Human Molecular Genetics, 2005, Vol.

14, Review Issue 1

doi:10.1093/hmg/ddi115

R41R46

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
Received January 23, 2005; Revised and Accepted February 23, 2005

INTRODUCTION
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 cells 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.
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

Upon encounter with a pathogen, a naive helper T cell undergoes cell division and further differentiation (reviewed in 15).
The maturing progeny can choose to become Th1 cells, which
express substantial amounts of IFN-g 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

*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@mail.med.upenn.edu

# The Author 2005. Published by Oxford University Press. All rights reserved.
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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.

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Human Molecular Genetics, 2005, Vol. 14, Review Issue 1

5 but not IFN-g (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
The effector cytokine genes Il4 and Ifng appear to exist in a
restrictive chromatin structure in the naive helper T cell

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.

(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 submaximal, 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).
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 lineagespecific 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

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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 2 CD82 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-g (Th1 cell) or IL-4, IL-13 and IL-5 (Th2 cell).

Human Molecular Genetics, 2005, Vol. 14, Review Issue 1

CONDITIONAL ROLES OF ACTIVATORS:

EPIGENETICS REDUX
T-bet seems to cooperatively induce IFN-g 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-g 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 MBD2deficient 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 misexpressed, 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-g, 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-g induction or GATA-less IL-4 induction.

ORGANIZATION AND MEMORIZATION OF

CELL FATE
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-g and IL-4 only in the presence of some

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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.

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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 remodeling 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

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

CONCLUDING REMARKS
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.

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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.

(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-g transcriptional activity.
Several mechanisms have been proposed to account for the
deeper restrictions on the forbidden cytokine. Nuclear repositioning 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 2 CD82 (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.

Human Molecular Genetics, 2005, Vol. 14, Review Issue 1

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

22.

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