Activation of Invariant NKT Cells Ameliorates Experimental
Ocular Autoimmunity by A Mechanism Involving Innate IFN-?
Production and Dampening of the Adaptive Th1 and Th17
Rafael S. Grajewski,2,3* Anna M. Hansen,2* Rajeev K. Agarwal,4* Mitchell Kronenberg,†
Stephane Sidobre,†Shao Bo Su,5* Phyllis B. Silver,* Moriya Tsuji,‡Richard W. Franck,§
Anne P. Lawton,†Chi-Chao Chan,* and Rachel R. Caspi6*
Invariant NKT cells (iNKT cells) have been reported to play a role not only in innate immunity but also to regulate several
models of autoimmunity. Furthermore, iNKT cells are necessary for the generation of the prototypic eye-related immune
regulatory phenomenon, anterior chamber associated immune deviation (ACAID). In this study, we explore the role of iNKT
cells in regulation of autoimmunity to retina, using a model of experimental autoimmune uveitis (EAU) in mice immunized
with a uveitogenic regimen of the retinal Ag, interphotoreceptor retinoid-binding protein. Natural strain-specific variation
in iNKT number or induced genetic deficiencies in iNKT did not alter baseline susceptibility to EAU. However, iNKT
function seemed to correlate with susceptibility and its pharmacological enhancement in vivo by treatment with iNKT TCR
ligands at the time of uveitogenic immunization reproducibly ameliorated disease scores. Use of different iNKT TCR ligands
revealed dependence on the elicited cytokine profile. Surprisingly, superior protection against EAU was achieved with
?-C-GalCer, which induces a strong IFN-? but only a weak IL-4 production by iNKT cells, in contrast to the ligands
?-GalCer (both IFN-? and IL-4) and OCH (primarily IL-4). The protective effect of ?-C-GalCer was associated with a
reduction of adaptive Ag-specific IFN-? and IL-17 production and was negated by systemic neutralization of IFN-?. These
data suggest that pharmacological activation of iNKT cells protects from EAU at least in part by a mechanism involving
innate production of IFN-? and a consequent dampening of the Th1 as well as the Th17 effector responses.
Immunology, 2008, 181: 4791–4797.
The Journal of
nvariant NKT cells (iNKT)7are considered to represent an
innate subset of T cells. iNKT cells have a semi-invariant
V?14-J?18 TCR repertoire specific for lipid Ags that are
presented on the MHC class I-like CD1d molecule. ?-Galacto-
sylceramide (?-GalCer), a synthetic glycolipid derived from
marine sponges, is a well known iNKT TCR ligand (1). Re-
cently, natural ligands of iNKT TCR have been described, such
as ?-glucuronosylceramide and glycosphingolipids from Sphin-
gomonadaceae (2–4), that resemble cell wall constituents of
some Gram-negative bacteria, supporting an innate role of
iNKT cells in some infectious diseases. Other studies also re-
vealed regulatory and protective properties in various models of
autoimmunity such as experimental autoimmune encephalomy-
elitis (EAE) and type-1 diabetes (reviewed in Ref. 1). Most of
these studies used synthetic ligands for iNKT cell activation
and therapeutic regimen.
Upon ligation of their invariant TCR with ?-GalCer, iNKT cells
rapidly produce large amounts of cytokines such as IFN-? and
IL-4 (5). Analogues of ?-GalCer prepared by total synthesis led to
the identification of ligands that induce a cytokine pattern that is
more biased toward either a Th1-type (IFN-?) or a Th2-type (IL-4)
response. An example of the former is ?-C-GalCer, whereas the
latter includes the ligand (2s,3s,4r)-1-O-(?-D-galactopyranosyl)-N-
tetracosanoyl-2-amino-1,3,4-nonanetriol) (OCH) (6, 7). Some
studies demonstrated altered biological effects of these ?-GalCer
analogues on autoimmunity and cancer that could be ascribed to
the cytokine profiles they elicit (6, 7).
Experimental autoimmune uveitis (EAU) induced in animals by
immunization with retinal Ags in CFA is a model for human au-
toimmune uveitis, a disease that accounts for ?10–15% of severe
visual handicap in the US. EAU is induced by immunization with
*Laboratory of Immunology, National Eye Institute, National Institutes of Health,
Bethesda, MD 20892;†La Jolla Institute for Allergy and Immunology, La Jolla, CA
92037;‡Aaron Diamond AIDS Research Center, The Rockefeller University, New
York, NY 10016; and§Hunter College of the City University of New York, New
York, NY 10021
Received for publication April 16, 2007. Accepted for publication July 29, 2008.
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.
1This work was supported by National Institutes of Health Intramural funding.
2R.S.G. and A.M.H. contributed equally to this study.
3Current address: Wu ¨rzburg University, University Eye Hospital, Josef-Schneider-
Strasse 11, 97080 Wu ¨rzburg, Germany.
4Current address: Organ Systems Branch, Office of Centers, Training and Resources,
Office of the Director, National Cancer Institute, National Institutes of Health, 6116
Executive Boulevard, Suite 7006, Rockville, MD 20852.
5Current address: Institute of Inflammation and Immune Diseases, Shantou Univer-
sity Medical College, 22 Xin Ling Road, Shantou City, 515041, People’s Republic of
6Address correspondence and reprint requests to Dr. Rachel R. Caspi, Laboratory of
Immunology, National Eye Institute, National Institutes of Health, 10 Center Drive,
10/10n222, Bethesda, MD 20892-1857. E-mail address: email@example.com
7Abbreviations used in this paper: iNKT, invariant NKT cell; EAU, experimental
autoimmune uveitis; IRBP, interphotoreceptor retinoid-binding protein; ?-GalCer,
?-galactosylceramide; OCH, (2s,3s,4r)-1-O-(?-D-galactopyranosyl)-N-tetracosanoyl-
2-amino-1,3,4-nonanetriol); ACAID, anterior chamber-associated immune deviation;
WT, wild type; KO, knockout; EAE, experimental autoimmune encephalomyelitis.
The Journal of Immunology
the same retinal Ags that are recognized by uveitis patients and
is dependent on CD4?Th1 and Th17 effector cells (8, 9). EAU
in mice is induced with the interphotoreceptor retinoid-binding
protein (IRBP) or with its pathogenic fragments emulsified in
The relationship between NKT cells and eye-related immune
responses is not well understood. Although iNKT cells have been
shown to play an important role in the eye-related regulatory phe-
nomenon known as anterior chamber-associated immune deviation
(ACAID) (10, 11), the possible role of iNKT cells in regulation of
EAU has not been established. In the present study, we examine
the role of iNKT cells and the effects of the iNKT cell ligands on
EAU. Our data show that although natural strain-specific varia-
tions or genetically induced lack of iNKT cells do not seem to
affect the threshold of susceptibility to EAU, iNKT function
seemed to correlate with susceptibility. Importantly, a pharmaco-
logical enhancement of these cells using glycolipid iNKT cell li-
gands was able to inhibit induction of disease. This appeared to be
due at least in part to iNKT-produced IFN-? and a consequent
dampening of the adaptive Th1 and Th17 pathogenic effector
Materials and Methods
B10.RIII, B10.A, C57BL/6, DBA/2, AKR, and BALB/c mice (wild-type
(WT) and CD1d-knockout (KO)) were purchased from The Jackson Lab-
oratory. All experiments were approved by the National Eye Institute An-
imal Care and Use Committee. Animal care and use conformed to Insti-
tutional guidelines and to the Association for Research in Vision and
Ophthalmology guidelines on the use of animals in ophthalmic and vision
Ags and reagents
Bovine IRBP was purified as described (12, 13). CFA was purchased from
Difco and was supplemented with additional Mycobacterium tuberculosis
H37RA to 2.5 mg/ml. Purified derivative of tuberculin (PPD) was pur-
chased from the Statens Seruminstitut. ?-GalCer (KRN7000) was provided
by the Kirin Brewery (14). ?-C-GalCer was synthesized as described pre-
viously (6). OCH was synthesized by Drs. Chi-Huey Wong and Douglas
Wu of the Scripps Research Institute, La Jolla, CA. Neutralizing anti-
IFN-? Abs (clone R4-6A2) were obtained from the Biological Resources
Branch, National Cancer Institute.
Isolation of lymphoid cells from liver
Livers were perfused in situ through the hepatic portal vein with PBS and
minced into small pieces in PBS with 2% FCS and 0.02% sodium azide
(PBS/FCS/Az). The tissue was then pressed though a 200-gauge mesh and
cells were suspended in cold (4°C) PBS/FCS/Az. Cells were washed twice
(500 g for 7 min). The pelleted cells were resuspended in 37.5% isotonic
percoll at room temperature (25 ml per liver) and spun at 680g for 12 min.
The cell pellet made up of lymphocytes and erythrocytes was collected,
washed once in PBS/FCS/Az, and erythrocytes were lysed with 2 ml red
cell lysis buffer (Sigma-Aldrich) for 4 min. Cells were then recovered by
centrifugation through a layer of FBS, resuspended in PBS/FCS/Az, fil-
tered through 100-?m mesh, counted, and prepared for immunophenotyp-
ing by flow cytometry.
Immunization, EAU induction, and EAU scoring
C57BL/6 and BALB/c mice were immunized with 150 ?g of bovine IRBP
emulsified in CFA supplemented with Mycobacterium tuberculosis, strain
H37RA from Difco to 2.5 mg/ml. Clinical disease was evaluated by fundus
examination in a masked fashion and was scored on a scale from 0 (no
inflammation) to 4 (complete destruction of the retina) in half-point incre-
ments, as described previously (8). Eyes were harvested for histopathology
21 days after immunization. Disease was scored by an ophthalmic pathol-
ogist (C.-C. Chan) in a masked fashion as described previously (8).
?-GalCer and other treatments
Unless otherwise noted, 5 ?g of either ?-GalCer, ?-C-GalCer, or OCH
were added to and emulsified with the uveitogenic Ag preparation (IRBP/
CFA 1:1 v/v). The emulsion was injected s.c., divided into three doses
(both thighs and base of tail). Systemic IFN-? neutralization was achieved
by treatment with monoclonal anti-IFN-? Ab, 150 ?g/mouse injected i.p.
on days ?2, 0, and 2).
Determination of immunological responses
Cytokine production to ?-GalCer analogues in culture was examined on
splenocytes obtained from naive mice. Cell suspensions of 2.5 ? 106
cells/ml were incubated with 100 ng/ml of the stimulant and supernatants
were collected after 48 h. Cytokine production in vivo to ?-GalCer ana-
logues was measured in sera collected at the indicated time points after i.p.
injection of 5 ?g of the analog. For determination of Ag-specific cytokine
production and proliferation, spleens and lymph nodes draining the site of
immunization (inguinal and iliac) were collected on day 21 and were
pooled within each group. Proliferation to the indicated doses of Ag was
assayed by [3H]thymidine uptake during the last 16 h of a 72-h culture on
triplicate cultures of 0.2 ml, as described (15). Cytokine responses were
determined using the Pierce Chemical multiplex SearchLight Arrays tech-
nology (Ref. 16 and http://www.endogen.com/services).
iNKT cells were enumerated by flow cytometry after exclusion of dead
cells by DNA staining with 7-amino-actinomycin D. Cells were reacted
with PE-labeled CD1d/?-GalCer tetramers, allophycocyanin-labeled
?-TCR, and FITC-labeled anti-CD4 Abs. ?-TCR-positive cells were
gated, and the percentage of ?-GalCer-positive cells (either CD4?or
CD4?) was determined by counting of 1 ? 105viable ?-TCR-positive
cells. Multiplication of percentages by absolute numbers of lympho-
cytes that were isolated from each organ (? counter) resulted in the total
iNKT numbers shown in Fig. 1.
Statistical analysis and data presentation
All experiments were performed at least twice and results were highly
reproducible. Figures show data from representative or from pooled ex-
periments, as specified. EAU severity is represented by fundoscopy scores
determined on days 19–21. All fundoscopy scores were confirmed by his-
topathology. Where appropriate, statistical analysis of EAU severity was
performed using the Snedecor and Cochran z test for linear trend in pro-
portions (17). This is a nonparametric, frequency-based test that takes into
account both disease severity and incidence. Probability values of ?0.05
were considered to be significant. Values determined to be significantly
different from controls are marked with an asterisk in the figures.
iNKT cytokine profile, but not their number, may correlate with
susceptibility to EAU
EAU is a disease model where susceptibility varies consider-
ably among different mouse strains. The strain with the highest
known susceptibility is B10.RIII. B10.A is another susceptible
strain that can develop high disease scores, but, unlike the
B10.RIII strain, it requires administration of pertussis toxin at
the time of immunization to develop EAU, as do all other sus-
ceptible strains. C57BL/6 and DBA/2 mice have mild to mod-
erate susceptibility, and AKR as well as BALB/c mice are re-
sistant to disease. We asked the question whether susceptibility
to disease in a series of EAU-characterized mouse strains cor-
related with their numbers of iNKT cells. Fig. 1a shows a sche-
matic representation of typical EAU scores for six mouse
strains, summarizing previously reported findings (18–20).
Representative pictures of severe, mild and no disease are
shown in the inset.
Most of the iNKT cells in the body are concentrated in the
thymus, liver and spleen. We enumerated invariant TCR-bear-
ing NKT cells in these three organs in the different mouse
strains using flow cytometry, by binding of PE-labeled CD1d/
?-GalCer-tetramers. Although there were strain-specific differ-
ences in iNKT cell numbers and variations in their content in
the different organs, there was no correlation with strain-spe-
cific differences in disease susceptibility (Fig. 1, b and c). In
addition, a low iNKT number in one organ (e.g., thymus B10.A
4792 NKT CELLS PROTECT FROM EAU
and spleen AKR) tended to be counterbalanced by a higher
number in another organ (e.g., liver of B10.A and AKR) in
some strains. Consequently, the total number of iNKT cells was
often similar between strains with different EAU susceptibilities
We next examined the percent of CD4?iNKT cells of the total
iNKT cells, as this subset plays a role in the eye-specific regulatory
phenomenon known as ACAID (11, 21). CD4?iNKT cells were
enumerated by double staining for CD4 and for the invariant TCR
using CD1d/?-GalCer tetramers. The data revealed that the differ-
ences in CD4?iNKT cells between the strains were even less
pronounced than total iNKT numbers and did not correlate with
disease susceptibility (Fig. 1d).
Lastly, we selected three strains that were either resistant, mod-
erately susceptible, or highly susceptible to EAU (BALB/c,
C57BL/6, and B10RIII, respectively) and tested their iNKT cells
in vitro using two different ligands, ?-GalCer and OCH, each
known to elicit differing cytokine profiles (4, 22). Single-cell sus-
pensions were made from whole spleens and were stimulated with
the indicated ligands for 48 h. Analysis of culture supernatants
revealed that iNKT cells in resistant BALB/c mice produce sig-
nificantly higher levels of IFN-?, IL-4, and IL-2, irrespective of the
ligand used for stimulation (Fig. 2). Interestingly, the opposite was
the case with IL-17, with the highest levels detected in supernatant
of B10RIII splenocytes. iNKT cells have recently been identified
as a source of innate IL-17 (23), although the significance of this
response for autoimmune disease is yet to be defined. No inter-
strain differences were found in other cytokines examined, such as
IL-10, IL-13, and IL-5 (not shown).
Genetic lack of iNKT cells does not enhance susceptibility to
If iNKT cells had a role in raising the threshold of susceptibility to
EAU, we would expect that iNKT deficiency would result in more
severe disease. However, mice deficient in CD1d (lacking CD1d-
dependent NKT cells) (24) did not show enhanced EAU suscep-
tibility compared with their WT counterparts (Fig. 3). The time of
onset as well as the course of disease as determined by periodic
fundus examinations were also not affected (data not shown). Mice
deficient in CD1d on the resistant BALB/c background remained
resistant (Fig. 3). These data are consistent with observations made
by others in the EAE model (25–27).
Activation of iNKT cells ameliorates EAU but analogues of
?-GalCer differ in their efficacy
We next examined whether functional triggering of iNKT cells
using invariant TCR ligands can affect EAU. Five micrograms of
?-GalCer incorporated into the IRBP/CFA emulsion ameliorated
EAU severity and incidence in C57BL/6 mice (Fig. 4a). To ex-
amine the effect of ?-GalCer analogues, mice were similarly
treated with ?-C-GalCer and OCH. The data showed that OCH
protected no better than ?-GalCer, whereas ?-C-GalCer was the
most effective (Fig. 4b).
bers and percentages of CD4?NKT
cells seem unrelated to susceptibility
to EAU. Six different mouse strains
(six mice per strain, 8 wk of age) were
immunized with 150 ?g IRBP in CFA
and an additional i.p. injection of 0.3
?g pertussis toxin. B10.RIII mice
were immunized with 10 ?g IRBP
without additional pertussis toxin. a,
Schematic representation of typical
strain-specific disease scores and
representative histopathology (in-
set), based on established data. Lym-
phocytes from naive livers, thy-
muses and spleens were isolated on a
density gradient, and iNKT cell
numbers were determined by flow
cytometry using labeled CD1d/?-
?-TCR Abs as described in Materi-
als and Methods. b, Average of
isolated iNKT cell numbers in thy-
mus, lymph node, and spleen, ex-
pressed as a total cell number col-
lected from each mouse. c, Average
of isolated NKT cell numbers in thy-
mus, lymph node and spleen, ex-
pressed as a percentage of the total
cell number collected from each
mouse. d, Proportion of CD4?iNKT
cells expressed as a percentage of
total iNKT cells. The data are
pooled from two identical experi-
ments of three mice each (total six
individual animals per point).
Total iNKT cell num-
4793The Journal of Immunology
This pattern of protection was unexpected because OCH devi-
ates the iNKT response to TCR ligation toward IL-4 production
(whereas ?-C-GalCer skews toward IFN-?) and has moreover
been shown in experimental models of arthritis, diabetes in the
NOD mouse, and encephalomyelitis (EAE) to be more protective
than ?-GalCer in its original form through an IL-4 dependent
mechanism (reviewed in Refs. 7, 22). We therefore examined
whether our ?-GalCer, ?-C-GalCer, and OCH preparations had
the expected effect on iNKT cytokine production. Data obtained
by measuring IL-4 and IFN-? in serum of mice injected with the
three ?-GalCer analogues confirmed that indeed the three ana-
logues elicited cytokine profiles that were in keeping with what
has been reported by others (Fig. 5). These data suggested that
are present in iNKT cells from sus-
ceptible, compared with resistant,
strains. Splenocytes from BALB/c,
C57BL/6, and B10RIII mice were
isolated and cultured in HL-1 media
containing 1% normal mouse serum
and were left either unstimulated or
were treated with 100 ng/ml of either
?-GalCer or OCH, as indicated in the
figure. Cell supernatants were col-
lected after 48 h and cytokines were
analyzed by Pierce Searchlight Tech-
nology. Data are representative of
two experiments with three individual
mice in each group. Supernatants
were pooled before analysis.
or the susceptibility to EAU. An EAU-susceptible (C57BL/6) and resistant
strain (BALB/c) was immunized as described in Fig. 1a. Likewise NKT-
deficient strains on these genetic backgrounds were immunized, and EAU
scores were compared with the WT mice. EAU scores of C57BL/6 WT and
J?18-KO mice. Representative experiment of two showing the same pat-
tern (data were not pooled due to inter-experiment variation in disease
severity). Positive mice of total are indicated within each bar.
Lack of NKT cells does not seem to alter the disease course
NKT cells ameliorates EAU. C57BL/6 WT mice were immunized with
IRBP as described in Fig. 1a with (black column) or without (white col-
umn) 5 ?g of the synthetic iNKT cell ligand ?-GalCer. EAU scores on day
19 after immunization are shown (p ? 0.005). b, Analogues of ?-GalCer
differ in their ability to ameliorate EAU. C57BL/6 WT mice were immu-
nized as described in Fig. 1a without (white column) or with 5 ?g of the
synthetic iNKT cell ligand ?-GalCer (black column, p vs control ?0.09) or
?-C-GalCer (gray column, p vs control ?0.02) or OCH (dark gray column,
p. vs control ?0.22). Data combined from three experiments.
Effect of iNKT cell activation on EAU. a, Activation of
4794 NKT CELLS PROTECT FROM EAU
an IFN-?-dominated cytokine profile elicited by ?-C-GalCer is
more efficient in protecting from EAU than a deviation toward
an IL-4 dominated profile in this model.
Protective effect of iNKT cells in EAU seems to require innate
IFN-? and is associated with reduced adaptive IFN-? and
As we have previously observed that systemically produced IFN-?
can have a protective role in EAU (19, 28, 29), we decided to
examine whether the enhanced protective effect of ?-C-GalCer
compared with the other analogues was due to its ability to induce
enhanced production of IFN-? by iNKT cells. We therefore in-
jected mice immunized for EAU in the presence of ?-C-GalCer
with neutralizing Abs to IFN-? at the time of immunization, when
innate production of IFN-? induced by ?-C-GalCer would be oc-
curring (according to the timeline established in Fig. 5). EAU and
adaptive IFN-? and IL-17, representing pathogenic effector Th1
and Th17 cell responses, were examined. Mice protected from
disease with ?-C-GalCer had strongly reduced adaptive IFN-? and
IL-17 responses (Fig. 6). Neutralization of IFN-? at the time of
immunization restored EAU scores in ?-C-GalCer-treated mice,
but had no effect on disease progression in the control group, dem-
onstrating a specific requirement for IFN-? in the protection. In
addition, subsequent Ag-specific production of IFN-? and IL-17
was restored (Fig. 6, b and c), supporting the notion that innate
production of IFN-? elicited by ?-C-GalCer had a role in the pro-
tection and in inhibition of adaptive responses induced by this
In the present study, we demonstrate that iNKT cells can have a
role in EAU regulation. Their role appears to be not in setting the
threshold of susceptibility to EAU, as do the natural CD4?CD25?
regulatory cells whose function in deterring development of ocular
autoimmunity we have characterized in recent studies (30, 31).
Rather, they can inhibit developing disease following a pharma-
cological enhancement of their activity at or around the time of
priming. In chronic autoimmunity, priming of new effector T cells
is believed to be occurring on a continuous basis. Since endoge-
nous ligands for iNKT cells exist in the body and can trigger iNKT
activity (4, 5), it is conceivable that iNKT cells can participate in
modulating the course of ocular autoimmune disease. Thus, there
appears to be a “division of labor” between the natural
CD4?CD25?regulatory T cells and iNKT cells, with the former
setting the threshold of susceptibility and the latter possibly reg-
ulating the autoimmune response after that threshold has been
The group of Stein-Streilein (10, 11, 21) demonstrated that
iNKT cells have a central role in ACAID, a prototypic regulatory
phenomenon elicited by injection of Ag into the anterior chamber
of the eye and its transport by eye-derived APC to the spleen.
?-C-GalCer, or OCH. Serum was collected from three mice at the indicated times (hours after injection). The assay was performed on pooled serum
samples; therefore, error bars could not be generated.
Differing cytokine profiles are elicited by ?-GalCer analogues in vivo. C57BL/6 WT mice were injected i.p. with 5 ?g of either ?-GalCer,
innate IFN-? and is associated with reduced adaptive IFN-? and IL-17
responses. C57BL/6 WT mice were immunized as described in Fig. 1a
with or without additional 5 ?g of the iNKT cell ligand ?-C-GalCer in the
emulsion. Mice were injected with anti-IFN-? or isotype on days?2, 0 and
2 relative to immunization. a, EAU scores on day 19 after immunization.
Positive out of total mice are shown within the bars. Only the ?-C-GalCer
treated group is significantly different from control (p ? 0.028); (b) IFN-?
and (c) IL-17 titers in pooled supernatants of splenocytes from the mice in
a obtained on day 21 after immunization and cultured for 48 h with 30 ?g
IRBP. Note that due to the fact that the assay was conducted on pooled
supernatants the results represent an average of the group but no error bars
could be generated. Cytokine levels were determined using Pierce Search-
light Technology. Control cultures were set up without IRBP (IFN-? ?200
pg/ml, IL-17 ? 15 pg/ml). Shown is a representative experiment of two.
Protective effect of iNKT cells in EAU seems to require
4795 The Journal of Immunology
iNKT cells are recruited into the spleen via a mechanism involving
MIP-2 and participate in priming the adaptive T regulatory cells
typically associated with ACAID. Although prior elicitation of
ACAID to IRBP can inhibit a subsequent episode of EAU (32), it
is unlikely that the protection from EAU by iNKT that we observe
here bears a relationship to their role in ACAID. In ACAID, the
eliciting Ag originates from the eye, which has to be perturbed
(injected with Ag) in order for this phenomenon to be observed,
and iNKT activation, if any, occurs without additional manipula-
tion. In contrast, in our study, pharmacological activation of iNKT
cells is needed and is applied when the eye is still intact. Thus, it
is conceivable that iNKT cells may regulate ocular immune re-
sponses at more than one level.
Studies in the models of experimental arthritis, NOD diabetes,
and EAE (reviewed in Ref. 22) had indicated that activation of
iNKT by OCH is more effective than by ?-GalCer, which was
attributed to its induction of IL-4 and Th2 skewing. We were there-
fore surprised to find that OCH was not more effective than ?-Gal-
Cer in protecting from EAU, and that the most efficient protection
followed administration of ?-C-GalCer, which induces an IFN-?
dominated iNKT cytokine response. Thus, effectiveness of protec-
tion paralleled the innate IFN-? inducing ability of the invariant
TCR ligand. The protection was accompanied by reduction in the
IRBP-specific adaptive Th1 and Th17 pathogenic effector re-
sponses, as judged by production of their respective hallmark cy-
tokines IFN-? and IL-17 to in vitro recall with IRBP. The func-
tional role of IFN-? in the protective and regulatory effects of
iNKT are strongly supported by direct evidence showing that neu-
tralization of innate IFN-? reversed the protective effect of ?-C-
GalCer and restored the subsequent proinflammatory cytokine pro-
duction of the adaptive response. This is not to say that the
mechanism of protection is the same for all the three analogues.
Our data do not negate the possibility that protection from EAU by
OCH and by ?-GalCer could involve IL-4, as was previously
demonstrated in several other autoimmune disease models (22).
Our data are in line with some previous reports, which revealed
that protection from autoimmune disease by iNKT may not always
involve IL-4 and Th2 skewing. Studies by Lehuen and her col-
leagues (33, 34) demonstrated that even in the absence of IL-4,
iNKT cells can control EAE and experimental type 1 diabetes.
This was associated with a decrease in Th1-associated pathogenic
autoimmune responses without inducing Th2 responses and was
due at least in part to induction of anergy in the autoreactive T cells
(35). Such a mechanism could also be involved in the prevention
of EAU observed here. Although these studies did not directly
implicate IFN-? in these effects, participation of IFN-? (rather than
IL-4) in protection from EAE was suggested by Furlan et al. (27).
It should be noted that high systemic levels of IFN-? early or
late in the disease can be protective, but likely by different mech-
anisms. Initial production of IFN-? would be mostly from NKT
and NK cells, whereas later in disease Ag-specific Th1 cells are a
major source of IFN-?. We previously showed that early up-reg-
ulation of IFN-? by injections of IL-12 inhibits development of
EAU and associated immunological responses by aborting prim-
ing, through a process that involves induction of NO and apoptosis
(29). The innate IFN-? produced by ?-C-GalCer-triggered NKT
cells may well work in a similar fashion. In contrast, protective
effects of IFN-? later in the disease appear to be due to its role in
elimination of spent effector cells by activation-induced cell death
(36, 37). Thus, neutralization of systemic IFN-? at that stage also
enhances disease (19), although at that point it is not possible to
distinguish between effects of IFN-? produced by Ag-specific T
cells and iNKT cells.
In summary, we have demonstrated that iNKT cells can actively
participate in regulating the autoimmune response to immunolog-
ically privileged retinal Ags. This apparently occurs at a different
level than their role in induction of ACAID. The mechanism in-
volves the induction of innate IFN-? production through ligation
of the invariant TCR and results in inhibited development of adap-
tive Th1 and Th17 responses that represent pathogenic effector
mechanisms in uveitis.
We thank Dr. S. Yamano of the Kirin Brewery, Tokyo, Japan for providing
?-GalCer (KRN7000) and Drs. Chi-Huey Wong and Douglas Wu of the
Scripps Research Institute, La Jolla, CA for synthesizing the OCH used in
The authors have no financial conflict of interest.
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4797The Journal of Immunology