Human Molecular Genetics

Human Molecular Genetics 2005 14(Review Issue 1):R41-R46; doi:10.1093/hmg/ddi115

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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org

Epigenetic control in the immune response

Steven L. Reiner^*

Abramson Family Cancer Research Institute and Department of Medicine, Division of Infectious Diseases, University of Pennsylvania, Philadelphia, PA 19104, USA

^* To whom correspondence should be addressed at: University of Pennsylvania, Building BRB II/III, Room 414, 421 Curie Boulevard, Philadelphia, PA 19104, USA. Tel: +1 2157465536; Fax: +1 2157465525; Email: sreiner{at}mail.med.upenn.edu

Received January 23, 2005; Accepted February 23, 2005

	ABSTRACT

TOP ABSTRACT INTRODUCTION HOW HELPER T CELLS... DEVELOPMENTAL GENETICS AND... CONDITIONAL ROLES OF ACTIVATORS:... ORGANIZATION AND MEMORIZATION OF... TURNING POTENTIALITY OFF CONCLUDING REMARKS REFERENCES

 
Helper T cells engaged in an immune response confront a prevalent challenge for developmentally regulated gene expression: How does a cell give rise to daughter cells with different fates? Additionally, lymphocyte function is intimately associated with the processes of cell division and migration. This imposes an additional burden for daughter cells, to remember inductive events from which they are temporally and spatially removed. An emerging view is that helper T cells use epigenetic mechanisms tied to the structure of chromatin and its covalent modifications to achieve at least two important features of their programed gene expression. Epigenetic effects organize the ability of signal transduction pathways to generate a restricted set of progeny from a multi-potent progenitor. In addition, epigenetic effects seem to allow dividing cells to memorize, or imprint, signaling events that occurred earlier in their development. Beyond helper T cells, the use of epigenetic effects is emerging as a common strategy in development and function of the mammalian immune system, suggesting that epigenetic effects may play a more prominent role in metazoan cell differentiation than previously appreciated. Lymphocytes are, thus, becoming a tractable system for genetic and biochemical dissection of the ways in which the genome is embedded with regulatory information to achieve developmental complexity.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION HOW HELPER T CELLS... DEVELOPMENTAL GENETICS AND... CONDITIONAL ROLES OF ACTIVATORS:... ORGANIZATION AND MEMORIZATION OF... TURNING POTENTIALITY OFF CONCLUDING REMARKS REFERENCES

 
Lymphocytes develop in distinct organs such as bone marrow in the case of B cells and thymus in the case of T cells (Fig. 1). During development, each lymphocyte must re-arrange its antigen receptor genes. This will allow the cell's eventual engagement in an immune response, defense against a microbe that possesses an antigenic ligand complementary to its antigen receptor. In addition, maturing cells acquire the potential for gene expression of lineage-restricted attributes that will be useful to mobilize upon encounter with the pathogen, but that would also be harmful to the host if prematurely or constitutively elaborated.

View larger version (18K): [in this window] [in a new window]   Figure 1. Developmental sequences of T cell ontogeny and function. Committed pro-T cells (far left, white) enter the thymus expressing neither CD4 nor CD8. After re-arrangement of one chain of their antigen receptor, they undergo massive proliferation and express both CD4 and CD8. At this stage, the other chain of the antigen receptor is re-arranged. Subsequently, one or the other of the two lineage markers (CD4 or CD8) is down-regulated in a non-mitotic transition. The repression of the Cd4 gene in CD4–CD8– cells is reversible. In contrast, the transition from CD4+CD8+ to mature CD8+ cell is characterized by heritable silencing of the Cd4 gene. CD4+ (shown) and CD8+ (not shown) thymocytes emigrate to peripheral lymphoid organs. A naive CD4+ T cell (middle, white) proliferates and its progeny undergo further (‘effector’) differentiation in response to pathogens. The mature progeny are categorized, on the basis of their secretion of non-overlapping patterns of cytokines such as IFN- (Th1 cell) or IL-4, IL-13 and IL-5 (Th2 cell).

 
The developing B and T lymphocyte is, in one sense, mature when it exits the bone marrow and thymus, respectively (Fig. 1). It is ready for ‘action’, albeit not actively engaged. However, in many respects, it is still developmentally immature. Prior to exposure to its antigen, the cell is referred to as ‘naive’. When the naive cell encounters its microbial antigen, it undergoes division and further differentiation. Its daughters will always be clonally recognizable because of the inheritance of specific recombination in the antigen receptor loci. However, they will differ markedly from the naive cell with regard to gene expression of key regulators of the immune response.

The sequence of development occurring in an immune response is referred to as effector differentiation, because it involves transcriptional induction of molecules that mediate the clearance of pathogens (Fig. 1). This review will focus on the problem of effector differentiation of helper T cells, because it deals with issues prevalent throughout metazoan development: how can a cell selectively limit or expand its potential gene activity to give rise to daughter cells with different fates and, later, how do these daughters remember the prior gene induction events during the course of their subsequent cell division.

	HOW HELPER T CELLS MAKE DECISIONS

TOP ABSTRACT INTRODUCTION HOW HELPER T CELLS... DEVELOPMENTAL GENETICS AND... CONDITIONAL ROLES OF ACTIVATORS:... ORGANIZATION AND MEMORIZATION OF... TURNING POTENTIALITY OFF CONCLUDING REMARKS REFERENCES

 
Upon encounter with a pathogen, a naive helper T cell undergoes cell division and further differentiation (reviewed in 1–5). The maturing progeny can choose to become Th1 cells, which express substantial amounts of IFN- but not interleukins 4, 13 and 5. In contrast, they can choose to become Th2 cells, which express substantial amounts of interleukins 4, 13 and 5 but not IFN- (Fig. 1). Th1 cells orchestrate the immune defenses led by phagocytic cells against intracellular pathogens, whereas Th2 cells regulate the non-phagocytic or humoral defenses involved in elimination of extracellular microbes. It is believed that each antigen-specific cell is multi-potent, capable of giving rise to a number of distinct progeny, depending on the nature of the immune response. The proper choice of Th1 or Th2 predominance can determine the outcome of many infectious diseases.

It has been suggested that helper T cell effector differentiation is linked to the process of cell division (6–8). In contrast to the non-mitotic decision of thymocytes that ultimately choose to down-regulate either CD4 or CD8 (Fig. 1), a naive T cell, itself, seems not truly able to differentiate into a mature Th1 or Th2 cell. Instead, the naive cell receives information that leads to a pattern of successive gene expression changes in its daughter, granddaughter and even later generation cells. The programed alteration and inheritance of gene expression changes from parent to daughter cell have been investigated intensively in the last several years. An emerging view of this process is that it is highly regulated by extrinsic signaling pathways and self-reinforcing gene regulatory networks such as the induction of transcription factor cascades. However, as important as the orchestrated appearance of lineage-specific transcription factors are epigenetic factors. This review will focus on why it seems that the way in which chromatin is configured, re-configured and stabilized during cell division is critical for the proper temporal and spatial organization of the important developmental process known as helper T cell effector differentiation.

	DEVELOPMENTAL GENETICS AND EPIGENETICS OF THE IMMUNE RESPONSE

TOP ABSTRACT INTRODUCTION HOW HELPER T CELLS... DEVELOPMENTAL GENETICS AND... CONDITIONAL ROLES OF ACTIVATORS:... ORGANIZATION AND MEMORIZATION OF... TURNING POTENTIALITY OFF CONCLUDING REMARKS REFERENCES

 
The effector cytokine genes Il4 and Ifng appear to exist in a restrictive chromatin structure in the naive helper T cell (Fig. 2). Although the loci are repressed by chromatin and DNA methylation-based constraints, they are not completely transcriptionally silent or not as silent as they will eventually become in the forbidden mature lineages. Instead, in the newly activated progenitor T cell, there is measurable, albeit sub-maximal, transcriptional activity (9) and a mixed-lineage pattern of promoter acetylation of Il4 and Ifng (10–12). This low-level multi-lineage behavior, referred to as the poised state, appears to be independent of the extrinsic signals associated with the final restriction of more mature Th1 and Th2 cells (Fig. 2). As cells mature in the appropriate environment, more restricted patterns of transcription emerge and permissive histone modifications encompass the loci encoding the active cytokines (13–16).

View larger version (21K): [in this window] [in a new window]   Figure 2. An epigenetic model of helper T cell differentiation. The Ifng and Il4 genes exist in a restrictive structure in the progenitor (naive) cell. After it has been activated by antigen, but before it divides, there is low-level transcription (dashed line) of the cytokine genes and induction of the genes encoding the lineage-determining activators, T-bet and GATA-3. During the initial cell divisions, T-bet and GATA-3 mediate chromatin remodeling of the Ifng and Il4 genes in Th1 and Th2 cells, respectively. Th1 cells also heritably silence the gene encoding GATA-3 and further repress or silence Il4, whereas Th2 cells heritably silence the gene encoding T-bet and further repress or silence Ifng. Not shown here, but detailed generically in Figure 3, is the feature that maturation of Th1 and Th2 cells is characterized by loss of CpG methylation in the Ifng and Il4 gene bodies, respectively. This may eventually result in a state of heritable re-modeling of the cytokine genes, which no longer requires the actions of T-bet and GATA-3.

 
Once a naive helper T cell becomes activated, it also begins to express substantial amounts of two important transcriptional activators, the T-box transcription factor, T-bet, and the zinc finger-containing transcription factor, GATA-3 (17,18) (Fig. 2). T-bet (18–21) and GATA-3 (17,22–26) have been dubbed master-regulatory proteins of Th1 and Th2 differentiation, respectively, on the basis of their ability to confer lineage-specific attributes in cells of the opposing lineage. T-bet and GATA-3 have also been implicated in chromatin re-modeling of their putative target genes, Ifng (10,13,16,18,27,28) and Il4 (11,14,25,29–35), respectively. In recent years, the epigenetic landscape of these genes has been investigated at the level of DNA methylation, DNase I hypersensitivity, histone tail modifications, intrachromosomal conformational arrangement and nuclear positioning (6,10–13,15,16,31,36–42).

Il4 is part of a locus of co-regulated cytokines, which also contains Il13 and Il5. Curiously, interspersed between the small region containing Il4 and Il13 and the distant Il5 gene is the Rad50 gene. Frequently, but not always, IL-4, IL-13 and IL-5 are coordinately expressed in Th2 cells. A mechanistic basis for this may rest in the recent finding that the promoters of all three cytokine genes, although located at some distance from each other, are looped together (35,43), around a locus control region (LCR), which resides in the Rad50 gene body (14,40,44). The Rad50 promoter is excluded from the looping, perhaps explaining its ubiquitous pattern of expression. The Th2 LCR seems to be more important for expression of IL-4 and IL-13 than for IL-5, possibly indicating that (i) the former genes are more dependent on chromatin re-modeling for activation and (ii) the long-range looping facilitates coordinated transcriptional activity. The looping configuration of the Th2 cytokine cluster, moreover, is not purely lineage-specific, because naive, Th1 and Th2 cells all exhibit this conformation. Therefore, it is likely that local changes in chromatin structure mediate specificity of the transcriptionally active or silent state (14,15). Thus, the looping of this locus seems to be necessary, but not sufficient to impart activity on the Th2 cytokine cluster.

One of the major themes to emerge from study of the Ifng and Il4 loci is that cis-acting regulatory regions, both positive and negative, are frequently conserved across mammalian species and are also hypersensitive to DNase I (13,16,31,32,36,37,44,45). Currently, our view of which activators, repressors and other chromosomal proteins are bound to these regions is somewhat primitive. However, it is anticipated that such detail is not long in coming and that it will aid greatly in illuminating the dynamic nature of epigenetic alteration, which seems to be occurring during helper T cell differentiation.

	CONDITIONAL ROLES OF ACTIVATORS: EPIGENETICS REDUX

TOP ABSTRACT INTRODUCTION HOW HELPER T CELLS... DEVELOPMENTAL GENETICS AND... CONDITIONAL ROLES OF ACTIVATORS:... ORGANIZATION AND MEMORIZATION OF... TURNING POTENTIALITY OFF CONCLUDING REMARKS REFERENCES

 
T-bet seems to cooperatively induce IFN- expression in maturing Th1 cells by inducing a co-factor, HLX, a homeobox transcription factor (46,47). T-bet and HLX appear to synergistically activate the Ifng locus, which somehow leads to a heritable state of IFN- activity. Somewhat unexpectedly, this heritably permissive state may become independent of T-bet activity, because a dominant negative form of T-bet becomes progressively incapable of antagonizing transcription and permissive chromatin changes when Th1 cells mature (46). Although this has not been formally tested in mature Th1 cells using conditional gene deletion of Tbet, these data suggest that the activities necessary for inducing chromatin re-modeling and competence for gene expression are not always equivalent to the factors that maintain competence of the re-modeled state. This behavior parallels epigenetic gene silencing. There are instances in single-celled and metazoan eukaryotes where an initial repressor may be necessary to establish restriction on the activity of a gene, but later the silenced or repressed state is propagated or maintained without the initiating repressor.

An inductive epigenetic behavior has also been reported in Th2 maturation. GATA-3 has been implicated in the formation of DNase I hypersensitivity sites, transcriptionally favorable histone modifications and the repulsion of repressors linked to methylated DNA at the Il4 locus (10,11,23,29–31,35,43). Deletion of Gata3 at the onset of Th2 development prevents the transcriptional induction of IL-4, but deletion of Gata3 in more mature Th2 cells does not appear to impair the maintenance of the permissive state of IL-4 transcription (24,26).

It is, thus, presumed that the actions of T-bet and GATA-3 might be to re-model a locus to its permissive state and not necessarily to be obligatory activators of the promoters of Ifng and Il4, respectively. Curiously, mature Th1 and Th2 cells do not cease to express T-bet and GATA-3, respectively. Instead, these factors are heritably maintained and are essential for ongoing activity of some lineage-restricted gene activity. Why some gene induction becomes activator independent, or seemingly locked-in, but some remains completely activator dependent is not presently understood.

An epigenetic basis for helper T cell differentiation was initially suggested by observations that CpG demethylation and stable DNase I hypersensitivity sites were induced in the Ifng and Il4 loci as Th1 and Th2 cells, respectively, developed heritable patterns of cytokine gene expression (6,36,37,48,49). In addition, use of small molecule inhibitors of histone deacetylases and maintenance methylation caused dramatic derepression of cytokine expression in developing Th1 and Th2 cells, even compensating for a lack of differentiation-inducing signals (6). Later, investigations using cells harboring deletion of Dnmt1 (50–52), the maintenance methyltransferase gene, or Mbd2 (23), a gene which potentially links methylated DNA and condensed chromatin, revealed derepression of cytokine expression during Th1 and Th2 maturation, which supported an important role for epigenetic effects in the organization of helper T cell differentiation.

On the basis of the phenotype DNMT1- and MBD2-deficient helper T cells, it became apparent that a loss of gene silencing leads to a disorganized state of differentiation in which high level effector cytokines were temporally mis-expressed, appearing in progenitor cells rather than being delayed until the daughter generations. In addition, the organized restriction of non-overlapping cytokine profiles was perturbed (50,51). Gene silencing mutant Th1 and Th2 cells would each express IL-4 and IFN-, respectively (23). Surprisingly, this mis-expression of the forbidden cytokines is not due to disorganized transcription factor pedigrees. MBD2 deficient Th1 and Th2 cells would each polarize into cells that express only T-bet or Gata-3, respectively (23). One of the key functions of activators, such as GATA-3, was found to be displacement of MBD2-bound repressive complexes from chromatin, even prior to the occurrence of CpG demethylation (23) (Fig. 3). In this situation, the role of the activators was revealed to be a conditional one, referable to the presence of chromatin-based repression in the progenitor cell. Without such repression, it is possible to get T-bet-less IFN- induction or GATA-less IL-4 induction.

View larger version (18K): [in this window] [in a new window]   Figure 3. A speculative model linking DNA methylation to heritable gene induction. In progenitor helper T cells, the gene bodies of cytokine loci (as depicted in Fig. 2) have CpG methylation (red circles) and chromatin-based repression nucleated by methyl-binding domain proteins (MBDs). An activator, which functions to re-model chromatin, causes displacement of repressive complexes from methylated DNA at the onset of gene induction. At this point, antagonism of the activator would reverse the chromatin changes and transcriptional competence. Later, when interference with maintenance methylation has erased the methyl-CpG marks (open green circles), there is no longer a beacon to recruit MBDs and the repressive forces. At this stage, loss of activator would no longer reverse the transcriptionally permissive state.

 

	ORGANIZATION AND MEMORIZATION OF CELL FATE

TOP ABSTRACT INTRODUCTION HOW HELPER T CELLS... DEVELOPMENTAL GENETICS AND... CONDITIONAL ROLES OF ACTIVATORS:... ORGANIZATION AND MEMORIZATION OF... TURNING POTENTIALITY OFF CONCLUDING REMARKS REFERENCES

 
The dispensability of activators in maintenance of gene expression in mature cells (24,26,46) and the mis-expression of cytokine genes in the absence of activators seen in gene silencing mutants (23,50,51) may be related phenomena. T-bet and GATA-3 seem to be essential for inducing competence for expression of IFN- and IL-4 only in the presence of some facultative gene silencing in the progenitor (naive) helper T cell. If the progenitor lacks gene silencing, then effector cytokines can be expressed without activity of T-bet and GATA-3. Likewise, if the cytokine loci have undergone developmental re-modeling of their silent structure, the cytokines can also be expressed without apparent need of the activators (Fig. 3).

It is, therefore, speculated that epigenetic effects mechanistically link two cardinal features of helper T cell differentiation, organization and memorization. Facultative repression allows a progenitor to be poised for multi-potential differentiation, yet restrict gene activity to its daughter cells in a manner that is specified by extrinsic signals. In essence, epigenetic repression prevents the progenitor from adopting a fate with mixed-lineage attributes, perhaps to enforce the principle of division-of-labor, which characterizes the vertebrate immune response.

The orderly dismantling of the facultative repression, moreover, might allow daughter generations of mature Th1 and Th2 cells to remember gene induction events that occurred in the progenitor. In such a model (Fig. 3), CpG methylation serves as a beacon for repressors such as MecP1 (MBD2 plus its associated repressive complex). A lineage re-modeling activator repels the repressor and induces chromatin changes and transcriptional competence. Although key CpG residues are still methylated, the inductive process remains reversible and activator dependent. When the chromatin changes eventually interfere with maintenance methylation, the beacon for the repressors is lost (Fig. 3). At this point, the permissive state is locked-in, no longer requiring the inductive activator. Although several predictions of this model have been borne out experimentally, it remains speculative that loss of CpG methylation is the sole explanation for the heritable quality of Th1 and Th2 differentiation.

	TURNING POTENTIALITY OFF

TOP ABSTRACT INTRODUCTION HOW HELPER T CELLS... DEVELOPMENTAL GENETICS AND... CONDITIONAL ROLES OF ACTIVATORS:... ORGANIZATION AND MEMORIZATION OF... TURNING POTENTIALITY OFF CONCLUDING REMARKS REFERENCES

 
As Th1 and Th2 cells mature, the transcriptional potential of each of the poised cytokine genes in the naive T cell becomes progressively more restricted in the forbidden lineage (8,9,41,53). In developing Th1 cells, it is a simple matter to introduce GATA-3 and trans-activate Il4 and induce chromatin re-modeling of the Th2 cytokine cluster. However, in more mature Th1 cells, GATA-3 appears to work with diminishing efficiency (29). Likewise, as Th2 cells mature, it becomes more difficult to induce IFN- transcriptional activity.

Several mechanisms have been proposed to account for the deeper restrictions on the forbidden cytokine. Nuclear re-positioning and de novo CpG methylation of both the cytokine loci and the trans-activator loci have all been reported (9,41,42). One of the emerging explanations for the progressive loss of potential to induce IL-4 expression as cells enter the Th1 lineage is the existence of a silencer element downstream of the Il4 gene body (53). Although this element does not undergo apparent heterochromatin-like marking (15,53), it seems to be essential for preventing Th1 cells from expressing IL-4. Whether it is a site of active repression or a nidus for heritable silencing is not yet known.

Although heritable silencing is not completely understood in helper T cells, this mode of regulation is pivotal for an important developmental process in CD8+ T cells (54) (Fig. 1). A silencer element in the Cd4 locus is the site of reversible repression in CD4–CD8– (double negative) thymocytes, which is relieved when cells progress to the CD4+CD8+ (double positive) stage. During the double-positive stage, those cells entering the CD8+ single-positive lineage utilize the same silencer element for nucleating an irreversible silencing, which is maintained in peripheral CD8+ T cells throughout their subsequent cell division history (55–59). The precise nature of this heritable silencing is not yet understood, but should serve as a useful model for how non-permissive chromatin structure is inherited during cell division in mammals.

	CONCLUDING REMARKS

TOP ABSTRACT INTRODUCTION HOW HELPER T CELLS... DEVELOPMENTAL GENETICS AND... CONDITIONAL ROLES OF ACTIVATORS:... ORGANIZATION AND MEMORIZATION OF... TURNING POTENTIALITY OFF CONCLUDING REMARKS REFERENCES

 
The last 7 years has witnessed exciting developments in the field of epigenetic gene regulation in helper T lymphocytes. Gene silencing seems to play at least two important roles in their differentiation. As an organizing principle, it helps to regulate temporal and spatial segregation of gene expression, such that the progenitor does not commit to an undesired state of heritable, mixed-lineage gene expression, a veritable ‘Jekyll and Hyde’ fate. In addition, the way in which facultative silencing is dismantled in an orderly fashion seems to provide an imprinting mechanism for gene induction, allowing daughter cells to re-iterate an induced program of gene expression despite being temporally and spatially removed from the inductive event.

Although many details of the epigenetic processes of lymphocytes are still undefined, the level of resolution is continually improving. The importance of epigenetic effects in the immune system (3,54,60–62) adds a new dimension to the biological significance of these pathways. Epigenetic effects are not simply ways to achieve allelic heterogeneity within the same cell. Instead, or in addition, epigenetic effects seem to allow developmental signaling events to achieve an unprecedented temporal and spatial complexity, which might be crucial in the organization of much of higher metazoan life.

	ACKNOWLEDGEMENTS

 
The author is grateful to many lab members past and present and to the NIH and the Abramson Family for their support.

	REFERENCES

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