| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
,
Departments of
*
Immunology,
Cell Biology and Genetics, and
Pulmonary Medicine, Faculty of Medicine, Erasmus University Rotterdam, Rotterdam, The Netherlands; and
Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
was
decreased. Moreover, CD4+ T cells manifested
rapid secretion of the Th2 cytokines IL-4, IL-5, and IL-10, reminiscent
of Th2 memory cells. In contrast to wild-type
CD4+ cells, which lost GATA-3 expression when
cultured under Th1-polarizing conditions,
CD2-GATA3-transgenic CD4+ cells
maintained expression of GATA-3 protein. Under Th1 conditions, cellular
proliferation of CD2-GATA3-transgenic
CD4+ cells was severely hampered, IFN-
production was decreased and Th2 cytokine production was increased.
Enforced GATA-3 expression inhibited Th1-mediated in vivo
responses, such as Ag-specific IgG2a production or a delayed-type
hypersensitivity response to keyhole limpet hemocyanin.
Collectively, these observations indicate that enforced
GATA-3 expression selectively inhibits Th1 differentiation
and induces Th2 differentiation. The increased functional capacity to
secrete Th2 cytokines, along with the increased expression of surface
markers for Ag-experienced Th2-committed cells, would argue for a role
of GATA-3 in Th2 memory formation. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and TNF-
, whereas Th2 cells typically produce IL-4, IL-5,
IL-10, and IL-13. Each subset mediates distinct effector functions in
vivo. Th1 cells are predominantly involved in immune responses against
intracellular pathogens and are associated with autoimmune disease. Th2
cells are of importance in the defense against extracellular pathogens
and are implicated in atopy and allergic diseases
(3, 4, 5). Both Th1 and Th2 cells are derived from a common naive precursor (4, 5, 6). Signaling pathways initiated by cytokines play a dominant role in driving the differentiation of activated naive CD4+ T cells into either effector phenotype (2, 7). For instance, IL-12 induces the differentiation of naive Th cells to the Th1 effector phenotype (8, 9, 10, 11) by activation of the transcription factor Stat4 (12, 13, 14). On the other hand, Th2 differentiation is mediated by Stat6 activation through IL-4 receptor engagement (15, 16, 17, 18, 19, 20, 21). In response to chronic antigenic stimulation in vivo, progressive polarization of the cytokine responses ultimately leads to the commitment of Th cells to mutually exclusive Th phenotypes, which are thought to be maintained independently of extrinsic factors (22, 23).
Stat6 induces the expression of the transcription factors GATA-3 and
c-Maf (24), which have been shown to be selectively
expressed in a Th2-specific fashion (25, 26, 27). Using
Stat6-deficient cells it has been shown that, although IL-4 and Stat6
signaling may initially direct Th2 development, GATA-3 and c-Maf are
capable of inducing the development of stabile Th2 commitment,
independent of Stat6 (28). In vitro differentiation into
Th1 cells induces chromatin remodeling of the IFN- locus
and, conversely, the differentiation into Th2 cells induces remodeling
of the IL-4/IL-5/IL-13 locus (29, 30).
Recently, GATA-3 has been shown to play an instructive role in
directing Th2 differentiation (31).
During early T cell development, GATA-3 gene expression is
required for the development of the earliest T cell progenitors
(32, 33, 34). GATA-3 levels are low during the two phases of
TCR gene rearrangement, but are high in the fraction of
rapidly proliferating cells that insulates these two periods of
TCR rearrangement (33). GATA-3 expression
remains high in CD4+ thymocytes, but
progressively declines in CD8+ thymocytes (see
accompanying paper). GATA-3 is detected in naive
CD4+ T cells and expression levels increase
substantially during Th2 differentiation (26, 27). GATA-3
expression has been shown to be indispensable for Th2 development and
is down-regulated in response to IL-12-mediated Stat4 activation
(27, 35). GATA-3 strongly transactivates the
IL-5 promoter, but appears to have only limited effects on
IL-4 gene transcription (27, 36, 37).
Retroviral introduction of GATA-3 during in vitro Th1 differentiation
of naive CD4+ T cells resulted in an inhibition
of IFN- production, independently of IL-4 (35, 38), and
a down-regulation of IL-12R2 (35), which normally
accompanies Th2 differentiation (39).
The manipulation of Stat6 and GATA-3 expression in Th1 and Th2 polarization cultures of wild-type or specific cytokine-deficient cells in vitro have added significantly to our understanding of the molecular basis of Th1/Th2 differentiation. However, limited data are available on the role of GATA-3 during immune responses in animal models, partly because the embryonic lethality of GATA-3 deficiency in mice precluded in vivo studies (40). Analysis of transgenic mice with T cell-specific expression of a dominant-negative mutant of GATA-3 indicated that inhibition of GATA-3 activity reduced the key features of asthma, including Th2 cytokine levels, eosinophilia, and IgE production (41).
To study the function of GATA-3 during T cell differentiation, we
generated transgenic mice in which the expression of GATA-3
is under the control of the human CD2 locus control region
(see accompanying paper). In these mice, the enforced GATA-3
expression induced the development of thymic lymphomas of
CD4+CD8+/low T cells and
inhibited the maturation of CD8 single-positive
(SP)3
cells in the thymus. Within the CD8 SP population in the thymus,
apoptosis was increased and the fraction of mature
CD69lowHSAlow cells was
significantly reduced. The numbers of peripheral
CD8+ T cells were 
50% of normal. These
observations supported a role for GATA-3 in the regulation of CD4/CD8
lineage commitment (see accompanying paper).
To investigate how the enforced expression of GATA-3 affected Th cell differentiation, we analyzed T cell-mediated immune responses in vivo in CD2-GATA3-transgenic mice. The observations of a selective deficiency of Ag-specific IgG2a production and severely reduced delayed-type hypersensitivity (DTH) responses in these mice showed that enforced GATA-3 expression inhibited the differentiation of Th1 cells in vivo. Furthermore, the expression of surface markers specific for Ag-experienced Th2 cells, along with the increased functional capacity to secrete the Th2 cytokines, indicated that transgenic GATA-3 expression induced Th2 commitment and pointed at a role for GATA-3 in Th2 memory formation.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The CD2-GATA3 mice are described in the accompanying paper and were crossed on a uniform FVB background. To determine the genotype of the mice, tail DNA was analyzed by Southern blotting as described in the accompanying paper.
Flow cytometric analyses
The preparation of single-cell suspensions, mAb incubations and
three- or four-color cytometry have been described previously
(42). The following mAb were purchased from BD
PharMingen (San Diego, CA): FITCconjugated anti-CD3
,
PE-conjugated anti-CD4 (L3T4), anti-CD24/heat-stable Ag,
anti-CD25 (clone 3C7), anti-CD62L and anti-CD69,
CyChrome-conjugated anti-CD4 (L3T4), anti-CD8 and
anti-CD44, biotinylated anti-CD4 (L3T4), and anti-CD8,
APC-labeled anti-CD3 and anti-CD4. Anti-CD45RB (MB23G2) was
a purified mAb conjugated to biotin according to standard procedures.
Secondary Abs used were PE-, TriColor-, or APC-conjugated streptavidin
(Caltag, Burlingame, CA). The Th2-selective surface marker T1/ST2
(3E10, rat IgG1, kindly provided by A. J. Coyle, Millenium
Pharmaceuticals, Cambridge, MA) was detected by secondary goat
anti-rat IgG-PE (Jackson Immuno-Research Laboratories,
West Grove, PA) (43).
For intracellular detection of GATA-3 protein, cells were fixed and permeabilized using paraformaldehyde and saponin as described previously (44) and subsequently incubated with the Hg-3-31 anti-GATA-3 mAb (Santa Cruz Biotechnology, Santa Cruz, CA) and FITC-labeled anti-mouse IgG1 (BD PharMingen) as a second step. For three-color analysis, 0.5–1 x 105 events were scored using a FACScan analyzer (BD Biosciences, Sunnyvale, CA). For four-color analysis, 105–2 x 106 events were scored using a FACSCalibur dual laser instrument (BD Biosciences).
For intracellular detection of cytokines, cells were stimulated for
40 h in the presence of mAb to CD28 (37.51; 5 µg/ml) in 96-well
plates (106 cells/well) precoated with mAb to
CD3 (145 2C11; 10 µg/ml in PBS). Subsequently, cells were
stimulated by adding PMA (50 ng/ml; Sigma, St. Louis, MO) and calcium
ionophore (500 ng/ml; Sigma) for 5 h. For the last 3 h of the
culture, brefeldin A (10 µg/ml; Sigma) was added to the cells.
Finally, cells were harvested and stained with CyChrome-labeled mAb to
CD4 or CD8 (BD PharMingen). Cells were fixed using 2% paraformaldehyde
and stored up to 3 days at 4°C. Intracellular cytokine staining was
performed using PE-labeled mAb to IL-2, IL-4, and IL-10 (BD PharMingen)
and APC-labeled mAb to IL-2, IL-5, and IFN- (BD PharMingen)
according to the manufacturer’s instructions.
Serum Ig detection and in vivo immunizations
Total serum Ig levels were determined by subclass-specific sandwich ELISA as described previously (45). Immunizations were done i.p. with 100 µg trinitrophenol-keyhole limpet hemocyanin (TNP-KLH) precipitated on alum. Serum levels of TNP-specific Ig subclasses were determined by ELISA, using TNP-specific standards (IgG1, IgG2a, and IgG2b) or TNP-specific reference serum samples (IgM and IgG3), as described elsewhere (46).
Purification of CD4+ T cells and in vitro cultures
Single-cell suspensions from spleen were incubated with biotinylated mAb to CD8 (YTS-169), CD11b/Mac-1 (M1/70), CD40 (FGK-45.5), B220 (RA3-6B2), and IgM (M41), followed by streptavidin-conjugated microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Using a Vario-MACS, CD4+ T cells were purified according to the manufacturer’s instruction to purity >95%. The CD4+ T cells were cultured for up to 5 days in the presence of IL-2 (50 U/ml) on 96-well plates precoated with 10 µg/ml anti-CD3 (145 2C11) mAb.
Purified CD4+ T cells were polarized into Th1 and
Th2 effector cells in a total volume of 200 µl for 4 days in the
presence of 5 µg/ml anti-CD28 (37.51) and 50 IU/ml IL-2 on
96-well plates, which were precoated with 10 µg/ml anti-CD3
(145 2C11) (33). Th1-polarizing cultures included 5 ng/ml
rIL-12 (R&D Systems, Minneapolis, MN) and 10 µg/ml neutralizing mAbs
to IL-4 (11B11). Th2-polarized cells were cultured in the presence of
10–50 ng/ml rIL-4 and 10 µg/ml neutralizing mAbs to IFN-
(XMG1.2). After 4 days of culture, the cells were thoroughly washed and
transferred to new anti-CD3-coated 96-well plates and cultured in
the presence of IL-2, without addition of further cytokines or
neutralizing Abs.
To measure DNA synthesis during T cell cultures, cells were pulsed with
[3H]thymidine for 16 h, harvested, and
counted using standard methods. Cytokine levels in culture supernatants
were determined by ELISA using the Opteia kit for IL-4, IL-5, IL-10,
and IFN- (BD PharMingen) according to the manufacturer’s
instructions. Expression of GATA-3 protein was evaluated using a
Western blotting procedure as described in the accompanying paper.
DTH responses
DTH responses were performed essentially as described by Cua et al. (47). In short, mice were immunized i.p. with 100 µg KLH in 250 µl PBS and on day 6 they were challenged with 150 µg KLH in 25 µl PBS in the left hind footpad. The right hind footpad was in injected with a vehicle control (25 µl PBS). Responses were quantified 24 and 48 h after the challenge by measuring the difference in footpad thickness between the KLH- and the PBS-injected footpads.
Immunohistochemistry
Tissue samples were embedded in OCT compound and frozen 5-µm cryostat sections were acetone fixed and single labelings were performed using standard procedures (45). The mAbs biotinylated anti-IL-4 (11B11) and alkaline phosphatase-conjugated anti-IL-5 (TRFK5) were purified hybridoma supernatants and conjugated according to standard procedures. Biotinylated anti-IL-10 (SXC1) was purchased from BD PharMingen.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In two independent CD2-GATA-3-transgenic lines expression of hemagglutinin (HA)-tagged GATA-3 was under the control of the CD2 locus control region. Due to the presence of this transgene, GATA-3 expression was significantly enhanced in the thymus, especially in the DP fraction. In these mice, the expression of transgenic GATA-3 in peripheral lymphoid organs was low (see accompanying paper). In the experiments described below, we did not detect any differences between the two independent lines TgA and TgB.
To determine the effect of enforced GATA-3 expression on the
development of the peripheral T cell compartments, we investigated the
sizes of the CD4+ and CD8+
populations in CD2-GATA3-transgenic mice and their
nontransgenic littermates at different ages (Fig. 1
A). In spleen and mesenteric lymph nodes, the numbers of
CD4+ T cells in CD2-GATA3 mice were
either increased (3 wk of age) or within normal ranges (12 wk of age).
In contrast, the CD8+ T cell populations were
consistently reduced in number, to 50% of normal, irrespective of
the age analyzed (Fig. 1A). Despite the observed increased
apoptosis in the fraction of mature SP cells in the thymus (see
accompanying paper), the CD2-GATA3-transgenic mice were not
found to be lymphopenic at any of the ages analyzed.
|
, B and C, in CD2-GATA3-transgenic mice
both the CD4+ and the CD8+
T cell populations contained increased proportions of
CD44high, CD45RBlow, and
CD62Llow cells. This was found both in spleen and
in lymph nodes, and pointed to the presence of elevated numbers of
either recently activated or Ag-experienced T cells (48).
The expression levels of CD69 and IL-2R
(CD25), which are markers of
recently activated T cells (49), were low in
CD2-GATA3 mice and nontransgenic littermates (shown for CD25
in Fig. 1, A and B), arguing against the presence
of major fractions of recently activated T cells in
CD2-GATA3 mice. It has been shown that the proportions of T
cells with an Ag-experienced phenotype increases with age, probably as
a result of progressive Ag experience (50, 51). When we
compared CD44 and CD62L expression at two different ages, 3 and 7 mo,
we indeed found that the conversion to the
CD44highCD62Llow
Ag-experienced T cell phenotype increased with age, both in
CD2-GATA3-transgenic mice and in control mice (shown
for CD62L in Fig. 1C). Taken together, these results indicated that in the CD2-GATA3 mice both the CD4+ and the CD8+ T cell population contained increased proportions of cells with an Ag-experienced phenotype.
Enforced expression of GATA-3 results in increased numbers of T1/ST2-positive CD4+ T cells in the periphery
The IL-1R family member T1/ST2 is preferentially expressed on the surface of murine Th2 cells (43, 52, 53, 54). It was recently shown that CD4+ cells become T1/ST2 positive after repeated antigenic stimulation under Th2-polarizing conditions and that Th2 cytokine production precedes T1/ST2 expression (55). Therefore, T1/ST2 expression appears to be a late event in Th2 commitment.
To investigate whether CD4+ T cells in
CD2-GATA3 mice exhibited preferential Th2 polarization and
advanced Th2 commitment in vivo, we evaluated surface expression of
T1/ST2 (Fig. 2). In nontransgenic controls, we found T1/ST2 expression on 5.1% ± 1.3
and 1.1% ± 0.1 (n = 5) of CD4+
T cells in spleen and mesenteric lymph nodes, respectively. In
four-color labelings with CD4, CD8, CD44, and T1/ST2, it was shown that
in nontransgenic mice T1/ST2 expression was largely confined to the
CD44high fraction of activated/memory
CD4+ T cells (Fig. 2).
|
).
When we analyzed T1/ST2 expression in the thymic subpopulations, we
found induction of T1/ST2 on a small fraction of the CD4 SP cells in
CD2-GATA3-transgenic mice: 6.0% ± 1.0 (n =
3) compared with 0.4% ± 0.05 in nontransgenic mice (n
= 5). In contrast, T1/ST2 expression was not significantly induced on
CD8 SP thymocytes (<0.5%). Consistent with the reported absence of
T1/ST2 on the surface of CD8+ cells (43, 52), we found very low numbers of T1/ST2-positive cells in
nontransgenic mice. However, some T1/ST2 expression was observed on
CD8+ T cells in spleen (3.0% ± 0.8) and lymph
node (1.7% ± 0.6) from CD2-GATA3-transgenic mice
(Fig. 2).
In summary, these data indicate that enforced expression of GATA-3 resulted in significantly increased numbers of peripheral CD4+ T cells with an advanced Th2-committed T1/ST2+ phenotype, not only in the CD44high activated/memory T cell compartment but also in CD44low naive T cells.
Increased ability to secrete Th2 cytokines in CD2-GATA3-transgenic T cells
As one hallmark of a memory cell population is the ability to
secrete a wider diversity of cytokines (48), we
investigated the ability of peripheral T cells to synthesize various
cytokines. After polyclonal in vitro stimulation of mesenteric lymph
node cells, significant differences in the cytokine production profiles
between wild-type and CD2-GATA3-transgenic mice were
observed. When intracellular cytokine expression was analyzed by flow
cytometry, CD2-GATA3-transgenic CD4+
and CD8+ T cells manifested decreased expression
of IL-2 and IFN- (Fig. 3A). In addition, the production of the Th2 cytokines IL-4,
IL-5, and IL-10 was increased in CD2-GATA3-transgenic T
cells after 40 and 60 h of culture (Fig. 3B).
|
C).
Collectively, the CD2-GATA3-transgenic T cells were
characterized by an increased production of Th2 cytokines and reduced
production of IL-2 and IFN- upon stimulation, suggesting the
presence of an increased memory compartment within the peripheral T
cell population in vivo.
Expression of GATA-3 in CD2-GATA3-transgenic T cell cultures under Th1-polarizing conditions
Next, we performed in vitro Th1/Th2 polarization culture
experiments on purified CD4+ T cells from spleen
and lymph nodes to investigate whether the differentiation potential
into T effector phenotypes was altered in the CD2-GATA3
mice. In these experiments, cells were stimulated with anti-CD3
mAbs, either under "default" conditions (without additional
cytokines or Abs), Th1-polarizing conditions (in the presence of IL-12
and anti-IL-4 mAbs), or under Th2-polarizing conditions (in the
presence of IL-4 and anti-IFN- mAbs) for 4 days. Subsequently,
the cells were washed and restimulated with anti-CD3 mAbs for 3
days, without additional Abs or cytokines.
First, we evaluated the GATA-3 expression in lymph node-derived T cell
cultures by Western blotting and intracellular flow cytometric analyses
using a mouse monoclonal antiserum specific for GATA-3 (Fig. 4, A and B). In nontransgenic and
CD2-GATA3-transgenic CD4+ T cell
cultures, GATA-3 protein was abundantly expressed both under default
and Th2-polarizing conditions. GATA-3 protein could not be detected in
nontransgenic Th1 cultures (Fig. 4A). In contrast, GATA-3
was expressed in CD2-GATA3-transgenic Th1 cultures, although
the levels of expression were lower than in the corresponding Th2 or
default cultures (Fig. 4A). The GATA-3 protein present had
the apparent molecular mass of endogenous GATA-3, and no transgene
encoded GATA-3 bands could be detected using Abs specific for the HA
tag present in the transgenic GATA-3 protein.
|
B). Therefore, we conclude that due to the presence of the CD2-GATA3 transgene, GATA-3 protein expression is maintained in CD4+ T cells that are cultured under Th1-polarizing conditions.
The Th1/Th2 differentiation potential of CD2-GATA3-transgenic T cells in vitro
Cytokine production was evaluated in the Th1/Th2-polarized
CD4+ T cell cultures from lymph nodes or spleen.
CD2-GATA3-transgenic CD4+ T cells
produced normal amounts of IL-4, but higher levels of IL-5 and IL-10 in
the default or Th2-polarized cultures when compared with nontransgenic
CD4+ T cells (Fig. 4C). In addition,
when CD2-GATA3-transgenic CD4+ T cells
were cultured under Th1-polarizing conditions, they produced
significantly increased amounts of IL-4, IL-5, and IL-10. Irrespective
of the culture conditions, the production of IFN- was significantly
reduced in the CD2-GATA3-transgenic
CD4+ T cells when compared with those from
nontransgenic littermates (Fig. 4C).
The reduced IFN- production by CD2-GATA3-transgenic
CD4+ T cells cultured under Th1-polarizing
conditions could either result from an inhibitory effect of GATA-3 on
the differentiation of naive cells into Th1 cells, or by an inhibition
of the amount of IFN- produced by differentiated Th1 effector cells.
To distinguish between these possibilities, we assessed cell viability
and proliferation in the T cell cultures. When analyzed by flow
cytometry using propidium iodide, CD4+ T cells
from CD2-GATA3-transgenic mice showed increased cell death
under Th1 culture conditions at day 7, as compared with nontransgenic
littermates (Fig. 4D). When
[3H]thymidine incorporation was assessed at day
7, we observed a specific inhibitory effect of enforced GATA-3
expression on cell proliferation in Th1 cultures (Fig. 4E).
By contrast, the presence of the CD2-GATA3 transgene
enhanced viability and proliferation of CD4+ T
cells in the Th2 cultures (Fig. 4, D and E).
Collectively, these observations demonstrate that although the presence of the CD2-GATA3 transgene inhibited the proliferation of Th cells under Th1-polarizing conditions, considerable production of Th2 cytokines was still present. Furthermore, the enforced GATA-3 expression significantly supported proliferation and differentiation of Th2 effector cells in a Th2 environment.
GATA-3-expressing lymphoblastoid tumors express Th2 cytokines
The coordinate expression of IL-4, IL-5. and IL-13 is thought to
be under the direct control of GATA-3, as GATA-3 specifically interacts
with an intergenic DNase I hypersensitivity site in the Th2 cytokine
locus that contains the IL-4/IL-5/IL-13 gene cluster
(30). However, the mechanism by which GATA-3 would
regulate IL-10 expression is unknown. The rapid production of IL-10
after anti-CD3 stimulation in vitro (Fig. 3C) would
suggest that GATA-3 is directly involved in the regulation of
IL-10 gene expression. To further address this question, we
examined lymphoblastoid tumor samples from
CD2-GATA3-transgenic mice. At the age of 9 mo, 50% of
these mice developed thymic lymphomas that were
CD4+CD8+/low and expressed
high levels of GATA-3 (see accompanying paper). Immunohistochemical
analyses of thymic tumor tissues showed that most of the tumors
contained areas where the lymphoblastoid cells had lost expression of
CD8 and sometimes also CD4 (data not shown). Particularly in such
areas, very high expression of the Th2 cytokines IL-4, IL-5, or IL-10
was found (Fig. 5, A–C). Moreover, when tumor cells were cultured in the
presence of anti-CD3, an extremely high production of Th2
cytokines, including IL-10, was observed (an example is shown in Fig. 5D).
|
Enforced GATA-3 expression inhibits switching to IgG2a in an Ag-specific immune response
Serum levels of individual Ig isotypes are generally dependent on
the Th1/Th2 balance. IL-4 primes mouse B lymphocytes for switching to
IgG1 and IgE, while IgG2a responses are induced by IFN-
(56). When total serum Ig levels were determined in
2–3-mo-old CD2-GATA3-transgenic mice and nontransgenic
littermates by ELISA, a selective increase in IgG1 was found in the
CD2-GATA3-transgenic animals (Fig. 6A). The levels of all other isotypes, including IgE, were
similar in the two groups of mice (Fig. 6, A and
C, IgE preimmune values).
|
-dependent isotype IgG2a
on day 7 after the booster injection, as measured in a TNP-specific
ELISA (Fig. 6B). The TNP-specific levels of the other Ig
isotypes elicited in this response were comparable between transgenic
animals and wild-type littermates (shown for IgG1 in Fig. 6B). Also the primary IgG2a response to TNP-KLH in vivo was
specifically decreased in the CD2-GATA3-transgenic animals
(to 15% of normal), as determined in the serum at day 7 and 14
after an i.p. immunization with 100 µg TNP-KLH precipitated on alum
(data not shown). The Th2-dependent induction of heavy chain class
switch to IgE was determined at day 11 after i.p. injection of 10 µg
TNP-KLH precipitated on alum. CD2-GATA3-transgenic mice
manifested elevated total serum IgE levels in this response (Fig. 6C).
Next, we directly tested the ability of CD2-GATA3-transgenic
T cells to respond to Ag after a previous i.p. immunization with
TNP-KLH. CD4+ T cells were purified from spleens
of immunized CD2-GATA3-transgenic mice and control
littermates 4 wk after i.p. injection with TNP-KLH precipitated on alum
and stimulated in vitro using wild-type irradiated APCs, which had been
preincubated with KLH for 4 h. Proliferative responses were
determined by [3H]thymidine incorporation and
showed that CD2-GATA3-transgenic mice had enhanced in vitro
recall responses (Fig. 6C).
In conclusion, the observed suppression of TNP-specific IgG2a
production indicated that transgenic GATA-3 expression is sufficient to
inhibit IFN--mediated Ag-specific class switching. In the
CD2-GATA3-transgenic mice, the Th2-dependent induction of
IgE was enhanced, whereas increased class switching to IgG1 was only
observed in the total serum levels. In addition,
CD2-GATA3-transgenic CD4+ T cells were
shown to have an elevated in vitro recall response.
Enforced GATA-3 expression diminishes the DTH response to KLH
To directly test whether enforced GATA-3 expression suppresses
Th1-dependent immune responses in vivo, we assayed DTH responses to the
protein Ag KLH. Two-month-old mice were primed by i.p. injection of 100
µg KLH and challenged on day 6 by injection of 25 µl PBS alone and
25 µl PBS containing 150 µg KLH in the left and right hind footpad,
respectively. Twenty-four hours after the injections, footpad thickness
was measured and the difference between the two footpads was calculated
(Fig. 7). The KLH-induced footpad swelling was significantly reduced in
CD2-GATA3-transgenic animals, as compared with the wild-type
littermates. This reduction of footpad swelling did not reflect delayed
kinetics of the DTH response, since also at 48 h after the
injections footpad swellings were still essentially absent in the
CD2-GATA3-transgenic mice.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production, independently of
IL-4 expression (35, 38).
To analyze the function of GATA-3 in an in vivo system, we investigated
transgenic mice that expressed GATA-3 under the control of the
CD2 locus control region. In these mice, the expression of
transgenic GATA-3 in the peripheral T cells of the spleen or lymph
nodes was very low (see accompanying paper). However, in contrast to
wild-type CD4+ cells, which lost GATA-3
expression when cultured under Th1-polarizing conditions,
CD2-GATA3-transgenic CD4+ cells
maintained expression of endogenous GATA-3 protein. The finding by
Ouyang et al. (28) that GATA-3 may, either directly or
indirectly, activate its own expression could be a mechanism by which
low amounts of transgenic GATA-3 enhance the expression of endogenous
GATA-3. This could explain our observation of increased GATA-3 protein
levels in T cell cultures, while transgenic GATA-3 could not be
detected in Western blotting analyses using Abs to GATA-3 or the HA tag
(Fig. 4, A and B).
Our analyses of the CD2-GATA3-transgenic mice support the
findings that GATA-3 expression inhibits Th1 development. The enforced
GATA-3 expression inhibited Th1-mediated responses in vivo, including
Ag-specific IgG2a production and DTH responses to protein Ag. In our
Th1/Th2 polarization cultures, enforced GATA-3 expression under
Th1-inducing culture conditions resulted in a reduction of cell
survival, proliferation, and IFN- production. The additional
findings of increased T1/ST2 expression in CD4+ T
cells and elevated total IgG1 serum levels suggest that the presence of
the CD2-GATA3 gene drives T cells preferentially toward
differentiation along the Th2 pathway. Therefore, we conclude that
GATA-3 plays a dual role in vivo in the differentiation of naive Th
cells into Th2 cells, since it both represses Th1 differentiation and
induced Th2 differentiation.
Various lines of evidence indicate that the enforced expression of
GATA-3 may enhance Th2 memory cell formation. First, in
CD2-GATA3-transgenic mice, the peripheral T cell compartment
contained a high proportion of cells with an Ag-experienced cell
surface profile, defined as
CD44highCD45RBlowCD62Llow
and negative for CD25 and CD69. The ratio of naive vs memory phenotype
cells decreased with age, as normally seen in wild-type mice. The
possibility that peripheral T cells obtained the Ag-experienced surface
phenotype because of a homeostatic proliferation mechanism
(57) in the CD2-GATA3-transgenic mice is
unlikely, because these mice were not lymphopenic at any of the ages
analyzed. Second, in CD2-GATA3-transgenic mice the
expression of the Th2-specific T1/ST2 marker within the
CD44high memory Th cell population in spleen and
lymph nodes was increased by a factor 6 and 20, respectively.
T1/ST2 marker expression is associated with advanced Th2 commitment, as
it was shown to be expressed only after repeated antigenic stimulation
under Th2-polarizing conditions in vitro, with delayed kinetics
compared with the kinetics of Th2 cytokines (55). Third,
CD2-GATA3-transgenic T cells were rapidly induced to
synthesize IL-4, IL-5, and IL-10 in vitro, whereas production of IL-2
was low, which is typical for memory Th2 cells (48).
Fourth, T cells from CD2-GATA3 mice exhibited an increased
recall response to TNP-KLH Ag in vitro. Finally, the selective increase
of the total levels of the IL-4-dependent isotype IgG1 in the serum
would also be consistent with increased Th2 memory formation.
It is presently not clear how GATA-3 would affect Th2 memory formation. GATA-3 may regulate the cell fate decision of activated CD4+ T cells, by reducing activation-induced cell death, in favor of Th2 memory cell formation. Alternatively, GATA-3 may facilitate the differentiation process of dividing effector T cells that are already committed to the memory cell fate. A third possibility is that GATA-3 would act as a survival factor for Th2 memory cells. This is not very likely, because survival alone does not appear to be sufficient for memory cell formation, as was shown by the absence of increased memory formation in Bcl-2-transgenic mice (58). Additional experiments are required to define GATA-3 targets that are involved in Th2 memory cell formation.
One of the molecules involved in Th2 memory development may be T1/ST2,
as it is normally specifically expressed in the Th2 lineage within the
compartment of CD44high activated/memory T cells
(Fig. 2). Because cross-linking of T1/ST2 enhanced proliferation of Th2
cells that were stimulated with suboptimal concentrations of
anti-CD3 mAb (55), it is possible that the increased
proliferation and cell survival of CD2-GATA3-transgenic
CD4+ T cells in the in vitro Th2 polarization
cultures originates from increased T1/ST2 expression. The increased
expression of T1/ST2 in CD2-GATA3-transgenic mice would
argue for a direct regulation of T1/ST2 transcription in T
cells by GATA-3. The identification of three GATA elements in the
minimal GATA-responsive T1/ST2 promoter in mast cells
(59) would support this hypothesis of a direct regulation
of T1/ST2 expression by GATA-3, independent of Th2-specific
cytokines. Therefore, we hypothesize that GATA-3 is not only essential
for instructive differentiation of naive Th cells into committed Th2
cells (26, 27, 28, 31, 35), but can also affect proliferation
and survival of GATA-3-expressing CD4+ T cells
through the induction of T1/ST2.
In conclusion, this study shows that enforced expression of GATA-3 inhibits Th1 function and induces Th2 commitment in vivo. Moreover, the increased expression of T1/ST2, the enhanced production of Th2 cytokines in response to T cell activation, and the elevated serum levels of IgG1 in CD2-GATA3-transgenic mice argue for a role of GATA-3 in the formation of Th2 memory.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Rudolf W. Hendriks, Department of Immunology, Faculty of Medicine, Room Ee853, Erasmus University Rotterdam, Dr. Molewaterplein 50, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. E-mail address: hendriks{at}immu.fgg.eur.nl
3 Abbreviations used in this paper: SP, single positive; DTH, delayed-type hypersensitivity; KLH, keyhole limpet hemocyanin; TNP, trinitrophenol; HA, hemagglutinin.
Received for publication April 12, 2000. Accepted for publication May 8, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(IFN-) production during differentiation of human T helper (Th) cells and transient IFN- production in established Th2 cell clones. J. Exp. Med. 179:1273. production and diminishes interleukin 4 inhibition of such priming. Proc. Natl. Acad. Sci. USA 90:10188. T-cell-receptor transgenic system. Proc. Natl. Acad. Sci. USA 89:6065. production from developing Th1 cells in addition to inducing IL-4 and IL-5 levels. Clin. Immunol. 91:134.[Medline]
2 subunit expression in developing T helper 1 (Th1) and Th2 cells. J. Exp. Med. 185:817. synthesis by mouse CD8+ T cells correlates with shifts in the frequencies of cell subsets defined by membrane CD44, CD45RB, 3G11, and MEL-14 expression. J. Immunol. 151:575.[Abstract]
and B cell stimulatory factor-1 reciprocally regulate Ig isotype production. Science 236:944.This article has been cited by other articles:
|
|
 |
|
  N. Yamashita, H. Tashimo, H. Ishida, Y. Matsuo, H. Tamauchi, M. Terashima, I. Yoshiwara, S. Habu, and K. Ohta Involvement of GATA-3-dependent Th2 lymphocyte activation in airway hyperresponsiveness Am J Physiol Lung Cell Mol Physiol, June 1, 2006; 290(6): L1045 - L1051. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Shoemaker, M. Saraiva, and A. O'Garra GATA-3 Directly Remodels the IL-10 Locus Independently of IL-4 in CD4+ T Cells J. Immunol., March 15, 2006; 176(6): 3470 - 3479. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Cannarile, F. Fallarino, M. Agostini, S. Cuzzocrea, E. Mazzon, C. Vacca, T. Genovese, G. Migliorati, E. Ayroldi, and C. Riccardi Increased GILZ expression in transgenic mice up-regulates Th-2 lymphokines Blood, February 1, 2006; 107(3): 1039 - 1047. [Abstract] [Full Text] [PDF]
|
|
|
|
, Wild-type mice; 
,
CD2-GATA3-transgenic mice. The levels of the indicated
cytokines were measured in the culture supernatants by ELISA.
C, Proliferation, determined by
[3H]thymidine incorporation, in response to
anti-CD3
, lymphoblastoid tumor cells from a
lymph node.
, n = 9) and
CD2-GATA3-transgenic mice (•, n =
6) 24 h after the injections.