Differentiation-dependent functional and epigenetic landscapes for cytokine genes in virus-specific CD8+ T cells Alice E. Dentona, Brendan E. Russa, Peter C. Dohertya,b,1, Sudha Raoc, and Stephen J. Turnera,1 a
Department of Microbiology and Immunology, University of Melbourne, Parkville 3010, Australia; bDepartment of Immunology, St Jude Children’s Research Hospital, Memphis, TN 38105; and cFaculty of Applied Science, Canberra University, Canberra 2601, Australia Contributed by Peter C. Doherty, July 31, 2011 (sent for review June 28, 2011)
Although the simultaneous engagement of multiple effector mechanisms is thought to characterize optimal CD8+ T-cell immunity and facilitate pathogen clearance, the differentiation pathways leading to the acquisition and maintenance of such polyfunctional activity are not well understood. Division-dependent profiles of effector molecule expression for virus-specific T cells are analyzed here by using a combination of carboxyfluorescein succinimidyl ester dilution and intracellular cytokine staining subsequent to T-cell receptor ligation. The experiments show that, although the majority of naive CD8+ T-cell precursors are preprogrammed to produce TNF-α soon after stimulation and a proportion make both TNF-α and IL-2, the progressive acquisition of IFN-γ expression depends on continued lymphocyte proliferation. Furthermore, the extensive division characteristic of differentiation to peak effector activity is associated with the progressive dominance of IFN-γ and the concomitant loss of polyfunctional cytokine production, although this is not apparent for long-term CD8+ T-cell memory. Such proliferation-dependent variation in cytokine production appears tied to the epigenetic signatures within the ifnG and tnfA proximal promoters. Specifically, those cytokine gene loci that are rapidly expressed following antigen stimulation at different stages of T-cell differentiation can be shown (by ChIP) to have permissive epigenetic and RNA polymerase II docking signatures. Thus, the dynamic changes in cytokine profiles for naive, effector, and memory T cells are underpinned by specific epigenetic landscapes that regulate responsiveness following T-cell receptor ligation. cytotoxic T cell
| influenza | naive T cell | histone
T
he determining characteristic of adaptive T-cell immunity is the capacity to acquire lineage-specific functions that mediate acute pathogen elimination (i.e., effector phase) while at the same time promoting the establishment of long-lived, readily reinduced cytotoxic T lymphocyte (CTL) memory. Naive, effector, and memory CTLs are characterized by distinct transcriptional and functional profiles (1, 2). For example, naive CD8+ CTLps are generally considered to be functionally inert before T-cell receptor (TCR) ligation, then proliferation leads to the acquisition of differentiated, lineage-specific, effector functions (1, 3). In contrast, memory T cells are known to acquire effector activity soon after reinfection, reflecting the induction of molecular pathways that may not require further cell division (2, 4– 6). Although this rapid recall of functional capacity is a key characteristic of CD8+ T-cell memory that promotes more rapid pathogen clearance, the molecular programs that facilitates such enhanced responsiveness are far from defined. Following TCR ligation, naive, peptide-MHC class I–specific (pMHCI) CTL precursors (CTLps) acquire the capacity to secrete proinflammatory cytokines and to form cytoplasmic granules containing serine esterases (i.e., granzymes) and the poreforming protein, perforin—the inducers of apoptotic cell death in virus-infected target cells. The progressive expression of IFN-γ (7), perforin (8), and various granzymes (8–10) is clearly linked to ongoing lymphocyte proliferation, supporting the notion that 15306e15311 | PNAS | September 13, 2011 | vol. 108 | no. 37
extended CTL division directly correlates with more potent Tcell effector function (8, 11). In agreement with this, multiparameter, single-cell analysis of granzyme and perforin profiles for virus-specific CTLs has established that, early on, there is substantial heterogeneity that decreases as more molecules are expressed with further cycling (12–14). In addition, functional profiling of effector and memory CTLs induced after primary influenza A virus infection of C57BL/6J (B6) mice demonstrated that profiles of intracellular cytokine expression (both mRNA and induced protein) followed a strict hierarchy and most likely reflected sequential acquisition of multiple effector functions as a result of progressive differentiation following activation (15). Of particular interest, and in contradiction to the accepted paradigm, it has recently been established that naive CTLps secrete TNF-α in response to TCR-mediated signals (16, 17). The molecular basis of multiple effector gene expression during CTL differentiation is therefore far from clear. The incorporation of covalently modified histone proteins within specific gene promoter regions is a key regulatory mechanism for activating, or repressing, gene transcription (18). For example, acetylation of the histone H3 at lysine 9 (H3K9ac) within gene promoters is considered a correlate of transcriptional activation (19). The same is true (20) for trimethylation of histone 3 at lysine 4 (H3K4me3). By contrast, trimethylation of lysine 27 (H3K27me3) is a correlate of transcriptional repression (19). Several studies have demonstrated that the acquisition of CTL function is associated with increases in the level of histone acetylation for effector gene promoters (20–24). More recent analysis has further shown that the extent of histone methylation can also play a role in effector gene expression for naive and memory CTL populations (25, 26). Although the combinatorial nature of specific histone modifications within specific gene loci is clearly a key driver for regulating gene transcription, just how this balance of histone acetylation/methylation contributes to the acquisition and maintenance of virus-specific CTL functional capacity requires clarification. The present analysis uses adoptive transfer of naive, carboxyfluorescein succinimidyl ester (CFSE)-labeled, TCR-transgenic CD44loCD62LhiCD8+ CTLps into virus-infected mice to track the acquisition of multiple cytokine function during the course of progressive, pMHCI-induced clonal expansion. We demonstrate here that there is a dynamic relationship between the expression of effector cytokines and cell division, with cytokine polyfunctionality being more a characteristic of less divided populations. Moreover, we identify key epigenetic signatures that determine the inverse relationship between TNF-α and IFN-γ
Author contributions: A.E.D., B.E.R., S.R., and S.J.T. designed research; A.E.D. and B.E.R. performed research; S.R. contributed new reagents/analytic tools; A.E.D., B.E.R., P.C.D., and S.J.T. analyzed data; and A.E.D., B.E.R., P.C.D., S.R., and S.J.T. wrote the paper. The authors declare no conflict of interest. 1
To whom correspondence may be addressed. E-mail:
[email protected] or pcd@ unimelb.edu.au.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1112520108/-/DCSupplemental.
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Results Naive OT-I CTLs Rapidly Express TNF-α but Not IFN-γ Following Stimulation. Recent experiments have demonstrated that naive
CD8+ T-cell populations can produce TNF-α immediately following TCR-mediated stimulation, before the initiation of cell division (16, 17). By using a standard ICS assay read at 5 h after stimulation with cognate peptide, this somewhat surprising conclusion was confirmed for naive, TCR-transgenic, CD44loCD62Lhi OT-I T cells taken 24 h after adoptive transfer into uninfected B6 recipients. The OT-I T cells were first “parked” to replicate the physiological situation for naive CTLps in normal, unifected mice. As found previously for polyclonal CD44loCD62Lhi CTLps from WT mice (16, 17), most of the reisolated, naive OT-I T cells produced TNF-α, and some made both TNF-α and IL-2 (Fig. 1 A and B), but few were positive for IFN-γ (Fig. 1A). Then, quantitative real time-PCR showed that naive OT-I CTLps had at least 50 times greater relative levels of pre-existing TNF-α versus IFN-γ mRNA (Fig. 1C). Such pre-existing TNF-α mRNA levels provide a mechanistic basis for the selective up-regulation of TNF-α (compared with IFN-γ) mRNA and protein levels within 5 h of stimulating naive, resting OT-I T cells with peptide (Fig. 1 A and D). Thus, the majority of naive OT-I CTLps have a capacity for immediate TNF-α production and a proportion can also make IL-2, but not IFN-γ or any of the molecules (i.e., granzymes/perforin) that mediate cytolysis (8). Cell Division-Linked Acquisition of IFN-γ and Loss of TNF-α. Naive, CFSE-labeled OT-I T cells were adoptively transferred into congenic B6 hosts that had been infected intranasally (i.n.) 2 d previously with the A/HKx31-OVA virus. The OT-I cells were then recovered at 60 h after transfer, stimulated for 5 h directly ex vivo with the OVA SIINFEKL peptide, and stained for intracellular cytokine production. As shown previously (27), no cytokine staining was detected in the absence of peptide stimulation (Fig. 2 A and B). Consistent with earlier reports (7, 28), the acquisition of IFN-γ expression by these OT-I CTLps was
Fig. 1. Naive OT-I T cells rapidly express TNF-α and IL-2, but not IFN-γ, after peptide stimulation. (A and B) Naive (CD44loCD62Lhi) OT-I T cells were taken 24 h after adoptive transfer into uninfected B6 recipients and stimulated with 1 μM SIINFEKL peptide for 5 h in the presence of 5 μg/mL brefeldin. Coexpression of intracellular cytokines is illustrated for TNF-α and IFN-γ (A) or TNF-α and IL-2 (B). Data are representative of five independent experiments. (C and D) IFN-γ and TNF-α mRNA levels within resting naive (CD44loCD62Lhi) OT-I CTLs (C) or after 5 h of peptide stimulation (D) by quantitative real-time PCR. Shown is the relative increase in mRNA levels for TNF-α compared with IFN-γ mRNA levels in naive resting OT-I CTLs.
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clearly time-dependent (Fig. 2A). In contrast, there was immediate TNF-α production (Fig. 2B). The CFSE-labeled OT-I cells were then gated on the basis of division number (Fig. 2C) and analyzed for TNF-α/IFN-γ coexpression (Fig. 2D). At low cycle number, the IFN-γ+ OT-I cells emerged within the TNF-α+ population, to increase in prevalence with progressive cycling (Fig. 2D). The frequency of polyfunctional (IFN-γ+TNF-α+) OT-I CTLs continued to increase, peaking at approximately division 4 to 5. The emergence of an IFN-γ+TNFα− set was observed at division 2, also peaking at division 4 to 5 (Fig. 2D). Surprisingly, the acquisition of IL-2 production by OT-I cells did not show a strong link to division (Fig. S1). Approximately 40% of cytokine+ OT-I cells in the spleen were IL-2+ within one division, compared with only 20% of naive OT-I, and the expression of IL-2 was maintained by 35% to 40% of activated OT-I throughout subsequent divisions. Thus, in contrast to earlier suggestions (15), the coexpression of multiple cytokines within virus-specific CTL populations likely reflects the continued presence of T cells that have not undergone extensive proliferation. Relating Cytokine Acquisition to Differential T-Cell Expansion.
Graded numbers of naive, OT-I cells (102, 104, 105, and 106) were adoptively transferred into naive B6 mice, which were infected i.n. 24 h later with 104 pfu of the x31-OVA virus. The fold expansion of lymphocytes in vivo was then determined for spleen populations as the ratio of the OT-I CTL counts on d 10 after infection relative to the input cell number, assuming a 10% engraftment (29). As expected, altering CTLp availability directly influenced the extent of antigen-induced expansion (Fig. 3A). Transferring 106 OT-I T cells led to a 10-fold increase in numbers. Then, between 106 and 104, every 10-fold decrease in the cell dose was associated with 10 times greater expansion (Fig. 3A). It thus seems that these adoptively transferred OT-I T cells proliferate until they reach an appropriate threshold level in spleen: the lower the input cell count, the greater the subsequent cell division. The extent of cytokine polyfunctionality for these adoptively transferred OT-I T cells correlated inversely with the degree of
Fig. 2. Gain of IFN-γ production in vivo is tightly coupled to division. CFSElabeled Ly5.1+OT-I cells (106) were injected into B6 mice 2 d after infection with 104 pfu of x31-OVA. Profiles of CFSE dilution and cytokine production following peptide stimulation for IFN-γ (A) or TNF-α (B) are shown for splenic OT-I cells 60 h after transfer. Cellular division was measured by CFSE dilution (C) and the coexpression of IFN-γ and TNF-α was determined for CTLs from the spleen and compared with that for naive OT-I cells from uninfected recipients (D). Data shown are mean ± SEM (n = 3) and are representative of three independent experiments.
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expression during virus-specific T-cell differentiation. Taken together, these results establish, and provide a mechanistic basis for, the diversity of functional outcomes following antigen recognition by naive, effector, and memory T cells.
Fig. 3. Development of the TNF-α−IFN-γ+ phenotype is dependent on in vivo expansion. Ly5.1+OT-I cells (102–106) were adoptively transferred into B6 mice 1 d before i.n. infection with 104 pfu x31-OVA. Ten days later, splenic OT-I cells were analyzed for expansion (A), the coexpression of IFN-γ and TNF-α within the cytokine-producing population (B), and the mean fluorescence intensity (MFI) of cytokine staining (C) for OT-I cells derived from the MLN and spleen. Data shown are mean ± SEM (n = 3–7) and are representative of three independent experiments. Significance tested with oneway ANOVA test with Tukey posttest (***P < 0.001) (A) or Student t test (*P < 0.05 and **P < 0.01) (C). OT-I expansion and effector function are influenced by antigen dose in vivo. Ly5.1+OT-I cells (104 or 106) were transferred into naive B6 mice and infected i.n. with 104 pfu x31-OVA or 102 pfu x31OVA (in x31-WT) 24 h later. The OT-I cells were isolated 60 h after transfer, and the fold expansion (D) and coexpression of IFN-γ and TNF-α (E) determined. Data are shown as mean ± SEM (n = 4).
expansion following x31-OVA virus challenge (Fig. 3B). Injecting large numbers (106) of naive OT-I CTLps resulted in more TNFα and IFN-γ coexpression following pMHCI stimulation, compared with the values for those given fewer (105 or 104) T cells (Fig. 3B). Single IFN-γ producers were observed predominantly in mice seeded with the lowest numbers (102 and 104) of naive CTLps (Fig. 3B), further establishing that continued OT-I T-cell expansion leads to diminished TNF-α production. Furthermore, the extensively divided CTL progeny from the “low-dose” (104) CTLp transfer produced less IFN-γ and TNF-α on a per-cell basis compared with those derived from the high CTLp frequency (106) set (Fig. 3C). Overall, these data establish that sustained CTL expansion results in effector populations that are less polyfunctional and have a diminished capacity to secrete effector cytokines following in vitro restimulation with peptide. To more precisely determine the role of antigen dose in the maintenance of multiple CTL effector functions, 104 OT-I cells were adoptively transferred into naive B6 hosts, which were then infected with 104 pfu of x31-OVA or 102 pfu x31-OVA mixed with x31-WT to give an equivalent (104 pfu) total viral dose. Controls were given 106 OT-I T cells and infected with 104 pfu of x31-OVA (Fig. S2). As before, the fold expansion of naive OT-I CTLps for mice given 104 pfu x31-OVA was inversely related to the input cell count (Fig. 3D), with TNF-α/ IFN-γ multifunctionality being best maintained in the mice given 106 (vs. 104) OT-I CTLps (Fig. 3E). The transferred T cells recovered from mice that were given low OT-I CTLp numbers (104) and challenged with a low antigen dose (102 pfu x31-OVA), but equivalent amount of influenza virus (+104 x31-WT), divided less (Fig. 3D) and maintained a greater level of IFN-γ/TNF-α coexpression (Fig. 3E). These experiments demonstrate that specific antigen load, and not necessarily inflammation (as a function of total virus exposure), can influence the degree of expansion and the subsequent functional capacity of virus-specific CTL effectors. 15308 | www.pnas.org/cgi/doi/10.1073/pnas.1112520108
We have shown previously that the effector and memory influenza-specific CTL sets exhibit differences in IFN-γ/TNF-α coexpression (15, 30). For instance, DbNP366-specific CTLs isolated at the peak of the response are comprised of distinct IFNγ+/TNF-α− and IFN-γ+/TNF-α+ subsets. In contrast, DbNP366specific memory CTLs tend to express an IFN-γ+/TNF-α+ phenotype (15). To examine whether comparable functional profiles were characteristic of effector and memory OT-I–specific CTLs, 104 OT-I cells were adoptively transferred into naive B6 hosts, which were then left uninfected or infected i.n. with 104 pfu of x31-OVA. Recovered naive, effector (d9), and memory (d120) OT-I populations were then analyzed for TNF-α/IFN-γ coexpression (Fig. S2). Although the naive OT-I CTLps only expressed TNF-α upon stimulation, effector (d9) and memory (d120) OT-I CTLs displayed differential IFN-γ/TNF-α coexpression, reminiscent of the DbNP366-specific CTL induced after virus infection (15). Taken together, these data demonstrate that the capacity of naive, effector, and memory CTLs to express IFN-γ and/or TNF-α is a reflection of a given differentiation state that likely reflect differences in transcriptional potential. Epigenetic Signatures for IFN-γ and TNF-α in Progressive CTL Differentiation States. Given that specific histone modifications
within gene promoter regions promote, or suppress, gene transcriptional potential (18), the present analysis showing varied spectra of cytokine inducibility at progressive stages of OT-I CTL differentiation provides a defined analytical system for dissecting such epigenetic control. To examine this, we compared the incorporation of H3K9ac, H3K4me3, and H3K27me3 modifications within the 200-bp promoter region upstream of the ifnG and tnfA transcriptional start sites (TSSs; Figs. S3 and S4), for naive, effector, and memory OT-I CTL populations. Nucleosome evacuation from within proximal gene promoters results in greater accessibility to key regulatory sequences, enabling subsequent gene activation (31). Such changes can be probed by determining the presence of the histone 3 (H3) protein within specific promoter regions, independent of specific histone modifications. Following activation, reduced H3 incorporation was observed at the proximal ifnG promoter region of effector, compared with naive, OT-I T cells (Fig. 4A). The loss of H3 is consistent with observed patterns of chromatin remodeling and increased accessibility at other CTL effector gene loci (20). Furthermore, this profile of H3 loss was also apparent for OT-I memory CTLs, indicating that any chromatin remodeling that occurs during the initial stage of effector CTL differentiation is maintained into the memory phase. In contrast to the dramatic changes observed for the ifnG promoter, the tnfA promoter showed a small decrease in H3 incorporation between naive and effector OT-I T cells, although further H3 loss at the tnfA promoter was characteristic of the OT-I memory set. Taken together, these data indicate that the activation-induced loss of H3 within the proximal promoter of effector loci following activation is likely to be a key factor in maintaining readily recalled functional capacity for memory CTL populations. Earlier analysis of granzyme expression profiles established that increased chromatin accessibility is necessary, but not sufficient, for the acquisition of lineage-specific effector gene functions within activated CTL populations (20). Importantly, the capacity to express lineage-specific effector genes correlates strongly with increased enrichment of the active epigenetic marks, H3K9ac and H3K4me3, and reduced levels of the repressive mark, H3K27me3 (20, 26, 32). Compared with naive CTLps, a pattern of increased levels of active H3K9ac and H3K4me3 modifications and reciprocal loss of the H3K27me3 repressive mark was also found for the proximal ifnG promoter of effector OT-I CTLs (Fig. 4B). Enrichment of these permissive epigenetic signatures extended a further 600 bp upstream of the TSS within effector CTLs, indicating extensive chromatin remodeling across the promoter region (Fig. S5A). In addition, the changes in H3K9ac, H3K4me3, and H3K27me3 enrichment observed for the naive OT-I CTLp to Denton et al.
Such loci are “poised,” with RNA pol II docking requiring histone H3 loss and removal of nucleosomes around the TSS, and the enrichment of permissive epigenetic marks like H3K9ac and H3K4me3. To determine whether the presence of permissive marks within the ifnG and tnfA loci correlated with the docking of RNA pol II, we performed ChIP by using RNA pol II-specific antibodies. As expected, RNA pol II was enriched at the tnfA, but not the ifnG, proximal promoters of naive OT-I CTLps (Fig. 5). By contrast, RNA pol II was detected at the ifnG and tnfA proximal promoters for the effector and memory OT-I CTL sets (Fig. 5). Epigenetic remodelling of the ifnG promoter during differentiation is thus required for the recruitment of RNA pol II, allowing the immediate expression of IFN-γ by effector and memory CTLs following antigen challenge. On the contrary, the tnfA promoter looks to be poised at all stages of CTL differentiation, providing a mechanistic basis for rapid TNF-α expression.
Fig. 4. Histone modifications present at the ifnG and tnfA promoters in memory OT-I cells. Sort-purified naive effector (5–10 pooled mice, day 10 after infection) and memory (20–30 pooled mice, day > 70 after infection) OT-I cells were fixed, sonicated, and subjected to ChIP with antibodies directed against H3 (A), H3K9ac (n = 2), H3K4me3 (n = 3), or H3K27me3 (n = 3) (B and C). The CHiP enrichment ratio is expressed as the extent of enrichment vs. input DNA (A) or enrichment vs. H3 ChIP (B and C) for the ifnG and tnfA set C primers only (data for n = 3).
effector CTL transition were maintained into the memory phase (Fig. 4B). In contrast to the ifnG promoter, naive OT-I CTLps showed enrichment of the permissive H3K9ac and H3K4me3 marks and lower levels of the repressive H3K27me3 modification within the tnfA proximal promoter (Fig. 4C). This epigenetic landscape is consistent with naive OT-I CTLps being capable of rapid (5 h) TNF-α production following peptide stimulation (Fig. 1). Although there was little change in the permissive H3K9ac and H3K4me3 marks at the tnfA proximal promoter for OT-I effector CTLs, a substantial increase in H3K27me3 incorporation was detected within the tnfA proximal promoter (Fig. 4C and Fig. S5). This change to a more repressive epigenetic landscape correlated with a decrease in TNF-α functional capacity for extensively divided OT-I effector CTLs (Figs. 2 and 3). Finally, OT-I memory CTLs demonstrated further enrichment of the permissive H3K9ac and H3K4me3 modifications within the tnfA proximal promoter, concomitant with a reduction in the repressive H3K27me3 modification, similar to that observed in naive OT-I CTLps (Fig. 4C).
Discussion Naive T cells are often thought of as being functionally inert in terms of specific effector activity, being required to undergo a program of proliferation and differentiation to acquire lineagespecific effector function (1, 7–9, 28). As such, current thinking suggests that extended differentiation is key to the expression of multiple effector functions, enabling more effective immune control (12, 14, 35). However, the present analysis confirms earlier, and often unappreciated, evidence that some effector pathways, specifically those leading to TNF-α (but not IFN-γ) production, are rapidly elicited following specific recognition by naive T cells before proliferation (16, 17). Polyfunctional potential (TNF-α+IFN-γ+) is then acquired within three to four divisions, but extended cycling leads to the loss of TNF-α production for a substantial set of activated CTLs, leading to a progressive decrease in functional capacity. Adoptive transfer of large numbers of TCR transgenic CD8+ T cells leads to the generation of CTL effectors that divide less and tend to coexpress IFN-γ and TNF-α. In contrast, transferring 10 to 100 fold less CTLps resulted in extended proliferation and the loss of TNF-α expression. When antigen dose but not inflammation was limited, maintenance of multiple effector mechanisms at the peak of the CD8+ T-cell response correlated with limited virus-specific CTL expansion. These data are supported by comparison with the results of Zehn and colleagues (36), who demonstrated that limited proliferation of OT-I CTL activated with “low-affinity” SIINFEKL variants was associated with coexpression of IFN-γ and TNF-α. Although these data confirm earlier reports that progressive differentiation is a key factor shaping phenotypic and functional heterogeneity for pathogen-specific CTL responses (11, 37), they provide the additional insight that polyfunctional cytokine production is associated with a limited versus greater degree of expansion. This is in contrast to the acquisition of cytolytic gene expression (perforin and the granzymes), whereby continued cell division leads to a broader spectrum of effector gene expression (8, 12, 14).
the epigenetic landscape underpin the varied capacity of naive, effector, and memory OT-I CTLs to transcribe and express IFNγ and/or TNF-α following TCR ligation. This is particularly true for memory CTLs, which demonstrate greater multifunctional capacity than naive or effector T cells (Figs. 2 and 3) (15). With many inducible loci, a key component of the transcriptional machinery, RNA polymerase II (RNA pol II), is already docked onto proximal promoters to enable rapid transcription (33, 34). Denton et al.
Fig. 5. Docking of RNA pol II at the TSS of effector genes. ChIP for RNA pol II binding at the proximal promoter of the ifnG (white bars) or tnfA (black bars) genes was performed as in Fig. 4. The ChIP enrichment ratio is expressed as enrichment vs. input DNA for the ifnG and tnfA set C primers only. Data shown are for n = 3.
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Permissive Epigenetic Signatures and Increased RNA Polymerase II Docking. The analyses detailed thus far suggest that changes in
Perhaps down-regulation of key inflammatory cytokines at the peak of the CTL response serves to limit the severity of immunopathology, especially in sensitive tissues such as the lung. To further examine the role of inflammation in driving CTL differentiation and effector function, it could be of interest to modulate viral replication by antiviral therapy, or to activate naive OT-I CTLs in the presence of graded doses of inflammatory mediators such as CpG or poly I:C. Such approaches may allow us to discriminate further between antigen load versus inflammation as drivers of CTL effector differentiation and function. The present analysis also provides a basis for the further dissection of the observed differences in cytokine production characteristic of the various influenza A virus, pMHCI-specific CTL responses in B6 mice. Despite being induced by the same infection, CTL responses to pMHCI epitopes derived from the nucleoprotein (NP366–374, ASNENMETM, binds H2-Db) and the acid polymerase (PA224–233, SSLENFRAYV, binds H2-Db) display different cytokine expression profiles. While most DbPA224specific CTLs produce both TNFα+ and IFN-γ+ following restimulation, only half the DbNP366-specific CTLs have the same phenotype, with the remainder being single IFN-γ producers (15, 30). The broader functional profile for the DbPA224specific CTL set was initially attributed to greater TCR/pMHCI avidity (15). However, this correlation between high TCR avidity and multifunctional cytokine production does not extend to other influenza-specific CTL responses. For example, dual expression of IFN-γ/TNF-α was equivalent for DbPA224-specific (high avidity) T cells and CTLs directed against the basic polymerase subunit 1-F2 epitope (PB1-F262–71, LSLRNPILV, binds H-2Db), despite the latter having a low TCR/pMHCI avidity profile (38). Subsequent enumeration of naive CTLp frequencies demonstrated a hierarchy whereby the required degree of expansion to reach the known magnitudes of CTL response following virus challenge correlated inversely with multifunctional cytokine potential. Compared with the naive CTLp sets specific for DbPA224 and DbPB1-F262, the DbNP366-specific population is smaller by a factor of at least three, but expands to a much greater extent after infection and loses multifunctional cytokine production (39). In contrast, both the DbPA224- and DbPB1F262-specific CTL repertoires start with a higher precursor frequency, proliferate less following antigen challenge, and tend to maintain an IFN-γ+TNF+ phenotype. Taken together with the findings presented here, it seems that IFN-γ/TNF-α coexpression by virus-specific CD8+ CTLs is—setting the subsequent memory phase aside—a characteristic of less differentiated cells present early during infection. It will be of interest to determine whether this extends to other cytokines/chemokines, to provide further insights into how multifunctional capacity is regulated within virus-specific CTL populations. The present analysis establishes that the capacity of naive, effector, and memory T cells to make IFN-γ and TNF-α is directly linked to the presence of defined epigenetic signatures within the proximal promoters. For example, the tnfA proximal promoter of naive OT-I cells has an overall permissive epigenetic landscape (increased chromatin accessibility, enrichment for H3K4me3/H3K9ac, and lack of H3K27me3), compared with the generally repressive epigenetic signature for the ifnG promoter (closed chromatin structure, lack of H3K4me3/H3K9ac, and enrichment of H3K27me3). Of note, the permissive epigenetic signature within the tnfA promoter in place before cellular division. A recent study has demonstrated that incorporation of the H3.3 variant occurs within permissive promoters before division, carrying with it permissive epigenetic modifications (40). Thus, rapid induction of TNF within naive T cells before division can be thought of as a consequence of active epigenetic signatures carried by H3.3 nucleosomes, and then subsequent assembly of a partial basal transcription complex (40). The ifnG promoter region undergoes significant remodeling in effector CTLs, becoming more accessible and acquiring a permissive epigenetic signature. The fact that the necessary chro15310 | www.pnas.org/cgi/doi/10.1073/pnas.1112520108
matin remodeling events required for the acquisition of effector gene expression take time to eventuate presumably explains the link between continued cell division and the acquisition of lineagespecific functional capacity within recently activated T cells (7, 8, 28). Although extended effector T-cell differentiation led to permissive epigenetic marks being deposited within the ifnG promoter to allow IFN-γ expression, the progression to a more repressive tnfA epigenetic signature shows that, for effector CTLs, distinct regulatory mechanisms act at these two cytokine gene loci. Although the precise mechanism is not clear, a likely explanation is that transcription factors determine distinct epigenetic processes at different loci within the same activated CTL. For example, lineage commitment of CD4+ TH2 T cells involves up-regulation of the transcription factor GATA-3, driving the expression of IL-4 but shutting down IFN-γ by altering the local epigenetic landscapes within the respective gene loci (41). It is thus of considerable interest to determine which, if any, transcription factors are binding to the tnfA promoter during effector CTL differentiation, and whether they can act as transcriptional repressors by altering the epigenetic landscape. A number of studies have shown that the presence of permissive epigenetic signatures, including the enrichment of H3K4me3 and H3K9ac, together with the recruitment of RNA pol II at proximal promoters is associated with genes that are poised for transcriptional activity (33, 42, 43). This epigenetic configuration is particularly characteristic of readily inducible (or poised) genes: RNA pol II seems to be “on hold” at the TSS so that, subsequent to the TCR ligation, the polymerase is released, allowing rapid gene expression (34). A cardinal feature of memory T cells is their capacity to elicit effector function soon after further antigen stimulation, without the need for extended differentiation (2, 4, 10). This capacity for rapid responsiveness appears to be underpinned by changes in the epigenetic landscape within various gene promoter regions (24, 26). Moreover, it has recently been suggested that the docking of RNA pol II is key to enabling the rapid transcription of effector genes within memory CTL populations (26). As the expression of effector molecules requires induction via the TCR (27), it seems that the immediate, differential expression of cytokines by naive, effector, and memory CD8+ T cells reflects that defined arrays of histone modifications provide permissive epigenetic signatures that allow RNA pol II to dock at the TSS, with subsequent release of RNA Pol II and the induction of gene transcription following the receipt of specific TCR-mediated signals. Treatment of activated memory CD8+ T cells with a histone acetyltransferase inhibitor has been shown to result in diminished H3K9ac expression within the granzyme B promoter and an associated decrease in granzyme B levels (22). It will be of particular interest to test whether specifically altering the epigenetic modifications within naive, effector, and memory CTLs by the use of pharmacological agents, such as histone methyltransferase inhibitors, will alter the RNA pol II docking, and subsequent transcriptional potential, of the TNF-α and IFN-γ genes. Materials and Methods Animals, Viruses, and Infection. Ly5.2+ C57BL/6J (B6) and congenic Ly5.1+ OT-I mice were bred and housed under specific pathogen-free conditions in the Department of Microbiology and Immunology Animal Facility at the University of Melbourne. For infection, mice were anesthetized and infected i.n. with 104 pfu of recombinant A/HKx31 virus engineered to express the OVA257–264 peptide (x31-OVA) in the neuraminidase stalk (44). All experiments were conducted according to approval obtained from the institutional animal ethics committee. CFSE Labeling, Adoptive Transfer, Tissue Sampling, and Intracellular Cytokine Staining. Pooled lymph nodes (107 cells/mL in sort buffer) from naive female OT-I mice were labeled with 5 μM CFSE (Sigma) for 10 min at 37 °C. Cells were washed in media containing 10% FCS and resuspended in PBS solution at the desired number of OT-I cells per 200 μL for i.v. injection via the tail vein 48 h after infection with x31-OVA, and lymphocytes were isolated from the spleen at 60 h after transfer. For cell sorting, lymphocyte prepa-
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Fifty nanograms equivalent of RNA was assayed by using TaqMan Gene MGB primer/probe sets for TNF-α and IFN-γ and the mitochondrial ribosomal protein, L32, as an internal standard. Samples were run on a Bio-Rad iQ5 real-time PCR detection system, and Ct values were calculated by using the iQ5 real-time PCR analysis software and subsequently analyzed for change in expression relative to the L32 control. ChIP. ChIP was carried out as previously described (20). The primer pair sets used to probe the IFN-γ and TNF-α proximal promoters are outlined in Table S1. Each primer pair was subjected to PCR with serially diluted, sonicated genomic DNA as template. The efficiency of all primer sets was approximately equivalent (90–105%), as determined by iQ5 software (Figs. S3 and S4).
RNA Extraction and Real-Time PCR. After bulk sorting, naive, effector, or memory (CD8+Ly5.1+) OT-I cells (0.9 × 106) were lysed in 1 mL TRIzol reagent (Invitrogen) followed by chloroform extraction and isopropanol precipitation. RNA (200–500 ng) was reverse-transcribed into cDNA by using the Omniscript RT kit (Qiagen) according to the manufacturer’s instructions.
ACKNOWLEDGMENTS. This work was supported by Australian National Health and Medical Research Council (NHMRC) Program Grant 5671222 (to P.C.D. and S.J.T.), NHMRC Project Grant 1003131 (to S.J.T. and S.R.), National Institutes of Health Grant AI70251 (to P.C.D.), an NHMRC Dora Lush Postgraduate Award (to A.E.D.), and a Pfizer Senior Research Fellowship (to S.J.T.); and by the American Lebanese Syrian Associated Charities.
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IMMUNOLOGY
rations (107/mL) were resuspended in PBS solution/0.1% FCS and stained with anti-CD8α–peridinin-chlorophyll protein–Cy5.5, anti-CD45.1–phycoerthyrin, CD62L–allophycocyanin, and CD44–FITC (to detect OT-I cells). Naive (CD44loCD62Lhi), effector (CD44hi, d10 after infection), and memory (CD44hiCD62Llo, day 60 after infection) OT-I T cells (CD8+Ly5.1+) were sorted by using a FACSAria sorter for subsequent RNA extraction or ChIP analysis. For measurement of cytokine production, lymphocytes preparations were stimulated for 5 h with 1 μM of SIINFEKL in the presence of GolgiPlug (BD Biosciences) and 25 U/mL IL-2. Cells were stained with anti-CD8α and anti-CD45.1 for analysis of transferred OT-I cells before fixation and permeabilization by using the Cytofix/Cytoperm kit (BD Biosciences), and subsequent staining with a combination of anti–IFN-γ–FITC or -PECy7, anti–IL-2– phycoerthyrin, and anti–TNF-α–allophycocyanin. Samples were then analyzed by using a FACSCanto and FlowJo software.
