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Loci in Memory CD8 T Cells Is Influenced by CD4 T Cells1
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* Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104;
Joseph Stokes, Jr. Research Institute, Children’s Hospital of Philadelphia, Philadelphia, PA 19104; and
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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. Our data show that this defect in IL-2 and IFN- production is independent of clonal selection, functional avidity maturation, and the integrity of proximal TCR signaling, but rather involves epigenetic modification of these cytokine genes. Activated Ag-specific CD8 T cells exhibit rapid DNA demethylation at the IL-2 and IFN- loci and substantial histone acetylation at the IFN- promoter and enhancer regions. These epigenetic modifications occur early after infection at the effector stage and are maintained through memory development. However, activated unhelped CD8 T cells, which fail to develop into functional memory and are incapable of rapid cytokine production, exhibit increased DNA methylation at the IL-2 promoter and fail to acetylate histones at the IFN- locus. Thus, CD4 T cell help influences epigenetic modification during CD8 TM differentiation and these epigenetic changes provide a molecular basis for the enhanced responsiveness and the maintenance of a "ready-to-respond" state in CD8 TM. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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The development of functional CD8 TM requires CD4 T cell help (CD4 Th) during the naive to CD8 TM transition. Unhelped CD8 TM are defective in all three cardinal features of fully functional CD8 TM, exhibiting poor survival, impaired secondary expansion, and a diminished capacity for immediate effector function upon restimulation (12, 13, 14, 15). In this study, we investigated the defect underlying the diminished cytokine production by unhelped CD8 TM to understand the molecular basis for the enhanced responsiveness of CD8 TM. We tested whether the unhelped CD8 TM population is altered in the distribution and selection of high-affinity CD8 TM clones and whether these cells are defective in functional avidity maturation and TCR signal transduction. We also examined whether CD4 Th influences the epigenetic modification of genes involved in CD8 T cell function, specifically IL-2 and IFN-. Our results show that the defect in unhelped CD8 TM is not due to aberrations in the TCR repertoire nor in functional avidity maturation. Rather, our studies establish that the differentiation of naive CD8 T cells into CD8 TM during infection is accompanied by demethylation of DNA and acetylation of histones at the regulatory regions of the IL-2 and IFN- genes, and that CD4 Th influences these epigenetic changes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Sex- and age-matched B6, B6-CD4–/–, and B6-MHC-II–/– mice purchased from The Jackson Laboratory were infected i.p. with 2 x 105 PFU of lymphocytic choriomeningitis virus (LCMV) Armstrong or 5 x 104 CFU of recombinant Listeria monocytogenes expressing gp33–41 of LCMV.
Adoptive transfer
Naive Thy1.1 P14 CD8 T cells were isolated from 5- to 8-wk P14 TCR-transgenic mice and purified by CD8+ MACS beads (Miltenyi Biotec), and 1–2 x 105 live cells were transferred i.p. into Thy 1.2 mice, which were then infected 12–16 h posttransfer.
Intracellular cytokine staining and flow cytometry
Single-cell suspensions were stimulated ex vivo either with gp33 (10–7 M), np396 (10–7 M) (or at the indicated concentration in avidity experiments), or PMA (25 ng/ml; Sigma-Aldrich) and ionomycin (1000 nM; Sigma-Aldrich), as indicated. Surface stain and intracellular cytokine staining was performed as previously described (14). Lck staining was performed by primary staining with polyclonal rabbit anti-Lck serum, followed by secondary staining with FITC-conjugated goat anti-rabbit. Preimmune rabbit serum followed by the same goat anti-rabbit secondary staining served as the control Ab. All Abs were purchased from BD Pharmingen.
DNA methylation and chromatin immunoprecipitation (ChIP) analysis
Naive, effector, and memory P14 cells were purified with a FACSVantage sorter (BD Biosciences) for CD8+ and Thy1.1+ cells. In some experiments, P14 CD8 T cells were purified by FITC-selective MACS beads (Miltenyi Biotec). Genomic DNA from purified cells was bisulfite converted as described previously (16). Nested primer PCR was performed for the IL-2 promoter and the IFN- promoter and intronic enhancer (primers shown in Table I, DNA methylation analysis) using the bisulfite converted DNA as template. The PCR product was cloned and then sequenced.
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promoter, IFN- enhancer, and CD3
promoter (primers shown in Table I, ChIP assay using a MyIQ thermal cycler (Bio-Rad). Specific enrichment is calculated using the cycle threshold (Ct): 2(Ct of control ChIP – Ct of control Input)/2(Ct of AcH3 ChIP – Ct of AcH3 Input). Statistical analysis
Values are given as the mean of the individual samples ± SD. Statistical significance was assessed by the unpaired Student’s t test and only values < p = 0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Rapid production of IFN- and IL-2 upon restimulation is characteristic of stable, protective CD8 TM, while CD8 TM primed without CD4 Th are defective in immediate cytokine production (14, 17). To study the molecular basis for the enhanced responsiveness of CD8 TM, we compared CD8 TM generated in the presence of absence of CD4 T cell help. We infected the control C57BL/6 (B6) mice and the CD4 T cell-deficient B6-CD4–/– mice with LCMV and then examined IFN- and IL-2 production by CD8 TE at day 8 postinfection (p.i.) and TM at day 105 p.i. by intracellular cytokine staining following 5 h ex vivo stimulation with gp33 peptide (LCMV-specific H-2Db-restricted epitope). CD8 TE isolated from B6 (helped) and B6-CD4–/– (unhelped) mice produced a similar level of IFN- per cell, as measured by FACS analysis of the geometric mean fluorescent intensity (MFI; Fig. 1). CD8 TM from B6-CD4–/– mice produced considerably less IFN- per cell than helped CD8 TM (Fig. 1). At the effector stage, <5% of the IFN-+ cells also produced IL-2 in either B6 or B6-CD4–/– mice (Fig. 1). Over time, the IFN- MFI and the frequency of IFN- and IL-2 coproducers increased in both the helped and unhelped CD8 TM; however, at no time studied (up to 4 mo p.i.) did the unhelped CD8 TM improve to the levels of cytokine production seen in the B6 controls (data not shown). Similar results were also observed with LCMV np396 peptide restimulation, in B6-MHC-II–/– mice, and after infection with recombinant strains of L. monocytogenes expressing either the gp33 epitope of LCMV or OVA (Ref. 17 and our data not shown). Naive CD8 T cells did not produce appreciable levels of IFN- or IL-2 immediately upon brief ex vivo stimulation (data not shown and see Fig. 5). Therefore, while CD4 Th was not required for maximal production of IFN- at the effector phase, unhelped CD8 TM exhibited a defect in the capacity to produce IFN- and IL-2 upon re-encounter with Ag.
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Previous studies have shown a progressive selection for higher affinity clones as a polyclonal population of naive T cells is activated to become effectors and further differentiate into memory cells (5, 6). Therefore, we asked whether the lack of CD4 Th results in a failure in the selection of the most fit clones among the effector populations to populate the pool of memory cells. To test this, we compared the variable 
(V-) chain usage in the Ag-specific populations of CD8 TE (8 days p.i.) and CD8 TM (>30 days p.i.) in LCMV-infected B6 and B6-CD4–/– mice. CD8 T cells were costained with a panel of Abs to various V- chains and Db/gp33 or Kb/gp34 tetramers, and the percent of the Ag-specific cells expressing a particular V- chain was determined (representative FACS plots, Fig. 2A). The distribution of V- chains in CD8 TM was equivalent between B6 and B6-CD4–/– mice in both Ag-specific populations examined (Fig. 2, B and C, and CD8 TE data not shown). Similarly, no difference in the V- chain distribution between helped and unhelped Ag-specific CD8 TM was observed following infection with a recombinant strain of L. monocytogenes expressing the gp33 epitope of LCMV (data not shown). These results show no difference between B6 and B6-CD4–/– mice in the selection of clones for the pool of CD8 TM.
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(Fig. 3). As the cells matured over time, the helped P14 CD8 TM produced more IFN- per cell and began producing IL-2 in response to restimulation, while unhelped P14 CD8 TM produced significantly less IFN- and 50–70% fewer cells coproduced IL-2 (Fig. 3). These differences were observed for CD8 TM obtained from spleens (Fig. 3), as well as lymph nodes and bone marrow (data not shown). Thus, monoclonal P14 CD8 TM cells fully recapitulated the defective cytokine production phenotype observed in polyclonal CD8 TM, consistent with published findings (17). These data together strongly argue against clonal selection as the basis for the functional defect in immediate cytokine production in unhelped CD8 TM.
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Although Ag-activated CD8 T cells do not undergo a change in TCR affinity, they progressively become more responsive to peptide stimulation through a process termed TCR functional avidity maturation (9). Therefore, we examined whether unhelped CD8 TM show evidence of impaired functional avidity maturation. Following restimulation with serial 10-fold dilutions of gp33 peptide, P14 CD8 TM from both B6 and B6-CD4–/– LCMV-infected hosts first produced IFN- in response to gp33 at 10–11 M and plateaued at 10–9 M (Fig. 4, A and B). When calculated, as the percent of maximal response, the response curves were identical for CD8 TM from both B6 and B6-CD4–/– mice and both populations exhibited half maximal response at 4 x 10–11 M of gp33 peptide (Fig. 4B). This was true when calculated either as percent of maximal number of IFN--producing cells or percent of maximal IFN- production per cells as measured by MFI (Fig. 4, B and C). Although we observed no difference in functional avidity between the helped and unhelped P14 CD8 TM, the unhelped P14 CD8 TM were still defective in per cell IFN- production even at the highest concentrations of gp33 (Fig. 4, A and C). The MFI of IFN- staining for unhelped CD8 TM never reached the level observed in helped CD8 TM (Fig. 4C), nor did the number of IL-2 coproducers (data not shown). Functional avidity maturation is associated with augmented signaling through components of the TCR-signaling apparatus (10). We found no differences in the levels of the proximal tyrosine kinase Lck between helped and unhelped CD8 TE and CD8 TM (Fig. 4D). Nor were there differences in the lipid rafts, as assayed by asialo-GM1 levels, or in the phosphorylation of ERK in response to peptide stimulation between helped and unhelped CD8 TM (data not shown). Although CD8 T cells underwent functional avidity maturation as they became activated (9), our results indicate that the defect in the unhelped CD8 TM is not due to measurable differences in TCR signaling and functional avidity maturation.
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per cell and fewer were positive for IL-2 than helped P14 CD8 TM (Fig. 5). It even failed to partially restore normal cytokine production in unhelped CD8 TM because P+I stimulation did not result in any increase in the MFI of IFN- or the percent of IFN-/IL-2 coproducers compared with those from peptide stimulation (Fig. 5). These results show that the defect responsible for reduced cytokine production in unhelped CD8 TM is downstream of proximal TCR signaling.
Demethylation of DNA at the IL-2 and IFN- loci during CD8 TM differentiation
Coordinate and heritable patterns of gene expression are often enforced epigenetically, through physical and chemical modifications of genomic DNA that do not alter its primary sequence. We investigated whether the differentiation of Ag-specific CD8 T cells involves epigenetic changes that might affect the potential for the expression of cytokine genes. We first characterized DNA methylation, an important type of epigenetic modification that imposes a direct physical constraint to transcription factor binding and represses gene expression. We examined DNA methylation patterns at the proximal region of the IL-2 promoter, the IFN- promoter, and the IFN- intronic enhancer in naive, effector, and memory CD8 T cells generated in the presence or absence of CD4 Th during acute LCMV infection in vivo.
In naive P14 CD8 T cells, 31% of all proximal IL-2 promoter CpG sites and 17% of all CpG sites at the IFN- promoter were methylated CpG (MeCpG) sites (Fig. 6A and Table II list the percent methylation for each CpG site). In both cases, methylation was most concentrated at the CpG most proximal to the first exon, where almost 90% of the IL-2 alleles and nearly 70% of the IFN- alleles were methylated (Fig. 6A). In the intronic enhancer of the IFN- gene, each of the 10 CpG sites exhibited >80% methylation with an average MeCpG level of 90% across the enhancer (Fig. 6A).
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locus exhibited a profound loss of DNA methylation at all CpG sites, with overall MeCpG decreasing from 17 to 0.4% at the promoter and from 90 to 1.0% at the enhancer (Fig. 6B). The IL-2 promoter also exhibited a loss of MeCpG in CD8 TE, but to a lesser extent than at the IFN- regulatory regions (from 31 to 14% in overall MeCpG and from 88 to 31% at the proximal CpG site, Fig. 6B). We next examined the MeCpG status of the IL-2 and IFN- loci in CD8 TM isolated 2 mo postinfection. We found that the largely demethylated state of the IFN- promoter and enhancer attained early in the response by CD8 TE was stably maintained throughout CD8 TM development (Fig. 6D). Moreover, the CD8 TE to CD8 TM transition was accompanied by a progressive decrease in the methylation status of the IL-2 promoter, such that the four CpG sites in this region were almost completely demethylated (4% overall and 12% at the proximal site, Fig. 6D). The almost complete loss of MeCpG at the IL-2 promoter in CD8 TM correlates with a gain of IL-2 gene expression during the CD8 TE to CD8 TM transition (Figs. 1 and 3).
Therefore, our results show that activation of naive CD8 T cells into effectors is associated with a profound loss of DNA methylation at all CpG sites in the IFN- promoter and enhancer. This largely demethylated state of the IFN- locus attained at the effector stage is stably maintained into CD8 TM. Although demethylation of CpG sites in the IL-2 promoter is not as profound as in the IFN- locus, there is a progressive decrease in the methylation status of the IL-2 promoter during the CD8 TE to CD8 TM transition.
CD4 Th influences DNA methylation at the IL-2 promoter in CD8 TM
With our finding of epigenetic changes at IL-2 and IFN- loci during effector and memory differentiation, we examined whether CD4 Th influences epigenetic tagging of the IL-2 or IFN- loci in CD8 TE or CD8 TM. At the IFN- promoter and enhancer, CD8 TE (day 8 p.i.) isolated from B6-CD4–/– recipients exhibited a similar pattern and degree of DNA demethylation as observed in CD8 TE from B6 recipients (Fig. 6C). Unhelped CD8 TM retained the same degree of DNA demethylation at the regulatory regions of the IFN- locus as observed in helped CD8 TM (Fig. 6D). However, unlike helped CD8 TM, which exhibited almost complete loss of MeCpG at the IL-2 promoter (Fig. 6D), the CD8 TM from B6-CD4–/– mice showed no further loss in DNA methylation and remained at the levels observed in CD8 TE (Fig. 6E). This failure of the unhelped CD8 TM to sustain progressive demethylation of the IL-2 promoter is associated with a defect in IL-2 production (Figs. 1 and 3).
Increased histone acetylation at the IFN- locus during CD8 TM differentiation
We also examined the AcH3 status, another important form of epigenetic modification that relaxes chromatin and favors gene expression. Naive CD8 T cells exhibited merely background levels of AcH3 at the IL-2 promoter (Fig. 7E) and the IFN- enhancer (Fig. 7C), but a low degree of AcH3 at the IFN- promoter (11-fold over background, Fig. 7, A and B) and strong AcH3 at the CD3 promoter (17-fold over background, Fig. 7D). These AcH3 levels, along with the MeCpG patterns, indicate that the cytokine genes of naive CD8 T cells are not strongly poised for rapid transcription (18, 19), consistent with the inability of naive cells to immediately produce IL-2 and IFN- upon either peptide or P+I restimulation (Fig. 5).
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promoter and a 40-fold increase at the IFN- enhancer in AcH3 levels relative to the levels in naive cells (Fig. 7, A–C). However, unlike the regulatory regions of the IFN- gene, the IL-2 promoter in CD8 TE exhibited no histone acetylation and remained hypoacetylated in CD8 TM (Fig. 7E). Strikingly, CD8 TM retained strong AcH3 at the IFN- locus, with average levels at the promoter 5-fold over naive levels (Fig. 7B), and 43-fold greater AcH3 at the enhancer (Fig. 7C). This level of AcH3 at the IFN- regulatory regions in the absence of active transcription is indicative of a poised locus that can be expressed immediately in response to extracellular signals (18, 19).
In brief, our results show that the IL-2 promoter exhibits no AcH3 and remains hypoacetylated during the differentiation of naive CD8 T cells to CD8 TE and CD8 TM. In contrast, there is a marked increase in AcH3 at the IFN- locus as naive CD8 T cells differentiate into CD8 TE and this status of high AcH3 at the IFN- locus is maintained in CD8 TM. These results, together with the DNA methylation data, show that CD8 T cell differentiation from naive to effector to memory is accompanied by epigenetic changes at both IL-2 and the IFN- loci and indicate that epigenetic modification is an integral part of CD8 TM differentiation.
CD4 Th is required for histone acetylation at the IFN- locus in CD8 TE and TM
Although demethylation of CpG sites within the IFN- promoter and enhancer in CD8 TE and TM occurred independently of CD4 Th, histone acetylation at these same regions was highly dependent upon the presence of CD4 Th. Unhelped CD8 TE exhibited only half the degree of AcH3 at the IFN- promoter and 3-fold less AcH3 at the IFN- enhancer, compared with those observed in helped CD8 TE (Fig. 7, B and C). Interestingly, unhelped CD8 TM isolated 2 mo after infection remained hypoacetylated at these regulatory regions (Fig. 7, B and C). Importantly, the lack of AcH3 at the IL-2 and IFN- regulatory regions in unhelped CD8 T cells is locus specific, as AcH3 at the CD3 promoter was strong and did not differ significantly in helped vs unhelped CD8 T cells (Fig. 7D). Greatly reduced AcH3 at the IFN- locus in unhelped CD8 TM is associated with the reduced capacity of these cells to produce IFN- (Figs. 1 and 3).
Our results show that full demethylation at the IL-2 promoter and histone acetylation at the IFN- locus in CD8 TE and CD8 TM requires CD4 Th. These deficiencies in epigenetic modifications at the cytokine loci in unhelped CD8 TM cells correlates strongly with the data showing that full cytokine production by CD8 TM requires CD4 Th (Figs. 1 and 3). Together, these results suggest that epigenetic modifications are a molecular mechanism by which CD4 Th promotes the development of functional CD8 TM.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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. Our data here show that the defect in IL-2 and IFN- gene expression in unhelped CD8 TM is not at the level of either clonal selection, functional avidity maturation, or the integrity of proximal TCR signaling, but rather involves epigenetic modification of these cytokine genes.
Methylation of genomic DNA inhibits gene expression by imposing a direct physical constraint to transcription factor binding, as well as by recruiting complexes of enzymes that promote chromatin compaction (20, 21, 22). Conversely, histone acetylation inhibits nucleosome compaction and generates binding sites for ATP-dependent chromatin remodeling enzymes that free the DNA from around the nucleosomes (23). Moreover, DNA methylation has been shown to specifically inhibit transcription of both the IL-2 and IFN- genes in CD4 and CD8 T cells activated in vitro (24, 25, 26, 27), while drugs that promote histone acetylation can augment IFN- gene expression in T cells (28, 29, 30, 31). A recent study has described demethylation of DNA at the IL-2 and IFN- promoters as naive CD8 T cells differentiate into CD8 TM in response to viral infection (31). In this study, we find that Ag-specific CD8 T cells activated in vivo during infection exhibit rapid demethylation at the IL-2 and IFN- and extensive histone acetylation at the IFN- promoter and enhancer regions. Furthermore, our results demonstrate that these epigenetic modifications occur early after infection at the effector stage and are maintained during the development of memory. More importantly, we find that activated CD8 T cells that fail to develop into functional memory cells due to the absence of CD4 Th also fail to acetylate histones at the IFN- locus, exhibit increased DNA methylation at the IL-2 promoter, and are incapable of rapid and high-level expression of the IFN- and IL-2 genes. Thus, epigenetic modification provides a molecular basis for the enhanced responsiveness and the maintenance of such a "ready-to-respond" state over a long period of time and through multiple generations in CD8 TM.
Why are CD4 T cells required for CD8 T cell memory development but not for early effector differentiation? The answer to this question may lie in the differential requirement of epigenetic modifications for expression of effector functions by activated, rapidly dividing effectors vs relatively quiescent memory cells. Chromosome decondensation during DNA replication in rapidly dividing effectors could serve to open and derepress differentiation-associated genes independently of histone acetylation (32, 33). In this case, the IFN- locus in CD8 TE may not require histone acetylation for transcriptional competence. Demethylation of CpG sites within the IFN- promoter and enhancer, which occurs independently of CD4 Th (Fig. 6, B and C), may cooperate with S-phase chromosome decondensation to allow for full IFN- gene expression in activated CD8 TE. This appears to be the case for CD4 T cells, which must undergo multiple cell divisions to achieve expression of differentiated cytokine genes, such as IL-4 and IFN- (34, 35, 36). Alternatively, it remains possible that chromatin remodeling at the IFN- gene could be mediated by the small amount of AcH3 that does accumulate at the enhancer region in effector cells even in the absence of CD4 Th (Fig. 7C), or it could be mediated by other histone modifications not measured in these studies (18, 19).
In contrast to effectors, memory T cells are mostly quiescent with a small percentage of the population undergoing slow homeostatic proliferation at any given time. Therefore, histone acetylation may play a much more important role in keeping the IFN- locus open in resting CD8 TM and allow immediate production of high-level IFN- by all CD8 TM cells upon restimulation. Consistent with this, we found extensive AcH3 at the IFN- locus in helped CD8 TM but considerably less in unhelped CD8 TM (Fig. 7, A–C), which are defective in immediate IFN- production (Figs. 1 and 3). In contrast, we were unable to detect significant AcH3 at the IL-2 promoter even in helped CD8 TM (Fig. 7E). At any given time, only a distinct subset of CD8 TM produce IL-2 (Figs. 1 and 3) and it is possible that these are the cells that have recently undergone homeostatic proliferation. In CD4 T cells, rapid production of IL-2 in response to restimulation only occurs in T cells that have undergone cell divisions, suggesting that S-phase chromosome decondensation may play an important role in keeping this locus accessible to transcription factors (36, 37). In this case, reduced DNA methylation at the promoter associated with the effector to memory transition in helped CD8 T cells (Fig. 6D) may likewise promote expression of the IL-2 gene in CD8 TM, as it does in CD4 T cells (26). Alternatively, it is possible that CD4 Th promotes histone acetylation at regions other than the IL-2 promoter that regulate IL-2 gene expression in CD8 T cells (38). It is also possible that nucleosomes are completely displaced from the proximal region of the IL-2 promoter in CD8 TM, a phenomenon observed following stimulation of the EL4 T cell line (39).
The effector to memory transition for CD8 T cells involves the cessation of Ag receptor signaling, increased expression of many genes involved in survival, proliferation, cytokine/cytokine receptor expression and protein translation, and repression of certain genes that oppose memory function (11). Epigenetic modification likely involves multiple processes that leave positive and negative imprints at many different loci during CD8 TM differentiation. Although these modifications clearly occur at the early effector stage, they may also occur at later stages and/or be modified continuously by extracellular signals during maintenance (17). Moreover, the defective epigenetic remodeling described here might be responsible for defective CD8 TM function found in other situations, such as chronic infections. In chronic infection with LCMV clone 13, CD8 TM function is impaired due to Ag persistence, though there is also a parallel decrease in functional CD4 T cells (40). Similarly, there is suggestive evidence for low-level Ag persistence in LCMV Armstrong-infected B6-MHC II–/– mice. Although our data suggests that these defects in epigenetic modifications contribute to the unhelped phenotype, regardless of the cause (Ag-persistence and/or absence of CD4 Th) the failure of proper epigenetic modification might be a common mechanism responsible for defective CD8 TM function. Additional studies of histone acetylation, histone methylation, and corepressor occupancy at various loci and at different time points will provide a full understanding of the epigenetic regulation of CD8 TM differentiation and the influence of CD4 Th in these processes.
In conclusion, our results show that epigenetic changes at the IL-2 and IFN- loci accompany activation of naive CD8 T cells to become effectors. These modifications are maintained as effectors differentiate into memory cells, and likely play an important role in keeping CD8 TM cells poised to respond immediately upon Ag re-encounter. These results suggest a model of epigenetic modification as an integral part of CD8 TM differentiation. We propose that these processes lead to the stable epigenetic modification of an entire program of genes involved in T cell survival, metabolism, proliferation, and effector function, which serve as a molecular basis for cellular memory during T cell responses.
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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 Institute of Health Grants AI45025 (to H.S.) and AI059881 (to A.D.W.), and the Biesecker Pediatric Liver Disease Center (to A.D.W.). 
2 Address correspondence and reprint requests to Dr. Andrew D. Wells, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104. E-mail address: adwells{at}mail.med.upenn.edu or Dr. Hao Shen, Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104. E-mail address: hshen{at}mail.med.upenn.edu
3 Abbreviations used in this paper: CD8 TE, effector CD8 T cell; CD8 TM, memory CD8 T cell; LCMV, lymphocytic choriomeningitis virus; ChIP, chromatin immunoprecipitation; AcH3, histone H3 acetylation; Ct, cycle threshold; p.i., postinfection; MFI, geometric mean fluorescent intensity; P+I, PMA and ionomycin; MeCpG, methylated CpG.
Received for publication March 14, 2006. Accepted for publication May 1, 2006.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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(IFN-) and interleukin 3 (IL-3) genes in newly activated primary CD8+ T lymphocytes: regional IFN- promoter demethylation and mRNA expression are heritable in CD44highCD8+ T cells. J. Exp. Med. 188: 103-117. promoter demethylation in CD44highCD8+ T lymphocytes. J. Immunol. 162: 5053-5057. loci accompany Th1/Th2 differentiation. J. Immunol. 169: 647-650. gene occurs in memory but not naive CD8 T cells. J. Immunol. 176: 4083-4093. This article has been cited by other articles:
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S. Fuse, C.-Y. Tsai, M. J. Molloy, S. R. Allie, W. Zhang, H. Yagita, and E. J. Usherwood Recall Responses by Helpless Memory CD8+ T Cells Are Restricted by the Up-Regulation of PD-1 J. Immunol., April 1, 2009; 182(7): 4244 - 4254. [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. K. Whitmire, N. Benning, B. Eam, and J. L. Whitton Increasing the CD4+ T Cell Precursor Frequency Leads to Competition for IFN-{gamma} Thereby Degrading Memory Cell Quantity and Quality J. Immunol., May 15, 2008; 180(10): 6777 - 6785. [Abstract] [Full Text] [PDF]
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J. A. Sacks and M. J. Bevan TRAIL Deficiency Does Not Rescue Impaired CD8+ T Cell Memory Generated in the Absence of CD4+ T Cell Help J. Immunol., April 1, 2008; 180(7): 4570 - 4576. [Abstract] [Full Text] [PDF]
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T. Wang, H. Dai, N. Wan, Y. Moore, and Z. Dai The Role for Monocyte Chemoattractant Protein-1 in the Generation and Function of Memory CD8+ T Cells J. Immunol., March 1, 2008; 180(5): 2886 - 2893. [Abstract] [Full Text] [PDF]
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N. Wan, H. Dai, T. Wang, Y. Moore, X. X. Zheng, and Z. Dai Bystander Central Memory but Not Effector Memory CD8+ T Cells Suppress Allograft Rejection J. Immunol., January 1, 2008; 180(1): 113 - 121. [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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 A. Singh and M. Suresh A role for TNF in limiting the duration of CTL effector phase and magnitude of CD8 T cell memory J. Leukoc. Biol., November 1, 2007; 82(5): 1201 - 1211. [Abstract] [Full Text] [PDF]
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M. R. Chandok, F. I. Okoye, M. P. Ndejembi, and D. L. Farber A Biochemical Signature for Rapid Recall of Memory CD4 T Cells J. Immunol., September 15, 2007; 179(6): 3689 - 3698. [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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K. A. Richards, F. A. Chaves, F. R. Krafcik, D. J. Topham, C. A. Lazarski, and A. J. Sant Direct Ex Vivo Analyses of HLA-DR1 Transgenic Mice Reveal an Exceptionally Broad Pattern of Immunodominance in the Primary HLA-DR1-Restricted CD4 T-Cell Response to Influenza Virus Hemagglutinin J. Virol., July 15, 2007; 81(14): 7608 - 7619. [Abstract] [Full Text] [PDF]
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P. H. Horne, M. A. Koester, K. Jayashankar, K. E. Lunsford, H. L. Dziema, and G. L. Bumgardner Disparate Primary and Secondary Allospecific CD8+ T Cell Cytolytic Effector Function in the Presence or Absence of Host CD4+ T Cells J. Immunol., July 1, 2007; 179(1): 80 - 88. [Abstract] [Full Text] [PDF]
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Z. Liu, H. Dai, N. Wan, T. Wang, S. Bertera, M. Trucco, and Z. Dai Suppression of Memory CD8 T Cell Generation and Function by Tryptophan Catabolism J. Immunol., April 1, 2007; 178(7): 4260 - 4266. [Abstract] [Full Text] [PDF]
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 C. Chen, E. A. Rowell, R. M. Thomas, W. W. Hancock, and A. D. Wells Transcriptional Regulation by Foxp3 Is Associated with Direct Promoter Occupancy and Modulation of Histone Acetylation J. Biol. Chem., December 1, 2006; 281(48): 36828 - 36834. [Abstract] [Full Text] [PDF]
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; B6-CD4–/–, 
). The bars show the mean ± SD of three independent experiments (each with pools of five mice). No significant differences between helped and unhelped populations were found for any of the V-
; B6-CD4–/–,). In the lower graphs, the results are normalized and expressed as the percentage of the maximal response, with the dashed lines indicating the concentration at which half-maximal response is achieved (3.2 x 10–11 M). D, Lck expression in P14 CD8 T cells from B6 (black line) and B6-CD4–/– (gray shading) host mice. Dashed lines indicate staining with control Abs. Data are obtained from three to five individual mice examined on day 8 (effector) and >day 45 p.i. (memory) and are representative of three to four separate experiments.
