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Distinct NKT Cell Subsets Are Induced by
Different Chlamydia Species Leading to
Differential Adaptive Immunity and Host
Resistance to the Infections
Antony George Joyee, Hongyu Qiu, Shuhe Wang, Yijun
Fan, Laura Bilenki and Xi Yang
J Immunol 2007; 178:1048-1058; ;
doi: 10.4049/jimmunol.178.2.1048
http://www.jimmunol.org/content/178/2/1048
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References
The Journal of Immunology
Distinct NKT Cell Subsets Are Induced by Different Chlamydia
Species Leading to Differential Adaptive Immunity and Host
Resistance to the Infections1
Antony George Joyee, Hongyu Qiu, Shuhe Wang, Yijun Fan, Laura Bilenki, and Xi Yang2
N
atural killer T cells are a unique subset of T lymphocytes
that express markers of NK cells and a semi-invariant
TCR. These cells have been identified as a novel lymphocyte population that acts in innate immune responses. Unlike
conventional T lymphocytes, NKT recognize glyco- and phospholipids, rather than peptide Ags, presented by the nonclassical MHC
class I molecule CD1 (1). The striking feature of NKT cells is that
these cells can rapidly secrete large amounts of IFN-␥ and/or IL-4
upon activation, by interaction with the appropriate CD1d1/Ag
complex (2). These two cytokines are important for the regulation
of type 1 and type 2 T cell responses, respectively (3, 4), thus,
NKT cells are believed to be able to modulate immune responses
following various infections or other disease conditions (5–16). It
has been well-established that virtually all mouse (V␣14) and human (V␣24) invariant NKT (iNKT)3 cells can recognize ␣-galactosylceramide (␣-GalCer), a glycolipid originally extracted from
marine sponges (17, 18). CD1-dependent NKT cells can be classified as two types based on their reactivity to ␣-GalCer. The “classical” or iNKT cells which express the semi-invariant ␣␤ TCR
with the use of V␣14 and J␣18 can be activated by ␣-GalCer in the
Laboratory for Infection and Immunity, Departments of Medical Microbiology and
Immunology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba,
Canada
Received for publication July 13, 2006. Accepted for publication October 30, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by operating grants from the Canadian Institutes for Health
Research (CIHR) and the Manitoba Health Research Council. X.Y. is Canada Research Chair in Infection and Immunity. A.G.J. is a recipient of Postdoctoral Fellowship Award from the International Centre for Infectious Diseases/CIHR National
Training Program. L.B. is a recipient of a CIHR Doctoral Studentship and a trainee
of the CIHR National Training Program in Allergy/Asthma.
2
Address correspondence and reprint requests to Dr. Xi Yang, Laboratory for Infection and Immunity, Department of Medical Microbiology, Faculty of Medicine, University of Manitoba, Room 523, BMSB, 730 William Avenue, Winnipeg, Manitoba
R3E 0W3, Canada. E-mail address: [email protected]
Abbreviations used in this paper: iNKT, invariant NKT; ␣-GalCer, ␣-galactosylceramide; MoPn, mouse pneumonitis; KO, knockout; IFU, inclusion-forming unit; EB,
elementary body; D-PBS, Dulbecco’s PBS; p.i., postinfection; WT, wild type.
3
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
www.jimmunol.org
context of CD1, while the nonclassical NKT cells with diverse
TCR are nonreactive to ␣-GalCer (19). In humans, differential cytokine production by distinct NKT cell subsets has been demonstrated (20, 21). NKT-1-like cells show predominant IFN-␥ production while NKT-2-like cells produce higher IL-4. Such NKT
subsets may influence the nature of adaptive immune responses
during different infections.
Chlamydiae are obligate intracellular bacteria, which include
two species, Chlamydia pneumoniae and Chlamydia trachomatis,
commonly causing human infections. C. pneumoniae causes a
wide spectrum of acute and chronic respiratory diseases like bronchitis, obstructive pulmonary disease, sinusitis, etc., while C. trachomatis causes ocular, respiratory, and sexually transmitted diseases. The prevalence of chlamydial infections is very high
worldwide. In particular, Abs specific for C. pneumoniae can be
detected in the serum of up to 70% of healthy human beings implying that most individuals have had contact with these organisms
(22). More recently, C. pneumoniae has been implicated in the
pathogenesis of diverse diseases such as atherosclerosis, Alzheimer’s disease, and multiple sclerosis (23–25). The pathogenesis of
these conditions is considered to be immunopathologically mediated. Given the role of C. pneumoniae in many human diseases, an
effective vaccine would be of significant public health benefit. However, vaccine development against this important human pathogen has
been hindered by the limited understanding of immune mechanisms
that lead to protective or adverse immune responses.
The experimental animal models, especially mouse models, to
study C. pneumoniae and C. trachomatis infections have been instrumental in studying the components of immune system that contribute to, or is essential for, the control of these intracellular
pathogens. Studies in both humans and mice have shown that the
activation of type 1 immune responses including cell-mediated immunity and IFN-␥ production are essential for protection against
chlamydial infection, while type 2 response may be associated
with immunopathology (26 –34). However, the precise immune
mechanisms involved in resistance or pathogenesis to chlamydial
infection has not been fully elucidated. In particular, the role of
cellular and soluble components in immune responses during the
innate phase of infection remains largely unknown.
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We investigated the role of NKT cells in immunity to Chlamydia pneumoniae and Chlamydia muridarum infections using a
combination of knockout mice and specific cellular activation approaches. The NKT-deficient mice showed exacerbated susceptibility to C. pneumoniae infection, but more resistance to C. muridarum infection. Activation of NKT reduced C. pneumoniae in
vivo growth, but enhanced C. muridarum infection. Cellular analysis of invariant NKT cells revealed distinct cytokine patterns
following C. pneumoniae and C. muridarum infections, i.e., predominant IFN-␥ in the former, while predominant IL-4 in the latter.
The cytokine patterns of CD4ⴙ and CD8ⴙ T cells matched those of NKT cells. Our data provide in vivo evidence for a functionally
diverse role of NKT cells in immune response to two intracellular bacterial pathogens. These results suggest that distinct NKT
subsets are induced by even biologically closely related pathogens, thus leading to differential adaptive immune response and
infection outcomes. The Journal of Immunology, 2007, 178: 1048 –1058.
The Journal of Immunology
Materials and Methods
Mice
Breeding pairs of J␣18 knock out (NKT-KO) mice with B6 background
were provided by Dr. M. Taniguchi (RIKEN Research Center for Allergy
and Immunology, Yokohama City, Japan). These mice were generated by
specific deletion of the J␣18 (formerly J␣281) gene segment using homologous recombination and aggregation chimera techniques (36). Breeding
pairs of CD1d KO mice were purchased from The Jackson Laboratory. The
mice were bred and maintained at the pathogen-free animal care facility
(University of Manitoba, Winnipeg, Manitoba, Canada). Control C57BL/6
and BALB/c mice were purchased from Charles River Laboratories or bred
at the University of Manitoba animal care facility. Eight- to 10-wk-old
mice were used in the study. All experiments were done in compliance with
the institutional guidelines issued by the Canadian Council of Animal Care.
Organism
C. pneumoniae (AR-39; provided by Dr. K. Wolf, University of Manitoba)
was propagated in HL cells in Eagle’s MEM containing 10% FBS and 2
mM L-glutamine. Infected cells were harvested with sterile glass beads and
ultrasonically disrupted. After centrifugation at 500 ⫻ g for 10 min to
remove cell debris, bacteria were concentrated by high-speed centrifugation at 30,000 ⫻ g for 25 min, resuspended in sucrose-phosphate-glutamic
acid (SPG) buffer, and stored frozen in small aliquots (frozen at ⫺80°C)
until used. The infectivity, as measured by inclusion-forming units (IFUs)
of the bacterial preparation, was determined in HL cell culture. The same
seed stock of C. pneumoniae was used throughout the study. For immunological in vitro assays and Ab ELISAs, highly purified elementary body
(EB) preparations were obtained by adopting a Renografin gradient separation method as described previously (37). In brief, the partially purified
EB suspension was passed through a 20 –50% (v/v) Renografin (Squibb)
gradient. The EB band from the interphase was collected, washed twice,
and resuspended in SPG buffer. For cytokine induction in culture, a sonicated killed preparation of C. pneumoniae was prepared as previously described (38). Briefly, renografin-purified C. pneumoniae EBs were sonicated for 10 min on ice (Sonic Vibra Cell VC 130; Sonics & Materials).
The nonviability of chlamydiae was confirmed by inoculation onto HL
cells. This sonicated-killed C. pneumoniae preparation was used for in
vitro antigenic restimulation assays for cytokine analysis.
Mice infection and quantitation of in vivo infectivity
Mice were mildly sedated with isoflurane and inoculated intranasally with
5 ⫻ 106 IFUs of C. pneumoniae in 40 ␮l of PBS. At predetermined days
after inoculation, the mice were euthanized and the lungs were aseptically
isolated and mechanically homogenized using a cell grinder in SPG buffer.
The tissue homogenates were centrifuged down at 1900 ⫻ g for 30 min at
4°C to remove coarse tissues and debris and the supernatants were stored
frozen at ⫺80°C until tested. For C. pneumoniae quantitation, 100 ␮l of
serially diluted organ tissue supernatants were inoculated onto HL cells
grown to confluence in 96-well flat-bottom microtiter plates. The plates
were centrifuged at 1100 ⫻ g followed by incubation at 37°C for another
30 min. The cell layers were then washed and 200 ␮l of MEM containing
cycloheximide (2 ␮g/ml), gentamicin (10 ␮g/ml), and vancomycin (25
␮g/ml) were added to each well. The plates were incubated for 72 h at 37°C
in 5% CO2. After the incubation, the culture medium was removed, cell
monolayers were fixed with methanol, and stained with Chlamydia-specific
murine mAb and HRP-conjugated goat anti-mouse IgG secondary Abs and
developed with substrate (4-chloro-1-naphthol; Sigma-Aldrich). The number of inclusions was counted under a microscope at ⫻100 magnification.
Five fields through the midline of each well were counted. C. pneumoniae
titers in the organs were calculated based on dilution titers of the original
inoculum.
Histological analysis
Lungs were aseptically removed and fixed in 10%-buffered formalin and
embedded in paraffin for histological assessment. The tissue sections were
stained with H&E and the histological changes and cellular infiltration
were determined by light microscopy. For the detection of C. pneumoniae
inclusions and analysis of cell types using immunofluorescence staining,
one lung was snap-frozen in liquid nitrogen. For the latter analysis, lung
sections cut (at a thickness of 10 ␮m) from the frozen tissue were treated
with 2% goat normal serum for blocking and further stained for epithelial
cells, macrophages, and chlamydial inclusions. For staining chlamydial
inclusions, sections were incubated with mouse anti-Chlamydia Ab
(Chemicon International) and then with FITC-conjugated goat anti-mouse
IgG secondary Ab (Sigma-Aldrich). Macrophages were stained with PE
labeled anti-mouse F4/80 Ab (eBioscience).To stain epithelial cells, tissue
sections were further treated with mouse antipan-cytokeratin Ab (Calbiochem-Novabiochem). The sections were washed, incubated with biotinylated goat anti-mouse Ab (DakoCytomation), and then finally developed
with AMCA (Vector Laboratories). The stained tissue sections were observed under a fluorescent microscope and analyzed.
In vitro restimulation assays and cytokine measurements
Mice were killed at various days postinfection (p.i.) and the spleens and
draining (mediastinum) lymph nodes were aseptically removed. To analyze
for cytokine production, single-cell suspensions were prepared from spleen
and lymph nodes and cultured at a concentration of 7.5 and 5.0 ⫻ 106
cells/ml, respectively, alone or with sonicated killed C. pneumoniae (104
IFU/ml). Duplicate cultures were established from the spleen and lymph
node cells of individual mice in each group. The cells were incubated at
37°C in complete culture medium: RPMI 1640 containing 10% heat-inactivated FBS, 25 ␮g/ml gentamicin, 2 mM L-glutamine, and 5 ⫻ 10⫺5 2-ME
(Kodak) for 72 h in a 5% CO2 atmosphere, after which the supernatants
were collected, frozen, and later analyzed for cytokines by ELISA using
purified (capture) and biotinylated (detection) Abs as previously described
(30, 31). Abs purchased from BD Pharmingen were used for ELISA measuring IFN-␥, IL-12, IL-4, and IL-5.
Determination of C. pneumoniae-specific Ab levels
Ab titers for C. pneumoniae-specific IgG1 and IgG2a responses were measured using an alkaline phosphatase-based ELISA as previously described
(31, 32). Briefly, microtiter plates were coated overnight with killed C.
pneumoniae EBs. After blocking and washing, serially diluted sera were
incubated for 2 h at 37°C. Biotinylated goat anti-mouse Ab was added after
washing. Following overnight incubation at 4°C, alkaline phosphatase-conjugated streptavidin (Jackson ImmunoResearch Laboratories; Bio/Can Scientific) was added for 45 min at room temperature. The enzyme substrate
p-nitrophenyl phosphate (Sigma-Aldrich) (in 0.5 mM MgCl2, 10% diethanolamine (pH 9.8)) was added and the reaction was allowed to proceed for
60 min. The plates were read by using an ELISA reader at 405 nm. Results
are expressed as ELISA titers using the endpoint (cutoff at OD 0.5) of the
titration curves compared with a constant internal standard run in each
assay.
Flow cytometric analysis of cell surface markers
Mouse-specific Abs were purchased from BD Biosciences. Anti-CD3-PECy7, anti-CD3-PE, anti-CD4-PE, anti-CD8-PE-Cy7, anti-NK1.1-PE, CD19-FITC, and corresponding isotype controls were purchased from BD
Biosciences. Cells were stained for surface markers in a staining buffer
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We recently reported that NKT cells promote C. trachomatis
infection in a murine lung infection model (35). Using a mouse
biovar of C. trachomatis, mouse pneumonitis (MoPn) strain (more
recently named as Chlamydia muridarum), we found that NKTdeficient CD1-knockout (KO) mice are much more resistant to
infection and that the activation of NKT cells by ␣-GalCer exacerbated host susceptibility to infection. In the present study, we
further investigated the role of NKT cells in C. pneumoniae infection using a combination of approaches. First, we used two
types of gene knockout mice that are deficient in NKT cells
(NKT-KO and CD1-KO mice) to identify the role of NKT in immunity to C. pneumoniae infection. Next, we analyzed the effect of
pharmacological activation of iNKT cells by ␣-GalCer, the NKT
ligand in the outcome of infection, and finally we performed a
direct cellular analysis on iNKT cells by using CD1d tetramers to
elucidate the nature of NKT responses following infections with
these two different chlamydial species. The results showed that
NKT cells, in contrast to what was seen during C. muridarum
infection, play a crucial role in mediating protective immune responses against C. pneumoniae. Further analyses showed that
NKT cells activated by C. pneumoniae exhibit very distinct cytokine patterns from those activated by C. muridarum infection.
These findings suggest that NKT cells may play diverse roles in
immune responses to even very closely related pathogens, which
may differentially influence adaptive immune responses and infection outcomes.
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DISTINCT ROLE OF NKT IN CHLAMYDIAL INFECTIONS
(Dulbecco’s PBS (D-PBS); Sigma-Aldrich) without Ca2⫹ and Mg2⫹ containing 2% FCS and 0.09% NaN3) on ice for 30 min in the dark. Staining
for NKT cells was done by using PE-mCDd/PBS57 ligand tetramer (provided by the National Institute of Allergy and Infectious Disease MHC
Tetramer Core Facility, Atlanta, GA). After incubation with PE-Ab tetramer and PE-Cy7-anti-CD3␧ Abs on ice for 30 min in the dark, the cells
were fixed using 2% paraformaldehyde for 1 h, washed twice, resuspended
in D-PBS with 2% FCS and 1 mM EDTA. NKT cells were identified as
tetramer-positive and CD3␧-positive cell population. Sample data was collected using a FACSCalibur flow cytometer (BD Biosciences) and the data
were analyzed using CellQuest software (BD Biosciences).
Intracellular cytokine staining
␣-GalCer administration and assessment of immune
responses p.i.
␣-GalCer was provided by Kirin Brewery. Mice received a single i.v. injection of 4 ␮g of ␣-GalCer diluted in a 0.2-ml volume of PBS. Control
mice were injected with an identical volume of vehicle solution alone
(0.025% polysorbate 20 in PBS). Mice were infected with C. pneumoniae
2 h after ␣-GalCer or polysorbate injection.
Statistical analysis
IFU and serum Ab titers were transformed to logarithmic values. The differences in IFU detection, cytokine production, and Ab levels between
groups were analyzed by the unpaired, two-tailed Student t test to determine significance with Prism software (PRISM 4; GraphPad).
Results
Increased susceptibility to C. pneumoniae infection in
NKT-KO mice
To assess whether NKT cell deficiency renders mice susceptible
to C. pneumoniae infection, groups of NKT-KO and control
C57BL/6 mice were intranasally infected with C. pneumoniae.
The results showed that, compared with wild-type (WT) mice,
the NKT-KO mice suffered much greater weight loss. The WT
mice lost ⬃18% of body weight at day 3 p.i. and started to
regain the lost weight from day 4 p.i. and recovered the original
body weight by 2 wk following the infection. In contrast, the
KO mice showed a continuous decline of body weight and lost
⬎30% of their body weight at day 9 following infection (Fig.
1A). We next quantified the in vivo growth of C. pneumoniae in
the lungs. The NKT-KO mice showed significantly higher bacterial loads in the lung than WT mice at day 9 p.i. (Fig. 1B). At
a later time point (day 15 p.i.), the WT mice showed a dramatic
reduction in the pulmonary bacterial load, which dropped
⬎99%, while the KO mice still had significant bacterial burden
FIGURE 1. More severe body weight loss and higher organism growth
in the lungs after C. pneumoniae infection in NKT-KO mice than in the
WT C57BL/6 mice. Mice were intranasally infected with C. pneumoniae
(5 ⫻ 106 IFUs) and were monitored daily for body weight changes (A).
Each point represents the mean ⫾ SD of four mice. The original body
weights of the mice were similar between the groups. Mice were sacrificed
on day 9 (B) or day 15 (C) p.i. and the lungs were collected and the
bacterial loads were quantitatively analyzed as described in Materials and
Methods. Results are shown as mean ⫾ SD. One of three independent
experiments with similar results is shown. ⴱ, p ⬍ 0.05, ⴱⴱⴱ, p ⬍ 0.001, WT
vs KO mice.
in lungs which was close to 1000-fold higher than that in the
WT mice (Fig. 1C). These data demonstrate that NKT-KO mice
experience more severe disease and in vivo pathogen growth,
thus more susceptible to C. pneumoniae infection.
More severe pathological changes in NKT-KO mice
The histological analysis of lung tissues showed very severe pathological changes and tissue damage in the NKT-KO mice, which
correlated with higher bacterial loads in the lungs. On day 9 p.i.,
the alveolar architecture in KO mice was severely disrupted and
showed much dense and diffused cellular infiltration characterized
by high proportion of neutrophils and other polymorphonuclear
cells (Fig. 2A). Most alveoli were filled with inflammatory exudates. In contrast, the control C57BL/6 mice showed much less
lung pathology and the tissue inflammation and cellular infiltration
was more localized. The cellular infiltrate mainly comprised macrophages and lymphocytes (Fig. 2B). On day 15 p.i., both groups
showed signs of recovery in the lung, but the NKT-KO mice to a
much less degree. Notably, some multinucleated giant cells were
observed in the lung tissue of WT mice, but not in NKT-KO mice
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Single-cell suspensions of the lung were prepared by collagenase and
DNase digestion of the lung tissue. Briefly, the lung tissues were minced
into small pieces and incubated in RPMI 1640 containing 2 mg/ml collagenase type XI (Sigma-Aldrich) and 100 ␮g/ml DNase I (Sigma-Aldrich)
for 60 min at 37°C. The digested tissue suspension was passed through a
70-␮M cell strainer (BD Biosciences). Splenocytes were obtained by pressing the spleen through 70-␮M strainer. For all cells, erythrocytes were
lysed with ACK lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM
EDTA) followed by two washes in RPMI 1640 with 5% FCS and resuspended in complete RPMI 1640 medium.
For intracellular cytokine staining, lung mononuclear cells and splenocytes were stimulated with PMA (50 ng/ml) and ionomycin (1 ␮g/ml) and
incubated for 6 h in complete RPMI 1640 medium at 37°C. For the last 4 h
incubation, monensin (eBioscience) was added to accumulate cytokines
intracellularly. Cell staining was performed in a volume of 100 ␮l in 96well plates. In brief, cultured cells (2 ⫻ 106) were washed twice and incubated with FcR block Abs (anti-16/32; eBioscience) for 15 min at 4°C to
block nonspecific staining. Cell surface staining was performed as described above. The cells were then fixed and permeabilized with Cytofix/
Cytoperm (BD Pharmingen) as per manufacturer’s instructions and stained
intracellularly with anti-IFN-␥-allophycocyanin (XMG 1.2) and anti-IL-4FITC (BVD6-24G2) mAbs (eBioscience) or with corresponding isotope
control Abs in permeabilization buffer (BD Pharmingen). Finally, the cells
were washed, resuspended in D-PBS containing 2% FCS and 1 mM
EDTA, and analyzed by flow cytometry.
The Journal of Immunology
at this stage (Fig. 2C). Consistent with the determination of chlamydial IFUs in lung homogenates, NKT-KO mice showed more
inclusions in the lung on both day 9 (Fig. 2D) and day 15 (Fig. 2E)
p.i. Importantly, it appeared that more inclusions were visible in
macrophages (Fig. 2, D and E, green color) in NKT-KO mice than
those in WT mice, which suggests the failure of the macrophages
in effectively controlling chlamydial growth within the cells.
NKT cells show an IFN-␥-polarized cytokine response following
C. pneumoniae infection
To elucidate the mechanism by which NKT cells contributed to
host defense during C. pneumoniae infection, we performed a direct cellular level analysis on NKT kinetics and cytokine patterns
using ligand-loaded CD1d tetramer. NKT cells showed an expansion of ⬃5-fold after C. pneumoniae infection. The number and
percentage of NKT in the lungs peaked at day 3 p.i., after which
they decreased and reached basal levels about days 9 and 10 p.i.
(Fig. 3A). Intracellular cytokine staining analysis of lung (Fig. 3B)
and spleen (Fig. 3C) NKT cells at 3 days p.i. from the WT mice for
IFN-␥ and IL-4 showed significantly increased proportion of IFN␥-producing cells compared with that in the uninfected mice. This
difference was more obvious in NKT cells from the lung than those
from the spleen (Fig. 3, B and C). Thus, these results suggest that
FIGURE 3. NKT show an IFN-␥-polarizing cytokine pattern following
C. pneumoniae infection. A, CD1-d tetramer staining. Expansion of NKT
cells after C. pneumoniae infection in vivo. C57BL/6 mice were infected
with C. pneumoniae intranasally as described in the legend to Fig. 1 and
killed at various days p.i. Lung lymphocytes were prepared as described in
Materials and Methods and stained with PE-Cy7-labeled anti-CD3e Abs
and PE-labeled mCD1d/PBS57 ligand tetramer. The NKT cells were identified as CD3⫹ tetramer (⫹) cells by flow cytometry. B and C, Intracellular
cytokine staining. Mice were killed at 3 days after intranasal infection with
C. pneumoniae (Cpn). The lung (B) and spleen (C) cells were analyzed for
NKT cytokine responses by four-color flow cytometry. Intracellular cytokine staining was performed using Cytofix/Cytoperm kit from BD Biosciences with allophycocyanin-conjugated anti-IFN-␥ Abs and FITC-conjugated anti-IL-4 Abs or corresponding isotope controls. The analysis was
performed on NKT cells gated as CD3⫹ tetramer (⫹) cells. D and E,
Graphs represent IFN-␥ production NKT cells from lungs (D) and spleen
(E) from uninfected and C. pneumoniae-infected mice. Data show the
mean ⫾ SD and represent three independent experiments with three mice
in each group. ⴱⴱ, p ⬍ 0.01, and ⴱⴱⴱ, p ⬍ 0.001, Student’s t test.
C. pneumoniae infection can skew the cytokine response of NKT
cells to IFN-␥ polarization.
NKT cells influence the adaptive immune responses and mediate
the development of type 1 T cell responses
The cytokine pattern of NKT cells in the innate phase during C.
pneumoniae infection may contribute to the activation and differentiation of the major T cell lineages, CD8⫹ and CD4⫹ T cells,
thus influencing the development of type 1 or type 2 immune responses. To further evaluate whether NKT cells influence T cells
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FIGURE 2. More serious pathological response in NKT-KO than in WT
mice. The lungs from mice intranasally infected with C. pneumoniae (5 ⫻
106 IFUs) at day 9 or 15 p.i. were analyzed for histological changes by
H&E staining (A–C) and immunofluorescence staining (D and E) as described in Materials and Methods. A, Day 9 p.i.; B, day 15 p.i.; C, day
9 p.i.; D, day 9 p.i.; and E, day 15 p.i. The H&E-stained slides were
analyzed in low (⫻200, A and B) and high power (⫻400, C) magnification
under light microscopy. White arrow indicates multinucleated giant cells;
black arrows indicate epithelioid cells; green arrows indicate macrophage
death due to necrosis in the KO mice. The immunofluorescence staining
slides were analyzed under fluorescence microscopy (⫻400). Green color,
Chlamydial inclusions; red color, macrophages; blue color, epithelial cells.
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FIGURE 4. NKT cells influence the development of IFN-␥-producing CD8⫹ and
CD4⫹ T cells. Mice were intranasally infected with C. pneumoniae (5 ⫻ 106 IFUs)
with or without ␣-GalCer pretreatment. At
day 10 p.i., splenocytes were analyzed for
Th1 (IFN-␥) and Th2 (IL-4) cytokines by
intracellular cytokine staining as described
in Materials and Methods. Cytokine production pattern of CD8⫹ and CD4⫹ T cells
in NKT-KO and WT mice in the adaptive
immune phase following C. pneumoniae infection with or without ␣-GalCer treatment.
A and B, IFN-␥-producing CD8⫹ and CD4⫹
T cells in NKT-KO and WT mice following
infection. C, Graph represents IFN-␥ production pattern by CD8⫹ and CD4⫹ T cells
from NKT-KO and WT mice following C.
pneumoniae infection. D and E, Effect of
␣-GalCer treatment on IFN-␥ production by
CD8⫹ and CD4⫹ T cells in the NKT-KO
and WT mice infected with C. pneumoniae.
F, Graph represents IFN-␥ production pattern by CD8⫹ and CD4⫹ T cells following
C. pneumoniae infection with prior ␣GalCer treatment. G and H, IL-4 production by CD8⫹ and CD4⫹ T cells in
NKT-KO and WT mice infected with C.
pneumoniae. I, Graph represents IL-4 production pattern by CD8⫹ and CD4⫹ T
cells following C. pneumoniae infection.
J and K, Effect of ␣-GalCer treatment on
IL-4 production by CD8⫹ and CD4⫹ T
cells in the NKT-KO and WT mice following C. pneumoniae infection. L, Graph
representing IL-4 production pattern by
CD8⫹ and CD4⫹ T cells following C.
pneumoniae infection with prior ␣-GalCer
treatment. Data show the mean ⫾ SD. At
least three independent experiments with
three mice in each group were performed
and one representative experiment is
shown. ⴱ, p ⬍ 0.05, ⴱⴱ, p ⬍ 0.01, and
ⴱⴱⴱ, p ⬍ 0.001, Student’s t test. WT vs
KO mice.
DISTINCT ROLE OF NKT IN CHLAMYDIAL INFECTIONS
The Journal of Immunology
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FIGURE 5. Altered cytokine and Ab
responses in NKT-KO mice. A, Mice
(four mice per group) were infected intranasally with C. pneumoniae as described in the legend to Fig. 1 and were
sacrificed 9 days p.i. Spleen and draining
lymph node cells were cultured with sonicated-killed C. pneumoniae as described
in Materials and Methods. Th1 (IFN-␥)
and proinflammatory (IL-12) cytokines
and Th2 (IL-4, IL-5) cytokines in 72-h
culture supernatants were determined by
ELISA. Data are presented as the mean ⫾
SD of each group. One representative experiment of three independent experiments is shown. B, Serum samples were
collected at day 16 p.i. and tested for C.
pneumoniae-specific IgG1 and IgG2a.
Ab titers were transformed to log 10
values. Pooled data for three experiments (12 mice in each group) are presented. Data show the mean ⫾ SD.
ⴱ, p ⬍ 0.05, ⴱⴱ, p ⬍ 0.01, and ⴱⴱⴱ, p ⬍
0.001. NKT-KO vs WT mice.
1053
1054
NKT activation by ␣-GalCer enhances the protective immune
responses following infection
The above results generated from studies using NKT-KO mice
demonstrated an important role of NKT in host resistance to C.
pneumoniae infection. To further confirm this conclusion, we used
an alternative approach, which tested NKT function in the condition of enhanced NKT activation. WT mice were treated with
␣-GalCer, which specifically activates NKT, and the effect of treatment on the outcome of infection was examined. As shown in Fig.
6A, ␣-GalCer pretreatment significantly reduced the number of
bacteria in the lungs of WT mice to ⬃10-fold when compared with
mice given vehicle treatment. Moreover, the reduction in the bacterial load in the former was correlated with enhanced type 1 cytokine response. As illustrated in Fig. 6B, ␣-GalCer treatment
strongly enhanced the production of IFN-␥ and IL-12 in the WT
mice in comparison with the vehicle-treated controls. Moreover,
significant reduction in the levels of type 2 cytokines, IL-4 and
IL-5, was also seen in these mice. Furthermore, as shown in Fig.
FIGURE 6. Effect of in vivo stimulation of NKT cells by ␣-GalCer on
host response to C. pneumoniae. A and B, Naive C57BL/6 and NKT-KO
mice (four mice per group) were injected i.v. with 4 ␮g of ␣-GalCer in PBS
or with polysorbate vehicle control. The mice were intranasally infected
with C. pneumoniae (Cpn; 5 ⫻ 106 IFUs), 2 h after ␣-GalCer or vehicle
injection. Mice were sacrificed on day 9 p.i. for quantification of C. pneumoniae growth in the lungs (A) and for the analysis of C. pneumoniaespecific cytokine production by spleen cells (B) using the methods described in Materials and Methods. C and D, WT C57BL/6 mice were
treated with ␣-GalCer (4 ␮g) followed by intranasal infection with C.
pneumoniae (Cpn; 5 ⫻ 106 IFUs) or mock treatment. The lung (C) and
spleen (D) cells were analyzed for NKT cytokine patterns using four-color
flow cytometry as described in the legend to Fig. 3. One of three independent experiments with similar results is shown. ⴱ, p ⬍ 0.05. ⴱⴱ, p ⬍ 0.01.
6, C and D, intracellular cytokine analysis showed that prior treatment with ␣-GalCer followed by C. pneumoniae infection enhanced the IFN-␥ production by NKT after infection, while ␣-GalCer
treatment alone typically enhanced both IFN-␥ and IL-4 production by NKT cells (in comparison with that in naive mice
and C. pneumoniae infected, but ␣-GalCer-untreated mice as
shown in Fig. 3, B and C). The IFN-␥ polarization effect on NKT
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(CD8⫹ and CD4⫹) in the context of cytokine response to C. pneumoniae infection, we analyzed the cellular cytokine patterns of
CD4⫹ and CD8⫹ T cells in the WT and NKT-KO mice in the
adaptive immune phase of infection. At day 10 p.i., mice were
killed and the cytokine profile of CD4⫹ and CD8⫹ T cells was
analyzed. Intracellular cytokine staining for IFN-␥ and IL-4
showed a distinct pattern in IFN-␥ production by CD8⫹ T cells
between the WT and KO mice. The WT mice showed a significantly higher proportion of IFN-␥-producing CD8⫹ T cells compared with the NKT-KO mice (Fig. 4A). In addition, CD4⫹ T cells
of WT mice showed a Th1 cytokine pattern with significantly
higher IFN-␥ (Fig. 4B) and lower IL-4 levels, while CD4⫹ T cells
from NKT-KO mice showed a Th2-like pattern with more IL-4
(Fig. 4D), but less IFN-␥ (Fig. 4B). Of note, CD8⫹ T cells were the
major producers of IFN-␥ compared with CD4⫹ T cells, which
supports previously published work (39 – 41) showing the importance of the CD8⫹ T cell response as a major protective mechanism against this pathogen. Overall, these results suggested that
NKT cells could modulate both CD8⫹ and CD4⫹ T cell responses
toward the establishment of the protective type 1 response during
C. pneumoniae infection.
To further confirm the effect of NKT on Chlamydia-driven cytokine production patterns in the population level, we examined the
cytokine production by spleen and draining lymph node cells in the
infected mice. As illustrated in Fig. 5A, WT mice displayed a
type 1 cytokine production pattern, characterized by significantly
higher levels of IFN-␥ and IL-12 compared with the NKT-KO
mice. In addition, they also showed significantly lower levels of
type 2 cytokines, IL-4 and IL-5. Conversely, the NKT-KO mice
exhibited a type 2 cytokine pattern with significantly higher levels
of IL-4 and IL-5, but lower levels of both IFN-␥ and IL-12. In
combination with the data from flow cytometry analysis, these
results confirm that NKT cells can modulate cytokine responses in
a population level during C. pneumoniae infection. We further
measured the levels of C. pneumoniae-specific IgG1 and IgG2a
Abs in serum samples from NKT-KO and C57BL/6 control mice
before and after infection. After infection (day 15), the WT mice
showed significantly higher levels of serum IgG2a Abs than
NKT-KO mice. In addition, WT mice also showed lower levels of
IgG1 Abs when compared with the KO mice (Fig. 5B). These
results suggest that NKT cells play an important role in regulating
the production of Th1-associated Ab isotope responses in this
model of infection. In aggregate, the results indicate that NKT
cells contribute to the development and/or promotion of the protective immune responses to C. pneumoniae infection.
DISTINCT ROLE OF NKT IN CHLAMYDIAL INFECTIONS
The Journal of Immunology
1055
by C. pneumoniae infection was observed in NKT from the lung
(Fig. 6C) and spleen (Fig. 6D). In addition, CD4⫹ and CD8⫹ T
cell cytokine pattern analysis showed that prior ␣-GalCer treatment enhanced IFN-␥ production by both types of cells in WT, but
not in NKT-KO, mice (Fig. 4, E and F). Moreover, histological
findings showed that ␣-GalCer treatment substantially reduced
pulmonary pathological changes in the infected mice (data not
shown). In contrast to these observations in the WT mice, the
protective effect of ␣-GalCer treatment was not detected in the
NKT-KO mice, confirming the specificity of ␣-GalCer on NKT
activation. Taken together, these results further confirm the direct
involvement of activated NKT cells in eliciting strong protective
type 1 responses against C. pneumoniae infection.
Similar outcome of infection in CD1-KO mice as in
NKT-KO mice
To further confirm the role of NKT in C. pneumoniae infection, we
next examined CD1-KO mice, another type of NKT-deficient
mice. The rationale for using CD1-KO mice is 2-fold. First, the
CD1-KO mice had a different genetic background (BALB/c) from
the NKT-KO mice, thus the role of NKT can be examined in a
different mouse strain. This is an important point because in certain
murine models of infection, the protective or pathological outcome
of infection varies in mouse strains with different genetic background, such as C57BL/6 vs BALB/c mice (42). Therefore, it was
also our aim to see whether the difference in genetic background
had an influence on the protective role of NKT in C. pneumoniae
infection. The other and more important rationale is that our previous finding on the role of NKT in C. muridarum infection was
based on a study using CD1-KO mice (35). To conclude that NKT
play distinct roles in C. pneumoniae and C. muridarum infection,
the potential variation due to the differences in the types of NKTdeficient mice should be excluded. In the present study, our results
showed that, similar to our finding in NKT-KO mice, CD1-KO
mice also showed increased susceptibility to C. pneumoniae infection. As shown in Fig. 7, CD1-KO KO mice showed greater
body weight loss compared with the control BALB/c mice (Fig.
7A), which was associated with significantly higher pathogen loads
in the lungs (Fig. 7B) and decreased type 1 cytokine responses
(Fig. 7C). Further, ␣-GalCer treatment had no beneficial effect on
the outcome of infection in CD1-KO mice, but showed significantly enhanced protective immunity against C. pneumoniae in
WT BALB/c mice (data not shown). These results confirm that
NKT/CD1d indeed play a protective role in the course of C. pneumoniae infection, which is not affected by the genetic backgrounds
of mice, at least in the tested strains. In addition, the results indicate that the distinct role of NKT in C. pneumoniae and C. muridarum infections observed in our studies was not due to the difference in the types of NKT-deficient mice used.
NKT cytokine response in C. muridarum infection is different
from that in C. pneumoniae infection
As mentioned earlier, our previous report on the role of NKT in C.
muridarum infection was based on the findings with CD1 KO mice
on BALB/c background (35). Moreover, in the previous study,
although a Th2 promoting role of NKT was observed in C. muridarum infection (35), the cytokine profile of NKT cells following
infection was not examined. Therefore, we next performed experiments with NKT-KO mice to exclude the possibility of mouse
strain difference reflecting in different outcomes to these two
pathogens. NKT-KO mice and the WT C57BL/6 control mice
were infected with C. muridarum and were analyzed for body
weight loss, in vivo organism growth in the lungs and cytokine
pattern. Our results showed that the WT, but not NKT-KO, mice
experienced more body weight loss (Fig. 8A), higher lung pathogen loads (Fig. 8B) and increased production of IL-4, whereas the
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FIGURE 7. Similar outcome in
CD1 KO mice as that of NKT-KO
mice after C. pneumoniae infection.
CD1-KO and BALB/c control mice
were intranasally infected with C.
pneumoniae (5 ⫻ 106 IFUs) and were
monitored for body weight changes
(A). At day 9 p.i. infection, chlamydial loads in the lungs (B) and organism-driven cytokine production by
spleen cells (C and D) were analyzed
using the methods described in Materials and Methods. ⴱ, p ⬍ 0.05,
BALB/c vs CD1-KO mice.
1056
DISTINCT ROLE OF NKT IN CHLAMYDIAL INFECTIONS
IFN-␥ levels were similar between the groups (Fig. 8, C and D).
These results are consistent with our previously reported findings
in CD1 KO mice (35).
To further examine whether the cytokine profile of NKT activated by C. trachomatis (MoPn) is different from those activated
by C. pneumoniae, we directly examined the NKT cytokine response following MoPn infection using CD1d tetramers. As shown
in Fig. 8E, intracellular cytokine staining of lung NKT cells from
MoPn-infected WT C57BL/6 mice showed a predominant IL-4
cytokine response. Further, prior treatment with ␣-GalCer increased the proportion of IL-4-producing NKT cells in C. muridarum-infected mice (Fig. 8). This cytokine pattern is in sharp
contrast to the cytokine profile of NKT (IFN-␥ dominant) following C. pneumoniae infection in the absence (Fig. 3) or presence of
␣-GalCer stimulation (Fig. 6). Taken together, these findings confirm that the initial cytokine response of NKT is different following
C. pneumoniae or C. muridarum infection, which may be the basis
for the role played by NKT cells in the development of adaptive
immune responses to, and the outcome of, the infection.
Discussion
The most significant finding in this study is the difference in NKT
cytokine patterns and disease outcomes following infection with
two different chlamydial species. It has provided in vivo evidence
that distinct NKT subsets (NKT-1 like or NKT-2 like) are activated in the same organ (lung) following infection with two
closely related intracellular pathogens, which contributes to the
development of differential adaptive immune responses to, and different outcomes of, the infections. Our previous study showed that
NKT promote susceptibility to C. muridarum lung infection (35).
The comparison of cytokine production patterns and the types of
immune response between CD1-KO and WT mice and the findings
on the effect of ␣-GalCer treatment suggested that NKT enhance
type 2 responses to C. muridarum infection (35). In sharp contrast,
the present data revealed a crucial protective role of NKT in C.
pneumoniae infection. Our analysis on both lung and spleen NKT
showed that these cells produced higher IFN-␥ than IL-4 following
C. pneumoniae infection, which was associated with type 1 cytokine production by both CD8 and CD4 T cells. To find an answer
to why the role of NKT is different in C. pneumoniae and C.
muridarum infection models, we also dissected the cellular cytokine response of NKT in C. muridarum infection using intracellular cytokine staining analysis. Interestingly, the data showed that
NKT display a distinct pattern of cytokine response depending on
the pathogen. Thus, our data provide in vivo evidence that depending on the nature of the infectious stimuli, distinct NKT subsets
can be generated, thus subsequently influencing the development
of cellular responses to either type 1 or type 2 directions and the
outcome of infections. Moreover, our data show that, similar to
that observed in human studies (20, 21), functionally distinct NKT
cell subsets in the mouse also have differential cytokine production
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FIGURE 8. Promoting role of NKT cells in
C. muridarum infection. A, Less body weight
loss following C. muridarum infection in
NKT-KO mice. B–D, In vivo organism growth
and cytokine pattern after C. muridarum infection. NKT-KO and C57BL/6 mice were intranasally infected with C. muridarum (MoPn; 1000
IFUs) sacrificed on day 12 p.i. and analyzed for
in vivo chlamydial growth in the lungs (B) and
cytokine production by spleen cells (C and D).
One representative experiment of three independent experiments with similar results is shown.
ⴱ, p ⬍ 0.05, NKT-KO vs C57BL/6 mice. E, IL4-dominant cytokine response of NKT cells after
C. muridarum infection. C57BL/6 mice were infected with C. muridarum with or without prior
␣-GalCer treatment (4 ␮g). The mice were killed
at day 3 p.i. and lung lymphocytes were analyzed by intracellular cytokine staining for NKT
cytokine pattern as described in the legend to
Fig. 3. The figures show analysis on gated CD3⫹
tetramer (⫹) cells (E). One of the three independent experiments with similar results is shown.
The Journal of Immunology
Our findings provide evidence on the role of NKT cells in influencing the direction of adaptive immune responses, including
CD4 and CD8 T cells, to C. pneumoniae infection in vivo. Thus,
these findings demonstrate that NKT cells function not only in the
innate immune phase, but also in bridging to the type 1 acquired
immune responses, which leads to the host protection against C.
pneumoniae infection. In terms of the mechanism by which NKT
manipulate adaptive immune responses, because it is known that
IFN-␥ has a potent activity on Ag processing and presentation by
APCs, it is likely that IFN-␥, secreted by NKT, enhances the function of APCs, such as dendritic cells to produce IL-12. Such an
activity would result likely in preferential activation/differentiation
of C. pneumoniae-specific type-1 T cells and would ultimately
increase the level of protection. Because the activation of NKT in
vivo may also trigger a prompt series of cellular activation events
including the activation of other innate cells such as NK cells
allowing a more functional network of immune components, it
need not be the case that NKT cells are the sole or even the major
source of the IFN-␥, although these cells may be critical for the
initial induction of IFN-␥ synthesis which is important for directing immune responses to control infection. It should also be noted
that although the NKT KO mice indeed showed slower clearance
of C. pneumoniae infection and more serious disease (greater body
weight loss) than WT mice, they also showed a trend of recovery
in later stage (Fig. 1). The result suggests that the role of NKT in
host resistance to C. pneumoniae infection may be mainly in the
early stage of infection for the rapid induction of protective immunity, but may not be essential for the resolution of the infection.
However, because the fast clearance of the pathogen is critical in
preventing pathological changes in chlamydial diseases, the important role played by NKT cells in the early stage should not be
underestimated.
Although showing that iNKT cells mediate protective immune
responses in C. pneumoniae infection, our data also provide evidence that the type of immune response enhanced by the same
NKT stimulator, ␣-GalCer, may vary depending on the nature of
the pathogen. These findings have important clinical and therapeutic implications. A single injection of ␣-GalCer before intranasal
infection substantially reduced C. pneumoniae loads in the lungs of
WT mice and enhanced type 1 immunological responses. Of note,
␣-GalCer treatment significantly alleviated the lung pathology and
promoted faster normalization of alveolar architecture of lungs in
the treated WT mice compared with the vehicle-treated controls
(data not shown). However, in both NKT-KO and CD1-KO mice
infected with C. pneumoniae, the beneficial effect of ␣-GalCer
treatment was seen to be abolished, indicating that ␣-GalCer displays its adjuvant-like action by specifically activating V␣14 NKT
cells in the context of CD1d molecules. This indicated that the
administration of ␣-GalCer substantially enhances the level of protective immunity to this infection. In contrast, following C. muridarum (MoPn) infection, the administration of ␣-GalCer appears
to enhance type 2 response characterized by increased IL-4 and
IL-5 production (35). These observations raise a potential challenge to the attempt to use ␣-GalCer as an adjuvant in immunization, i.e., how to appropriately use ␣-GalCer to enhance the preferable protective type immunity against a particular infection.
In conclusion, the results of the present study unveiled the important role of iNKT cells in host protection against C. pneumoniae lung infection. Moreover, these findings provide in vivo
evidence that even biologically, closely related pathogens may activate different functional NKT subsets which can further polarize
the immune cells in adaptive immune responses, such as CD4⫹
and CD8⫹ cells. Further studies revealing the nature of the pathogens/stimuli which involve the development and/or promotion of
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profiles. This finding is particularly significant because it came
from a study using two biologically related pathogens.
In light of these findings, our results pose significant implications on the functional properties of NKT in host response to infections. From the chlamydial infection point of view, these results
suggest that the lung infection models of C. muridarum may not
reflect the innate and/or adaptive immune responses to C. pneumoniae infection. Even though both pathogens share many biological features, the cellular response and immunological course after
infection are different apart from the differences in growth characteristics and host specificity of these two pathogens. Using the
method of T cell intracellular cytokine staining, we confirmed the
reported findings on the difference of adaptive immune responses
between C. muridarum and C. pneumoniae infections. For example, in C. muridarum, both CD4⫹ and CD8⫹ T cells have been
reported to be protective, with the role of CD4⫹ T cells being more
important (43– 45). In contrast, CD8⫹ T cells have a prominent
role in protective responses that has been demonstrated both in the
clearance of primary infection (28) and against reinfection with C.
pneumoniae (29), while CD4⫹ T cells, in the absence of CD8⫹ T
cells, may even enhance the bacterial growth in the lungs (28).
Indeed, our present results showed that CD8⫹ T cells are the major
cell type producing a substantial amount of IFN-␥ during the adaptive immune phase of C. pneumoniae infection. Taken together,
our present and previous data showing a differential NKT response
suggest that adequate attention should be paid to potential differences in immunity to various chlamydial infections in designing
strategies against Chlamydia.
The exact mechanism of how different NKT responses are generated to these two Chlamydia infections remains elusive. At least
three possibilities, which are not necessarily exclusive, may exist:
1) C. pneumoniae and C. muridarum may preferentially induce
differentiation of NKT0-like cells into NKT1-or NKT2-like cells,
although this may be against the current dogma in mouse NKT cell
biology, which holds that NKT cells cannot be polarized; 2) the
same set of NKT is activated, but they produce skewed cytokine
profiles because of the different nature of the two chlamydial species (DNA homology of these species is under 10%); and 3) distinct subsets of NKT cells producing different sets of cytokines are
recruited to the lung. From a broader view, these data suggest that
NKT activation effect or the activation itself is pathogen or even
strain specific. This may be helpful in reconciling some inconsistent findings in previous studies (15, 46). For example, one previous study showed that NKT play a protective role in HSV infection (15). However, more recently, another study reported that
NKT cells are not critical for host protection against this pathogen,
because the absence of NKT in the NKT-KO did not affect the
outcome of infection when compared with the WT mice (46). Although both of these studies have analyzed the role of NKT in
HSV-1 infection, the strains of HSV-1 used were different. Thus,
it is likely that NKT cells are activated by certain epitopes specific
for pathogens or even strains of the same pathogen species. This
antigenic variability may account for differential functional roles
of NKT cells in infectious diseases. To address this issue more
precisely, characterization of glycolipid Ags from various microbial pathogens and identification of antigenic effects on NKT may
throw more light on immunological activity exerted by NKT cells.
It is probable that some microbial glycolipid Ags preferentially
activate NKT cells to produce more IFN-␥, while some activate
IL-4-producing NKT cells. Thus, the different functional NKT
subsets, such as NKT-1-like or NKT-2-like cells, may be activated
depending on the type or extent of activation stimuli or antigenic
epitopes, which in turn may reflect in the variability of NKT-mediated immunological outcomes.
1057
1058
differential NKT responses will greatly enhance our understanding
on the linkage between innate and adaptive immune responses in
infectious diseases.
Acknowledgments
We thank Dr. Katerina Wolf for initially providing us the AR-39 strain;
Dr. Y. Koezuka (Kirin Pharmaceuticals) for ␣-GalCer; and the National
Institute of Allergy and Infectious Disease MHC Tetramer Core Facility
for Cd1d tetramers for our experiments. We are grateful to Dr. Masaru
Taniguchi (RIKEN Research Center for Allergy and Immunology, Yokohama City, Japan) for providing the J␣18 KO mice.
Disclosures
The authors have no financial conflict of interest.
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DISTINCT ROLE OF NKT IN CHLAMYDIAL INFECTIONS