cytosines not contained in CpG sequences were mutated to thymidines, indicating that the bisulfite treatment was efficient (data not shown). CpG methylation at each site is displayed such that each line represents the methylation status of one DNA strand.
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transcription start site.
The IFN- locus was almost completely unmethylated in effector cells at days 7 and 8 postinfection with LCMV (Fig. 3B). As expected, this correlated with high mRNA levels (Fig. 2). Thus, IFN- gene methylation is distinctly different between naive and effector cells.
In contrast to effectors, memory P14 cells harvested 6–12 wk postinfection were partially methylated at CpG sites +17, +97, and + 120, but to a lesser degree than naive cells (Fig. 3B). The coding and noncoding strand showed similar methylation patterns. In comparison to naive cells, methylation was significantly less focused on sites +17, +97, and +117 after the transcription start site in memory cells, but appeared more evenly distributed (Fig. 3C). The average number of methylated CpGs per DNA strand was 2.7, very similar to 2.8 in naive cells. However, a larger number of memory cell DNA strands had no methylation, as 23% remained unmethylated, in stark contrast to 2% in naive cells (Fig. 3D). Thus, IFN- gene methylation in memory CD8 T cells is more heterogeneous than in naive cells in two ways: it is distributed more heterogeneously within the 300-bp region, and individual DNA strands differ more widely. The relative methylation patterns of naive, effector, and memory P14 cells were also observed in nontransgenic LCMV-specific CD8 T splenocytes from normal B6 mice (data not shown).
CpG methylation of H19, an unrelated, imprinted gene
Is the demethylation of the IFN- gene in effector P14 cells locus specific? Because effector cells might undergo global loss of methylation, a process that might contribute to their vulnerability to apoptosis (41), a control for this system was required. The H19 gene is a paternally imprinted gene whose methylation is modulated during oogenesis and spermatogenesis (42), but is not expected to specifically change during immune responses. Thus, we performed bisulfite sequencing using FACS-sorted P14 cells from female mice, examining a previously studied region upstream of the H19 gene with 11 CpG sites (Fig. 4A) (35, 36). All P14 cells showed significant methylation of this genetic locus (Fig. 4B). Quantification of the data showed that the average methylation of all sites was 62, 59, and 64% for naive, effector, and memory cells, respectively (Fig. 4C). Thus, there was no extensive global methylation change of this imprinted gene. Therefore, changes in DNA methylation were not observed at all genetic loci during CD8 T cell differentiation.
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For comparison, the CpG methylation of another cytokine gene, IL-2, which is regulated by gene methylation in CD4 T cells (28), was also examined during memory CD8 T cell differentiation. The IL-2 promoter is methylated at several sites in naive CD4 T cells, and this specifically silences transcription (28). CpG methylation of the five CpG sites closest to the transcription start were examined during CD8 T cell differentiation (Fig. 5A). In CD4 T cells, these sites are methylated, and Ag stimulation leads to the rapid demethylation of sites –380, –262, and –217, but not sites –69 and +64 (28). The methylation of the sites in CD8 T cells is unknown, as is their methylation in effector and memory T cells.
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and IL-2 promoters are demethylated in effector CD8 T cells. In contrast to the IFN- promoter, however, the IL-2 promoter was also demethylated in memory CD8 T cells, and the low level of methylation observed in effector cells was even further reduced. Thus, IL-2 and IFN- gene methylation are both specifically but differently regulated by DNA methylation during memory CD8 T cell differentiation, and the state of DNA methylation of both genes is highly locus specific at all of the examined developmental stages.
IFN- gene methylation changes rapidly during restimulation of memory P14 CD8 T cells
The CpG methylation of cytokine loci can rapidly change after T cell stimulation in a 4–7 h time frame (28). To examine the methylation status during the rapid induction of IFN- expression during restimulation of memory CD8 cells, IFN- gene methylation was determined in naive and memory P14 cells following 5 h of stimulation with Ag (Fig. 6A). During this short period of Ag stimulation, memory CD8 T cells express exceedingly high levels of IFN- mRNA whereas naive CD8 T cells contain significantly less IFN- mRNA (20,000-fold less) (Fig. 2, C and D). CpG methylation at the IFN- transcription start site did not change significantly in stimulated naive P14 cells (Fig. 6B). These stimulated naive P14 cells were similarly methylated for both extent and distribution of CpG methylation of the IFN- gene when the data were quantified, and no statistically significant methylation differences were found (Fig. 6D). In addition, the coding and noncoding DNA strands remained symmetrically methylated.
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gene methylation during 5 h of stimulation (Fig. 6, C and D). For resting and stimulated memory P14 cells, a total of four independent experiments were performed with P14 cells from the same LCMV immune mice. Demethylation was nearly complete on both the coding and noncoding strands. Thus, the IFN- gene loses CpG methylation at an extremely rapid rate, suggesting that this loss of methylation is a transcriptional control mechanism that is differentially regulated in naive and memory CD8 T cells.
In contrast to the IFN- gene, the IL-2 gene underwent some methylation changes during the stimulation of naive cells, as previously reported for CD4 T cells (28) (data not shown). Methylation at site –217 was completely lost in naive P14 cells during short-term stimulation for 5 h (data not shown), while methylation of sites –69 and +64 remained constant as it does in CD4 T cells (28). At this time point, naive P14 CD8 T cells increased IL-2 mRNA production 2500-fold over the resting state (not shown), a much greater increase than the 200-fold increase observed for IFN- mRNA in naive CD8 T cells at this time point (Fig. 2C). Stimulated memory CD8 T cells increased IL-2 mRNA levels 5000-fold and remained demethylated at the IL-2 gene during the 5-h incubation time (data not shown). In summary, rapid gene demethylation can be observed at both the IFN- and the IL-2 locus during short-term stimulation, but at different developmental states, demonstrating the specific regulation of this process.
5-Azacytidine induces IFN- expression in naive but not memory CD8 T cells
5-Azacytidine is a nucleoside analog of cytidine that specifically inhibits DNA methylation by trapping Dnmts during replication (43). This effectively leads to demethylation and the expression of genes silenced by DNA methylation. The effect of 5-azacytidine on IFN- production in naive and memory P14 cells was determined by intracellular FACS analysis (Fig. 7). P14 cells from the spleen were cultured with peptide gp33 and 5-azacytidine for 72 h. This led to a dose-dependent increase in the number of naive P14 cells able to produce IFN- (Fig. 7). The percentage of IFN--producing naive P14 cells rose from 12 to 56% with the highest dose of 5-azacytidine. The amount of intracellular IFN- also increased, as the MFI of IFN- cells shifted (Fig. 7) (35). The effect was first noticeable after 48 h of cell culture (data not shown), consistent with a need for cell replication, during which 5-azacytidine blocks Dnmt action. Labeling with CFSE revealed that most naive cells only produced IFN- after cell division (data not shown). Thus, DNA methylation directly silences the expression of IFN- in naive CD8 T cells. As expected, 5-azacytidine did not significantly increase the IFN- production of memory cells, as these cells were already rapidly undergoing IFN- gene demethylation and already efficiently produced IFN- (Fig. 7). This suggests that IFN- expression is silenced by DNA methylation in naive CD8 T cells, but not in memory cells, and further illustrates the differential regulation by gene methylation in the two cell types.
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mRNA induction and IFN- gene demethylation
Because a DNA demethylase enzyme has not been identified (20, 44), it is generally assumed that methylated CpG sites become demethylated due to lack of maintenance methylation during replication, when existing cytosine methylation is copied from the parent DNA strand onto the newly synthesized daughter strand by the maintenance methyltransferase, Dnmt1. If Dnmt1 activity is absent or cannot keep pace with newly synthesized DNA, CpG methylation should disappear by 50% with each cell division. During the first cell division, it should furthermore remain intact on one DNA strand but not the other. We did not observe this expected behavior for the IFN- gene in stimulated memory cells, as IFN- CpG methylation disappeared by >50% on both DNA strands within the first 5 h of stimulation (Fig. 6C). Memory CD8 T cells start to divide 18–24 h post restimulation, as measured with CFSE (data not shown). However, DNA replication might begin before cell division is completed at 18 h post restimulation of memory CD8 T cells. To address the level of DNA replication within 5 h of memory T cell restimulation, [3H]thymidine incorporation in P14 spleen cultures from LCMV immune mice was measured. P14 cells stimulated with or without 1 µM gp33 peptide in vitro in 12 duplicate wells showed no significant [3H]thymidine incorporation within the first 5 h of activation (Fig. 8A). Similar results were seen using BrdU incorporation to measure DNA replication (data not shown). Therefore, during 5 h of restimulation, P14 memory cells did not proliferate or synthesize new DNA to an extent that could be measured with these techniques. A small, but reproducible increase in [3H]thymidine incorporation was observed at 7 h after activation, indicating the beginning of measurable DNA synthesis. There was massive replication at 24 h of culture. As expected, replication was blocked by DNA synthesis inhibitors mitomycin C and aminopterin (Fig. 8A).
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protein and mRNA production. DNA synthesis was blocked with a variety of inhibitors that act through different mechanisms. If inhibiting DNA synthesis in turn inhibits DNA demethylation, we expected a lack of new IFN- transcription and also protein production. Memory P14 splenocytes were stimulated in culture in the presence of the DNA synthesis inhibitors mitomycin C (cross-links DNA), aminopterin (inhibits thymidine synthesis), and mizoribine (inhibits nucleotide synthesis), and then examined for intracellular IFN- production by FACS analysis (Fig. 8B) and mRNA (Fig. 8C). All three drugs were unable to block IFN- protein or mRNA production in memory P14 cells. Preincubation of the above cultures with the drugs for 3 h before the experiment to allow for drug uptake and depletion of endogenous nucleotides did also not block IFN- transcription (data not shown). Additionally, fluorouracil (inhibits pyrimidine synthesis), cytosine-b-D-arabinofluranoside (nucleotide analog), amethopterin (blocks thymidine synthesis), and hydroxyurea (blocks nucleotide synthesis) were used as DNA synthesis inhibitors, and they too did not block IFN- production (data not shown). Thus, blocking DNA synthesis had no effect on IFN- transcription. In contrast, IFN- transcription and protein production were severely affected with drugs inhibiting T cell activation (cyclosporin A), transcription (actinomycin D), or protein synthesis (cycloheximide).
To test directly whether DNA synthesis was necessary for IFN- gene demethylation in memory P14 T cells, memory P14 T cells were stimulated with peptide in the presence or absence of mitomycin C, a DNA synthesis inhibitor. Stimulated memory cells were demethylated regardless of the presence of mitomycin C (Fig. 8D). Thus, mitomycin C did not block demethylation. Therefore, IFN- gene demethylation in memory CD8 T cells appeared not to be dependent on DNA replication and cell division.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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while naive cells require proliferation before they can do so. Our current findings identify changes in DNA methylation as a novel mechanism that differentially controls the expression of this critical gene in naive vs memory CD8 T cells. We found specific changes in IFN- gene methylation during the differentiation of naive into effector cells, and of effector cells into memory cells. Importantly, although naive CD8 T cells are largely methylated at three CpG sites within the IFN- promoter and memory CD8 T cells are also partially methylated at these sites, exposure to Ag induces rapid demethylation of these sites only in memory cells. We contrast this to the IL-2 gene, which is rapidly demethylated in naive cells upon stimulation, and then remains demethylated during differentiation. Thus, there is both gene-specific and differentiation state-specific regulation of DNA methylation in CD8 T cells.
Our study has revealed a novel mechanism by which the timing and magnitude of IFN- gene expression can be controlled across different CD8 T cell developmental stages. Methylation in memory cells followed by rapid demethylation after antigenic stimulation ensures that memory CD8 T cells have a strict requirement to see Ag if they are to produce IFN-, a potent proinflammatory cytokine. In addition, rapid demethylation, specifically in memory, not in naive cells, reflects a basic immunologic principle: naive cells need to expand in numbers before they can become an effective defense factor, whereas the primary objective of memory cells with already increased precursor frequency is the rapid elimination of reinfecting pathogens.
Our most surprising result was the rapid IFN- gene demethylation in memory cells upon restimulation. The mechanism appears to not require cell division, as demethylation and IFN- mRNA production cannot be blocked by DNA synthesis inhibitors. This suggests that demethylation is probably not due to lack of maintenance methylation during DNA replication, but rather could be achieved by an enzymatic factor. The existence of such an enzyme has been previously postulated, when the rapid demethylation of the IL-2 gene was reported (28). There is currently no enzyme known with confirmed DNA demethylase activity. The recent report of a long-sought histone demethylase (45), whose existence had been doubted (46), makes the discovery of a CpG-demethylating enzyme more likely. However, other possibilities remain: cell restimulation could induce selective apoptosis of cells with a methylated IFN- locus or lead to the selective loss of these cells during cell sorting. In addition, DNA replication could originate close to the IFN- gene in memory CD8 T cells, and replication of the IFN- DNA could occur soon after restimulation in a time frame before DNA synthesis inhibitors act.
Our results suggest that the molecular machinery that controls gene methylation and demethylation operates differently in naive and memory CD8 T cells. This machinery is currently incompletely understood. In addition, different signals might be generated at the cell surface of naive and memory CD8 T cells. Signaling through the IL-7R has been shown to regulate gene methylation in thymocytes (47). Therefore, differences in cytokine receptor signaling could result in differential regulation of gene methylation in naive vs memory CD8 T cells.
The passing of genetic information other than through DNA sequence information has been called epigenetic regulation. It ensures long-term inheritance of gene expression patterns in differentiated cells. Therefore, it was surprising to find such rapid changes in IFN- gene methylation. Memory CD8 T cells are a unique cell type, however, because they are highly but not terminally differentiated (reviewed in Ref.48). Restimulation of memory cells marks the beginning of a rapid differentiation process into secondary effector cells. Thus, rapid CpG demethylation reflects the ability of memory cells to rapidly differentiate into effector cells.
NK cells like memory CD8 T cells are an early source of IFN-. Both cells do not need to proliferate before making IFN- (12, 49). Despite these similarities, IFN- gene methylation is regulated differently. Freshly isolated NK cells do not have a methylated IFN- gene locus (49). This is perhaps why freshly isolated NK cells have a higher level of constitutive IFN- mRNA than CD8 memory cells. NK cells express 200-fold more IFN- mRNA than naive T cells (50) while we found only 20-fold more in memory T cells. However, irrespective of the IFN- gene methylation status, we now demonstrate that memory CD8 T cells are not limited by their basal IFN- gene methylation because they can quickly lose it upon stimulation and start transcribing IFN- very rapidly.
Changes in DNA methylation have been implicated in cell lineage decisions (reviewed in Ref.8). The IFN-promoter is demethylated in effector CD8 T cells, but partially methylated again in memory cells. It is not clear whether demethylation and remethylation are sequential events in the linear differentiation of cells from naive to effector and memory cells. Alternatively, a subset of effector cells with higher methylation or faster remethylation might selectively survive to become memory cells. It will next be important to determine the IFN- gene methylation profile of the recently identified subset of effector cells that selectively develops into memory cells (51, 52). These memory precursor cells express higher levels of the IL-7R than other effector cells during the height of the CD8 T cell response to LCMV infection (51, 52). Further exploring of intracellular transcriptional control mechanisms such as DNA methylation will improve the molecular understanding of memory CD8 T cell differentiation processes. This might ultimately allow the selective induction of more potent memory responses to pathogens and vaccines.
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Acknowledgments |
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 This work was supported by National Institutes of Health Grant AI30048 (to R.A.), Primary Caretaker Technical Assistance Supplement 3 U19 AI057266-02S1 (to R.A. and E.N.K.), and National Cancer Institute Grant RO1 CA096810 (to J.M.B.). 
2 Address correspondence and reprint requests to Dr. Rafi Ahmed, Emory Vaccine Center, Rollins Research Building, Room G211, 1510 Clifton Road, Atlanta, GA 30322. E-mail address: ra{at}microbio.emory.edu
3 Abbreviations used in this paper: Dnmt, DNA methyltransferase; LCMV, lymphocytic choriomeningitis virus; MFI, mean fluorescence intensity.
Received for publication October 14, 2005. Accepted for publication January 19, 2006.
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References |
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 T. K. Kim, L. S. St. John, E. D. Wieder, J. Khalili, Q. Ma, and K. V. Komanduri Human Late Memory CD8+ T Cells Have a Distinct Cytokine Signature Characterized by CC Chemokine Production without IL-2 Production J. Immunol., November 15, 2009; 183(10): 6167 - 6174. [Abstract] [Full Text] [PDF]
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A. D. Wells New Insights into the Molecular Basis of T Cell Anergy: Anergy Factors, Avoidance Sensors, and Epigenetic Imprinting J. Immunol., June 15, 2009; 182(12): 7331 - 7341. [Abstract] [Full Text] [PDF]
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K. Aoki, N. Sato, A. Yamaguchi, O. Kaminuma, T. Hosozawa, and S. Miyatake Regulation of DNA Demethylation during Maturation of CD4+ Naive T Cells by the Conserved Noncoding Sequence 1 J. Immunol., June 15, 2009; 182(12): 7698 - 7707. [Abstract] [Full Text] [PDF]
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G. Lal, N. Zhang, W. van der Touw, Y. Ding, W. Ju, E. P. Bottinger, St. P. Reid, D. E. Levy, and J. S. Bromberg Epigenetic Regulation of Foxp3 Expression in Regulatory T Cells by DNA Methylation J. Immunol., January 1, 2009; 182(1): 259 - 273. [Abstract] [Full Text] [PDF]
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J. K. Northrop, A. D. Wells, and H. Shen Cutting Edge: Chromatin Remodeling as a Molecular Basis for the Enhanced Functionality of Memory CD8 T Cells J. Immunol., July 15, 2008; 181(2): 865 - 868. [Abstract] [Full Text] [PDF]
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 J. Liu, M. Ballaney, U. Al-alem, C. Quan, X. Jin, F. Perera, L.-C. Chen, and R. L. Miller Combined Inhaled Diesel Exhaust Particles and Allergen Exposure Alter Methylation of T Helper Genes and IgE Production In Vivo Toxicol. Sci., March 1, 2008; 102(1): 76 - 81. [Abstract] [Full Text] [PDF]
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R. M. Thomas, N. Chunder, C. Chen, S. E. Umetsu, S. Winandy, and A. D. Wells Ikaros Enforces the Costimulatory Requirement for IL2 Gene Expression and Is Required for Anergy Induction in CD4+ T Lymphocytes J. Immunol., December 1, 2007; 179(11): 7305 - 7315. [Abstract] [Full Text] [PDF]
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M. Szyf The Dynamic Epigenome and its Implications in Toxicology Toxicol. Sci., November 1, 2007; 100(1): 7 - 23. [Abstract] [Full Text] [PDF]
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 A. C. D'Alessio, I. C. G. Weaver, and M. Szyf Acetylation-Induced Transcription Is Required for Active DNA Demethylation in Methylation-Silenced Genes Mol. Cell. Biol., November 1, 2007; 27(21): 7462 - 7474. [Abstract] [Full Text] [PDF]
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E. B. Day, W. Zeng, P. C. Doherty, D. C. Jackson, K. Kedzierska, and S. J. Turner The Context of Epitope Presentation Can Influence Functional Quality of Recalled Influenza A Virus-Specific Memory CD8+ T Cells J. Immunol., August 15, 2007; 179(4): 2187 - 2194. [Abstract] [Full Text] [PDF]
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H.-R. Kim, K.-A Hwang, K.-C. Kim, and I. Kang Down-Regulation of IL-7R{alpha} Expression in Human T Cells via DNA Methylation J. Immunol., May 1, 2007; 178(9): 5473 - 5479. [Abstract] [Full Text] [PDF]
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 F. Zhang, J. H. Pomerantz, G. Sen, A. T. Palermo, and H. M. Blau Active tissue-specific DNA demethylation conferred by somatic cell nuclei in stable heterokaryons PNAS, March 13, 2007; 104(11): 4395 - 4400. [Abstract] [Full Text] [PDF]
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E. N. Kersh Impaired Memory CD8 T Cell Development in the Absence of Methyl-CpG-Binding Domain Protein 2 J. Immunol., September 15, 2006; 177(6): 3821 - 3826. [Abstract] [Full Text] [PDF]
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J. K. Northrop, R. M. Thomas, A. D. Wells, and H. Shen Epigenetic Remodeling of the IL-2 and IFN-{gamma} Loci in Memory CD8 T Cells Is Influenced by CD4 T Cells J. Immunol., July 15, 2006; 177(2): 1062 - 1069. [Abstract] [Full Text] [PDF]
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, unmethylated CpGs; •, methylated CpGs. P14 CD8 T cells from the spleen were purified by FACS sorting to 96% purity or greater; naive cells were CD44low, effector and memory cells were CD44high. CpG methylation was analyzed by "bisulfite sequencing". We performed multiple independent PCRs separately for the coding and noncoding strand, and sequenced multiple PCR-derived subclones. Duplicates were eliminated because they could potentially have arisen from the same PCR subclone. For the coding strands, we examined 5 (N, naive), 4 (E, effector), and 5 (M, memory) independent cell samples. We performed 7 (N), 14 (E), and 14 (M) independent PCRs, and sequenced 53 (N), 57 (E), 91 (M) DNA strands. For the noncoding strand, the numbers were: 3 (N), 3 (E), and 4 (M) independent cell samples; 6 (N), 6 (E), and 7 (M) independent PCRs; 44 (N), 10 (E), 19 (M) sequenced DNA strands. C, The percentage of DNA strands with methylation at the indicated CpG site was determined from data shown in B of coding and noncoding strands combined. The differences between naive and memory cells and between effector and both other cell types in the extent of methylation at sites + 17, +97, and +120 were statistically different (p < 0.05 using Fisher’s exact test). D, The number of methylated sites per individual DNA strand was determined from data shown in panel B of coding and noncoding strands combined. The average number of mCpGs per DNA strand was 2.8, 0.3, and 2.7 for naive, effector, or memory P14 cells, respectively.
