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*
Institut National de la Santé et de la Recherche Médicale (INSERM), U25, and Centre de l’Association Claude Bernard, Hôpital Necker, Paris, France; and
Howard Hughes Medical Institut, Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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immediately upon TCR cross-linking. We previously
showed in the thymus that the NK T subset requires IL-7 for its
functional maturation. In this study, we analyzed whether IL-7 was
capable of regulating the production of IL-4 and IFN- by the
discrete NK T subset of CD4+ cells in the periphery. Two
hours after injection of IL-7 into mice, or after a 4-h exposure to
IL-7 in vitro, IL-4 production by CD4+ cells in response to
anti-TCR-
ß is markedly increased. In contrast, IFN-
production remains essentially unchanged. In
ß2-microglobulin- and CD1-deficient mice, which lack NK T
cells, IL-7 treatment does not reestablish normal levels of IL-4 by
CD4+ T cells. Moreover, we observe that in wild-type mice,
the memory phenotype (CD62L-CD44+)
CD4+ T cells responsible for IL-4 production are not only
NK1.1+ cells, but also NK1.1- cells. This
NK1.1-IL-4-producing subset shares three important
characteristics with NK T cells: 1) Vß8 skewing; 2) CD1 restriction
as demonstrated by their absence in CD1-deficient mice and relative
overexpression in MHC II null mice; 3) sensitivity to IL-7 in terms of
IL-4 production. In conclusion, the present study provides evidence
that CD4+MHC class I-like-dependent T cell populations
include not only NK1.1+ cells, but also NK1.1-
cells, and that these two subsets are biased toward IL-4 production by
IL-7. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and
TNF-ß, which are involved in cell-mediated inflammatory reactions.
Th2 cells secrete mainly IL-4, IL-5, IL-6, IL-10, and IL-13, which
mediate B cell activation, Ab production, and negative regulation of
Th1 responses. This developmental pathway is influenced by several
factors (2, 3): the Ag dose, the type of APCs, and/or the costimulatory
pathways. However, the most effective inducer of CD4+ T
cell differentiation appears to be the local cytokine environment. It
is clear that the cytokines IL-12 and IFN- direct differentiation to
a Th1 phenotype. In many Th2 responses, IL-4 itself plays an essential
role in the induction of naive T cells to subsequently produce IL-4 and
other Th2-specific cytokines in response to Ag in vitro (4, 5) and in
vivo (4). It is still unknown, however, which cell population provides the earliest source of IL-4 required for specific Th2 responses in vivo (3). With regard to the mainstream CD4+ cell compartment, initial IL-4 could be produced by two distinct populations: 1) naive T cells in response to a primary stimulation in the presence of IL-6 (6); 2) already differentiated T cells that act on nearby naive T cells.
Recently, a subset of CD4+ T cells that express the NK1.1
molecule (7, 8), termed NK T cells, has been shown to rapidly secrete
large amounts of IL-4 in the spleen upon activation with anti-CD3
Abs injected i.v. (9). CD4+ NK1.1+ T cells
express intermediate level of TCR-ß, have a strongly biased
repertoire skewed toward Vß8 (7, 8, 10), and are selected and
restricted by nonclassical MHC class I molecules (7, 8, 11, 12),
principally CD1 (13, 14, 15). Thus, the number of TCR-ßint
NK1.1+ T cells is greatly reduced in the thymus and spleen
of ß2-microglobulin-deficient
(ß2m-/-)4
mice (11), and it has been proposed that this deficiency explains their
low production of IL-4 and IgE upon anti-CD3 and anti-IgD
injection (9, 16). Due to their capacity to rapidly produce IL-4 upon
activation, it has been speculated that CD4+
NK1.1+ T cells may induce Ag-specific Th2 responses in vivo
by providing significant amounts of IL-4 at the onset of an immune
response (3, 7, 8, 16). However, interestingly, NK1.1+ T
cells appear to possess a dual functionality. Besides IL-4, they
secrete significant amounts of IFN- via the activation of the
TCR/CD3 complex (15, 17). Moreover, they may be induced to produce
large amounts of IFN- (but not IL-4) upon NK1.1 receptor
cross-linking (18) or by proinflammatory cytokines such as IL-12 (18, 19).
It is likely that IL-4 dominates over IFN- (or IL-12), suggesting
that Th2 cell development is favored in the presence of both cytokines
(3, 5, 20). However, quantitative differences in IL-4 production during
the initial stimulus may be an important factor influencing Th cell
development and its stabilization (5, 21). In some circumstances,
administration of IL-12 induces a marked inhibition of IL-4-secreting
cells and may be indispensable in Ag-induced Th1 differentiation (22).
Altogether, these observations support the idea that factors increasing
the early IL-4 source would play a key role in promoting Th2
polarization.
Recent evidence from our laboratory suggests that IL-4 production by
thymic NK T cells depends on or is facilitated by IL-7 (23, 24). First,
thymic NK T cells from IL-7-deficient mice require IL-7 for their
functional maturation into IL-4-producing cells (23). Second, a short
2-h preincubation of wild-type mouse thymic NK T cells with IL-7 is
sufficient to significantly increase both the expression of IL-4 mRNA
and IL-4 production capacity, and IL-4 has no effect in the same
experimental conditions (24). Finally, the maintenance of IL-4
production by thymic NK T cells in vitro requires the specific presence
of IL-7 (23, 25). Collectively, these findings suggest that IL-7 is
important at a late stage of NK T cell development, i.e., to reach a
stage of full maturation. Thus, from the standpoint of designing new
effective vaccines, it will be relevant to determine whether, in the
periphery, IL-7 has the ability to direct NK T cells toward a Th1 or a
Th2 profile. We thus analyzed the modulatory effects of IL-7 in vivo
and in vitro on direct IL-4 and IFN- production by CD4+
splenocytes. We show that IL-7 contributes to efficiently increase
initial production of IL-4 (but not that of IFN-) by the
CD4+ splenic T cell compartment. Cell sorting experiments
using wild-type, CD1-deficient mice, ß2m-deficient mice,
or I-Aß-deficient mice revealed that IL-7-responder CD4+
splenic T cells responsible for IL-4 production include, as expected,
the NK T subset, but also an NK1.1- subset selected by
class I-related molecules. Thus, by amplifying the early IL-4 sources,
notably nonconventional CD4+ T cells, IL-7 may play a key
role in promoting Th2 polarization.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three- to ten-week-old female mice were used in this study. Wild-type and mutant (ß2m-/- or I-Aß-/-) C57BL/6 mice and wild-type BALB/c mice were bred and maintained in our animal facilities under specific pathogen-free conditions. Mutant ß2m-/- mice of the BALB/c background were kindly provided by J. C. Guery (INSERM U28; CHU Purpan, Toulouse, France). Mutant CD1-/- mice (C57BL/6 and mixed C57BL/6 x 129 backgrounds) have been described previously (14).
Treatment of mice with IL-7
Human rIL-7, produced in Escherichia coli and
purified by Immunex (Seattle, WA), was generously supplied by Sanofi
(Labège, France). The IL-7 had a specific biological activity of
8.8 x 106 U/mg, as measured by the proliferation of a
murine pre-B cell line. The endotoxin levels were 7.5 U/mg of IL-7. To
test the effect of IL-7 on the production of IL-4 and IFN- by
CD4+ T cell subpopulations, mice received s.c. one single
injection of 2 µg of IL-7 diluted in a pyrogen-free solution
containing BSA (500 µg/ml; Life Technologies, Gaithersburg, MD).
Control mice were injected with an identical volume of the BSA solution
alone. The in vivo biological effect of IL-7 was controlled by
measuring the increase in both the absolute number of hemopoietic cells
and the relative number of pre-B cells in the spleen of treated mice
(26). For this, a 4-day treatment (2 µg twice daily) of IL-7 was
performed and the mice were sacrificed 12 h after the last
injection.
Abs and FACS analysis
FITC anti-CD44 (clone 1 M7.8), PE anti-NK1.1 (clone PK136), PE anti-CD4, FITC anti-IgM, and PE anti-B220 were obtained from PharMingen (San Diego, CA). Biotinylated anti-CD62L (clone Mel-14) and FITC anti-CD3 (clone 145-2C11) were kindly provided by F. Lepault (CNRS URA 1461, Institut Necker, Paris, France) and by L. Chatenoud (INSERM U25, Institut Necker), respectively. Biotinylated anti-IL-7R (clone A7R34) was a kind gift from T. Sudo (Toray Industries, Kanagawa, Japan). Staining was performed as described previously (12). After incubation with the biotinylated Abs, cells were incubated with the appropriate FITC- and PE-labeled mAbs plus streptavidin-Tricolor (SAv-Tri; Caltag, So. San Francisco, CA). Control staining with irrelevant Abs was always performed in parallel. A FACScan flow cytometer (Becton Dickinson, Mountain View, CA) was used. A minimum of 1 x 104 events gated on viable cells was acquired with CellQuest software. Results were analyzed using Mac CellQuest software.
Cell preparation
Spleen cell suspensions were prepared using a homogenizer and
washed twice in PBS 1x supplemented with 5% FCS (Techgen, Les Ulis,
France), and RBCs were lysed in a hemolyse buffer (NH4Cl,
KHCO3, EDTA). Splenocytes were incubated for 45 min with
anti-CD4-coated magnetic beads (Miltenyi Biotech,
Bergisch-Gladbach, Germany) and positively sorted on a MACS positive
selection column. In some experiments, enriched CD4+
splenocytes were stained with FITC anti-CD44, PE anti-NK1.1,
and biotinylated anti-CD62L plus SAv-Tri. The
CD44+CD62L-NK1.1+ (NK T cells),
CD44+CD62L-NK1.1-
(NK1.1- memory phenotype T cells), and
CD44-CD62L+NK1.1- (naive
phenotype T cells) populations were then sorted using a FACS Vantage
cell sorter (Becton Dickinson). Purity of each enriched cell fraction
was about 98% after reanalysis (see Fig. 3
).
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To assess cytokine production, cells in RPMI 1640 Glutamax
culture medium (Life Technologies) supplemented with 10% FCS
(Techgen), 0.05 mM 2-ME, penicillin (100 IU/ml), and streptomycin (100
µg/ml) were plated in triplicate (2.5 x 104 to
1 x 105/well; 200 µl final volume) in 96-well
round-bottom microplates (Nunc, Roskilde, Denmark) coated with 10
µg/ml anti-TCR-ß (H57-597). The supernatants were harvested
48 h later and stored at -80°C until IL-4 and IFN- assays.
In some experiments, enriched CD4+ cells (4 x
105/well), NK1.1+ T cells (2 x
105/well), NK1.1- memory phenotype (2 x
105/well), or naive phenotype T cells (4 x
105/well) in 24-well plastic plates (0.5 ml final volume)
were pretreated for 4 h with murine rIL-2 (50 U/ml; Genzyme,
Cambridge, MA), murine rIL-4 (25 ng/ml; R&D Systems, Abingdon, U.K.),
human rIL-7 (35 ng/ml), or culture medium alone. Cells were then
extensively washed, and cultured with anti-TCR-ß for 48 h
in 96-well round-bottom microplates, as indicated above. Alternatively,
naive phenotype T cells were cultured in 24-well plastic plates (2
x 105/well; 0.5 ml final volume) for 4 days in the
presence of immobilized anti-TCR-ß mAb and IL-2 (50 U/ml),
with or without IL-4 (50 ng/ml). The cells were recovered and further
incubated (4 x 105 cells/well) for 4 h in the
presence or the absence of IL-7 (35 ng/ml). After extensive washing,
the cells were rechallenged (1 x 105 cells/well) with
immobilized anti-TCR-ß mAb for 48 h in 96-well
round-bottom microplates, as described above.
Cytokine assays
IL-4 content in supernatants of splenocyte cultures was
quantitated in a bioassay using the IL-4-dependent CT.4S cell line
(27), or with a two-site sandwich ELISA method (24) using 11B11 mAb for
capture and biotinylated BVD6 mAb for detection. Responses from serial
dilutions of the supernatants were compared with those elicited by
known amounts of murine rIL-4 (R&D Systems). IFN- was measured by
using a two-site sandwich ELISA (24): AN18 mAb was used for capture,
and biotinylated R46A2 mAb was used for detection. IFN- and IL-4
concentrations are expressed in ng/ml, and were derived from
calibration curves established with serial dilutions of mouse
recombinant standards (R&D Systems) in each assay.
Statistical analysis
Differences in cytokine production were analyzed using Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study was initiated to evaluate the role that IL-7 plays in
influencing the primary T cell developmental pathway. To address this
question, the effects of IL-7 on the production of IL-4 and IFN- by
several spleen subpopulations of CD4+ T cells were analyzed
using a simple in vitro system of primary stimulation with immobilized
mAb to TCR-ß. First, IL-4 and IFN- production by total splenic
CD4+ T cells was determined after a 4-h exposure in vitro
to IL-7. Splenic CD4+ T cells were isolated using
anti-CD4 mAb-coated paramagnetic beads and the MACS device.
As illustrated in Fig. 1A,
CD4+ splenocytes exposed to medium alone secreted
significant IL-4 and IFN- upon TCR cross-linking. IL-7 treatment
of splenic CD4+ cells in vitro resulted in a
significant increase in IL-4 secretion, but had no effect on
IFN- secretion. In contrast, both IL-2 and IL-4 failed to
increase IL-4 secretion under the same experimental conditions.
Because the proliferation rates were superimposable for IL-2, IL-4,
and IL-7, we assume that the effect of IL-7 was not simply related to
increased proliferation (see legend to Fig. 1).
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B). In these mice, IL-4 secretion was increased 2.5-
to 6-fold, as compared with control mice treated with vehicle alone. In
contrast, IFN- production remained essentially unchanged. Taken
together, these results indicate that a short-term treatment of mice
with IL-7 increases the IL-4/IFN- ratio in the spleen. Class I-like/CD1-dependent selection is required for IL-7-driven IL-4 up-regulation in CD4+ T cells
Among potential IL-4-producing cells in the spleen, NK T cells are
of particular interest because of their sensitivity to IL-7 in the
thymus (12, 24, 25). To determine whether NK T cells are the target of
IL-7 in our experimental system, we first analyzed the effect of
IL-7 in ß2m- and CD1-deficient mice, known to be
defective in their selection of NK T cells (11, 12, 13, 14, 15). Splenic
CD4+ T cells from C57BL/6 and BALB/c ß2m- or
CD1-deficient mice were found to be severely impaired in their capacity
to rapidly release IL-4. Importantly, the in vivo administration of
IL-7 did not normalize IL-4 production by CD4+ splenocytes
from these mice (Fig. 2). To exclude the
possibility that this effect was due to insufficient bioavailability of
IL-7 or to insensitivity to IL-7 in the spleen, we tested a
well-characterized surrogate marker, IL-7-induced B cell lymphopoiesis
(26), for IL-7 activity in vivo. After a 4-day IL-7 treatment, we
observed increased cellularity (data not shown) and frequency of the
particular IgM- B220+ pre-B cell subset in all
mice tested, including ß2m- and CD1-deficient mice (Table I).
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CD4+ NK1.1+ T cells are a major but not the only target of IL-7 for IL-4 production
To directly test whether CD4+ NK T cells were the
exclusive target of IL-7, we purified this minor cell subset comprising
less than 5% of total CD4+ splenic T cells. We took
advantage of its particular phenotype showing an intermediate
TCR-ß surface density, the presence of the NK1.1 and the CD44
markers, and the absence of the CD62L marker (28). Electronic cell
sorting of MACS-separated CD4+ splenic cells using
biotinylated Mel-14 (anti-CD62L mAb), PE-PK136 (anti-NK1.1
mAb), and FITC anti-CD44 mAb led routinely to approximately
95–98% pure
CD44+CD62L-NK1.1+TCR-ßint
cells (see Fig. 3A). The
CD4+NK1.1- subset included both
CD44+CD62L- and
CD44-CD62L+ cells (see Fig. 3 for purification
procedure). As shown in Tables II and
III, highly purified CD4+
CD44+CD62L-NK1.1+ cells produced
considerable amounts of IL-4 upon TCR cross-linking. However, purified
CD4+
CD44+CD62L-NK1.1- cells also
produced significant amounts of IL-4, although to a lesser degree than
CD4+NK1.1+ cells. In contrast, CD4+
CD44-CD62L+NK1.1- cells, namely
naive phenotype T cells (3, 29), produced virtually no IL-4 (<0.02
ng/ml) in the same culture conditions. Unlike CD4+
CD44+CD62L- cells, CD4+
CD44-CD62L+ cells acquired their potential to
produce IL-4 only upon restimulation, and such a production depended on
the presence of an initial IL-4 source during their priming (4, 5)
(Fig. 5). Thus, the data strongly suggest that the IL-4-producing assay
used in the present work reveals exclusively direct IL-4
production.
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). These results were
confirmed in vitro, indicating a direct effect of IL-7 on each of the
NK1.1- and the NK1.1+ subsets sharing the
CD44+CD62L- phenotype (Table III). Indeed,
exposure of these subsets for 4 h to IL-7 before
anti-TCR-ß stimulation was sufficient to lead to a significant
2- to 3-fold increase in IL-4 production (Table III). In contrast,
purified CD4+
CD44-CD62L+NK1.1- T cells
produced low amounts of IL-4 (<0.02 ng/ml) upon TCR cross-linking
following exposure to IL-7 in vivo as well as in vitro (Tables II and
III). FACS analysis using an anti-IL-7R indicates that the lack of
responsiveness to IL-7 was not due to lower IL-7R expression by
CD44-CD62L+NK1.1- T cells, as
compared with the two other CD4+ populations tested (data
not shown). Finally, both the
CD44+CD62L-NK1.1+ and the
CD44+CD62L-NK1.1- subsets
produced low amounts of IFN- in response to anti-TCR-ß
(less than 0.33 ng/ml per 2.5 x 104 cells), and IL-7
had no effect (less than 0.25 ng/ml per 2.5 x 104
cells).
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To determine the origin of the IL-4-producing NK1.1-
CD4+ T cells that respond to IL-7, we analyzed mice lacking
CD1 molecules, MHC class I molecules (i.e.,
ß2m-/- mice), or MHC class II molecules
(i.e., I-Aß-/- mice). In contrast to
ß2m-deficient mice that displayed a 2-fold reduction in
the size of the CD4+
CD44+CD62L-NK1.1- T compartment
(about 10–15% of the splenic CD4+ compartment instead of
25–30% in wild-type mice), I-Aß-/- mice contained a
large majority of these cells (approximately 50–60% of the splenic
CD4+ compartment), suggesting the ß2m
dependence of this subset for its selection. Furthermore,
CD4+ CD44+NK1.1- T cells, like
CD4+ CD44+NK1.1+ T cells, are
highly skewed toward Vß8 TCR usage in wild-type and
I-Aß-/- mice when compared with their naive
counterpart, CD4+ CD44-NK1.1-
cells (Table IV). Vß8 overexpression
was not observed in the CD4+
CD44+NK1.1- cell compartment from
ß2m-/- mice, confirming the existence of a
class I-dependent NK1.1- T cell population expressing a
TCR repertoire biased to Vß8. Finally, the absence of Vß8 bias in
CD4+ CD44+NK1.1- T cells from
CD1-/- mice provides direct evidence for the CD1
dependence of this population (Table IV).
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, splenic CD4+
CD44+CD62L-NK1.1- T cells from
I-Aß-/- mice treated with IL-7 produced more IL-4 upon
TCR cross-linking than their counterparts from untreated mice. This
finding definitively confirms the direct activity of IL-7 on the
NK1.1- MHC class I-like-dependent CD4+ T cell
population. Finally, as in wild-type mice (Table II), no modification
in IFN- production by CD4+
CD44+CD62L-NK1.1- T cells was
evidenced in response to IL-7 treatment of I-Aß-/- mice
(Fig. 4).
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To determine the effect of IL-7 on conventional IL-4-producing
cells, we took advantage of the fact that the presence of IL-4 during
in vitro priming determines the IL-4-producing potential of
CD4+ naive T cells upon restimulation (4, 5). In our hands,
memory CD4+ T cells from ß2m-deficient mice
produced IL-4 upon rechallenge provided they were primed in the
presence of IL-4 (Fig. 5). Pretreatment
with IL-7 led to a dramatic (5-fold) increase in IL-4 production, as
compared with pretreatment with medium alone. This suggests that IL-7
is able to increase IL-4 production in conventional memory
CD4+ T cells provided their IL-4-producing potential was
acquired during their development.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the context of T lymphocyte-dependent immune responses (36, 37, 38, 39, 40, 41), IL-7 supports proliferation of mature CD8+ T cells (32, 42, 43), and enhances the cytolytic activity of antiviral (44, 45) and antitumoral CD8+ CTL (46) in humans as well as in mice. IL-7 also supports proliferation of CD4+ T cells (32, 47), but the role of this cytokine on CD4+ Th cell differentiation pathways is not well understood (38, 40, 48).
We show herein that IL-7 has the ability to increase rapidly and
efficiently the IL-4-producing capacity of splenic CD4+ T
cells. A 4-h preincubation with IL-7 renders CD4+ spleen
cells capable of producing significantly more IL-4 upon stimulation by
TCR cross-linking than untreated CD4+ cells. Likewise,
CD4+ spleen cells can be sensitized in vivo by IL-7. It is
noteworthy that IL-7 treatment both in vivo and in vitro does not
modify the IFN- production by CD4+ spleen cells.
Moreover, 2 h after the s.c. administration of IL-7, both the
production of IL-4 in response to TCR in vitro and the IL-4 burst,
which follows a challenge with anti-CD3 Abs in vivo, were improved.
To our knowledge, these results represent the first successful use of a
cytokine in vivo other than IL-4 for the up-regulation of direct IL-4
secretion by CD4+ T cells.
Three lines of evidence favor the involvement of CD4+ NK T cells as a target for IL-7: 1) The action of IL-7 on IL-4 production by CD4+ spleen cells (stimulated either by anti-CD3 in vivo or anti-TCR in vitro) cannot be demonstrated in ß2m- and in CD1-deficient mice that lack NK T cells; 2) freshly sorted CD4+ NK1.1+ T cells from mice treated for 2 h with IL-7 produce more IL-4 upon TCR cross-linking than CD4+ NK1.1+ T cells from mice treated with excipient alone; 3) similarly, a single in vitro preincubation of CD4+ NK1.1+ T cells from unmanipulated mice with IL-7 induces an increase in IL-4 production as compared with control cells.
Analysis of sorted populations indicates that the CD4+ T
cells responsible for IL-4 production and its increase in response to
IL-7 are not only NK1.1+ T cells, but also
NK1.1- T cells that share both a memory phenotype
(CD44+CD62L-) and Vß8 skewing with
NK1.1+ T cells. NK1.1-CD44+
CD4+ T cells from class II-deficient mice exhibited higher
IL-4 production and Vß8 frequency when compared with normal mice.
Conversely, IL-4-secreting CD4+ T cells were not detected
in ß2m- and CD1-deficient mice for which no Vß8 skewing
could be demonstrated in the NK1.1-CD44+
CD4+ T population. These results indicate that a large
proportion of NK1.1- IL-4-producing cells are CD1
restricted and include NK T cells that have lost NK1.1 Ag expression.
In accordance with our findings, Cheng and Paul (49) have recently
identified a subset of NK1.1- CD4+ T cells
that exhibit a Vß8 skewing and that produce high levels of IL-4 upon
stimulation in vitro. However, in class II-deficient mice as in
wild-type mice, the frequency of cells expressing Vß8 is lower in the
CD44+NK1.1- CD4+ population, when
compared with the NK1.1+ CD4+ population (see
Table IV). One possible explanation for this finding might be the
existence of CD1-selected NK1.1- IL-4-producing cells that
are distinct from NK T cells. Our demonstration of an enhancing effect
of IL-7 on IL-4 production by the CD4+
CD44+CD62L-NK1.1- splenic
compartment in class II-deficient mice, but not in
ß2m-deficient mice, reinforces the view that IL-7 acts
not only on NK1.1+ cells, but also on NK1.1-
cells that are specific for class I-related molecules.
Along with CD1-dependent NK.1.1- and NK1.1+ T
cells, conventional (class II-restricted)
CD4+-differentiated Th2 cells may also be a source of early
IL-4. From our experiments, we cannot exclude that IL-4 production did
not result in part from MHC class II-restricted memory
(CD44+CD62L-) CD4+ T cells
previously primed by environmental or self Ags in vivo. This is in
accordance with the lower frequency of cells expressing Vß8 in the
wild-type CD4+ CD44+NK1.1-
population, as compared with that of the CD4+
CD44+NK1.1- population from
I-Aß-/- mice (see Table IV). Due to the difficulty in
discriminating between conventional and nonconventional
NK1.1- cells among CD4+ memory phenotype T
cells in normal mice ex vivo, we addressed the question of whether IL-7
regulates IL-4 production by mainstream CD4+-differentiated
Th2 lymphocytes using in vitro generated cells. The finding that a 4-h
IL-7 treatment in vitro is sufficient to drastically increase IL-4
production by these cells leads us to propose that IL-7 plays an
important role in Th2 differentiation.
Another important question is the mechanism for the putative IL-7
action on NK1.1+ and NK1.1- IL-4-producing T
cells. We previously showed that IL-7 was particularly effective in
promoting NK T cell expansion in vitro (12). Herein, the action of IL-7
cannot be explained only by an increase in the generation of
IL-4-producing cells since other cytokines such as IL-2 and IL-4, known
to induce the proliferation of T cells (see legend to Fig. 1), failed
to increase IL-4 production by CD4+ splenocytes. This
suggests a key role for the IL-7R -chain since the -chain is
common to IL-2, IL-4, and IL-7 receptors. Such a conclusion is
consistent with a recent report by Boesteanu et al. (50) showing
distinct roles for signals relayed through the common cytokine receptor
-chain and IL-7R -chain in NK T cell development. Although IL-7
stimulation alone does not result in detectable IL-4 secretion (data
not shown), it significantly increases IL-4 mRNA levels in NK T subsets
(24). Whether IL-7 has the ability to stabilize IL-4 mRNA needs further
investigation.
A possible role of NK T cells in the regulation of both Th1 and Th2
immune responses has been recently proposed (16, 51, 52, 53, 54). Cytokine
production by these cells, either of the Th1 or Th2 type, is partially
dependent on the cytokine profile present in their microenvironment.
IL-12 induces IFN- production by NK T cells (18, 19), suggesting
that IL-12 is important in driving NK T-dependent immune reactions
toward a Th1-type response (53, 54). In contrast, IL-7 drives NK T
cells toward the Th2-type phenotype. However, the possibility that the
action of IL-7 on CD1-dependent CD4+ T cells is sufficient
alone to drive Th2 differentiation is speculative since it has been
shown that mice selectively deficient in this population are capable of
normal Th2 responses (55, 56, 57). However, the fact that the action of
IL-7 is not confined to class I-like-dependent CD4+ T
populations, and may also involve conventional CD4+ Th2
populations reinforces the putative role of IL-7 as an important factor
for Th2 differentiation.
In conclusion, the present study provides evidence that a large spectrum of CD4+ splenic cells, including NK1.1+ and NK1.1- MHC class I-like-dependent CD4+ T cells, as well as conventional NK1.1- MHC class II-restricted CD4+ T cells, are biased toward IL-4 production by IL-7. This observation supports the idea that IL-7 favors a Th2-type profile, by potentiating early IL-4 sources, and facilitating the ongoing IL-4 secretion by primed CD4+ T cells. Given the above, IL-7 could be a useful tool in developing future immune deviation strategies.
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Acknowledgments |
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Footnotes |
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2 Current address: Laboratoire d’Immunologie-immunopathologie, Centre Hospitalier Universitaire, BP 577, 86021 Poitiers Cedex, France.
3 Address correspondence and reprint requests to Dr. André Herbelin, INSERM U25, Hôpital Necker, 161 rue de Sèvres, 75743 Paris Cedex 15, France. E-mail address:
4 Abbreviation used in this paper: ß2m, ß2-microglobulin.
Received for publication November 20, 1998. Accepted for publication April 1, 1999.
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M. C. Leite-de-Moraes, A. Hameg, A. Arnould, F. Machavoine, Y. Koezuka, E. Schneider, A. Herbelin, and M. Dy A Distinct IL-18-Induced Pathway to Fully Activate NK T Lymphocytes Independently from TCR Engagement J. Immunol., December 1, 1999; 163(11): 5871 - 5876. [Abstract] [Full Text] [PDF]
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