Transcriptional Activators of Helper T Cell Fate Are Required
for Establishment but Not Maintenance of Signature
Gisla ˆine A. Martins, Anne S. Hutchins, and Steven L. Reiner2
The stability of helper T cell fates is not well understood. Using conditional introduction of dominant-negative factors, we now show that
T-bet and GATA-3 are far more critical in establishment than maintenance of IFN-? and IL-4 activity during Th1 and Th2 maturation,
respectively. We also show that a genetic interaction between T-bet and its target Hlx seems to be required for Th1 maturation, but that
Hlx may also be dispensable for maintenance of a transcriptionally permissive ifng gene. In parallel to progressive activator indepen-
dence in the permissive lineage, the ifng gene becomes more recalcitrant to switching as the forbidden lineage matures. T-bet plus Hlx
can disrupt ifng silencing when introduced into developing Th2 cells, but they fail to perturb ifng silencing in mature Th2 cells. In
contrast, a hypermorphic allele of T-bet can reverse silencing of the ifng gene in mature Th2 cells. These results suggest that signature
gene activity of helper T cells is initially plastic but later becomes epigenetically fixed and offer an initial strategy for inducing mature
cells to switch their fate. The Journal of Immunology, 2005, 175: 5981–5985.
differentiation process that can generate at least two distinct effec-
tor subpopulations, Th1 and Th2 cells. Th1 cells secrete IFN-? and
lymphotoxin-?, whereas Th2 secrete IL-4, IL-5, and IL-13. Th1
cells mediate protective responses against intracellular pathogens
and tumors, whereas Th2 cells are necessary for the resistance to
extracellular microbes (1).
Generation of Th1 and Th2 cells depends on the fine regulation
of transcription, allowing activation of cytokine genes in a subset-
specific manner (1–3). T-bet is a T-box transcription factor that
specifies Th1 lineage commitment, by inducing lineage-restricted tar-
get genes such as ifng and il12rb2 (4–8). GATA-3 is a zinc finger-
including the induction of the Il4, Il13, and Il5 genes (9–13).
Numerous studies have suggested that activation of the ifng and
il4 loci involves derepression of chromatin-based gene silencing
that is mediated by the effects of T-bet and GATA-3, respectively
(reviewed in Refs. 3, 14, 15). Despite evidence for the essential
roles of T-bet and GATA-3 in the induction of lineage-restricted
gene activity, their contributions to maintaining heritable patterns
of Th1 and Th2 gene expression have remained largely undefined.
In developing Th1 cells, induction of IFN-? is blocked by a dom-
inant-negative (DN)3form of T-bet (7). In a Th1 clone, the reit-
he differentiation of helper T cells is crucial for effective
immune responses. Upon encounter with Ag/MHC, naive
helper T lymphocytes undergo clonal expansion and a
eration of IFN-? expression is remarkably unaffected by DN T-bet,
despite the antagonism of IL-12R?2 expression by DN T-bet in the
same clone. Conditional gene deletion of Tbet has not yet been
performed, but conditional deletion of gata3 indicates some par-
allel behavior (12, 13). IL-4 induction is sensitive to loss of
GATA-3 developing Th2 cells. In mature Th2 cells, reiteration of
IL-4 becomes less GATA-3 dependent, even though IL-5 and
IL-13 remain highly dependent on GATA-3.
Using a series of DN transcription factors, we now report that
the induction of heritable gene activity can be experimentally sep-
arable from the maintenance of that activity. The signature genes
of mature helper T cells, ifng and il4, appear to transit from a state
of genetically sensitive plasticity to a state of epigenetic fixation.
Using a hypermorphic form of T-bet, we now also show that the
progressive inability to switch on the ifng gene during Th2 matu-
ration can be readily overcome with a strong variant trans-activa-
tor. Together, these results provide critical mechanistic insight into
why lineage commitment of helper T cells is so responsive to
extrinsic signals and their downstream mediators but why mature
cells become recalcitrant to loss or gain of the same inductive
signaling. These studies also provide an initial proof-of-concept
that genetic strategies may be capable of switching what were
previously regarded as fixed states of differentiation.
Materials and Methods
C57BL/6, Sv129, and DO11 TCR transgenic mice were purchased from
commercial sources (Taconic Farms). B6 ? 129 T-bet-deficient (T-bet?/?)
mice were generated by deleting exons 2–6. ?exon 2–6 mice are pheno-
typically identical with mice carrying a deletion of exon 1 (Refs. 8 and 16 and
data not shown). Animal work was performed in accordance with guidelines
of the University of Pennsylvania Animal Care and Use Committee.
T cell differentiation
CD4?T cells from naive mice were stimulated with syngeneic APCs plus
soluble anti-CD3 (1 ?g/ml), anti-CD28 (0.5 ?g/ml), and human rIL-2 (20
U/ml; Roche), as described (9). Experimental Th1 conditions included rIL-12
(5 ng/ml; BD Biosciences) plus ?IL-4 (10 ?g/ml). Experimental Th2 condi-
tions included rIL-4 (5 U/ml; Roche) and ?IL-12 (10 ?g/ml). Restimulation
was performed using PMA/ionomycin or plate-bound anti-CD3 where indi-
cated, as previously described (7, 16). Th1 clone PGL2 was originally used to
Abramson Family Cancer Research Institute and Department of Medicine, University
of Pennsylvania, Philadelphia, PA 19104
Received for publication June 1, 2005. Accepted for publication August 30, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by the National Institutes of Health (Grants AI42370 and
AI53827) and the Abramson Family.
2Address correspondence and reprint requests to Dr. Steven L. Reiner, Abramson
Family Cancer Research Institute, University of Pennsylvania, Building BRB II/III,
Room 414, 421 Curie Boulevard, Philadelphia, PA 19104. E-mail address:
3Abbreviations used in this paper: DN, dominant negative; RV, retrovirus; dEnR,
Drosophila engrailed repressor; MFI, mean fluorescence intensity.
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc.0022-1767/05/$02.00
identify Hlx as a Th1-specific transcript by microarray (7). Th1 clone 9.1-2
protects BALB/c mice from Leishmania infection (17).
Retroviral gene transduction
Bicistronic retrovirus (RV) vectors were constructed as described previ-
ously (7). In some experiments, HLX bicistronic RV with truncated hCD8
was used to mark cells (18). Construction of DN Hlx, DN GATA-3, and
T-bet-VP16 were performed with PCR mutagenesis. For DN Hlx, the cen-
tral portion of the Hlx homeoprotein (aas 266–346), including the DNA
binding domain, was fused to the amino-terminal repression domain of the
Drosophila engrailed repressor (dEnR; aas 1–299). For DN GATA-3, the
central portion of the protein (aas 259–385), including the zinc finger-
containing DNA binding domain (aa 263–341), was fused to the amino-
terminal repression domain of dEnR (aas 1–299). For T-bet-VP16, the
carboxy-terminal region of T-bet was deleted, and the NH2-terminal re-
gion, including the T-box (aas 1–371) of T-bet, was fused to the trans-
activation domain of HSV protein VP16 (aas 410–490). Infection of T
cells was performed as described (7).
Real-time PCR and Northern blotting
RT-PCR assays were performed as described (7) using an ABI Prism 7900
BioDetector (Applied Biosystems). All PCR data are levels of test gene
over hypoxanthine phosphoribosyltransferase levels, with the lowest value
standardized at 1. Northern blotting was performed as described (16).
Mammalian two-hybrid assay
Full-length T-bet and Hlx were fused to Gal4 DNA binding domain and
VP16 trans-activation domains, respectively (CheckMate Mammalian
Two Hybrid assay kit; Promega). pGal4-T-bet, pVP16-Hlx, and controls
were cotransfected into 293T cells with a plasmid-encoding luciferase
driven by five Gal4-response elements (pG5Luc). Forty-eight hours after
transfection, Firefly and Renilla reniformis luciferase were measured using
the Dual-Glo luciferase assay system (Promega).
T-bet and Hlx physically and genetically interact during Th1
Attempts to dissect the genetic program that culminates in Th1 cell
maturation led to the identification of the homeobox gene, Hlx (for
H 2.0-like homeobox) as a Th1-specific transcription factor that
appeared to drive maturation of IFN-? activity in cooperation with
T-bet (7). Hlx mRNA was found to be preferentially expressed in
Th1 cells (Fig. 1A), as suggested (7, 19), but is strictly dependent
on (Fig. 1B) and quantitatively regulated by (Fig. 1C) T-bet. This
is the first gene ablation evidence to directly demonstrate that T-
bet is essential to induce Hlx, as prior evidence relied on overex-
pression of wild-type and DN factors (7). Once T-bet induces Hlx,
they cooperate to induce maturation of IFN-? activity in a cell-
intrinsic manner. This can be modeled in developing Th2 cells
transduced with either T-bet alone or Hlx plus T-bet, all cultured
within the same well (Fig. 1D). This effect is best demonstrated by
the dramatic increase in amount of IFN-? produced per cell, as
reflected in mean fluorescence intensity (MFI), or by secretion of
IFN-? from sorted cells (Fig. 1D). Similar results were obtained
with enriched naive cells from D011 TCR transgenic Rag2?/?
mice (data not shown).
To test whether the genetic interaction between T-bet and Hlx
that results in synergistic induction of IFN-? might be mediated by
a physical interaction, we used the mammalian two-hybrid assay
(20), which monitors the ability of two proteins to bring tethered
trans-activation and DNA binding domains in proximity. Cotrans-
fection of Gal4-T-bet and VP16-Hlx (Fig. 1E) resulted in a specific
and significant increase in luciferase activity (Fig. 1F). T-bet and
Hlx might, therefore, physically interact to mediate their synergis-
tic effects on transcription (Fig. 1D), a behavior described in other
T-box/homeobox factor partnerships (21–23).
Hlx may be required for establishment but not maintenance of
The foregoing data support a model of feed-forward gene induc-
tion, whereby T-bet induces Hlx, which then cooperates with its
inducer, T-bet, to promote IFN-? maturation in a cell-intrinsic
manner. Hlx gene deletion results in embryonic lethality (24). To
address the requirement for Hlx in mediating IFN-? production,
we, instead, used a strategy that has been used to successfully
antagonize the function of other homeodomain proteins (25, 26).
We constructed a RV consisting of the Hlx DNA binding domain
fused to the repression domain of the dEnR protein (Fig. 2A).
Introduction of DN Hlx 1 day after stimulation resulted in a
significant impairment in IFN-? synthesis, manifest primarily as
reduced MFI of intracellular staining of IFN-? per cell (Fig. 2B).
DN T-bet (7), however, resulted in a more severe defect, manifest
primarily as reduced percentage of cells with intracellular staining
of IFN-?. Analysis of IFN-? secretion by ELISA also revealed
is a Th1 gene, downstream of T-bet. A, Naive CD4?T cells were stimu-
lated in Th1 or Th2 conditions for 4 days before Northern analysis for
indicated transcripts. B, Northern blot of Hlx mRNA from T-bet?/?and
T-bet?/?cells cultured in Th1 conditions for 7 days. C, Hlx mRNA levels
were analyzed by real-time RT-PCR from cells stimulated in Th1 condi-
tions for 7 days. D, Hlx enables maturation of T-bet-mediated IFN-? ex-
pression. Flow cytometry panels (left): cells were stimulated in Th2 con-
ditions (plus anti-IFN-?) and transduced after 24 h with empty GFP and
empty hCD8 RVs (left) or T-bet GFP and Hlx hCD8 RVs (right). After 4
days, cells were restimulated, and IFN-? staining was assessed. Only
CD4?GFP?events are depicted. Horizontal and vertical values are the
percentage of IFN-??cells and the MFI of the transduced cells, respec-
tively. Bar graph (right): cells were stimulated in Th2 conditions (plus
anti-IFN-?) and transduced with indicated RVs after 24 h. Cells were
sorted and restimulated with immobilized anti-CD3 after 1 wk of primary
culture. After an additional 48 h, IFN-? secretion was measured by two-site
ELISA. E and F, T-bet and Hlx interact in a two-hybrid assay. E, Sche-
matic of fusion of yeast Gal4 DNA binding domain to T-bet (Gal4T-bet)
and of Herpes simplex VP16 trans-activation domain to Hlx (VP16Hlx). F,
Cotransfection of 293T cells with the Gal4-responsive luciferase reporter
(pG5Luc) and indicated expression plasmids was followed by evaluation of
relative luciferase activity at 48 h.
T-bet and Hlx genetically and physically interact. A–C, Hlx
5982 CONDITIONAL ROLES OF T-bet AND GATA-3 IN HELPER T CELL FATE
defects from both constructs (Fig. 2C). Identical results were ob-
tained in DO11.10 transgenic T cells (data not shown). As further
demonstration that this loss-of-function approach mirrors gain-of-
function experiments, we evaluated expression of IL12R?2. An-
tagonism of T-bet but not of Hlx resulted in impaired IL12R?2
mRNA expression (Fig. 2D). These observations are consistent
with the synergistic cooperation between T-bet and Hlx when in-
ducing IFN-? production, but not IL12R?2 expression in devel-
oping Th2 cells (Fig. 1D; Ref. 7). Curiously, neither DN Hlx nor
DN T-bet significantly altered the expression of IL-4 or GATA-3
in developing Th2 cells (data not shown).
Using a long-term Th1 clone, we had previously suggested that
T-bet might not be required for maintenance of IFN-? activity (7).
We wished to further test this model by asking whether T-bet or
Hlx is responsible for maintaining the heritably permissive state of
IFN-? activity in newly maturing Th1 cells. We therefore per-
formed a kinetic analysis. DO11.10 transgenic helper T cells were
activated with APC/peptide in Th1-polarizing conditions. Cells
were transduced with control, DN Hlx, or DN T-bet RVs at days
2, 9, 16, or 23, and IFN-? expression was analyzed 5 days after
transduction. As Th1 maturation progressed, expression of IFN-?
became recalcitrant to the effects of the DN factors (Fig. 2E). An-
tagonism of Hlx was still evident after 2 days, as reflected in re-
duced MFI of IFN-? staining, but was negligible after 1 wk. Co-
transduction of DN T-bet and DN Hlx also had no effect on the
ability to reiterate IFN-? activity (data not shown). Thus, estab-
lishment and maintenance of IFN-? activity in Th1 cells are ex-
perimentally distinguishable by their sensitivity and recalcitrance
to DN transcription factors, respectively.
The DN T-bet construct may influence multiple targets (il12rb2,
ifng, hlx, tbet). Conditional gene deletion or RNA interference will
therefore be important follow-up approaches to test the validity of
the model that T-bet and Hlx exhibit a hit-and-run behavior in
activating the ifng gene. Despite the relatively high levels of Hlx
during the first few days of Th1 priming (Refs. 7, 19; Fig. 3A), it
has been shown that the levels of Hlx fall in primary Th1 cell
cultures after the first few days (19). It was also reported that levels
of Hlx in a Th1 clone were significantly lower than in primary Th1
cultures (19). Despite the persistent expression of substantial
amounts of T-bet in Th1 clones (Fig. 3A; (4)), we also found that
the levels of Hlx in two other Th1 clones drops below the level of
early Th1 cultures (Fig. 3, A and B). Analysis of two different Th1
clones revealed that Hlx levels may drop slightly below develop-
ing Th1 cells or may even fall to levels below a Th2 clone (Fig. 3,
A and B), despite maintaining heritable IFN-? activity (Fig. 3C).
The lack of defect in IFN-? activity from delayed introduction of
DN Hlx (Fig. 2E) is thus quite consistent with the ability to ex-
perimentally separate Hlx expression from heritable IFN-? activity
in a mature Th1 clone. The persistent expression of T-bet without
Hlx in a Th1 clone is also consistent with the apparent lack of a
sufficient or necessary role for Hlx in IL-12R?2 expression (Ref.
7; Fig. 2D). Although IL-12R?2 expression continues to remain
dependent on T-bet in mature Th1 cells (7), the absence of Hlx in
such cells would not be predicted to be of consequence, based on
the negligible effect of overexpression or antagonism of Hlx on the
IL-12R?2 target (Ref. 7; Fig. 2D).
Decreasing plasticity of gene activity during Th2 cell maturation
The dispensability of an inducing activator for the maintenance of
gene expression has also been recently addressed in the Th2 fate.
Deletion of Gata3 during Th2 development perturbed establish-
ment of transcriptional competence of IL-4 activity (12, 13). In
contrast, deletion of Gata3 in mature Th2 cells had a moderate (12)
or minimal (13) effect on maintenance of IL-4 activity. The ap-
parent differences in those studies may have been attributable to
the timing of deletion. To explore this, we fashioned a DN
itable IFN-? activity. A, Northern analysis of developing Th1 cells (day 0–5)
compared with Th1 clone PGL2. B, Th1 clone 9.1-2 expresses lower Hlx
mRNA than a Th2 clone, by real-time RT-PCR. C, Despite low levels of Hlx,
Th1 clone 9.1-2 can rapidly (within 4 h) reiterate expression of IFN-?.
Hlx expression in mature Th1 cells can dissipate despite her-
A, Schematic of DN Hlx, containing the DNA binding domain fused to the dEnR repression domain. B, Cells were stimulated in Th1 conditions for 1 day and
transduced with control (Ctrl), DN Hlx, or DN T-bet RVs. IFN-? expression was evaluated after 4 days of Th1 differentiation. Horizontal and vertical values are
the percentage of IFN-??cells and the MFI of the transduced cells, respectively. C, Developing Th1 cells transduced with indicated RVs were sorted and
restimulated with immobilized anti-CD3 after 4 days. After an additional 48 h, IFN-? secretion was measured by two-site ELISA. D, Hlx is not required for
expression of IL12R?2. Developing Th1 cells transduced with indicated RVs were sorted, and IL12R?2 mRNA levels were evaluated by real-time RT-PCR 4
days after initial stimulation. E, Antagonism of Hlx or T-bet does not impair IFN-? production in more mature Th1 cells. DO11 transgenic T cells were activated,
and restimulated weekly in Th1 conditions. Cells were transduced with indicated RVs at indicated times. Five days after transduction, cells were restimulated with
PMA/ionomycin. Horizontal and vertical values are the percentage of IFN-??cells and MFI of the transduced cells, respectively.
T-bet and Hlx may be required for establishment but not maintenance of IFN-? maturation. A and B, Antagonism of Hlx impairs IFN-? expression.
5983 The Journal of Immunology
GATA-3 construct (Fig. 4A) and performed a kinetic analysis. In-
deed, introduction of DN GATA-3 at day 1 resulted in a significant
decrease in the frequency and intensity of IL-4-expressing cells
(Fig. 4B). By day 21 of Th2 maturation, however, DN G3 had
minimal effect on the expression of IL-4 (Fig. 4B). Intermediate
time points exhibited intermediate defects consistent with a pro-
gressive stability in il4 (data not shown). Thus, establishment and
maintenance of IL-4 activity in Th2 cells are experimentally dis-
tinguishable by their sensitivity and recalcitrance to DN GATA-3,
The preceding results suggest there is a limited window during
Th1 and Th2 differentiation in which the activity of the ifng and il4
genes is still plastic, capable of being antagonized by interference
with T-bet/Hlx and GATA-3. During the progression of naive to
mature helper T cells, forbidden effector cytokine genes undergo
even deeper silencing, through mechanisms such as centromeric
repositioning of the genes or de novo DNA methylation (27, 28).
Likewise, introduction of inducing trans-acting factors into the
forbidden lineages induces the forbidden cytokine gene with di-
minishing efficiency as cells mature (4, 29). We also found that
activation of the ifng gene in mature Th2 cells is progressively
restricted (data not shown). We therefore fashioned a putative hy-
permorphic form of T-bet to test its effect on the silencing of the
ifng gene in mature Th2 cells. T-bet-VP16 (Fig. 5A) has the car-
boxy-terminal trans-activation domain of T-bet replaced by the
HSV VP16 trans-activation domain (30). This domain was chosen
because of the ability of VP16 to recruit both histone acetyltrans-
ferases and ATP-dependent nucleosome remodeling complexes,
the two major enzymatic activities responsible for chromatin re-
modeling-dependent gene induction (31).
Cells were stimulated in Th2 conditions for 3 wk and transduced
with control, T-bet, T-bet plus Hlx, or T-bet-VP16 RVs. As as-
sessed by intracellular cytokine staining (Fig. 5B) and ELISA (Fig.
5C), expression of T-bet or T-bet plus Hlx resulted in only modest
increases in IFN-? expression in mature Th2 cells. T-bet-VP16,
however, could induce substantial secretion of IFN-? and con-
verted the majority of transduced cells into brightly staining, IFN-
?-positive cells. None of the interventions significantly repressed
IL-4 expression (data not shown). The dual expression of IL-4 and
IFN-? also suggested that we were not simply providing a selec-
tion mechanism for contaminating Th1 cells, but rather de-repress-
ing IFN-? in maturing Th2 cells. Thus, mature Th2 cells become
recalcitrant to the inducing factors, T-bet plus Hlx, which can me-
diate efficient IFN-? maturation in developing Th2 cells. By tar-
geting strong activators, however, the progressive restrictions that
are imposed on the forbidden cytokine gene may be capable of
being more efficiently overcome.
We now show that an activator of heritable patterns of gene ex-
pression might be conditionally critical, being essential for the
establishment but not necessarily the maintenance of cytokine
gene activity. Using a standard genetic approach that has success-
fully antagonized the function of other homeodomain proteins (25,
26), we provide the first loss-of-function experiments with Hlx.
These studies offer initial evidence that Hlx is indeed required to
cooperate with T-bet to establish a heritably permissive state of the
ifng gene. Moreover, our data show for the first time that Hlx and
T-bet can physically interact in a mammalian two-hybrid assay,
suggesting a potential molecular mechanism for their synergistic
activation of the ifng gene. Later, the active state of the ifng gene
becomes insensitive to the loss of both T-bet and Hlx activity (Fig.
6). The concept that a transcriptional activator that is essential to
induce a state of gene activity may not be required to maintain that
gene activity is an important, yet poorly understood, feature of
gene regulation in helper T cells that will require further study.
In parallel with this progressive stability of activity in the Th1
lineage, the ifng gene becomes progressively more difficult to ac-
tivate by ectopic T-bet and Hlx in maturing Th2 cells. A similar
behavior may be occurring in the il4 locus (Fig. 6), as GATA-3
seems to become progressively dispensable for IL-4 activity in
maturing Th2 cells, and the il4 gene becomes progressively more
difficult to trans-activate in maturing Th1 cells (29). Although we
have not yet been able to silence a heritably active cytokine locus,
we offer initial evidence that derepressing a more permanently
silenced locus might be achieved with strategies that target strong
chromatin activators to the locus (Fig. 5). The success of the T-bet-
VP16 construct in achieving substantial induction of IFN-? pro-
vides novel evidence for the viability of genetic strategies to in-
tervene during disease processes mediated by fully differentiated
helper T cells.
It is curious that within the same cell type, a single activator
might become dispensable for one target but remain continually
during Th2 maturation. A, Schematic of DN GATA-3 (DN G3), containing the
zinc finger DNA binding domain fused to the dEnR repression domain. B,
Antagonism of GATA-3 does not impair IL-4 expression in more mature Th2
cells. Cells cultured in Th2 conditions were transduced with control (Ctrl) or
DN G3 RVs at indicated times. IL-4 expression was evaluated 6 days after
transduction. Only transduced (GFP?) events are displayed.
IL-4 activity becomes resistant to the effects of DN GATA-3
a strong agonist T-bet variant. A, Schematic of T-bet-VP16, with its en-
dogenous trans-activation domain (trans) replaced by Herpes simplex
VP16 trans-activation domain (V). B and C, Expression of IFN-? in mature
Th2 cells transduced with T-bet, T-bet plus Hlx, or T-bet-VP16 RVs.
DO11.10 transgenic T cells were stimulated in Th2 conditions for 21 days
and transduced with indicated RVs expressing GFP or hCD8 markers.
Cells were restimulated 5 days after transduction with immobilized anti-
CD3, and expression of IFN-? was analyzed by flow cytometry at 6 h (B)
and by ELISA at 48 h (C). Numbers indicate percentage of transduced cells
that express IFN-?.
Overcoming ifng silencing during Th2 maturation requires
5984 CONDITIONAL ROLES OF T-bet AND GATA-3 IN HELPER T CELL FATE
required for another. In mature Th1 cells, T-bet seems to be es-
sential to maintain IL-12R?2 (7), whereas GATA-3 seems to be
essential for ongoing IL-5 and IL-13 expression in mature Th2
cells (12, 13). Whether the behavior of becoming activator inde-
pendent reflects the need to have more rapid expression of one
gene compared with another, or is simply a feature of the structural
elements of the gene remains unresolved. Likewise, the mecha-
nisms responsible for locking in the activity or silencing of cyto-
kine genes during helper T cell maturation are still incompletely
defined. We do not yet know whether the stability we observe is
truly epigenetic, maintained by self-propagating chromatin marks,
or is more accurately genetic, maintained by trans-activators other
than T-bet, Hlx, or GATA-3, and conventional repressors acting at
negative cis-elements (32). In either case, it is remarkable, al-
though not fully explicable, that recruitment of an engrailed re-
pression domain has such a minor effect on cytokine expression in
mature cells. Understanding the genetic and biochemical basis for the
transition between plasticity and inflexibility in gene activity or si-
lencing should, however, enable the rational design of approaches to
switch what were previously thought to be irrevocable cell fates.
We are grateful to A. Intlekofer for making constructs and for critical
advice, and A. Mullen, F. High, F. Schambach, C. Gasink, N. Takemoto,
C. DiCioccio, M. Banica, and M. Bogumil for assistance.
The authors have no financial conflict of interest.
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of gene activity during helper T cell differentiation. Left side, In developing
Th1 cells, the transcription factors T-bet and Hlx induce maturation of
IFN-? activity. This is initially plastic insofar as DN forms are capable of
reversing the derepression. Later, plasticity gives way to stability, because
IFN-? activity becomes unperturbed by DN factors. In developing Th2
cells, ifng gene repression is initially plastic, reversible by ectopic T-bet
and Hlx. Later in Th2 maturation, the plasticity gives way to stability in
ifng gene silencing, because T-bet/Hlx become less efficient in reversing
silencing. At the stage of stability, the hyperagonistic T-bet VP-16 can
reverse heritable ifng gene silencing in Th2 cells, whereas a modality that can
repress/silence IFN-? activity in mature Th1 cells has not yet been defined.
Right side, Th2 associated-IL-4 activity is initially antagonized by Gata3 gene
deletion or DN factor, and is enforced by ectopic GATA-3 provision of Th1
cells. These interventions work with diminishing efficiency as cells mature.
Interventions to reverse stability have not yet been defined.
Speculative model of the sequential plasticity and stability
5985 The Journal of Immunology