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*
Immunobiology Section, Laboratory of Parasitic Diseases, and
Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-, IL-2-) develop in significant
numbers in helminth-infected mice deficient in either IL-4R 
-chain
or Stat6. While an expanded population of Th1 (IL-4-,
IL-5-, IFN-+, IL-2+)
lymphocytes was observed in the same animals, surprisingly, cells with
a mixed Th0 cytokine pattern were rare. The cytokine production
phenotypes of the Th1 and Th2 subpopulations generated in infected
Stat6-deficient mice were unaffected by in vitro neutralization of
endogenous IL-4 or IFN-. Nevertheless, while addition of exogenous
rIL-12 resulted in transitory IFN- production by Th2 lymphocytes
from both wild-type and Stat6-deficient mice, IL-4 synthesis was
preserved in the former, but temporarily ablated in the latter cells.
Importantly, IL-4+ IFN-- and
IL-4- IFN-+ populations similar to those
arising in helminth-infected Stat6-deficient mice could also be
generated in vitro by repetitive polyclonal stimulation of
CD4+CD62Lhigh lymphocytes from uninfected mice
of the same strain. Together, the results of these single cell analysis
experiments demonstrate that IL-4R/Stat6 signaling, while influencing
the final frequency of Th2 lymphocytes, is not essential for Th2 cell
development, and suggest that this pathway has a previously
unrecognized function in stabilizing Th2 populations once they have
emerged. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As part of the above concept, it has been proposed that the early
induction of IL-12 or IL-4 from a non-T cell source provides a key
determinant of Th1/Th2 induction (reviewed by O’Garra
(7)). Thus, the triggering of IL-12 from dendritic cells
or macrophages has been shown to provide a strong Th1 bias to the
CD4+ T cell response in both in vitro and in vivo
systems (8, 9, 10). In the opposite direction, a number of
different cell types, including mast cells (11), basophils
(12), NK1.1+ T cells
(13), 
T cells (14), and eosinophils
(15), can serve as a source of early IL-4. Nevertheless,
none of these cell types appear to be obligatory for Th2 induction
(16, 17, 18, 19, 20) and, in some cases, may even inhibit Th2
development (21). Instead, there is now abundant evidence
that Th2 differentiation can be supported solely by IL-4 produced by
Ag-primed (22, 23) or memory CD4+
lymphocytes (24). Furthermore, recent experiments using
cytokine- or STAT-deficient mice indicate that Th1 or Th2 responses can
emerge even in the absence of IL-12/IL-4 or STAT signaling. Thus, in a
number of in vivo systems, significant Th2 cytokine production can be
detected in mice genetically deficient in IL-4 (25, 26, 27, 28).
In addition, while Stat4 knockout
(KO)3 (1)
mice are defective in Th1 cytokine production, double Stat4/Stat6 KO
animals are able to mount significant Th1 responses in vitro
(29), indicating the presence of a Stat4-independent
pathway for Th1 development.
During this multistep process in which naive cells acquire either Th1 or Th2 phenotype, CD4+ T lymphocytes that produce a mixture of Th1 and Th2 cytokines also may arise (30, 31). However, it is not clear whether these cells of intermediate (Th0) phenotype represent an obligatory differentiation step (32) or are in themselves final effector stages (33). Moreover, if these cells do indeed play a role as transitional intermediates in Th1/Th2 development, the extent to which they are dependent and/or susceptible to IL-4/IL-12 differentiation signals is poorly defined.
We have been studying the regulation of Th2 development in an in vivo
model involving infection with the helminth Schistosoma
mansoni. At 5 wk postinfection, the parasite begins to deposit in
host tissues large numbers of eggs that serve as a potent and
persistent stimulus of CD4+ T cell activation.
This response is dominated by a Th2 cytokine secretion profile
characterized by high levels of IL-4, IL-5, IL-10, and IL-13, and
importantly, fails to result in appreciable IL-12 production (34, 35). Previous studies revealed that the schistosome-induced
Th2-type response is partially retained in IL-4-deficient mice
(26), raising the possibility of IL-4-independent Th2
induction or the involvement of alternative IL-4R ligands. To
distinguish between these two hypotheses, we recently studied Th2
cytokine production in S. mansoni-infected IL-4R KO
animals. These mice displayed Th2 (IL-5, IL-10, and IL-13) as well as
Th1 cytokine levels comparable with those found in IL-4 KO animals
(36). In addition, in the data of Kaplan et al.
(37) on S. mansoni-infected Stat6 KO mice,
residual Th2 cytokine expression is apparent, although the significance
of these low level responses was not determined.
In the present study, we have formally analyzed at the single cell
level the source of the residual Th2 cytokine production occurring in
schistosome-infected IL-4R as well as Stat6 KO animals. Our findings
establish that the observed lymphokines originate from conventional
CD4+ Th2 cells. Interestingly, these Th2
lymphocyte populations arise simultaneously with Th1 cells generated as
a result of the absence of cross-regulation by IL-4. Nevertheless,
lymphocytes with a mixed Th1/Th2 cytokine secretion profile are only
rarely observed in the KO mice, suggesting that Th0 cells are not
obligatory precursors in Th subset differentiation. Because similarly
polarized Th2 and Th1 populations could be generated from uninfected
Stat6 KO mice by repeated in vitro polyclonal stimulation of naive
CD4+ cells, their occurrence is not dependent on
stimulation by helminth exposure. Taken together, these observations
are consistent with a model of Th1/Th2 differentiation in which the two
subsets arise independently of each other and in which IL-4R/Stat6
signaling is not essential for Th2 development, but nevertheless plays
a major role in determining the final frequency of cells that express a
Th2 lymphokine secretion profile.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-4R KO and Stat6 KO mice were generated, as previously
described, by homologous recombination in BALB/c-I and 129-derived CCE
embryonic stem cells, respectively (38, 5). The IL-4R
KO animals were bred as homozygotes on the original BALB/c background,
whereas the Stat6 KO mice were partially backcrossed to C57BL/6. Both
mouse strains were bred and maintained at the American Association for
the Accreditation of Laboratory Animal Care (AAALAC) accredited animal
facility of National Institute of Allergy and Infectious Diseases
(NIAID). Control BALB/cJ and C57BL/6 animals were purchased from The
Jackson Laboratory (Bar Harbor, ME) and Division of Cancer Treatment
(National Cancer Institute, Frederick, MD), respectively. C57BL/6 IL-4
KO mice (39) were obtained from Taconic Farms
(Germantown, NY).
Age (8–12 wk)- and sex-matched mice were used in each experiment. Mice were percutaneously infected with 35 S. mansoni cercariae (NMRI strain) and sacrificed 8 wk later (36). A soluble extract of schistosome eggs (SEA) was prepared as described (40).
Cell preparations
Mesenteric LN were pooled from 8-wk schistosome-infected mice
(n = 4–6/group), and single cell suspensions were
prepared in tissue culture medium (RPMI 1640 with 10% heat-inactivated
FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 20
mM HEPES, and 50 µM 2-ME). CD4+ cells were
purified from infected mice by staining LN cells with PE-labeled
anti-CD4 mAb (RM4-5; PharMingen, La Jolla, CA), followed by sorting
on a FACStar sorter (Becton Dickinson, San Jose, CA). The purity of
sorted CD4+ populations was 
97%.
Using the same approach, CD4+ cells were purified from splenocytes from uninfected wild-type (WT) and Stat6 KO mice, with the exception that RBC were lysed by osmotic treatment before staining. To isolate naive CD4+ cells, FITC-labeled anti-CD62L mAb (MEL-14; PharMingen) was added during the staining procedure and double-positive cells were collected. The purity of sorted CD4+CD62Lhigh populations was 95 ± 3%.
Spleen cells from uninfected mice were used as a source of APC. For primary cultures of sorted CD4+ cells from infected mice, splenocytes were depleted of T cells by anti-Thy-1.2 mAb (Cedarlane Laboratories, Hornby, Canada) and Low-Tox-M rabbit C (Cedarlane Laboratories) treatment (18). In subsequent stimulations, total splenocytes were used as APC. For anti-CD3 stimulation of CD4+ and CD4+CD62Lhigh cells from uninfected WT and Stat6 KO mice, splenocytes from IL-4 KO mice were used throughout the experiment. All preparations of APC were initially depleted of RBC by osmotic treatment and finally irradiated with 3000 rad.
Proliferation assay
Mesenteric LN cells from infected mice (3 x 106/ml) were cultured in 0.2 ml in flat-bottom 96-well plates with medium alone or SEA (20 µg/ml). Anti-class II (M5/114, rat IgG2b) (41), anti-CD4 (GK1.5, rat IgG2b) (42), or anti-CD8 mAb (2.43, rat IgG2b) (43) were added to cultures 2 h before SEA. After 48 h, cultures were pulsed with [3H]TdR (0.5 µCi/well; New England Nuclear, Boston, MA; sp. act., 2 Ci/mmol) for 18 h, and incorporated isotype was measured.
Cytokine assays
Mesenteric LN cells from infected animals (3 x 106/ml) were cultured in 24-well plates in 1 ml/well, while sorted CD4+ cells (2.5 x 105/well) from the same LN populations were cultured with irradiated T-depleted syngeneic splenocytes as APC (7.5 x 105/well) in flat-bottom 96-well plates in 0.2 ml/well in the presence or absence of SEA (20 µg/ml). Sorted CD4+ cells from uninfected WT and Stat6 KO mice (2 x 105/well) were cultured with irradiated IL-4 KO splenocytes as APC (7.5 x 105/well) in 0.2 ml/well in flat-bottom 96-well plates in the presence of anti-CD3 mAb (145-2C11; 1 µg/ml; PharMingen) with or without anti-IL-4R mAb (M1; 20 µg/ml; R&D Systems, Minneapolis, MN).
After 72 h, supernatants were collected for cytokine
determinations. IFN- was measured by ELISA (44), while
IL-4 was detected using a commercial ELISA kit from Endogen (Woburn,
MA). The limit of detection was 125 pg/ml and 62 pg/ml for the IFN-
and IL-4 assays, respectively.
Culture conditions before intracellular cytokine staining
Analyses of intracellular cytokine expression were
performed on cells from the same SEA-stimulated cultures used in the
cytokine secretion assays. Mesenteric LN cells from infected mice were
incubated for an additional 18 h in fresh medium added to replace
the culture supernatant collected at 72 h. In the case of sorted
CD4+ cells, cultures were expanded by adding
medium containing human rIL-2 (10 U/ml; Cetus Oncology, Chiron,
Emeryville, CA) for a 7-day period, followed by restimulation with
SEA (20 µg/ml) in the presence of irradiated splenocytes as APC.
On day 3 of this second SEA stimulation, supernatants were
collected and cells were cultured for an additional 4 days in medium
with rIL-2 before intracellular staining. When indicated, rIL-4
(20 ng/ml; R&D Systems), rIL-12 (10 ng/ml; kindly provided by
Hoffman-LaRoche, Nutley, NJ), anti-IL-4 (11B11; 20 µg/ml),
anti-IL-4R (M1; 20 µg/ml; R&D Systems), anti-IL-12 (C17.8;
20 µg/ml) (45), or anti-IFN- (XMG-6; 20 µg/ml)
(46) mAb were added to parallel SEA cultures during both
Ag stimulations performed. Staining patterns were also compared after a
third SEA stimulation done in the absence of exogenous cytokines
or Ab.
CD4+CD62Lhigh cells from uninfected Stat6 KO mice were analyzed for intracellular cytokine expression 7 days after each of three consecutive anti-CD3 stimulations performed on days 0, 7, and 14. Following each stimulation, 72-h culture supernatant was replaced with medium containing rIL-2 (10 U/ml).
Intracellular cytokine staining and flow cytometry
For intracellular staining, cells were stimulated in 1-ml
cultures in 24-well plates coated overnight with 2.5 µg/well of
anti-CD3 mAb (145-2C11). Two hours later, brefeldin A (BFA; 10
µg/ml; Sigma, St. Louis, MO) was added, and after 5 h, cells
were stained using a modified protocol based on that described by
Prussin and Metcalfe (47). Briefly, cells were washed once
in RPMI, stained with Cy-chrome-labeled anti-CD4 mAb (RM4-5;
PharMingen), and fixed for 15 min in 2% paraformaldehyde (Sigma) at
room temperature. After 30-min incubation in permeabilization buffer
(PBS containing 0.1% Saponin (Calbiochem-Novabiochem, La Jolla, CA),
0.1% FCS, and 20 mM HEPES) with 10% normal mouse serum and
anti-FcRII/III mAb (2.4G2; 5 µg/ml; PharMingen) at 4°C,
cells were stained for 30 min with different combinations of the
following, pretitrated flourochrome-labeled mAb at 4°C: PE
anti-IL-2 (JES6-5H4), PE anti-IL-4 (BVD4-1D11), FITC
anti-IL-4 (BVD4-1D11) (all from BioSource International, Camarillo,
CA), PE anti-IL-5 (TRFK5), FITC anti-IL-10 (JES5-16E3), and
FITC anti-IFN- (XMG1.2) (all from PharMingen), washed twice with
permeabilization buffer, and resuspended in PBS + 0.5% FCS. Cell
fluorescence was measured using a FACScan flow cytometer and data were
analyzed using CellQuest software (Becton Dickinson). The specificity
of the cytokine staining was confirmed in initial experiments in which
preincubation with corresponding unlabeled mAb completely inhibited the
signal obtained with labeled anti-cytokine mAb (not shown).
To determine the Vß8 usage of the IL-4+ CD4+ lymphocytes, cells were stained with Cy-chrome-labeled anti-CD4 and FITC-labeled anti-Vß8 mAb (MR5-2; PharMingen), fixed, and subsequently stained with PE-labeled anti-IL-4, as described above.
Isolation of T cell clones
SEA-specific T cell clones from infected Stat6 KO mice were obtained by limiting dilution of CD4+ cells twice stimulated with SEA, as described for the intracellular cytokine-staining procedure. Briefly, 7 days after a second Ag stimulation, cells were cultured (1 or 0.3 cells/well) in 0.1 ml/well in round-bottom 96-well plates with 3 x 105 irradiated syngeneic spleen cells and SEA (20 µg/ml). One day later, 0.1 ml/well of rIL-2 (20 U/ml) was added. After 2 wk, fresh medium with APC and Ag was added, followed by rIL-2 48 h later. During the fourth week, wells containing proliferative cells were identified visually and further expanded. Cloning efficiency was 12 and 4% for cultures with 1 and 0.3 cell/well, respectively. Cells from positive wells were characterized for their cytokine secretion profile by ELISA and by intracellular staining.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Stat6 KO
mice are conventional CD4+ T lymphocytes
We have previously shown that LN cells from IL-4R KO mice
infected with S. mansoni develop diminished yet significant
IL-4 responses when restimulated in vitro with parasite Ag
(36). We began the present study by performing a series of
experiments in both IL-4R- and Stat6-deficient mice to more
precisely define the cell populations producing IL-4 in
schistosome-infected mice of both types. Initial studies in infected
IL-4R KO mice indicated that in vitro treatment with anti-CD4,
but not anti-CD8 mAb abolished the residual Th2 cytokine production
observed (not shown). Similarly, in vitro addition of anti-CD4 but
not anti-CD8 mAb caused a dramatic reduction in the proliferative
responses of mesenteric LN cells from 8-wk schistosome-infected
IL-4R and Stat6 KO animals to soluble egg Ag (SEA) (Fig. 1
). Moreover, an even greater inhibition
in blastogenesis was detected when anti-class II mAb was added to
parallel cultures (Fig. 1). Thus, the T cell responses to SEA in
infected IL-4R and Stat6 KO mice are both CD4 and class II
dependent.
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KO mice
and compared their secretion of the cytokine in response to SEA. As
shown in Fig. 2,
CD4+ cells isolated from KO animals retained the
ability to secrete IL-4, albeit in lower amounts than equivalent
populations from WT mice. In contrast, whereas LN cells from WT animals
failed to produce appreciable amounts of IFN-, cells from IL-4R
KO mice secreted high levels of the cytokine when stimulated with SEA.
Again, this altered cytokine profile was retained in the
CD4+ T cells purified from these populations
(Fig. 2). IL-4/IFN- cytokine secretion patterns similar to those
described above in IL-4R KO mice were also obtained when purified
CD4+ T lymphocytes from infected Stat6 KO animals
were stimulated in vitro under the same conditions (data not
shown).
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and Stat6 KO mice. The frequency of cytokine-positive cells in ex vivo
LN populations from both WT and KO animals was generally <1% (data
not shown). To facilitate the detection of cytokines by this procedure,
we stimulated the LN populations with SEA for 72 h in vitro before
intracellular staining. As shown in Table I, the cytokine production pattern of the
CD4+ lymphocytes detected by flow cytometry was
found to closely resemble that observed in supernatants from
SEA-stimulated cultures (Fig. 2
) in that the majority of the Th cells
in the WT animals stained positively for IL-4 and not IFN-, while in
KO mice the reverse pattern was observed.
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0.2) were comparable with those of the equivalent
IL-4+ populations from WT mice (Table II) and very different from the ratio
(1.2) found among NK T cells (48). Thus, the
IL-4-producing cells arising in the absence of IL-4R signaling appear
to be conventional CD4+ T lymphocytes.
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are produced by distinct CD4+
subpopulations in LN cell cultures from infected IL-4R and Stat6 KO
animals
In the intracellular cytokine staining experiments described above
we noticed that the small population of IL-4+
CD4+ T cells arising in the KO mice appeared to
be negative for IFN-. To more accurately classify the phenotype of
these cells, we expanded the sorted CD4+
population by performing an additional stimulation with SEA in vitro
and repeated the intracellular staining for IL-4 and IFN- as well as
IL-2 and IL-5. As expected, the frequency of cytokine-producing cells
increased as a result of stimulation, but to the same degree in WT and
KO (Stat6) cultures (Fig. 3).
Importantly, the majority of the IL-4+ cells from
the KO mice was negative for both IL-2 and IFN-, while the
IFN-+ and IL-2+
populations were also predominantly IL-4 negative (Fig. 3, two
top right panels). Only minor subpopulations (3%) were found
to be double positive for IL-4 and either IL-2 or IFN-. The same
segregation in phenotype was observed between IFN-- and
IL-5-producing cells (Fig. 3, third right panel). In
contrast, IL-2 was heavily expressed by the
IFN-+ population, supporting their
classification as Th1 cells (Fig. 3, bottom right panel). As
expected, WT cells expressed a predominantly Th2 cytokine pattern
(IL-4+, IL-5+,
IFN-- , IL-2-) (Fig. 3, left panels), while the staining profile for
CD4+ cells from infected IL-4R KO animals
(data not shown) was similar to that found in Stat6 KO cells. Again,
these CD4+ populations from infected KO mice were
negative for NK1.1 and displayed
Vß8+/Vß8- ratios
indistinguishable from WT cultures (data not shown).
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, although the percentage
of CD4+ cells staining positive for IL-5 or
IFN- increased over time, no increase in the proportion of cells
with a mixed phenotype (IL-4+
IFN-+ or IL-5+
IFN-+) was observed in cultures from either
Stat6 or IL-4R KO animals over an additional 14-h period.
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(top panels), in both WT and Stat6 KO cultures, most
of the IL-5-producing cells were also positive for IL-4, as expected
for Th2 lymphocytes. In cultures from both WT and, to a lesser extent,
KO mice, large numbers of IL-4+
IL-5- cells were also observed, as expected from
the delayed production of IL-5 vs IL-4 by CD4+
cells from both WT and KO cultures (data not shown and Fig. 4). In
contrast, while most of the IL-4+ cells from WT
animals stained positively for IL-10, only 26% of the
IL-4+ cells from Stat6 KO mice were positive for
this second Th2 cytokine (Fig. 5, lower panels), which was
produced with the same kinetics as IL-4 (data not shown).
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T cell cloning was performed as a final step in the
characterization of the Th2 cells arising in parasite-infected Stat6 KO
mice. The sort-purified and restimulated CD4+
cells shown in
Figs. 3–5 were cloned by limiting dilution in the
presence of SEA plus IL-2. Equal numbers of clones with a Th1
(IL-4- IFN-+) or a Th2
(IL-4+, IL-5+,
IL-13+, IFN--)
phenotype were obtained. Consistent with the intracellular staining
results (Fig. 5), 3 of 10 of these Th2 clones produced IL-10. The
SEA-specific Stat6-negative Th2 cells derived have been established as
long-term clones and have maintained their cytokine profile over a 3-mo
period of testing.
To test the stability of the Th2 population generated in Stat6 KO mice,
sorted CD4+ cells were stimulated twice with SEA
in the presence of different cytokine and anti-cytokine reagents
previously reported to influence the differentiation of Th cells in WT
animals. Because the IL-4+ cells from Stat6 KO
mice develop in the presence of significant amounts of IFN-, we
first tested the effects of anti-IFN- or anti-IL-12 mAb on
the distribution of cells staining positive for IL-4 or IFN-, and no
significant changes were observed (data not shown). Similarly, addition
of either rIL-4, anti-IL-4, or anti-IL-4R mAb failed to
influence the frequency of the different Th cell populations in
cultures from both Stat6 KO and WT mice (Fig. 6 and data not shown). Consistent with
previously published findings that Th2 lymphocytes can develop the
capacity to produce IFN- (49, 50), addition of rIL-12
induced a fraction of the IL-4+ cells in WT
cultures to start coexpressing IFN-+ (Fig. 6).
Interestingly, rIL-12 triggered a quite different change in the
cultures obtained from Stat6 KO animals. Thus, while the frequency of
IFN-+ CD4+ lymphocytes
increased, these cells were not double IL-4+
IFN-+ producers (Fig. 6). Moreover, the number
of IL-4+ CD4+ lymphocytes
was dramatically reduced. The same cultures also showed marked
reductions in levels of IL-5, IL-10, and IL-13 (not shown), indicating
a wholesale decrease in the expression of Th2 cytokines. Thus, in the
absence of IL-4R/Stat6 signaling, Th2 cells appear to be more
susceptible to the destabilizing effects of IL-12. Nevertheless, the
expression of IFN- by IL-12-treated cells from both WT and
Stat6-deficient mice was transient, returning to almost baseline after
one restimulation in the absence of that cytokine (Fig. 6, right
panels). Importantly, removal of IL-12 also resulted in recovery
of IL-4 synthesis in cultures from Stat6 KO mice, confirming the
phenotypic identity of the Th2 population present.
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- phenotype can also be derived from uninfected
Stat6 KO mice
Since it could be argued that our observation of Th2 cells in
IL-4R- or Stat6-deficient animals depends on stimulation by
helminth infection, we attempted to generate cells with the same
phenotype from noninfected KO donors. Splenic
CD4+ T lymphocyte populations were purified from
uninfected Stat6 KO or WT control mice and stimulated with anti-CD3
mAb in the presence of irradiated splenocytes from IL-4 KO donors as
APC. As expected, IL-4 was not detected in 72-h supernatants from
either WT or Stat6 KO anti-CD3-stimulated cultures. However, when
anti-IL-4R-blocking mAb was added to parallel cultures to prevent IL-4
consumption, significant amounts of IL-4 were found in both WT and
Stat6 KO supernatants (Table III).
Indeed, comparable levels of the cytokine were produced by cells from
both strains, suggesting that the higher frequency of IL-4-producing
CD4+ cells seen in vivo in helminth-infected WT
vs Stat6 KO mice may reflect the enhancing effect of prolonged
IL-4/IL-4R interaction on Th2 development. Consistent with their
known Th1 bias, lymphocytes from Stat6 KO mice produced higher levels
of IFN- than control cells from WT animals when stimulated with
anti-CD3.
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). These cells were also found to
produce IL-5 (data not shown). In agreement with the intracellular
staining results obtained in the analysis of CD4+
lymphocytes from S. mansoni-infected IL-4R and Stat6 KO
mice, only a very small proportion of the IL-4+
CD4+ cells coexpressed IFN-, confirming the
segregation of differentiating cells into Th1 or Th2 in the absence of
IL-4R signaling. As expected, when similar anti-CD3 experiments
were performed with
CD4+CD62Lhigh lymphocytes
from WT animals, IL-4+ cells could be detected in
significant proportion (1/3 cytokine-producing cells) even after a
single round of stimulation (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although Th2 cytokine production by both IL-4 and IL-4R KO mice has
been observed in a number of experimental systems, it was possible that
such responses originate from Th0 lymphocytes or unconventional
CD4+ T cells (e.g., NK T cells) (25, 38). The data presented in this work demonstrate that the
CD4+ IL-4-producing cells that arise in
schistosome-infected IL-4R and Stat6 KO animals are conventional Th2
cells based on several criteria. First, these cells possess a pure Th2
(IL-4+, IL-5+,
IFN--, IL-2-)
cytokine as opposed to mixed Th1/Th2 cytokine secretion profile of Th0
cells and NK T cells. Moreover, their response to schistosome Ag is
class II restricted and they do not selectively display the TCR Vß8
chain characteristically expressed by NK T cells. One unusual
characteristic of this population, however, is the low frequency of
cells expressing IL-10. This finding is, nevertheless, consistent with
recent work demonstrating a role for IL-4 in the generation of
IL-10-producing CD4+ cells (51).
Because the observed in vivo generation of CD4+
cells with a Th2 lymphokine pattern occurred in schistosome-infected
IL-4R and Stat6 KO mice, it could be argued that the results
obtained are uniquely dependent on this helminth stimulus.
Nevertheless, it should be noted that residual Th2 cytokine synthesis
in mice deficient in IL-4 or IL-4R signaling has been documented now in
response to a variety of different helminth and protozoan infections,
as well as to injected soluble protein Ag (25, 26, 27, 28, 52, 53, 54, 55). More importantly, as demonstrated in this work (Fig. 7), CD4+ cells with a Th2 lymphokine profile can
be generated in vitro from naive Th precursors from uninfected Stat6 KO
mice by repeated polyclonal stimulation with anti-CD3. Thus, it is
clear that Th2 development in the absence of IL-4R/Stat6 signaling is
not an isolated phenomenon dependent on a particular form of T cell
triggering.
In addition to low numbers of Th2 cells, the infected IL-4R and
Stat6 KO mouse strains also developed high numbers of Th1
(IFN-+ IL-2+) cells, as
predicted from the absence of IL-4R-dependent cross-regulation by IL-4
(29, 56, 57). Interestingly, however, this induction of
Th1 cytokine activity occurs despite the absence of detectable IL-12 in
vitro (<30 pg/ml of p40) and, moreover, has also been demonstrated to
take place in schistosome-infected double IL-4/IL-12 KO animals (E.
Patton, L. Rosa Brunet, and E. Pearce, personal communication).
Nevertheless, we failed to detect significant numbers of cells with a
mixed Th0 cytokine secretion pattern in the CD4+
populations from the same infected IL-4R or Stat6 KO mice from which
both Th1 and Th2 cells were recovered. Thus, in this situation in which
neither IL-4R signaling nor IL-12 production occurs, the potent T cell
activation provided by S. mansoni infection leads directly
to emergence of cells with a pure Th1 or Th2 phenotype. The above
conclusion is supported by the results of Reiner and colleagues, who
found that in the presence of pharmacologic epigenetic derepression,
anti-CD3-stimulated CD4+ T cells from Stat6
KO mice develop into distinct populations expressing IFN- or IL-4
(58). As shown in the present study, a similar segregation
into IL-4- or IFN--producing cells can be achieved in the absence of
pharmacologic derepression by repetitive anti-CD3 stimulation of
CD4+CD62Lhigh cells from
uninfected Stat6 KO mice (Fig. 7).
The results of the present study have several implications for the mechanism of Th subset development. Most importantly, they formally confirm that generation of Th2 cells can indeed take place in the absence of IL-4R/Stat6 signaling, arguing against models in which this triggering event is essential for instructing naive cells to develop a Th2 cytokine secretion profile. Our findings are equally inconsistent with models of Th differentiation in which cytokine genes are expressed independently of each other at the single cell level, since we fail to detect significant numbers of cells with a mixed Th0 phenotype. Finally, the same observations imply that Th0 cells are not necessary precursors for Th1 and Th2 lymphocytes, and instead may represent a later stage of CD4+ cell differentiation. Although these conclusions are based on results obtained with mice deficient in IL-4R signaling, findings from previous studies in IL-4R/Stat6-sufficient animals suggest that they are generally applicable. For example, in analyses of cytokine gene expression at early time points during differentiation of transgenic CD4+ T lymphocytes, most cells were found to express either Th1 or Th2 cytokine mRNAs, with Th0 cells being rare (59). The generation of Th1 and Th2, in the absence of Th0 cells, has also been noted in related studies involving intracellular staining of in vitro differentiating transgenic CD4+ lymphocytes (60), as well as in polyclonally activated human peripheral blood T cells (61).
Nevertheless, stable clones with a mixed Th0 cytokine profile are frequently encountered in both mouse and human studies (30, 31, 62, 63). We speculate that such clones, while stable in vitro, do not persist in vivo due to the continuing influence of the cytokine microenvironment. Based on our finding that Th0 cells are rare in cultures from animals with defective IL-4R signaling, we argue that their development is also dependent on cytokine cross-talk, but that this step occurs after the initial generation of cells with a pure Th1 and Th2 phenotype.
While, as demonstrated in this study, IL-4R signaling is not essential
for Th2 induction, this pathway is clearly involved in determining the
outcome of CD4+ T cell differentiation in an
IL-4R-sufficient setting. Thus, two key questions raised by our
findings concern the role of IL-4 in the acquisition and maintenance of
the Th2 phenotype and how Th2 cells are generated in its absence.
Several possible, but not mutually exclusive, explanations exist. One
alternative is that there is a small but significant probability that
commitment to the Th2 phenotype may occur as a result of epigenetic
derepression in a population of cells stimulated solely through their
TCR. That probability may vary depending upon the conditions of such
stimulation (e.g., high vs low Ag concentration (64, 65);
level of B7/CD28 costimulation (66); presence of BCL-6
expression (67)), but is markedly augmented in the
presence of IL-4. An additional mechanism by which IL-4 may increase
the frequency of cells with a Th2 phenotype is by restricting Th1
development. This concept is supported by the observation of Kaplan et
al. (29), that while Stat4 KO mice are defective in
CD4-dependent IFN- production, such responses are restored in double
Stat4/Stat6 KO animals in which IL-4R signaling is now ablated. Our
observation of persistent Th1 cells in schistosome-infected IL-4R
and Stat6 KO mice in the absence of significant IL-12 production (Fig. 3) is also consistent with the latter hypothesis.
A second possible mechanism that would explain IL-4R-independent
acquisition of the Th2 phenotype is that this process normally depends
upon both TCR-mediated stimulation and IL-4R triggering, but that in
its absence, other as yet undefined signals exist that can functionally
substitute for that provided by IL-4. One important version of this
hypothesis is that the absence of IL-12 in itself provides the critical
secondary element required for Th2 differentiation. Indeed, lack of
IL-12 induction is a common feature in all of the settings discussed
above, in which residual Th2 cytokine synthesis was observed in mice
deficient in IL-4 or IL-4R signaling.
In a third model of Th2 differentiation, TCR engagement is the major
factor that leads to competence for the transcription of Th2 cytokines,
but IL-4 acts after this initial event. That IL-4 acts purely as a
selective agent and leads to the preferential growth or survival of
cells that have already committed to the Th2 phenotype is highly
unlikely. Although Th1 and Th2 cells do have different growth
properties and different responsiveness to IL-12 and IL-4, one can
employ tissue culture conditions in which there is no apparent growth
advantage of the responding cells and still find that IL-4 favors the
development of Th2 cells from naive precursors (68).
Instead, we favor the hypothesis that autocrine IL-4R signaling plays a
major role in stabilizing cells with a Th2 phenotype, protecting them
against IL-12-mediated inhibition, as exemplified by the experiment
shown in Fig. 6. IL-4R signaling may also promote Th2 stabilization
through its involvement in IL-10 induction (Fig. 5), known to suppress
Th1 cytokine expression (69). Indeed, priming Th1 or Th2
cells in the absence of their differentiative cytokines may render them
generally more labile to subsequent alterations in the cytokine
microenvironment because they have not been locked in by genetic
remodeling. This system may be asymmetrical with paracrine IL-12 acting
early in CD4+ differentiation to enhance Th1
generation, and in its absence, autocrine IL-4 acting at a later stage
to promote and stabilize Th2 development.
Regardless of the precise mechanisms involved, it is clear that IL-4R/Stat6-independent Th2 development exists and may reflect a more general pathway for CD4 T cell subset diversification. Further studies on the regulatory effects of IL-4 and IL-12 on early Th1/Th2 populations generated from mice deficient for these two cytokines should offer a powerful approach for both delineating this process and assessing its overall contribution to CD4+ lymphocyte differentiation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Dragana Jankovic, Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 4, Room 126, 9000 Rockville Pike, MD 20892-0425. E-mail address:
3 Abbreviations used in this paper: KO, knockout; BFA, brefeldin A; LN, lymph node; SEA, soluble egg Ag; WT, wild type.
Received for publication November 8, 1999. Accepted for publication January 13, 2000.
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M. J. Czar, E. N. Kersh, L. A. Mijares, G. Lanier, J. Lewis, G. Yap, A. Chen, A. Sher, C. S. Duckett, R. Ahmed, et al. Altered lymphocyte responses and cytokine production in mice deficient in the X-linked lymphoproliferative disease gene SH2D1A/DSHP/SAP PNAS, June 7, 2001; (2001) 131193098. [Abstract] [Full Text] [PDF]
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J. D. Farrar, W. Ouyang, M. Lohning, M. Assenmacher, A. Radbruch, O. Kanagawa, and K. M. Murphy An Instructive Component in T Helper Cell Type 2 (Th2) Development Mediated by Gata-3 J. Exp. Med., March 5, 2001; 193(5): 643 - 650. [Abstract] [Full Text] [PDF]
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A. P. Mountford, K. G. Hogg, P. S. Coulson, and F. Brombacher Signaling via Interleukin-4 Receptor {alpha} Chain Is Required for Successful Vaccination against Schistosomiasis in BALB/c Mice Infect. Immun., January 1, 2001; 69(1): 228 - 236. [Abstract] [Full Text] [PDF]
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S. Ostrand-Rosenberg, M. J. Grusby, and V. K. Clements Cutting Edge: STAT6-Deficient Mice Have Enhanced Tumor Immunity to Primary and Metastatic Mammary Carcinoma J. Immunol., December 1, 2000; 165(11): 6015 - 6019. [Abstract] [Full Text] [PDF]
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N. Noben-Trauth, J. Hu-Li, and W. E. Paul Conventional, Naive CD4+ T Cells Provide an Initial Source of IL-4 During Th2 Differentiation J. Immunol., October 1, 2000; 165(7): 3620 - 3625. [Abstract] [Full Text] [PDF]
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 L. H. Glimcher and K. M. Murphy Lineage commitment in the immune system: the T helper lymphocyte grows up Genes & Dev., July 15, 2000; 14(14): 1693 - 1711. [Full Text]
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M. J. Czar, E. N. Kersh, L. A. Mijares, G. Lanier, J. Lewis, G. Yap, A. Chen, A. Sher, C. S. Duckett, R. Ahmed, et al. Altered lymphocyte responses and cytokine production in mice deficient in the X-linked lymphoproliferative disease gene SH2D1A/DSHP/SAP PNAS, June 19, 2001; 98(13): 7449 - 7454. [Abstract] [Full Text] [PDF]
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