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Versus IL-4 and IL-10: Selective Induction of IL-10 by Sequential Stimulation of Naive Th Cells with IL-12 and IL-41
*
Institute for Genetics, University of Cologne, Zentrum für Molekularbiologische Medizin, Cologne, Germany, and Deutsches Rheuma-Forschungszentrum, Berlin, Germany; and
Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, may be
critically involved in the establishment of chronic inflammation and
inflammatory autoimmune disease. To date, it has been shown that in
IL-12-stimulated murine Th cell lines containing a major fraction of
Th1 cells, Th2 cells can be induced by IL-4 until about 2 wk after
initial activation, but not later. Here we analyze, based on the
magnetic isolation of viable Th1 cells according to their specific
expression of IFN-, the cytokine commitment of individual Th1 cells.
After activation of naive Th cells with Ag and IL-12 for up to 5 wk,
isolated IFN--producing cells were restimulated with Ag and IL-4.
Within the first 3 to 4 wk of IL-12 stimulation, some
IFN-+ cells stopped expression of IFN- when
restimulated with IL-4. However, within only 1 to 2 wk of IL-12
stimulation, few IFN-+ cells could be converted to
produce IL-4. Others continued to express IFN- and thus were already
committed to a proinflammatory, Th1-like phenotype. Surprisingly,
within 3 wk of IL-12 stimulation, many of the IFN--producing cells
responded to IL-4 restimulation by expression of IL-10, but neither
IFN- nor IL-4, i.e., by conversion to a suppressive,
anti-inflammatory phenotype. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-4, contributes to resistance or
susceptibility to particular pathogens (1, 2, 3, 4, 5, 6). Expression of distinct
Th1 or Th2 cytokines is also associated with and may contribute to
pathologic immune reactions against allergens or autoantigens, i.e.,
allergy and autoimmunity (7, 8, 9). Understanding plasticity and stability
of expression of cytokines in Th cells is thus of obvious importance
for the development of vaccination strategies and therapeutic concepts
as well as our basic understanding of chronic immune reactions.
Th differentiation can be efficiently polarized into a Th1- or a
Th2-like phenotype in vitro (10, 11, 12, 13) as well as in vivo by IL-12 or
IL-4, respectively (14, 15, 16). Treatment of Leishmania
major-infected BALB/c Th2-type responder mice with anti-IL-4
mAb or IL-12 at the time of infection prevents the development of a
fatal Th2 response and instead induces a protective Th1 reaction
(15, 16, 17). However, already about 1 wk after infection, IL-12 or
anti-IL-4 can no longer convert the Th2 into a protective Th1
response (16). After 1 wk of stimulation with IL-4, murine Th2
populations polarized in vitro can no longer be converted to the Th1
phenotype by restimulation with IL-12 (18, 19, 20), probably due to a
defect in IL-12 signal transduction (18). IFN- seems to be able to
restore IL-12 responsiveness under these conditions (21, 22, 23). Polarized
murine Th1 populations can be converted to Th0/Th2-like populations by
IL-4 after 1 wk of stimulation with IL-12 in vitro (18, 19, 20). After
about 3 wk, however, Th1 populations become committed as well and no
longer respond to IL-4 (20).
Until now, conversion of Th1 and Th2 responses has been analyzed on the level of polarized populations of Th cells. However, even highly polarized Th1 and Th2 populations are still heterogeneous with respect to cytokines they express, in that at least 10% and often >20% of the Th cells do not produce the cytokines in question (24, 25, 26). Therefore it has not been clear whether conversion of Th cell populations is the result of conversion of individual Th1 or Th2 cells or of selective outgrowth and differentiation of cells previously not expressing cytokines. Conversely, the stability of Th1 and Th2 populations observed after repeated polarization at later time points could be the result of selective survival of cells responding to the initial polarization.
Here, we have generated in vitro and isolated viable Th1 cells,
producing IFN-, and analyzed the stability of their cytokine
expression under converting conditions. Naive murine Th cells were
stimulated for up to 5 wk with Ag and IL-12 in vitro. From such
polarized populations, still containing about 30% of cells expressing
neither IFN- nor IL-4; 70% producing IFN-, but not IL-4; and
<1% expressing IL-10, IFN--producing cells were isolated according
to expression of surface IFN- (suIFN-), using magnetofluorescent
liposomes for its detection (27, 28). The IFN--producing cells were
restimulated with Ag and IL-4 and then analyzed for expression of
IFN-, IL-4, IL-5, and IL-10.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice homozygously transgenic for the DO11.10 
ß-TCR
(OVA-TCRtg) (29) on BALB/c background were a gift from
Dennis Y. Loh (Washington University School of Medicine, St. Louis, MO)
and were bred under specific pathogen-free conditions in laminar flow
incubators. Specific pathogen-free BALB/cJ mice (8–12 wk) were
obtained from the Bomholtgard Breeding and Research Center (Ry,
Denmark). Mice were killed by cervical dislocation for the isolation of
spleen cells (SC).4
Isolation of CD62L+ CD4+ T cells by MACS MultiSort
OVA-TCRtg SC were stained with FITC-conjugated anti-CD4 mAb (GK1.5/4 (30)) and MultiSort anti-FITC microbeads (Miltenyi Biotec). CD4+ cells were isolated by positive selection on VS+ columns using the high gradient magnetic cell separation system MACS (Miltenyi Biotec), as described previously (31). Then, microbeads were cleaved off enzymatically and the CD4+ cells were stained for CD62L (L-selectin) with digoxigenized (DIG) anti-CD62L mAb (MEL-14 (32)) and anti-DIG MACS microbeads. CD62L+ CD4+ cells were then purified to 99% by positive MiniMACS selection on MS+ columns.
Stimulation of OVA-TCRtg Th cells in vitro
Cells were cultured at 1 to 2 x 106 cells/ml in complete RPMI 1640 (Life Technologies, Grand Island, NY) containing 100 U/ml penicillin, 0.1 mg/ml streptomycin, 0.3 mg/ml glutamine, 10 µM 2-ME, and 5% FCS (PAA, Linz, Austria).
The antigenic peptide OVA323–339 (Neosystem, Strasbourg, France) was used at a 1 µM concentration. OVA-TCRtg SC or congenic BALB/c SC were depleted of T cells with CD4-, CD8-, and Thy1.2- or CD5-microbeads by MACS (Miltenyi Biotec). Ninety-nine percent pure T cell-depleted SC (T-SC) were used as APC for OVA-TCRtg T cells at a 5:1 ratio. Recombinant murine IL-12 (gift from Maurice Gately, Hoffmann-La Roche, Nutley, NJ) was added at 100 U/ml, IL-4 (culture supernatant of NIH-3T3, transfected with murine IL-4 cDNA; gift from Werner Müller, Institute for Genetics, University of Cologne, Cologne, Germany) at 30% (v/v), and neutralizing anti-IL-4 mAb 11B11 (33) at 10 µg/ml as indicated. For restimulation, PMA and ionomycin (both from Sigma, St. Louis, MO) were used at 5 ng/ml and 1 µg/ml, respectively. Brefeldin A (Sigma) was added in some experiments at 5 µg/ml, 2 h before fixation.
Abs and flow cytometry
For intracellular staining, surface staining, or ELISA the
following rat anti-mouse cytokine mAb were used: anti-IL-2
JES6-5H4 and JES6-1A12 (34, 35); anti-IL-4 11B11 (33), BVD4-1D11
and BVD6–24G2 (34, 35); anti-IL-5 TRFK4 and TRFK5 (36); and
anti-IFN- AN18.17.24 (37), R4-6A2 (38), anti-IL-10 JES5-2A5
(34), and SXC1 and SXC2 (39).
FACScan and CellQuest research software (Becton Dickinson, Mountain View, CA) were used for flow cytometry. Dead cells were excluded according to light scatter and staining with propidium iodide (0.3 µg/ml).
Detection and isolation of IFN--producing cells according to
suIFN-
suIFN-, as specific marker for IFN--expressing cells, was
detected on activated Th cells using magnetofluorescent liposomes, as
described previously (27, 28). Briefly, cells were incubated with
purified rat IgG (0.1 mg/ml; Nordic, Tilburg, The Netherlands) in
PBS/BSA/NaN3 at 4°C for 10 min to block unspecific
binding of rat mAb. Then the cells were labeled with DIG-conjugated
AN18.17.24 (5 µg/ml) in PBS/BSA/NaN3 at 4°C for 10 min.
Cells were washed twice and stained with sheep anti-DIG Fab
fragments conjugated to magnetofluorescent liposomes in
PBS/BSA/NaN3 for 30 min at 4°C with gentle agitation.
Cells were washed, resuspended, and analyzed by flow cytometry.
suIFN--positive cells were enriched to 99% purity on
VS+ columns by MACS.
Intracellular staining of cytokines
Cells were fixed, permeabilized, and stained for intracellular
cytokines and surface markers as described previously (24). For
detection of IL-4, IFN-, and IL-10, the mAb 11B11, R4-6A2,
AN18.17.24, and JES5-2A5 were used as DIG- and/or
nitrophenylacetyl-conjugated Ab with FITC-conjugated sheep anti-DIG
Fab (Boehringer Mannheim, Mannheim, Germany) and/or
phycoerythrin-conjugated anti-nitrophenylacetyl mAb S43-10 (40) as
secondary reagents. Stained cells were analyzed by flow cytometry.
ELISA for murine IL-2, IL-4, IL-5, IFN-, and IL-10
Concentrations of IL-2, IL-4, IL-5, IFN-, and IL-10 in the
culture supernatants were determined by sandwich ELISA as described
previously (41, 42), using mAb JES6-5H4, BVD4-1D11, TRFK5, R4-6A2, and
SXC2 as coating Ab and biotinylated mAb JES6-1A12, BVD6-24G2, TRFK4,
AN18.17.24, and SXC1 as developing Ab, respectively. The detection
limits of the ELISA were 0.1 ng/ml for IL-2, 0.2 to 0.4 ng/ml for IL-4,
0.6 to 3 ng/ml for IL-5, 1.6 to 2 ng/ml for IL-10, and 0.1 to 0.2 ng/ml
for IFN-, respectively.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Naive Th cells were isolated from DO11.10 TCR transgenic
(OVA-TCRtg) mice by positive selection of
CD62L+ CD4+ splenic lymphocytes by
multiparameter magnetic cell sorting with MACS MultiSort. First,
CD4+ T cells were specifically labeled with
superparamagnetic MultiSort microbeads and enriched up to 99% purity
(Fig. 1
). The microbeads were then
cleaved off from the cell surface enzymatically. From the purified
CD4+ cells, CD62L+ cells were isolated in a
second magnetic separation step (Fig. 1). Thus, CD62L+
CD4+ T cells were enriched to a purity of 99%.
CD62L+ CD4+ cells expressed IL-2, but no
detectable IL-4, IL-5, IL-10, or IFN-, upon stimulation with PMA and
ionomycin for 4 h, as analyzed by intracellular cytokine staining
(data not shown).
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-expressing (Th1) cells
CD62L+ CD4+ cells from
OVA-TCRtg mice were stimulated for 1 wk with Ag and APC (T
cell-depleted SC) in the presence of IL-12 to induce polarized Th1
populations. In one experiment (Expt. 3), anti-IL-4 mAb was also
added to neutralize any endogenous IL-4. After 1 wk, all viable T cells
expressed CD4 and the transgenic TCR, detectable with the
clonotype-specific mAb KJ1-26.1 (43) (data not shown). As has been
described previously (20, 26), no IL-4-producing cells (<0.1%) were
detectable by intracellular immunofluorescence (Fig. 2) and ELISA (data not shown) upon
restimulation with PMA and ionomycin. IL-10-positive cells were
detectable at very low frequencies (<1%; data not shown), even in the
presence of anti-IL-4 mAb. While secreting high amounts of IFN-
into the culture supernatant (11, 44, 45) (data not shown), such
polarized Th1 populations are still heterogeneous with respect to
expression of IFN- (20, 24, 26) (Fig. 2). At most, 75% of the cells
expressed IFN- as detectable by intracellular staining (Fig. 2).
Twenty to thirty percent of the Th cells did not produce IFN-. We
have shown previously that only those cells staining for IFN-
intracellularly also secrete IFN-, by correlation of intracellular
staining with suIFN- expression, isolation of suIFN-+
cells, and analysis of secretion (28).
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-expressing cells from a
polarized Th1 population and analyzed the stability of cytokine
expression (Fig. 3).
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-expressing cells
CD62L+ CD4+ T cells from
OVA-TCRtg mice were stimulated with the antigenic peptide
OVA323–339 and APC in the presence of IL-12 (and
anti-IL-4 mAb in Expt. 3) for 1 wk, as described above. The cells
were then restimulated for 4 to 5 h with PMA and ionomycin, and
stained for suIFN- with digoxigenized AN18.17.24 mAb and
anti-DIG magnetofluorescent liposomes (27, 28). By high gradient
magnetic cell sorting with MACS, suIFN-+ cells were
enriched from 37 to 60% to 99% (Fig. 3
and Table I). The separation was controlled for
enrichment of IFN--expressing cells by staining of intracellular
IFN- (inIFN-). Among the 99% pure suIFN-+ cells,
>96% also stained for inIFN- when fixed and analyzed directly
after the sort (Table I). Isolated suIFN-+ cells, when
stimulated for 2 h more with PMA and ionomycin in the presence of
the secretion inhibitor brefeldin A, stained to 99.5% for inIFN-
(Table I).
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-expressing Th
cells were cultured for 5 to 6 days with IL-2 in the presence or the
absence of IL-4, IL-12, or IL-12 plus anti-IL-4. Unseparated and
isolated IFN-+ Th cells were then restimulated again
with the antigenic peptide OVA323–339 and T cell-depleted
SC as APC in the presence or the absence of IL-4, IL-12, or IL-12 plus
anti-IL-4, i.e., under neutral (no external cytokine), converting
(IL-4), or preserving (IL-12 with or without anti-IL-4) conditions
for 4 days (Expt. 1 and 3) or 7 days (Expt. 2).
The expression of IL-4, IL-10, and IFN- in OVA-TCRtg Th
cells was analyzed by intracellular immunofluorescence after
restimulation with PMA and ionomycin for 4 h, with brefeldin A
added for the last 2 h before fixation (Figs. 3 and 4A, and Table II). After 24 h of PMA and ionomycin
stimulation, the concentrations of IL-2, IL-4, IL-5, IL-10, and IFN-
in the culture supernatant were analyzed by ELISA (Fig. 4B).
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, Table II), i.e., after 4 days of stimulation with Ag,
indicating that some cells might have been refractory to restimulation
early after activation (46). In addition to cytokine expression, expansion and proliferation of sorted and unsorted OVA-TCR+ Th cells, as detected with the clonotype-specific mAb KJ1-26.1 (43), were analyzed (data not shown). On the average, the number of OVA-TCR+ Th cells increased about threefold after restimulation with Ag. In Expt. 1 and 3 no significant difference between cultures with or without exogenous IL-4 added was found, while in Expt. 2 OVA-TCR+ Th cells expanded better in the presence of IL-4 than in its absence (sixfold compared with threefold). The average number of cell divisions corresponded directly to the expansion of OVA-TCR+ Th cells when proliferation was analyzed using PKH26 membrane labeling (Sigma; data not shown). No indication of massive cell death was found.
Stability of IFN- expression upon neutral or preserving
restimulation
When unseparated cells and isolated IFN--expressing cells were
restimulated under neutral (medium alone) or preserving (IL-12 with or
without anti-IL-4) conditions, most of them (60–98%) continued to
produce IFN- (Fig. 4 and Table II). In all experiments, the
frequency of IFN-+ OVA-TCRtg cells was
higher among the isolated IFN--expressing cells than among the
unseparated cells.
In the first experiment, no IL-4-expressing cells were detectable in
neutrally or preservingly restimulated, unseparated or isolated
IFN--producing cells (Table II). In Expt. 2, we could detect a low
number of IL-4-expressing cells (0.6%) in the unseparated population
when restimulated with IL-12 (Table II). To neutralize endogenous IL-4,
which might have been produced by T cell-depleted SC used as APC, in
the third experiment we added anti-IL-4 mAb during the initial Th1
stimulation as well as to the preserving restimulation. No
IL-4-expressing cells were detectable then (Table II).
In contrast to IL-4, low numbers of IL-10-expressing cells (1.5–4%)
were consistently detectable in preserving restimulations (Table II).
In the primary Th1 population after 1 wk of antigenic stimulation with
IL-12, in the presence of anti-IL-4 mAb, IL-10-positive cells were
detectable, although they were few in number (<1%; data not shown).
Conversion of individual Th1 cells into IL-4- or IL-10-expressing cells
When unseparated IL-12-polarized Th cells and purified
IFN--producing Th cells from such cultures were restimulated under
converting conditions, i.e., in the presence of exogenous IL-4,
IL-4-expressing cells were readily detectable in both cultures (up to
25 and 12%, respectively; Figs. 3 and 4, and Table II), demonstrating
that individual IFN-+ Th1 cells had been converted to
IL-4 production. In the second experiment, the frequency of
IL-4-positive cells induced in the unseparated cells was twice as high
as that in the isolated IFN--expressing cells (Table II). In the
other two experiments, the frequencies of IL-4-positive cells induced
in the unseparated and in the isolated IFN-+ Th1 cells
were similar (Table II).
Fifteen to fifty percent of the 6- to 7-day IL-12-stimulated
IFN--producing Th cells were induced to express IL-10 after
restimulation with IL-4 (Figs. 3 and 4, and Table II). Again, in two
experiments the frequencies of IL-10-positive cells generated in the
unseparated and sorted IFN-+ populations were very
similar, while in the third experiment 40% more IL-10 producers were
generated in the unsorted population (Table II).
In the presence, but not in the absence, of exogenous IL-4, we could
detect IL-5 in the culture supernatant of both IFN-+ and
unsorted cells (Fig. 4B). Due to limitations in cell
numbers, the production of IL-5 was analyzed only by ELISA.
The presence of exogenous IL-4 further resulted in a strong reduction
of the expression of IFN- (Figs. 3 and 4, and Table II). Only 5 to
20% of the unsorted cells and 6 to 23% of the sorted cells still
produced IFN- after converting restimulation, compared with 60 to
98% under preserving conditions.
By intracellular costaining of IL-4, IL-10, and IFN- in isolated
IFN-+ cells after converting restimulation, we analyzed
whether suppression of IFN- production and induction of IL-4 and
IL-10 expression were independent effects of IL-4 on individual Th1
cells. The analysis shows that IL-4- and IL-10-positive cells were
induced with similar frequencies among cells still expressing IFN-,
namely 13% of all IFN--producing cells coexpressed IL-4, and 42%
coexpressed IL-10; those cells that stopped to express IFN-, namely
12% of those produced IL-4, and 36% of those produced IL-10
(Table III). Thus, induction of IL-4 and
IL-10 production is apparently independent of suppression of IFN-
expression in individual Th1 cells.
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+ cells
Differential inducibility of IL-4 and IL-10 expression was
analyzed further in isolated IFN-+ cells polarized for
various times by repeated restimulations in the presence of IL-12. To
this end, IFN-+ cells were isolated by MACS after 1 wk
of primary activation with Ag and polarization with IL-12 and
anti-IL-4. After purification, IFN-+ Th cells were
cultured for 7 more days with IL-2 in the presence of IL-12 and
anti-IL-4. IFN-+ Th cells were then restimulated
three times with the antigenic peptide OVA323–339 and T
cell-depleted SC as APC in the presence of IL-12 plus anti-IL-4 or
in the presence of IL-4 for 4 to 6 days each time. The expressions of
IL-4, IL-10, and IFN- in OVA-TCRtg Th cells were
analyzed by intracellular immunofluorescence after recall stimulation
with PMA and ionomycin for 4 h, and brefeldin A was added for the
last 2 h (Fig. 5A). After
48 h of PMA and ionomycin stimulation, the concentrations of IL-2,
IL-4, IL-5, IL-10, and IFN- in the culture supernatant were analyzed
by ELISA (Fig. 5B).
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production
by IL-4 were largely lost upon repeated conserving stimulations,
although with different kinetics (Fig. 5). Inducibility of IL-4
expression by exogenous IL-4 was nearly completely lost after 3 wk of
IL-12 polarization, while inducibility of IL-10, although reduced to
about 40%, as well as inducibility of IL-5 were still clearly
detectable then. At this time point, suppression of IFN- and IL-2
expression in Th1 cells was still comparable to IFN- and IL-2
suppression after 2 wk of IL-12 polarization. However, 1 wk later,
after about 4 wk of IL-12, only expression of IL-5, but not that of
IL-4 or IL-10, was induced by IL-4. At this point, production of
IFN- and IL-2 could only be partially suppressed in
IFN-+ (Th1) cells upon restimulation with IL-4. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-expressing polarized Th1 cells or
from Th cells not responding to IL-12 with expression of IFN-. Such
cells are present in polarized Th1 populations at frequencies of 20 to
30%. Here we directly address the fundamental question of plasticity
of cytokine expression in Th1 cells by isolation and functional
analysis of live IFN--producing cells from polarized Th1
populations. IFN--expressing Th cells can be isolated according to
low abundant suIFN-, detectable with magnetofluorescent liposomes.
Expression of suIFN- is precisely linked to secretion of IFN-
(28). By sorting of cells according to the expression of suIFN-, we
could also demonstrate that inIFN- immunofluorescence detects all
cells secreting IFN-, as detectable by ELISA (28). Apart from
establishing the validity of intracellular immunofluorescence for
detection of cytokine-expressing cells, this result confirms the
apparent heterogeneity of polarized Th1 populations with respect to
expression of IFN-.
To assess stability and plasticity of cytokine expression in
IFN-+ Th1 cells, purified IFN-+ cells
were restimulated under neutral, i.e., first IL-2-, then Ag-,
preserving (i.e., same plus IL-12) or converting (i.e., same plus IL-4)
conditions. While under neutral and preserving conditions, most
IFN-+ cells retain their phenotype, restimulation with
IL-4 converts a considerable fraction of cells to expression of IL-4
(5–12% of IFN-+ and 5–25% of unseparated Th cells),
IL-5, and/or IL-10 (15–50% of IFN-+ and 19–56% of
unseparated Th cells). It can be excluded that the converted cells are
derived from contaminating IFN-- cells contained in the
population of sorted IFN-+ cells. First,
IFN-+ cells had been enriched to 99.5%. If the
remaining 0.5% IFN-- cells had selectively survived
and/or expanded within the 1 to 2 wk of culture and restimulation after
sorting, either 90% of the IFN-+ cells would have had
to die or the IFN-- cells would have had to divide
three or four times more frequently than IFN-+ cells to
increase their frequency from 0.5 to 5%. However, we did not observe
massive death of IFN-+ cells, and proliferation rates
were similar in both sorted (99.5% pure IFN-+, 0.5%
IFN-- cells) and unsorted (50–70% pure
IFN-+, 30–50% IFN-- cells)
populations. The frequencies of IL-4- and IL-10-expressing cells as
well as the amount of secreted IL-5 induced by IL-4 were at most
twofold different in the separated and unseparated cell populations.
Our results show that IL-4 induces the expression of IL-4, IL-5, or
IL-10 in individual IFN-+ Th1 cells activated for 1 wk
in the presence of IL-12 with about the same efficiency as in
IFN-- cells of that same population.
While IL-4 induces up to 12% of IL-4-expressing cells and 50% of
IL-10-expressing cells in isolated IFN-+ Th1 cells
activated for 1 wk with Ag plus IL-12, stimulation of naive Th cells
for 1 wk with Ag plus IL-4 results in up to 35% IL-4-producing cells
and 70% IL-10-producing cells (Fig. 2; data not shown). The difference
of about 20% more IL-4- and/or IL-10-expressing naive vs converted
IFN-+ Th1 cells corresponds roughly to the 20% of Th1
cells in the converted population that still produce IFN-,
suggesting that after exposure to IL-12 for 1 wk, about one-third of
IFN-+ cells is stably committed to expression of
IFN-, while two-thirds have retained the plasticity of naive Th
cells. The molecular basis for this dichotomy is not clear, nor is that
for the loss of plasticity.
Nevertheless, it is surprising that, after 1 wk of priming with IL-12,
most individual IFN-+ cells are not stably committed to
expression of IFN-, unlike at later time points, and stop to express
IFN- upon converting restimulation with IL-4. Costaining of these
cells for IL-4, IL-10, and IFN- reveals that expressions of IL-4 and
IL-10 are induced with similar patterns and efficiencies in early Th1
cells, which either still do or no longer express IFN-, after
converting restimulation with IL-4. This suggests that suppression of
IFN- production and induction of IL-4 and IL-10 expression are
independent effects of IL-4 on individual Th1-like cells. Our results
show that the shift from stimulation with IL-12 to stimulation with
IL-4 after 1 wk of antigenic stimulation has a drastic effect on
IFN-+ cells, most of which at that stage still respond
to IL-4 by stopping to express IFN-, upon antigenic challenge. The
induction of IL-10 in quite a few cells should indirectly further
support this suppression of IFN- expression (47, 48). The shift from
Th1- to Th2-inducing conditions thus seems a reasonable strategy to at
least suppress if not convert immune reactions in the early phase of
immune responses. The in vitro experiments described here suggest that
IFN- expression can be switched off efficiently by IL-4 within the
first 3 wk of primary activation in the presence of IL-12.
In IL-12-mediated differentiation of IFN-+ Th1 cells,
inducibility of IL-4 expression by IL-4 is lost about 1 wk earlier
(after about 3 wk with IL-12) than inducibility of IL-10 expression and
suppression of IFN-, while IL-5 could still be induced by IL-4 even
after about 4 wk of IL-12 polarization. For both effects of IL-4,
suppression of IFN- and induction of IL-4 expression, STAT-6
activation seems to be required (49, 50, 51), which is detectable in early
Th1 cultures at the population level (52) and in all cells analyzed
here by fluorescence microscopy and flow cytometry (T. Stamm, M.
Löhning, and A. Radbruch, unpublished observation). Whether in
individual cells the production of IFN- is suppressed and/or the
expression of IL-4, IL-5, or IL-10 is induced, has to be regulated by
additional transcription factors. For induction of IL-4 expression,
specific transcription factors, such as c-maf (53) and
GATA-3 (54), were shown to be required. Interestingly, inhibition of
GATA-3 seems to have a strong effect on IL-4, a less strong effect on
IL-10, and only a weak effect on IL-5 expression, reflecting different
thresholds (or alternative pathways) for induction of IL-4 vs IL-10 vs
IL-5 (54), which could explain the different kinetics of inducibility
of IL-4, IL-10, and IL-5 expression observed here in the course of Th1
cell differentiation.
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Acknowledgments |
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Footnotes |
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2 Both authors contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Andreas Radbruch, Deutsches Rheuma-Forschungszentrum Berlin, Hannoversche Str. 27, D-10115 Berlin, Germany. E-mail address:
4 Abbreviations used in this paper: SC, spleen cells; CD62L, CD62 ligand; MACS, magnetic-activated cell sorting; DIG, digoxigen(ized); suIFN-, surface IFN-; inIFN-, intracellular IFN-.
Received for publication December 19, 1997. Accepted for publication May 21, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in response to Leishmania antigens in vitro. J. Immunol. 136:655.[Abstract]
production and diminishes interleukin 4 inhibition of such priming. Proc. Natl. Acad. Sci. USA 90:10188.ß T-cell-receptor transgenic system. Proc. Natl. Acad. Sci. USA 89:6065.-independent mechanism. J. Exp. Med. 171:115. in stabilizing the T helper cell type 1 and 2 phenotype. J. Immunol. 158:2648.[Abstract]
production is cytokine-dependent. Proc. Natl. Acad. Sci. USA 94:3189. and in interleukin-4-expressing cells. Eur. J. Immunol. 24:1097.[Medline]
on interferon- producing T cells from mouse and man. Eur. J. Immunol. 26:263.[Medline]
interferon inhibits lymphokine-induced antiviral and macrophage tumoricidal activities. J. Exp. Med. 159:1560.This article has been cited by other articles:
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  F. Blumenthal-Barby, K. Eulenburg, A. Schrage, M. Zeitz, A. Hamann, and K. Klugewitz In vivo modulation of antigen-experienced cells in response to high-dose oral antigen: deletion but no evidence for alterations in the cytokine phenotype Int. Immunol., July 1, 2008; 20(7): 893 - 900. [Abstract] [Full Text] [PDF]
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 S. Langer, D. Langnickel, P. Enghard, R. Undeutsch, G. R. Burmester, F. Hiepe, A. Radbruch, and G. Riemekasten The systemic and SmD183-119-autoantigen-specific cytokine memory of Th cells in SLE patients Rheumatology, February 1, 2007; 46(2): 238 - 245. [Abstract] [Full Text] [PDF]
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 T. Negishi, Y. Kato, O. Ooneda, J. Mimura, T. Takada, H. Mochizuki, M. Yamamoto, Y. Fujii-Kuriyama, and S. Furusako Effects of Aryl Hydrocarbon Receptor Signaling on the Modulation of Th1/Th2 Balance J. Immunol., December 1, 2005; 175(11): 7348 - 7356. [Abstract] [Full Text] [PDF]
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 L.-O. Tykocinski, P. Hajkova, H.-D. Chang, T. Stamm, O. SOzeri, M. LOhning, J. Hu-Li, U. Niesner, S. Kreher, B. Friedrich, et al. A Critical Control Element for Interleukin-4 Memory Expression in T Helper Lymphocytes J. Biol. Chem., August 5, 2005; 280(31): 28177 - 28185. [Abstract] [Full Text] [PDF]
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 M. Lohning, A. Richter, T. Stamm, J. Hu-Li, M. Assenmacher, W. E. Paul, and A. Radbruch Establishment of memory for IL-10 expression in developing T helper 2 cells requires repetitive IL-4 costimulation and does not impair proliferation PNAS, October 14, 2003; 100(21): 12307 - 12312. [Abstract] [Full Text] [PDF]
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M. J. Loza and B. Perussia Peripheral Immature CD2-/low T Cell Development from Type 2 to Type 1 Cytokine Production J. Immunol., September 15, 2002; 169(6): 3061 - 3068. [Abstract] [Full Text] [PDF]
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J. E. Wigginton and D. Kirschner A Model to Predict Cell-Mediated Immune Regulatory Mechanisms During Human Infection with Mycobacterium tuberculosis J. Immunol., February 1, 2001; 166(3): 1951 - 1967. [Abstract] [Full Text] [PDF]
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 H. Jonuleit, E. Schmitt, G. Schuler, J. Knop, and A. H. Enk Induction of Interleukin 10-Producing, Nonproliferating Cd4+ T Cells with Regulatory Properties by Repetitive Stimulation with Allogeneic Immature Human Dendritic Cells J. Exp. Med., November 6, 2000; 192(9): 1213 - 1222. [Abstract] [Full Text] [PDF]
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Z. Yin, D.-H. Zhang, T. Welte, G. Bahtiyar, S. Jung, L. Liu, X.-Y. Fu, A. Ray, and J. Craft Dominance of IL-12 Over IL-4 in {gamma}{delta} T Cell Differentiation Leads to Default Production of IFN-{gamma}: Failure to Down-Regulate IL-12 Receptor {beta}2-Chain Expression J. Immunol., March 15, 2000; 164(6): 3056 - 3064. [Abstract] [Full Text] [PDF]
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A. Richter, M. Lohning, and A. Radbruch Instruction for Cytokine Expression in T Helper Lymphocytes in Relation to Proliferation and Cell Cycle Progression J. Exp. Med., November 15, 1999; 190(10): 1439 - 1450. [Abstract] [Full Text] [PDF]
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A. G. Doyle, K. Buttigieg, P. Groves, B. J. Johnson, and A. Kelso The Activated Type 1-Polarized Cd8+ T Cell Population Isolated from an Effector Site Contains Cells with Flexible Cytokine Profiles J. Exp. Med., October 18, 1999; 190(8): 1081 - 1092. [Abstract] [Full Text] [PDF]
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M. Lohning, J. L. Grogan, A. J. Coyle, M. Yazdanbakhsh, C. Meisel, J.-C. Gutierrez-Ramos, A. Radbruch, and T. Kamradt T1/ST2 Expression Is Enhanced on CD4+ T Cells from Schistosome Egg-Induced Granulomas: Analysis of Th Cell Cytokine Coexpression Ex Vivo J. Immunol., April 1, 1999; 162(7): 3882 - 3889. [Abstract] [Full Text] [PDF]
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