Plasticity of Invariant NKT Cell Regulation of Allergic Airway Disease Is Dependent on IFN-γ Production

Article (PDF Available)inThe Journal of Immunology 185(1):253-62 · July 2010with41 Reads
DOI: 10.4049/jimmunol.0902301 · Source: PubMed
Abstract
Invariant NKT cells (iNKT cells) play a pivotal role in the development of allergen-induced airway hyperresponsiveness (AHR) and inflammation. However, it is unclear what role they play in the initiation (sensitization) phase as opposed to the effector (challenge) phase. The role of iNKT cells during sensitization was examined by determining the response of mice to intratracheal transfer of OVA-pulsed or OVA-alpha-galactosylceramide (OVA/alphaGalCer)-pulsed bone marrow-derived dendritic cells (BMDCs) prior to allergen challenge. Wild-type (WT) recipients of OVA-BMDCs developed AHR, increased airway eosinophilia, and increased levels of Th2 cytokines in bronchoalveolar lavage fluid, whereas recipients of OVA/alphaGalCer BMDCs failed to do so. In contrast, transfer of these same OVA/alphaGalCer BMDCs into IFN-gamma-deficient (IFN-gamma(-/-)) mice enhanced the development of these lung allergic responses, which was reversed by exogenous IFN-gamma treatment following OVA-BMDC transfer. Further, Jalpha18-deficient recipients, which lack iNKT cells, developed the full spectrum of lung allergic responses following reconstitution with highly purified WT liver or spleen iNKT cells and transfer of OVA-BMDCs, whereas reconstituted recipients of OVA/alphaGalCer BMDCs failed to do so. Transfer of iNKT cells from IFN-gamma(-/-) mice restored the development of these responses in Jalpha18-deficient recipients following OVA-BMDC transfer; the responses were enhanced following OVA/alphaGalCer BMDC transfer. iNKT cells from these IFN-gamma(-/-) mice produced higher levels of IL-13 in vitro compared with WT iNKT cells. These data identify IFN-gamma as playing a critical role in dictating the consequences of iNKT cell activation in the initiation phase of the development of AHR and airway inflammation.
The Journal of Immunology
Plasticity of Invariant NKT Cell Regulation of Allergic
Airway Disease Is Dependent on IFN-g Production
Hiroyuki Matsuda,*
,1
Katsuyuki Takeda,*
,1
Toshiyuki Koya,* Masakazu Okamoto,*
Yoshiki Shiraishi,* Nobuaki Miyahara,* Azzeddine Dakhama,* Jennifer L. Matsuda,
Laurent Gapin,
and Erwin W. Gelfand*
Invariant NKT cells (iNKT cells) play a pivotal role in the development of allergen-induced airway hyperresponsiveness (AHR)
and inflammation. However, it is unclear what role they play in the initiation (sensitization) phase as opposed to the effector
(challenge) phase. The role of iNKT cells during sensitization was examined by determining the response of mice to intratracheal
transfer of OVA-pulsed or OVA–a-galactosylceramide (OVA/aGalCer)-pulsed bone marrow-derived dendritic cells (BMDCs)
prior to allergen challenge. Wild-type (WT) recipients of OVA-BMDCs developed AHR, increased airway eosinophilia, and
increased levels of Th2 cytokines in bronchoalveolar lavage fluid, whereas recipients of OVA/aGalCer BMDCs failed to do so.
In contrast, transfer of these same OVA/aGalCer BMDCs into IFN-g–deficient (IFN-g
2/2
) mice enhanced the development of
these lung allergic responses, which was reversed by exogenous IFN-g treatment following OVA-BMDC transfer. Further, Ja18-
deficient recipients, which lack iNKT cells, developed the full spectrum of lung allergic responses following reconstitution with
highly purified WT liver or spleen iNKT cells and transfer of OVA-BMDCs, whereas reconstituted recipients of OVA/aGalCer
BMDCs failed to do so. Transfer of iNKT cells from IFN-g
2/2
mice restored the development of these responses in Ja18-deficient
recipients following OVA-BMDC transfer; the responses were enhanced following OVA/aGalCer BMDC transfer. iNKT cells from
these IFN-g
2/2
mice produced higher levels of IL-13 in vitro compared with WT iNKT cells. These data identify IFN-g as playing
a critical role in dictating the consequences of iNKT cell activation in the initiation phase of the development of AHR and airway
inflammation. The Journal of Immunology, 2010, 185: 253–262.
T
he characteristic features of bronchial asthma include var-
iable airflowo bstruction,airwayhyperresponsi veness(AHR),
mucus hypersecretion, and airway inflammation (1). Many
types of cells are involved in the development of airway in-
flammation in the asthmatic lung, including lymphocytes, mast cells,
eosinophils (Eos), and dendritic cells (DCs) (1–3). Much of the
supporting data identify Th2 cells that produce Th2 cytokines, such
as IL-4, -5, and -13, as being essential in the development of allergic
airway inflammation and AHR in humans (4) and mice (5, 6).
Differentiation of Th2 cells from naive T cells is an essential
component of the allergic response. Naive T cells require in-
teraction with mature APCs, such as DCs, to initiate the expansion
and acquisition of Th2 effector cell functions in response to Ag
exposure (7, 8). As a result, DCs play a pivotal role in asthma
development, regulating downstream responses to allergen expo-
sure (7). In the lung, DCs may represent the most important APCs
and play an essential role in the induction of allergic airway in-
flammation and AHR (7, 9). Following intratracheal transfer of
OVA-pulsed bone marrow-derived dendritic cells (BMDCs), mice
develop AHR and eosinophilic airway inflammation after OVA
challenge alone (10, 11). In such studies, the Ag-pulsed BMDCs
replace the active sensitization phase, priming the airways to sub-
sequent allergen challenge.
Invariant NKT cells (iNKT cells) represent a distinct lymphocyte
subpopulation that has important immunoregulatory functions (12,
13). iNKT cells express a semi-invariant TCR that recognizes
glycolipid Ags presented by the nonpolymorphic MHC class I-
like molecule CD1d (14, 15). a-galactosylceramide (aGalCer),
a specific ligand for iNKT cells isolated from a marine sponge
(12), rapidly induces the production of Th1 and Th2 cytokines,
including IFN-g and IL-4, by iNKT cells. Through the release of
these cytokines, iNKT cells modulate a variety of immune
responses, such as tumor immunity, autoimmune disease, and in-
fection (12–14).
The role of iNKT cells in the initiation of asthma has been
intensively studied but remains controversial (14, 15). In humans,
Akbari et al. (16) reported that the percentages of iNKT cells
strikingly increase in the airways of asthmatics. Although other
investigators found that the number of iNKT cells was not in-
creased or increased only marginally in the airway lumens or
airways of patients with asthma (17–19), recent studies indicated
*Division of Cell Biology, Department of Pediatrics, National Jewish Health, Denver,
CO 80206; and
Integrated Department of Immunology, University of Colorado
Health Sciences Center, Denver, CO 80217
1
H.M. and K.T. contributed equally to this work.
Received for publication July 17, 2009. Accepted for publication April 19, 2010.
This work was supported by National Institutes of Health Grants HL-36577, HL-
61005, and AI-77609 (to E.W.G.).
The content of this publication is solely the responsibility of the authors and does not
necessarily represent the official views of the National Heart, Lung, and Blood In-
stitute or the National Institutes of Health.
Address correspondence and reprint requests to Dr. Erwin W. Gelfand, National
Jewish Health, 1400 Jackson Street, Denver, CO 80206. E-mail address: gelfande
@njhealth.org
Abbreviations used in this paper: aGalCer, a-galactosylceramide; aGC, BMDCs
cultured with aGalCer; AHR, airway hyperresponsiveness; AM, alveolar macrophage;
BAL, bronchoalveolar lavage;BMDC,bone marrow-derived dendritic cell; DC, dendritic
cell; Eos, eosinophils; IFN-g
2/2
,IFN-g deficient; IFN-gKO, IFN-g
2
mice;IFN-g+OVA/
aGC, BMDCs cultured with OVA and a GalCer prior to exogenous IFN-g administration;
iNKT cells, invariant NKT cells; Ja18
2/2
,Ja18 deficient; Ja18KO, Ja18
2/2
mice; Lym,
lymphocytes; MCh, methacholine; Medium, BMDCs cultured without OVA or aGalCer;
mIFN-g,mouseIFN-g; MNC, mononuclear cell; Neut, neutrophils; OVA, BMDCs
cultured with OVA; OVA/aGC, BMDCs cultured with OVA and aGalCer; PAS,
periodic acid-Schiff; PBLN, peribronchial lymph node; RL, lung resistance; TC, total
cell; WT, wild-type.
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902301
that the numbers of iNKT cells in the airways of severe asthmatics
tend to be increased (20, 21). However, their role in the initiation
or amplification of asthma pathogenesis is not fully defined. In the
mouse, two reports showed that iNKT cells play an essential role
in the development of allergic airway inflammation and AHR (22,
23), whereas other groups did not find these effects (24–26). The
reasons for such discrepancies are unclear. They might suggest
that iNKT cell regulatory activities have a certain plasticity that
might be subject to a number of regulatory factors under different
experimental conditions.
We and other investigators showed that a single i.p. administration
of aGalCer prior to Ag challenge of sensitized mice inhibits allergic
airway inflammation and AHR through iNKT cells and in an IFN-g
dependent manner (25, 27, 28). It is unclear whether such effects
are restricted to the challenge phase or whether activation of
iNKT cells during the sensitization (initiation) phase also regulates
development of allergic inflammation and AHR, because they were
shown to be a potent producers of Th1- and Th2-type proinflamma-
tory cytokines (29). Intratracheal transfer of Ag-pulsed BMDCs leads
to the full development of lung allergic responses on allergen chal-
lenge alone, 10 d later (10, 11). In this study, we show that transfer
of BMDCs treated with aGalCer, a specific ligand of NKT cells,
prevented the development of lung allergic responses, and this was
dependent on IFN-g production by recipient iNKT cells. In the
absence of IFN-g in recipients, the OVA-pulsed BMDCs retained
the ability to induce allergic airway inflammation and AHR, and
these responses were further enhanced following transfer of OVA/
aGalCer BMDCs.
Materials and Methods
Animals
Eight- to 12 wk-old female C57BL/6 wild-type (WT) mice were purchased
from The Jackson Laboratory (Bar Harbor, ME) and used throughout the
study. IFN-g–deficient (IFN-g
2/2
) and Ja18-deficient (J a18
2/2
) mice on
a C57BL/6 background were bred in the animal facility at National Jewish
Health. The animals were maintained on an OVA-free diet. Experiments
were conducted under a protocol approved by the Institutional Animal
Care and Use Committee of National Jewish Health.
Reagents
Recombinant murine GM-CSF and murine IL-4 were purchased from R&D
Systems (Minneapolis, MN). aGalCer was obtained from Axxora (San
Diego, CA), and recombinant mouse IFN-g (mIFN-g) was obtained
from eBioscience (San Diego, CA). FITC-conjugated anti-mouse CD3ε
mAb (145-2C11), PE-PerCP–conjugated anti-mouse CD4 mAb (RM4-5),
allophycocyanin-conjugated anti-mouse IFN-g mAb (XMG1.2),
allophycocyanin-conjugat ed anti-mouse IL-4 mAb (11B11), and streptavid in-
allophycocyanin conjugate were purchased from BD Biosciences (San Jose,
CA). Biotinylated anti-mouse IL-13 Ab was obtained from R&D Systems.
PBS57-loaded CD1d tetramer was provided by the National Institute of
Allergy and Infectious Disease MHC Tetramer Core Facility (Atlanta, GA).
Generation of BMDCs
BMDCs were generated from bone marrow cells of naive C57BL/6 WT
mice, according to the procedure described by Inaba et al. (30), with some
modification. In brief, bone marrow cells obtained from femurs and tibias
of mice were placed in 75-ml flasks at 37˚C in culture medium (RPMI 1640
containing 10% heat-inactivated FCS, 50 mM2-ME,2mM
L-glutamine,
penicillin [100 U/ml], streptomycin [100 mg/ml; Invitrogen, Carlsbad,
CA]), recombinant murine GM-CSF (10 ng/ml), and recombinant murine
IL-4 (10 ng/ml). Nonadherent cells were collected by aspirating the medium
and transferring them into fresh flasks. On day 8, cells were pulsed with
OVA (grade V, 150 mg/ml; Sigma-Aldrich, St. Louis, MO) and aGalCer
(150 ng/ml) or OVA alone for 24 h and washed three times with PBS.
In vitro assay of BMDCs
BMDCs (1 3 10
6
cells) were incubated with or without OVA and/or aGalCer
for 24 h at 37˚C. After harvesting BMDCs, cytokine levels in culture
supernatants were measured by ELISA, and surface Ags of BMDCs were
analyzed by flow cytometry. The surface phenotype of BMDCs was analyzed
using FITC-conjugated anti–I-A
b
(AF6-120.1), FITC-conjugated anti-
CD11b (M1/70), allophycocyanin-conjugated anti-CD11c (HL3), PE-
conjugated anti-CD80 (16-10A1), and PE-conjugated anti-CD86 (GL1) (all
obtained from BD Pharmingen, San Diego, CA). For control staining, simi-
larly labeled, isotype-matched control Abs were used.
Transfer of allergen-pulsed BMDCs into naive mice
OVA-, aGalCer-, or OVA- and aGalCer-pulsed BMDCs (1 3 10
6
) were
instilled intratracheally into naive WT or IFN-g
2/2
mice on day 1; mice
that received nonpulsed BMDCs served as controls. Ten days after transfer
of BMDCs, animals were challenged with nebulized OVA (1% in saline)
for 20 min on days 11–13. Forty-eight hours after the last OVA chal-
lenge (day 15), AHR was assessed, and bronchoalveolar lavage (BAL)
fluid, serum, and tissues were obtained for further analyses. A group of
IFN-g
2/2
mice received 1 mg mIFN-g in 25 ml PBS, intratracheally, 1 d
after OVA/aGalCer BMDC transfer, followed by OVA challenge via
the airways.
Determination of airway responsiveness
Airway function was assessed, as previously described, measuring changes
in lung resistance (RL) in response to increasing doses of inhaled meth-
acholine (MCh) (31). Data are expressed as the percentage of change from
baseline RL values obtained after inhalation of saline. There were no
significant differences in baseline RL values among the groups.
Bronchoalveolar lavage
Immediately after assessment of airway function, lungs were lavaged via the
tracheal tube with 1 ml HBSS at room temperature. Total leukocyte numbers
were measured using a Coulter Counter (Coulter, Hialeah, FL). Cytospin
slides were stained with Leukostat (Fisher Diagnostics, Pittsburgh, PA) and
differentiated by standard hematological procedures in a blinded fashion.
Lung histology
Lungs were fixed in 10% formalin and processed into paraffin. Mucus-
containing goblet cells were detected by staining of paraffin sections
(5-mm thick) with periodic acid-Schiff (PAS). Sections were also stained
with H&E to analyze inflammatory cell infiltration. Histological analyses
were performed in a blinded manner under a light microscope linked to an
image-capture system. The numbers of PAS
+
goblet cells were determined
in cross-sectional areas of the airway wall. Eight to 10 sections were
evaluated per animal. The measurements were averaged for each animal,
and the mean value 6 SE was determined for each group.
Measurement of cytokines
Levels of cytokines in BAL fluid and cell culture supernatants were de-
termined using commercially available ELISAs, following the manu-
facturers’ instructions. ELISA kits for the detection of IL-4, -5, -10, and
-12 (p70) and IFN-g were obtained from BD Pharmingen. The IL-13
ELISA kit was purchased from R&D Systems. ELISA kits for mouse
IL-18 were obtained from Bender Medbioscience (Burlingame, CA), and
the IL-6 kit was from eBioscience. The limits of detection for each assay
were as follows: 4 pg/ml for IL-4, -5, and -6; 10 pg/ml for IL-10, -12, and
-18 and IFN-g; and 1.5 pg/ml for IL-13.
Lung leukocyte isolation
Lung leukocytes were isolated, as previously described (32), using colla-
genase digestion, followed by centrifugation on 35% Percoll density gra-
dients (Sigma-Aldrich).
Intracellular cytokine staining
Intracellular cytokine staining was performed as previously described (33).
Briefly, lung mononuclear cells (MNCs) were stimulated for 3 h with PMA
and ionomycin (10 and 500 ng/ml, respectively) in the presence of bre-
feldin A (10 mg/ml). After washing, cells were stained for cell surface
markers with mAbs against CD3, CD4, and CD1d tetramer. After fixation
and permeabilization, cells were stained with allophycocyanin-conjugated
anti–IFN-g or anti–IL-4 mAb or biotin-conjugated anti–IL-13. In parallel,
cells were similarly labeled with isotype-matched control Ab. After wash-
ing, staining was analyzed by flow cytometry on a FACSCalibur using
CellQuest software (BD Biosciences).
254 ACTIVATION OF iNKT CELLS BY DCs IN ASTHMA
In vitro cytokine production in peribronchial lymph nodes
Peribronchial lymph nodes (PBLNs) were removed and subsequently
passed through a stainless steel sieve. Single-cell preparations were sus-
pended in complete RPMI 1640 with 10% heat-inactivated FCS, 50 mM
2-ME, 2 mM
L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomy-
cin. PBLN MNCs (4 3 10
5
cells) were cultured for 24 h and 5 d in 96-well
round-bottom plates in the presence of OVA (100 mg/ml). Levels of IL-4,
-5, and -13 and IFN-g in culture supernatants were measured by ELISA.
In vitro activation of iNKT cells
Livers from WT or IFN-g
2/2
mice were harvested and subsequently passed
through a stainless steel sieve. After washing with PBS, MNCs were
isolated by 35% Percoll gradient centrifugation (Sigma-Aldrich). Liver
MNCs were cocultured with aGalCer WT BMDCs. Liver MNCs were
adjusted to 1 3 10
6
cells/ml of iNKT cells following tetramer staining
and mixed with 0.33 3 10
6
BMDCs/ml. After 24 h, culture supernatants
were collected, and the levels of IL-4 and -13 were measured by ELISA.
Cells were also analyzed by intracellular cytokine staining.
Adoptive transfer of iNKT cells into Ja18
2/2
mice
Liver cells from WT, IFN-g
2/2
,orJa18
2/2
mice were harvested and
subsequently passed through a stainless steel sieve. After washing with
PBS, MNCs were isolated by 35% Percoll density gradient centrifugation
(Sigma-Aldrich). Enrichment of iNKT cells was carried out by negative
selection using the CD4 isolation kit (CD4 Cellect Immunocolumn Kit
Mouse, Cedarlane Laboratories, Burlington, Ontario, Canada), in accordance
with the manufacturer’s instructions. Purity of iNKT cells from WT and IFN-
g
2/2
mice after isolation was 35–40%, as assessed by flow cytometry. iNKT
cell-enriched liver MNCs (0.8 3 10
6
cells) were transferred into Ja18
2/2
mice via the tail vein 1 d before the intratracheal instillation of allergen-
pulsed cell isolation, as described above. Isolated CD4
+
cells were stained
with PE-conjugated PBS57-loaded CD1d tetramer and purified with anti-PE
MicroBeads (Miltenyi Biotec, Bergisch-Gladbach, Germany). To further pu-
rify iNKT cells, PE-positive cells were sorted on MoFlo (DakoCytomation,
Fort Collins, CO) following MicroBeads separation. Purified spleen
iNKT cells (.95% were CD1d tetramer
+
and CD3
+
)(0.33 10
6
cells) were
transferred into Ja18
2/2
mice via the tail vein 1 d before the intratracheal
instillation of OVA BMDCs. Control mice received PBS prior to OVA
BMDCs. Both groups of recipient mice were challenged with OVA for 3 con-
secutive days and assayed 48 h after the last challenge, 10 d after injection
of BMDCs.
Statistical analysis
The t test was used to compare differences between two groups, whereas
ANOVA and the Tukey–Kramer multiple-means comparison tests were
used for comparisons among three or more groups. Statistical analyses
using nonparametric analysis (Mann–Whitney U test or Kruskal–Wallis
test) were also performed. The p values for significance were set to 0.05
for all tests with statistical software (JMP, SAS Institute, Cary, NC). The
data were pooled from three independent experiments with four
mice/group in each experiment (n = 12). Values for all measurements
are expressed as mean 6 SEM.
Results
Effect of aGalCer on BMDCs in vitro
To determine whether incubation with aGalCer alters the function
of BMDCs in vitro, we measured cytokine levels in culture
supernatants and analyzed the expression of several surface Ags
by flow cytometry. As shown in Fig. 1A, OVA, but not aGalCer,
induced IL-6 release from BMDCs. Other cytokines (i.e., IL-10,
-12, -13, and -18 were not detected (data not shown).
There were few differences in the levels of expression of CD80,
CD86, CD40, and MHC class II on aGalCer BMDCs and non-
pulsed BMDCs (Fig. 1B). OVA BMDCs expressed higher levels of
these surface Ags than did BMDCs cultured without OVA. OVA/
aGalCer BMDCs expressed the same levels of these Ags as did
OVA BMDCs. Collectively, aGalCer added to BMDCs did not
seem to alter the phenotype (cytokine profile, surface Ag ex-
pression) of the BMDCs in vitro.
Transfer of allergen-pulsed BMDCs in vivo into WT mice
To determine whether incubating BMDCs with aGalCer prior to
transfer into allergen- challenged recipients could alter the allergic
phenotype, we transferred OVA BMDCs, aGalCer BMDCs, or
OVA/aGalCer BMDCs intratracheally into naive WT mice prior
to challenge with OVA on three consecutive days. As shown in Fig.
2A, mice administered OVA BMDCs and challenged with OVA
developed significant increases in RL in response to increasing
doses of inhaled MCh. However, mice receiving aGalCer BMDCs
or OVA/aGalCer BMDCs failed to develop AHR to MCh; RL
levels were the same as in mice that received nonpulsed BMDCs.
Cell-composition analysis of BAL fluid demonstrated that airway
eosinophilia developed in WT mice that received OVA BMDCs. In
contrast, WT mice that received aGalCer BMDCs or OVA/
aGalCer BMDCs had decreased numbers of Eos in the BAL fluid
(Fig. 2B).
Examination of cytokine levels in the BAL fluid showed that IL-
4, -5, and -13 were elevated in recipients of OVA BMDCs, whereas
these cytokine levels were significantly lower in recipients of OVA/
aGalCer BMDCs. IFN-g levels in the BAL fluid of OVA/a GalCer
BMDC recipients were significantly increased compared with
recipients of OVA BMDCs (Fig. 2C).
Lung histology (Fig. 2D) revealed that recipients of OVA
BMDCs developed a marked infiltration of inflammatory cells,
including Eos, around the airways and vessels. However, these
lung inflammatory responses were not observed following transfer
of OVA/aGalCer BMDCs.
Animal models of allergic airway inflammation are accompanied
by goblet cell metaplasia and mucus hypersecretion in the airways
(34), which is a prominent feature of asthma. As shown in Fig. 2E
and 2F, challenge with OVA in recipients of OVA BMDCs
resulted in marked increases in the numbers of PAS
+
cells. In the
mice that received OVA/aGalCer BMDCs, few PAS
+
goblet cells
could be detected.
Intracellular cytokine staining of lung iNKT cells
In previously sensitized mice, IFN-g was shown to be critical to the
inhibition of allergic airway inflammation and AHR induced
by aGalCer (25, 27, 28). To determine whether transfer of OVA
BMDCs exposed to aGalCer modulated the numbers of iNKT cells
in the lung and their capacity for IFN-g production, we quantified
the number of iNKT cells in the lungs and the numbers of IFN-g
producing iNKT cells by intracellular cytokine staining. In mice
that received OVA/aGalCer BMDCs, a significant increase in the
number of CD3
+
CD1d-tetramer
+
cells (Fig. 3A,3B) and CD3
+
CD1d-tetramer
+
IFN-g
+
cells was observed in the lung compared
with mice that received OVA BMDCs (Fig. 3C).
Transfer of allergen-pulsed BMDCs in IFN-g
2/2
mice
Together, these data suggested that activation of iNKT cells by
aGalCer during the initiation phase (i.e., before allergen challenge)
attenuates the development of allergic airway inflammation and
AHR through increasing numbers of recipient IFN-g–producing
iNKT cells in the lung. To directly determine the role of IFN-g
in this inhibition, we examined the effects of administering
aGalCer BMDCs or exogenous mIFN-g to IFN-g
2/2
recipients.
In contrast to WT recipients, in which the development of AHR
was inhibited following administration of OVA/aGalCer BMDCs
(Fig. 2A), IFN-g
2/2
recipients of OVA/aGalCer BMDCs showed
a striking increase in AHR compared with IFN-g
2/2
mice that
received OVA BMDCs or aGalCer (non–OVA-pulsed) BMDCs
(Fig. 4A). Analysis of the cell composition of BAL fluid demon-
strated that airway eosinophilia was also significantly enhanced in
The Journal of Immunology 255
IFN-g
2/2
recipients of OVA/aGalCer BMDCs compared with
recipients of OVA BMDCs (Fig. 4B). The development of AHR in
IFN-g
2/2
recipients of OVA/aGalCer BMDCs was prevented by
exogenous IFN-g administration (Fig. 4A,4B). Examination of
cytokines in the BAL fluid demonstrated that levels of IL-4 and
-13 were also significantly higher in IFN-g
2/2
recipients of OVA/
aGalCer BMDCs; these cytokines, as well as IL-5, were decreased
by mIFN-g administration (Fig. 4C).
On histological analysis, IFN-g
2/2
recipients of OVA/aGalCer
BMDCs showed a greater inflammatory cell accumulation
compared with IFN-g
2/2
recipients of OVA BMDCs (Fig. 4D),
and the number of PAS
+
cells was also increased in IFN-g
2/2
recipients of OVA/aGalCer BMDCs (Fig. 4E).
These changes in IFN-g
2/2
recipients of OVA/aGalCer
BMDCs were accompanied by similar increases in the numbers
of CD3
+
CD1d-tetramer
+
cells in their lungs, as observed in WT
recipients (Fig. 3B). However, unlike WT recipients, the numbers
of lung CD3
+
CD1d-tetramer
+
IL-4
+
and CD3
+
CD1d-tetramer
+
IL-13
+
cells were markedly increased compared with IFN-g
2/2
mice that received OVA BMDCs (Fig. 5AC). These findings
associated with transfer of OVA/aGalCer BMDCs into IFN-g
2/2
mice identified a conversion of the responses with enhancement
FIGURE 1. aGalCer does not alter the phenotype of
BMDCs in vitro. BMDCs (1 3 10
6
cells) were incubated
with or without OVA and/or aGalCer for 24 h at 37˚C.
Cytokine levels in culture supernatants were measured by
ELISA, and surface Ag analyses were done by flow
cytometry. IL-6 levels (A) and expression of surface Ags
(B) in BMDCs in vitro. Data are representative of three
independent experiments (n = 12). pp , 0.05, comparing
O VA-pulsed BMDCs and OVA/aGalCer-pulsed BMDCs
versus medium only and aGalCer-pulsed BMDCs. aGC,
BMDCs cultured with aGalCer; Medium, BMDCs cul-
tured without OVA or aGalCer; OVA, BMDCs cultured
with O VA; OVA/aGC, BMDCs cultured with OVA and
aGalCer.
FIGURE 2. Transfer of OVA/aGalCer-pulsed BMDCs to WT mice prior to OVA challenge prevents development of allergic airway inflammation and AHR.
A, Airway resistance. B, BAL cell composition. C, Cytokine levels in BAL fluid. D, Representative photomicrographs (original magnificaion 3200). The tissues
were obtained 48 h after the last challenge and stained with H&E (ac) or PAS (df). Shown are photomicrographs of WT recipients of nonpulsed DCs (a, d),
OVA-pulsed BMDCs (b, e), and OVA and aGalCer-pulsed BMDCs (c, f). E, Quantitative analysis of PAS
+
cells in the lung tissue. Data represent mean 6 SEM
(n = 12). pp , 0.05, comparing WT recipients of OVA-pulsed BMDCs versus medium, aGalCer-pulsed BMDCs, or OVA/aGalCer-pulsed BMDCs. aGC,
BMDCs cultured with aGalCer; AM, alveolar macrophages; Eos, eosinophils; Lym, lymphocytes; Medium, BMDCs cultured without OVA or aGalCer
stimulation; Neut, neutrophils; OVA, BMDCs cultured with OVA; OVA/aGC, BMDCs cultured with OVA and aGalCer; TC, total cell.
256 ACTIVATION OF iNKT CELLS BY DCs IN ASTHMA
of AHR, airway eosinophilia, and Th2 cytokine production in
association with changes in the numbers and pattern of cytokine-
producing iNKT cells in the lung.
Further, to identify a conversion of the cytokine profile of T
cells in regional lymph nodes, PBLNs were recovered from WT or
IFN-g
2/2
mice following OVA or OVA/aGalCer BMDC transfer
and allergen challenge, and in vitro cytokine production was
analyzed. As shown in Fig. 5D, the levels of IL-4, -5, and -13
were increased and IFN-g was decreased in WT recipients of OVA
BMDCs compared with recipients of OVA/aGalCer BMDCs.
Conversely, in IFN-g
2/2
recipient mice, the levels of IL-4, -5,
and -13 were increased in recipients of OVA/aGalCer BMDCs.
IFN-g plays a pivotal role in the phenotype of iNKT cells and
development of allergic airway inflammation and AHR
The data suggested that IFN-g production by recipient iNKT
cells was pivotal in dictating the outcome of OVA/aGalCer
BMDC transfer on the development of lung allergic responses.
FIGURE 3. WT recipients of OVA- and
aGalCer-pulsed BMDCs have increased num-
bers of iNKT cells and production of IFN-g
in the lung. Lung MNCs were isolated and
stimulated with phorbol/ionomycin, fixed,
permeabilized, and stained with anti-mouse
CD3, CD1d tetramer, and IFN-g Ab and quan-
tified as described in Materials and Methods.
CD3
+
CD1d
+
tetramer
+
T cells were gated on
and analyzed for intracellular IFN-g (A), numb-
er of CD3
+
CD1d
+
Tcells(B), and number of
CD3
+
CD1d tetramer
+
IFN-g
+
Tcells(C).
Means 6 SEM from three independent ex-
periments are shown (n = 12). pp , 0.05.
OVA, BMDCs pulsed with OVA; OVA/aGC,
BMDCs pulsed with OVA and aGalCer.
FIGURE 4. Transfer of OVA/aGalCer-pulsed BMDCs prior to OVA challenge enhances allergic airway inflammation and AHR in IFN-g
2/2
recipients;
exogenous IFN-g inhibited this enhancement. A, Airway resistance. B, BAL cell composition. C, Cytokine levels in BAL fluid. D, Representative
photomicrographs from IFN-g
2/2
recipients of OVA-pulsed BMDCs (a, c)orOVA/aGalCer-pulsed BMDCs (b, d) (H&E, a, b; PAS, c, d). E, Quantitative
analysis of PAS
+
cell number. Data represent mean 6 SEM from three independent experiments. (n = 12). pp , 0.05, comparing IFN-g
2/2
recipients of
OVA/aGalCer-pulsed BMDCs versus recipients of OVA-pulsed BMDCs and OVA-pulsed BMDCs in IFN-g recipients. aGC, BMDCs cultured with
aGalCer; AM, alveolar macrophages; Eos, eosinophils; Lym, lymphocytes; OVA, BMDCs cultured with OVA; OVA/aGC, BMDCs cultured with OVA
and aGalCer; IFN-g+OVA/aGC, BMDCs cultured with OVA and aGalCer prior to exogenous IFN-g administration; Neut, neutrophils; TC, total cell.
The Journal of Immunology 257
To determine whether iNKT cells represented the primary source
of IFN-g production in dictating the outcome, CD4
+
T cells were
enriched from the liver of WT, IFN-g
2/2
,orJa18
2/2
mice and
adoptively transferred into Ja18
2/2
recipients before OVA or
OVA/aGalCer BMDC transfer prior to OVA challenge. J a18
2/2
recipients of cells purified from Ja18
2/2
mice did not develop
AHR, and the numbers of Eos in BAL fluid were reduced after
transfer of OVA BMDCs (Fig. 6). Ja18
2/2
mice that received
WT cells followed by OVA BMDCs exhibited significantly
increased AHR and airway eosinophilia. However, if these mice
received OVA/aGalCer BMDCs, AHR and airway eosinophilia
were markedly reduced. In contrast, Ja18
2/2
recipients of cells
from IFN-g
2/2
mice and OVA BMDCs developed levels of AHR
and airway eosinophilia comparable to the WT recipients. Ja18
2/2
recipients of iNKT-enriched cells from IFN-g
2/2
mice and OVA/
aGalCer BMDCs demonstrated the highest level of AHR and the
greatest number of Eos in the BAL fluid (Fig. 6). These data
indicate that IFN-g production from iNKT cells plays a pivotal
role in determining the outcome of BMDC transfer in naive mice
exposed to allergen challenge.
To determine the capacity for Th2 cytokine production in IFN-
g
2/2
iNKT cells, liver MNCs from naive IFN-g
2/2
or WT mice
were cultured with aGalCer BMDCs and cytokine levels were
examined. As shown in Fig. 7, IFN-g
2/2
iNKT cells were more
capable of producing IL-13 compared with WT iNKT cells. There
were no significant differences in IL-4 production levels between
WT and IFN-g
2/2
iNKT cells (data not shown).
Adoptive transfer of iNKT cells purified from spleen triggers
allergic airway inflammation and AHR
The functions of iNKT cells may differ when obtained from dif-
ferent tissues with distinct effects in a tumor model (35). To de-
termine whether iNKT cells from different tissues are capable of
initiating allergic airway inflammation and AHR, we examined
the activity of iNKT cells from the spleens of Ja18
2/2
mice. As
shown in Fig. 8A, iNKT cells were purified to .95% and trans-
ferred into Ja18
2/2
mice prior to OVA BMDC transfer and OVA
challenge. Mice that received spleen iNKT cells developed AHR
and eosinophilic airway inflammation (Fig. 8B,8C).
Discussion
Systemic administration of aGalCer, a specific ligand for iNK-
T cells, was shown to prevent the development of allergic airway
inflammation and AHR under certain conditions (25, 27, 28).
However, these findings could not distinguish whether the effects
were manifested during the initiation phase of the response or spe-
cifically altered the subsequent airway response to allergen chal-
lenge. Because DCs are important APCs in the lung and play
a critical role in the induction or the initiation phase of allergic
airway inflammation and AHR (7, 8), we sought to define whether
this ligand for iNKT cells could modify DC function and, in turn,
iNKT cell function. To focus on the initiation phase, we showed
that transfer of OVA BMDCs intratracheally could initiate the
development of AHR and airway inflammation in response to
OVA challenge in the absence of prior sensitization with adjuvant
(10, 11). In this way, allergen-pulsed BMDCs that are exposed to a
ligand for iNKT cells may be used to determine the potential role
for iNKT cells in the initiation phase, prior to allergen challenge.
First, we examined whether incubation of BMDCs with aGalCer
or allergen altered some of the characteristics of these cells. Al-
though pulsing of BMDCs with OVA increased IL-6 production and
levels of certain surface markers (CD80, CD86, CD40, and I-A
b
),
no significant differences were found in vitro when comparing the
responses with the addition of OVA/aGalCer. IL-6 production from
OVA BMDCs was likely induced through the small amounts of
LPS contaminating the OVA preparation (36). Because IL-6 was
shown to induce a polarization toward Th2 differentiation and sup-
pression of T regulatory cell function (37), OVA BMDCs may be
potent inducers of allergic airway responses.
However, when using these tw o populations of allergen-pulsed
BMDCs, we found important di fferences in vivo. Intratra cheal in-
stillation of allergen-pulsed BMDCs incubated with aGalCer pre-
vented the development of allergen-specific airway infla mmation
and AHR in response to allergen challenge in WT recipients. The
decreases in airway resp ons iveness to inhale d MCh and airwa y
eosinophi lia were as sociat ed with decreases in the levels of Th2
cytokines, including IL-4, -5, and -13, in BAL fluid and goblet cell
metaplasia and incre ases in IFN-g levels. R ecipients of OVA/
FIGURE 5. IFN-g
2/2
recipients of
OVA/aGalCer-pulsed BMDCs have in-
creased numbers of iNKT cells and
IL-4– and -13–producing cells in the
lung. Lung MNCs were isolated and
stimulated with phorbol/ionomycin,
fixed, permeabilized, and stained with
anti-mouse CD3, CD1d tetramer, and
IL-4 or -13 Ab and quantified as de-
scribed in Materials and Methods. A,
CD3
+
CD1d tetramer
+
T cells were gated
on and analyzed for intracellular IL-4
and -13. B, Numbers of CD3
+
CD1d
+
tetramer
+
T cells. C, Numbers of CD3
+
CD1d tetramer
+
IL-4
+
or -13
+
T cells.
OVA, BMDCs cultured with OVA;
OVA/aGC, BMDCs cultured with OVA
and aGalCer. D, PBLN cells from
WT or IFN-g
2/2
mice, which received
OVA or OVA/aGC BMDC followed by
allergen challenge were cultured with
OVA (100 ng/ml) and supernate cyto-
kine levels were determined. Means 6
SEM from three independent experi-
ments are shown (n = 12). pp , 0.05.
258 ACTIVATION OF iNKT CELLS BY DCs IN ASTHMA
aGalCer BMDCs also demonstrated significant increases in levels
of IFN-g in PBLNs, where allergen-captured DCs migrate and
present Ag to r ecirculating naive CD4
+
and CD8
+
cells (7, 10,
11). In the lungs of recipients of OVA/aGalCer BMDCs, the
number of iNKT cells that produced IFN-g was significantly
increased compared with the numbers in recipients of OVA
BMDCs.
However, in contrast to WT recipients, the transfer of OVA/
aGalCer BMDCs into IFN-g–deficient recipients prior to allergen
challenge markedly augmented development of airway inflam-
mation and AHR accompanied by increases in the levels of
BAL Th2 cytokines and goblet cell metaplasia. Transfer of
OVA/aGalCer BMDCs also resulted in increases in the number
of lung iNKT cells that produced IL-4 and -13, as demonstrated by
intracellular cytokine staining in tetramer
+
cells. These data in-
dicated that activation of iNKT cells by DCs treated with aGalCer
in the initiation phase played a pivotal role in the regulation of
the host response to allergen challenge, and central to this out-
come was whether host cells produced IFN-g.
Tο further address the role of IFN-g and iNKT cells, Ja18
2/2
mice, which were deficient in iNKT cells, received iNKT cells
enriched from the liver of WT, IFN-g
2/2
,orJa18
2/2
mice prior
to BMDC transfer and allergen challenge. Notably, Ja18
2/2
mice
did not develop AHR and airway inflammation following OVA-
BMDC transfer and allergen challenge unless they received WT
iNKT cells. Similar to WT recipients, Ja18
2/2
recipients showed
decreased airway responses to allergen challenge following trans-
fer of iNKT cells from WT mice prior to OVA/aGalCer BMDC
transfer. However, in the Ja18
2/2
mice that received iNKT cells
from IFN-g
2/2
mice, where only the iNKT (or donor) cells were
incapable of producing IFN-g in the recipient mice, transfer of
OVA/aGalCer BMDCs significantly enhanced AHR and increased
the number of Eos in BAL fluid. Although the transferred cells
were only enriched for iNKT cells, these findings suggest that
IFN-g production by iNKT cells can act as a “brake” on an
otherwise Th2-biased response. It is unclear whether the IFN-g
produced by iNKT cells directly antagonizes the Th2 response or
whether IFN-g produced by iNKT cells acts on some undefined
host cells that then block the development of a Th2 response.
Fujita et al. (38) suggested that IL-27 together with IFN-g
secreted by iNKT cells played a role in the suppression of
allergen-induced airway inflammation and Th2-type cytokine pro-
duction. Future experiments are needed to resolve this issue.
The present study demonstrated that activation of iNKT cells
prior to allergen challenge can prevent or enhance the development
of allergic airway inflammation and AHR, depending on whether
iNKT cells can produce IFN-g. These results share some features
with previous studies indicating that activation of iNKT cells in the
initial phase was critical to the development of allergic airway
inflammation and AHR (39, 40). Kim et al. (39) demonstrated
that aGalCer, coadministered intranasally with OVA on three
consecutive days, led to the development of AHR and airway
inflammation, whereas OVA priming alone did not result in
airway inflammation and AHR. Bilenki et al. (40) showed that
in vivo stimulation of NKT cells by systemic administration of
aGalCer in the initial phase enhanced ragweed-induced airway
eosinophilia. Because they administered aGalCer i.v., iNKT cells
were likely activated systemically, whereas in the current study,
activation was likely restricted to lung iNKT cells as a result of
the intratracheal administration of aGalCer-treated DCs. Unlike
the report of Meyer et al. (41), which showed that intranasal in-
stillation of aGalCer enhanced AHR and airway eosinophilia, we
were unable to alter these responses in WT or IFN-g
2/2
recipients
of aGalCer-treated DCs.
Some of the inconsistencies among the various studies may be
related to the number of treatments with aGalCer and/or the mode
of delivery (systemic versus local). Recent experiments demon-
strated that iNKT cells with different cytokine-secretion capacity
seemed to segregate in a tissue-specific manner. In a tumor model,
Crowe et al. (35) compared NKT cells from liver, spleen, and
thymus for their ability to mediate rejection of a sarcoma cell line
in vivo and showed that only liver-derived NKT cells could pre-
vent tumor growth. They concluded that iNKT cells exist in func-
tionally distinct subpopulations among different tissues. In our
model, we demonstrated that iNKT cells from at least two organs
showed similar function; iNKT purified from spleen played a role
in the initiation phase of the development of AHR and allergic
inflammation similar to that of iNKT cells isolated from liver.
aGalCer-primed mice re-exposed to the same Ag in vivo
retained the ability to produce systemic IL-4 rapidly, whereas
IFN-g could not be detected in the serum (29). Earlier priming
with aGalCer enhanced systemic cytokine secretion, especially
serum levels of IL-4 by 17-fold, 4 h after injection compared with
naive mice (42). A number of reports demonstrated that repeated
administration of aGalCer favors Th2 activation, skewing
responses to IL-4 production rather than IFN-g (29, 43–45).
However, it is unclear how this Th2 polarization is achieved; it
was reported that a single injection of aGalCer while first
stimulating iNKT cells led to a state of unresponsiveness upon
FIGURE 6. Levels of AHR and allergic airway inflammation in Ja18
2/2
mice are decreased after receiving iNKT cells from the livers of WT mice
but are enhanced following reconstitution with iNKT cells from the livers
of IFN-g
2/2
mice prior to transfer of OVA/aGalCer BMDCs and
OVA challenge. Ja18
2/2
mice received iNKT cells from Ja18
2/2
,WT,or
IFN-g
2/2
mice prior to OVA or OVA/aGC BMDC transfer followed by
aerosolized OVA challenge. A, Airway resistance. B, BAL cell com-
position. Mean 6 SEM from three independent experiments are shown (n =
12). pp , 0.05, comparing Ja18
2/2
recipients of iNKT cells from WT mice
and transfer of OVA BMDCs;
#
p , 0.05, comparing Ja18
2/2
recipients
reconstituted with iNKT cells from IFN-g
2/2
mice and transfer of OVA/
aGalCer BMDCs and OVA BMDCs. AM, alveolar macrophages; Eos,
eosinophils; IFN-gKO, IFN-g
2
mice; Ja18KO, Ja18
2/2
mice; Lym,
lymphocytes; Neut, neutrophils; OVA, BMDCs pulsed with OVA; OVA/
aGC, BMDCs pulsed with OVA and aGalCer; TC, total cell.
The Journal of Immunology 259
rechallenge with this Ag (45). In the current study, iNKT cells
were stimulated in vivo by aGalCer-pulsed DCs, and these DCs
are known to induce prolonged IFN-g–producing NKT cell
responses (46). As shown in this study, upregulation of IFN-g,
but not IL-4, was associated with inhibition of eosinophilic airway
inflammation, AHR, and Th2 responses.
It is of interest that OVA/aGalCer BMDCs inhibited the de-
velopment of lung allergic responses in WT recipients, but these
same cells augmented allergic inflammation and AHR in IFN-g
2/2
recipients. This suggested that the effect of aGalCer in IFN-g
2/2
mice may be the result of activation of a default pathway of
cytokine production by activated (recipient) iNKT cells. In WT
FIGURE 7. iNKT cells from the livers of IFN-g
2/2
mice produce higher levels of IL-13 compared with
WT iNKT cells. Liver MNCs from naive IFN-g
2/2
or WT mice were isolated and stimulated with
aGalCer-pulsed BMDCs for 24 h. IL-13 production
in the cytoplasm of iNKT cells or culture supernatants
was determined. A, Representative scattergram with
IL-13 cytoplasmic staining in CD3
+
CD1d tetramer
+
cells. B, IL-13 levels in supernatants from culture of
naive liver MNCs with aGalCer-pulsed BMDCs. pp ,
0.05, comparing IL-13 production from IFN-g
2/2
and
WT iNKT cells.
FIGURE 8. Ja18
2/2
recipients of spleen iNKT cells from WT mice prior to OVA-pulsed BMDC transfer and OVA challenge developed allergic airway
inflammation and AHR. A, WT spleen iNKT cells were purified using three steps: initially, CD4
+
T cells were enriched following negative selection. Aa,
These cells were stained with CD1d-PE tetramer and isolated by magnetic bead sorting. Ab, The CD1d
+
cells were further purified by cell sorting. Purity of
the cell suspensions was analyzed using the Accuri (C6, Ann Arbor, MI) flow cytometer. Purified iNKT cells were transferred into Ja18
2/2
mice followed
by OVA-pulsed BMDCs and allergen challenge. B, Airway resistance. C, BAL cell composition. Data represent mean 6 SEM (n = 12). pp , 0.05,
comparing Ja18
2/2
mice treated with PBS prior to OVA-pulsed BMDCs and OVA challenge versus Ja18
2/2
recipients of purified spleen iNKT cells prior
to OVA-pulsed BMDCs and OVA. AM, alveolar macrophages; Eos, eosinophils; Ja18KO, Ja18
2/ 2
mice receiving PBS prior to OVA-pulsed BMDCs;
Ja18KO+WT spleen iNKT, Ja18
2/2
mice receiving spleen iNKT cells prior to OVA-pulsed BMDCs; Lym, lymphocytes; Neut, neutrophils; TC, total cell.
260 ACTIVATION OF iNKT CELLS BY DCs IN ASTHMA
mice, iNKT cells activated by aGalCer-pulsed DCs preferentially
produced IFN-g rather than IL-4. In IFN-g–deficient mice, these
same DCs stimulated Th2 cytokine production in the iNKT cells.
The effects of iNKT cells on the allergic phenotype may be direct
(22) but more likely are indirect, modulating the activity of other
cells. IFN-g production from iNKT cells can affect bystander
cells, such as NK cells, CD4
+
T cells, and CD8
+
T cells (29,
47–49), and inhibit the development of Th2 responses, AHR, and
eosinophilic airway inflammation (28). iNKT cells from IFN-g
2/2
mice, while failing to produce IFN-g did produce IL-4 and -13.
IL-4 from iNKT cells can prime several cell types (50–52), resulting
in an upregulation of Th2 responses, IL-13 production, and the en-
hancement of AHR and airway inflammation.
The hygiene hypothesis suggests that early-life environmental
exposure to microbes or other pathogens and their products pro-
motes innate immune responses that protect against the development
of atopy and asthma (53). Many microorganisms have the ability to
indirectly activate iNKT cells during infection (14, 54), and some
microbial glycolipid Ags were shown to directly activate
iNKT cells (55–60). Thus, IFN-g plays a critical role in deter-
mining the consequences of activated iNKT cells in the development
of airway inflammation and AHR. As a result, early exposure of
IFN-g–sufficient hosts to microorganisms or pathogen- or microbe-
associated products activates iNKT cells and preferentially induces
IFN-g production, protecting against the development of atopy
and asthma. However, in individuals who have a lower capacity for
IFN-g production, potentially on a genetic basis or under certain
conditions, the activation of iNKT cells might induce the pro-
duction of IL-4 and -13 and enhance the development of atopy and
allergic responses. This concept gains support from findings in infants
at genetic risk for developing atopy who have weaker neonatal IFN-g
responses compared with low-risk infants (61), perhaps because of
differential patterns of methylation of the IFN-g promoter (62). It is
under such conditions that iNKT cell activation may play a significant
role in directing T cell differentiation and Th2 polarization, in-
creasing the risk for developing atopy and asthma.
Acknowledgments
We thank Diana Nabighian for expert help in preparing the manuscript and
Lynn Cunningham for performing the immunolabeling studies.
Disclosures
The authors have no financial conflicts of interest.
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262 ACTIVATION OF iNKT CELLS BY DCs IN ASTHMA
    • "As a consequence, the development of AHR in a murine model using OVA was blocked [70]. Another study demonstrated that transfer of bone marrow dendritic cells (BMDCs) treated with α-GalCer, prevented the development of lung allergic responses, and this was dependent on IFN-γ production by recipient type I NKT cells [71]. All these findings suggest novel therapeutic strategies for asthma by eliminating or skewing type I NKT cells toward Th1 functions. "
    [Show abstract] [Hide abstract] ABSTRACT: Natural killer T (NKT) cells are a heterogeneous group of T cells that share properties of both T cells and natural killer (NK) cells. Many of these cells recognize the non-polymorphic CD1d molecule, an antigen-presenting molecule that binds self and non-self glycolipids and lipids. NKT cells are able to influence autoimmune and cancer diseases, thanks to their innate and adaptative immune properties. A hallmark of NKT cells is their ability to express pro-and anti-inflammatory T helper(h)1 and Th2 cytokines upon antigenic stimulation, and to modify both inflammatory mediators and late stage T cell responses. Thus, NKT cells are considered critical cells in regulatory events that direct the inflammatory response. Additionally, NKT cells can regulate the functions of other NKT subsets. The purpose of this review is to highlight the importance of NKT cells as regulators of autoimmunity disorders and cancer.
    Full-text · Article · Jan 2013
  • [Show abstract] [Hide abstract] ABSTRACT: T helper 2 (Th2) cells play crucial roles in the development of allergic asthma, while various distinct cell populations also contribute to the pathogenesis of the disease. Invariant natural killer T (iNKT) cells produce large amounts of cytokines such as IL-4 and IFNγ upon stimulation with a ligand, α-galactosylceramide, and regulate various immune responses. Recently, a critical role of iNKT cells in the mouse model of asthma and also in asthma patients has been reported, while some contradictory results have also been described. Here, we summarize the experimental results in mouse and human systems, and discuss the current understanding of the role of NKT cells in the pathogenesis of asthma, including a possible mechanism by which iNKT cells are activated in asthma patients.
    Article · Dec 2010
  • [Show abstract] [Hide abstract] ABSTRACT: Invariant NK T (iNKT) cells are a subset of T lymphocytes that recognize glycolipid antigens bound with the antigen-presenting molecule CD1d. iNKT cells have potent immunoregulatory activities that can promote or suppress immune responses during different pathological conditions. These immunoregulatory properties can be harnessed for therapeutic purposes with cognate glycolipid antigens, such as the marine sponge-derived glycosphingolipid α-galactosylceramide. Preclinical studies have shown substantial promise for iNKT cell-based treatments of infections, cancer and autoimmune and inflammatory diseases. Translation of these preclinical studies to the clinic, while faced with some obstacles, has already had some initial success. In this article, we review the immunodulatory activities of iNKT cells and the potential for developing iNKT cell-based prophylactic and curative therapies of human disease.
    Article · Jan 2011
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