A rapid fluorescence-based assay for classification of iNKT cell activating glycolipids.
ABSTRACT Structural variants of α-galactosylceramide (αGC) that activate invariant natural killer T cells (iNKT cells) are being developed as potential immunomodulatory agents for a variety of applications. Identification of specific forms of these glycolipids that bias responses to favor production of proinflammatory vs anti-inflammatory cytokines is central to current efforts, but this goal has been hampered by the lack of in vitro screening assays that reliably predict the in vivo biological activity of these compounds. Here we describe a fluorescence-based assay to identify functionally distinct αGC analogues. Our assay is based on recent findings showing that presentation of glycolipid antigens by CD1d molecules localized to plasma membrane detergent-resistant microdomains (lipid rafts) is correlated with induction of interferon-γ secretion and Th1-biased cytokine responses. Using an assay that measures lipid raft residency of CD1d molecules loaded with αGC, we screened a library of ∼200 synthetic αGC analogues and identified 19 agonists with potential Th1-biasing activity. Analysis of a subset of these novel candidate Th1 type agonists in vivo in mice confirmed their ability to induce systemic cytokine responses consistent with a Th1 type bias. These results demonstrate the predictive value of this novel in vitro assay for assessing the in vivo functionality of glycolipid agonists and provide the basis for a relatively simple high-throughput assay for identification and functional classification of iNKT cell activating glycolipids.
- SourceAvailable from: Tony W Ng[Show abstract] [Hide abstract]
ABSTRACT: Recombinant Mycobacterium bovis bacillus Calmette-Guèrin (rBCG) has been explored as a vector for vaccines against HIV because of its ability to induce long lasting humoral and cell mediated immune responses. To maximize the potential for rBCG vaccines to induce effective immunity against HIV, various strategies are being employed to improve its ability to prime CD8 + T cells, which play an important role in the control of HIV infections. In this study we adopted a previously described approach of incorporating glycolipids that activate CD1d-restricted natural killer T (NKT) cells to enhance priming of CD8 + T cells by rBCG strains expressing an SIV Gag antigen (rBCG-SIV gag). We found that the incorporation of the synthetic NKT activating glycolipid a-galactosylceramide (a-GC) into rBCG-SIV gag significantly enhanced CD8 + T cell responses against an immunodominant Gag epitope, compared to responses primed by unmodified rBCG-SIV gag. The abilities of structural analogues of a-GC to enhance CD8 + T cell responses to rBCG were compared in both wild type and partially humanized mice that express human CD1d molecules in place of mouse CD1d. These studies identified an a-GC analogue known as 7DW8-5, which has previously been used successfully as an adjuvant in non-human primates, as a promising compound for enhancing immunogenicity of antigens delivered by rBCG.vectors. Our findings support the incorporation of synthetic glycolipid activators of NKT cells as a novel approach to enhance the immunogenicity of rBCG-vectored antigens for induction of CD8 + T cell responses. The glycolipid adjuvant 7DW8-5 may be a promising candidate for advancing to non-human primate and human clinical studies for the development of HIV vaccines based on rBCG vectors.PLoS ONE 09/2014; · 3.53 Impact Factor
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ABSTRACT: Many hematopoietic cell types express CD1d and are capable of presenting glycolipid antigens to invariant natural killer T cells (iNKT cells). However, the question of which cells are the principal presenters of glycolipid antigens in vivo remains controversial, and it has been suggested that this might vary depending on the structure of a particular glycolipid antigen. Here we have shown that a single type of cell, the CD8α(+) DEC-205(+) dendritic cell, was mainly responsible for capturing and presenting a variety of different glycolipid antigens, including multiple forms of α-galactosylceramide that stimulate widely divergent cytokine responses. After glycolipid presentation, these dendritic cells rapidly altered their expression of various costimulatory and coinhibitory molecules in a manner that was dependent on the structure of the antigen. These findings show flexibility in the outcome of two-way communication between CD8α(+) dendritic cells and iNKT cells, providing a mechanism for biasing toward either proinflammatory or anti-inflammatory responses.Immunity 01/2014; · 19.80 Impact Factor
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ABSTRACT: Abstract NKT cells are a subpopulation of T lymphocytes with phenotypic properties of both T and NK cells and a wide range of immune effector properties. In particular, one subset of these cells, known as invariant NKT cells (iNKT cells), has attracted substantial attention because of their ability to be specifically activated by glycolipid antigens presented by a cell surface protein called CD1d. The development of synthetic α-galactosylceramides as a family of powerful glycolipid agonists for iNKT cells has led to approaches for augmenting a wide variety of immune responses, including those involved in vaccination against infections and cancers. Here, we review basic, preclinical and clinical observations supporting approaches to improving immune responses through the use of iNKT cell-activating glycolipids. Results from preclinical animal studies and preliminary clinical studies in humans identify many promising applications for this approach in the development of vaccines and novel immunotherapies.Immunotherapy 03/2014; 6(3):309-20. · 2.39 Impact Factor
Published:March 22, 2011
r2011 American Chemical Society
dx.doi.org/10.1021/ja200070u|J. Am. Chem. Soc. 2011, 133, 5198–5201
A Rapid Fluorescence-Based Assay for Classification of iNKT Cell
Pooja Arora,†Manjunatha M. Venkataswamy,†Andres Baena,†Gabriel Bricard,†,rQian Li,||,O
Natacha Veerapen,^Rachel Ndonye,§Jeong Ju Park,#Ji Hyung Lee,#Kyung-Chang Seo,#Amy R. Howell,§
||Petr A. Illarionov,^Gurdyal S. Besra,^Sung-Kee Chung,#and Steven A. Porcelli*,†,‡
†Department of Microbiology and Immunology and‡Department of Medicine, Albert Einstein College of Medicine, Bronx, New York
10461, United States
§Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269-3060, United States
Department of Chemistry and Medicinal Chemistry Programme, National University of Singapore, and Singapore Bioimaging
Consortium, Agency for Science, Technology and Research (A*STAR), Biopolis, Singapore
^School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.
#Department of Chemistry, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea
S Supporting Information
ABSTRACT: Structural variants of R-galactosylceramide
(RGC) that activate invariant natural killer T cells (iNKT
cells) are being developed as potential immunomodulatory
agents for a variety of applications. Identification of specific
tion of proinflammatory vs anti-inflammatory cytokines is
central to current efforts, but this goal has been hampered by
the lack of in vitro screening assays that reliably predict the
in vivo biological activity of these compounds. Here we
describe a fluorescence-based assay to identify functionally
showing that presentation of glycolipid antigens by CD1d
molecules localized to plasma membrane detergent-resistant
microdomains (lipid rafts) is correlated with induction of
interferon-γ secretion and Th1-biased cytokine responses.
Using an assay that measures lipid raft residency of CD1d
molecules loaded with RGC, we screened a library of ∼200
synthetic RGC analogues and identified 19 agonists with
potential Th1-biasing activity. Analysis of a subset of these
novel candidate Th1 type agonists in vivo in mice confirmed
their ability to induce systemic cytokine responses consistent
value of this novel in vitro assay for assessing the in vivo
functionality of glycolipid agonists and provide the basis for a
relatively simple high-throughput assay for identification and
functional classification of iNKT cell activating glycolipids.
against a wide variety of infectious diseases and tumors and also
plays a role in regulating allergic and autoimmune diseases.1,2
These cells express surface markers associated with both con-
ventional thymus-derived (T) cells and natural killer (NK) cells.
Theyalsoexpress an invariant R chain as a component of their T
cell antigen receptors (TCRs).1The TCRs of these cells are
specific for a range of different glycolipid antigens, which are
recognized as noncovalent complexes with CD1d, a cell surface
nvariant natural killer T cells (iNKT cells) constitute a unique
lymphocyte subset that participates in immune responses
glycoprotein that is homologous to class I antigen presenting
molecules encoded by the major histocompatibility complex
iNKT cells is a form of R-galactosylceramide (RGC) designated
as KRN7000 (1).4Recognition of this glycolipid is highly
conserved between mouse and human, although studies suggest
subtle differences in responses by iNKT cells in these species.5
The capability of iNKT cells to activate or regulate NK cells,
dendritic cells (DCs), neutrophils, macrophages, and B and
conventional T cells makes them an attractive target for immu-
notherapeutics. Indeed, activation of iNKT cells with KRN7000
has been shown to augment beneficial host immunity in some
models and to suppress autoimmune diseases in others.2,6
Administration of 1 to miceinduces a mixed cytokineresponse
that is characterized by secretion of both IFNγ and IL-4, the
prototypical cytokines defining anti-inflammatory (Th2) versus
proinflammatory (Th1) cellular immune responses.7The agonist
1 is therefore classified as inducing a nonpolarized or Th0
response. The secretion of IL-4 peaks at 2 h post-injection, while
glycolipid administration. The majority of this systemic IFNγ
or unsaturated acyl chains (e.g., RGC C20:2; compound 2 in
Figure 1) can bias the response to favor production of Th2-type
reduced IFNγ production due to the failure to transactivate NK
cells.SuchTh2-biased iNKTcellactivatorshave been found to be
superior for treatment in several mouse models of autoimmune
to stronger and more sustained production of Th1-type cytokines
are more effective as adjuvants for inducing protective immunity
against infections and for anticancer immunity. The most exten-
R-C-GC(3),inwhichtheoxygenof theglycosidic bondhasbeen
replaced with a one-carbon linker.3,11This agonist induces
Received:January 4, 2011
dx.doi.org/10.1021/ja200070u |J. Am. Chem. Soc. 2011, 133, 5198–5201
Journal of the American Chemical Society
activation that results in higher and more sustained IFNγ secre-
tion. Variants of this agonist containing an olefin linkage also
display enhanced Th1 bias in cytokine responses.12
A commonly used screening assay to identify new RGC
analogues is based on the culture of splenic cells in the presence
of candidate agonists followed by quantification of cytokines in
Many Th2-biasing iNKT agonists have been successfully identi-
fied in this way. Unfortunately, this type of screening assay has
proved to be less effective for identifying Th1-biasing agonists.
Generally, the prototypical Th1-biasing R-C-GC analogue 3 has
been found to display little or no activity in such cell culture
assays, despite the Th1-biased response that is observed in vivo
IFNγ-inducing or Th1-biasing agonists is possible only by
of candidate agonists, an approach that is not amenable to high-
throughput screening. This represents a major limitation in the
identification of additional and potentially more potent IFNγ-
a wide variety of potential clinical applications.
We recently showed that RGC agonists that induce strong
IFNγ secretion from NK cells have differences in CD1d loading
and presentation relative to Th2-biasing agonists.13The former
agonists load CD1d in an endocytic compartment and are
selectively trafficked to lipid raft microdomains in the plasma
membrane. In contrast, Th2-biasing agonists are loaded directly
into CD1d proteins present on the cell surface. Initial studies
indicated that CD1d complexes formed with Th2-biasing ago-
nists tend to be pH-labile and therefore dissociate at the low pH
is required for lipid raft localization, these complexes are
excluded from lipid rafts (Figure 1). Localization in lipid rafts
is probably responsible for optimal stimulation of iNKT cells,
which ensures that transactivation of NK cells takes place. We
therefore evaluated the possibility of using quantitation of lipid
raft residency of CD1d/glycolipid complexes as a method of
identifying new RGC agonists that have the potential to stimu-
Here we describe the development of a rapid two-step flow
cytometry-based assay employing this principle to identify
potent RGC analogues and predict their functional properties.
To screen a large library of RGC analogues, we first selected
RGC candidate agonists that showed efficient binding and
presentation by CD1d. Individual glycolipids were incubated at
II15followed by quantitation of the binding of monoclonal
antibody (mAb) L363 to cell surface CD1d/glycolipid com-
plexes by flow cytometry. This mAb is specific for complexes
formed by the binding of RGC to murine CD1d.16Using this
approach, we screened a library of 193 synthetic RGC analogues
linkages. This library, provided in Table S1 in the Supporting
Information (SI), included a large diversity of fatty acyl mod-
ifications as well as all eight stereoisomers of KRN7000 and
agonists have been tested in vitro and in vivo for their iNKT
stimulatory activities (Table S2). In the initial stage of screening,
we selected all of the agonists that gave an increase in L363
staining of at least 4 standard deviations above the median
fluorescence intensity (MFI) of untreated control cells
(Figures S1 and S2 in the SI). This represented a relatively
stringentcriterion forCD1d bindingthatactuallyexcludedsome
of the known weak iNKT cell agonists (e.g., the weak Th2-
biasing compound known as OCH).7This selection reduced the
candidate agonists to 71, which were then investigated for lipid
raft localization using the detergent extraction method.
For quantitation of the extent of lipid raft residency, we
exploited the fact that lipid raft microdomains are resistant to
extraction with nonionic detergents such as Triton X-100 (Tx-
100). Thus, JAWS II cells were incubated with each RGC
analogue at 100 nM concentration and stained with Alexa Fluor
647-conjugated mAb L363. Since plasma membrane lipid rafts
are detergent-resistant, CD1d/glycolipid agonist complexes
localized in lipid rafts cannot be extracted, and a minimal
decrease in fluorescence intensity is observed over time. In
contrast, for the CD1d/glycolipid agonist complexes that are
excluded from lipid rafts, the MFI decreases sharply following
Tx-100 addition. We also considered that a decrease in MFI
following Tx-100 treatment could be caused by the dislodge-
ment of fluorescently conjugated mAb from the CD1d/RGC
complexes. This possibility was excluded by studies of the effect
of Tx-100 concentration on the binding of L363 with its ligand,
Tx-100 (0.06%) used for this assay did not cause dissociation of
L363 mAb from CD1d/RGC complexes (Figure S3). To per-
form the analysis of Tx-100 stability of cell surface CD1d/RGC
complexes, the MFI of cellswas first recorded for ∼5 s to obtain
a baseline starting value, and then Tx-100 was added to a final
concentration of 0.06%. The sample was vortexed briefly to
ensure uniform mixing, and data were acquired using flow
cytometry in kinetic mode to visualize changes in MFI over
time (Figure 2A). To validate the screening assay, we chose
agonists 1?3 as representative Th0, Th2, and Th1 cytokine-
biasing glycolipid agonists. CD1d complexes containing agonist
2wererapidlyeluted from thecellsurface by Tx-100(Figure2),
consistent with their exclusion from lipid rafts. This agonist
the 24 h time point. In contrast, both 1 and 3 were presented by
IFNγ responses in mice with 1?3 orders of magnitude greater
Th1 bias than for 2.
Since several known Th2-biasing RGC analogues in the
library showed reductions of up to 70% or more in the initial
fluorescence during a 30 s exposure to Tx-100, we classified
agonists as negative for lipid raft localization if they showed
Figure 1. Prototype RGC activators of iNKT cells and a schematic
dx.doi.org/10.1021/ja200070u |J. Am. Chem. Soc. 2011, 133, 5198–5201
Journal of the American Chemical Society
more than 50% reduction in MFI within 30 s after addition of
Tx100. Out of the 71 compounds analyzed, 51 were rapidly
extracted with Tx100, consistent with their being bound pre-
dominantly to CD1d proteins localized outside of lipid rafts
(Figure S4A,B). Of these 51 agonists, four had been previously
studied for biological activity in vivo by injection into mice,7,10
In all cases Th2-biased serum cytokine responses were elicited
with high secretion of IL-4 relative to IFNγ (Table S2). These
findings were consistent with the predictions of our lipid raft
presentation model and indicated that our assay was able to
accurately identify Th2-type iNKT cell agonists. In addition, we
identified 17 new agonists in our library that formed CD1d
complexes that were resistant to extraction with detergent
(Figure 3 and Figures S4C and S5) and were thus predicted
to induce strong and sustained IFNγ secretion following
glycolipid administration in vivo.
To test this prediction, we chose a random sample of seven
agonists (5, 6, 8, 9, 10, 12, and 16) from this group and tested
their activities in vivo by injecting them individually into mice
biasing agonists (4) was included as a reference standard. 4
induced relatively low IFNγ at the 2 h time point, and this was
undetectable by the 24 h time point. In contrast, all seven
compounds that were presented by non-Tx-100-extractable
CD1d molecules induced significantly higher levels of IFNγ
(Figure 4A) and sustained measurable serum levels of this
cytokine for extended time periods of up to 36 h (Figure S6).
A useful indicator of Th1 bias in the cytokine response is the
ratio ofIFNγobserved24 hafter glycolipidinjectionto thatfor
peakIL-4secretionat2h. This ratiohastypicallybeenfoundto
be 0.1?0.2 for well-characterized Th2-biasing agonists such as
2 (Figure 2D). In contrast, the prototypical agonist 1 gives a
ratio of ∼1, while the strongly Th1-biasing agonist 3 induces
very high IFNγ with very low IL-4 secretion, resulting in
∼2 fold increase in this ratio (Figure 2D). The 4-dehydroxy-
sphingoid base variant of 2 (agonist 5) behaved like a strongly
Th1-biasing agonist in vivo in mice, showing the highest
secretion of IFNγ at 24 h with minimal IL-4 induction relative
to all of the other agonists tested (Figure 4B).
Our data also showed that the extent of localization of CD1d/
RGC complexes into lipid rafts was directly related to the fatty
a comparison of the extents of lipid raft localization for six
different agonists from our library that differed only in their fatty
acyl chain lengths (Figure 5A). All of the agonists containing
saturated fatty acyl moieties with < 16 carbons were excluded
from lipid rafts, whereas all of the agonists containing saturated
lipid rafts. Introduction of diunsaturation in the C20 acyl chain
abolished lipid raft localization of CD1d-loaded complexes for
agonist 2, suggesting that the hydrophobicity of the acyl chain
rather than simplythe chain length is the feature that determines
the extent of trafficking and localization of CD1d/RGC com-
plexes to lipid rafts. Further increasing the hydrophobicity of 1
replacing the carbonyl oxygen with a sulfur atom (agonist 12),
to 1 (Figure 5B). Furthermore, the carbasugar variant of 2 was
still lipid-raft-excluded and showed a Th2-biased cytokine re-
sponse in vivo (data not shown). Importantly, the screening
has been demonstrated to correctly predict the Th1-biasing
ability of 3 using an in vitro method. However, given the similar
levels of raft localization for all Th1/Th0-biasing glycolipids, the
very pronounced IFNγ responses and Th1 biases of compounds
3 and 5 cannot be explained solely by enhanced lipid raft
association. The other factors that further augment the Th1 bias
for these compounds remain to be established.
Figure 2. Validation of the rapid in vitro fluorescent antibody staining
assay to predict the functional activity of RGC analogues. (A) Plot of
detergent extraction from JAWS II dendritic cells of CD1d/glycolipid
complexes formed with known Th1- and Th2-biasing RGC agonists (i.
e., compounds 1?3 in Figure 1). (B) In vivo serum IFNγ levels at
various time points following administration of 4 nmol of RGC
analogues. (C) Serum IL-4 secretion measured 2 h after injection of
RGC analogues. (D) Ratio of the IFNγ level observed at 24 h with that
of peak IL-4 secretion 2 h after glycolipid administration.
Figure 3. Structures of predicted cytokine-biasing agonists. 4 was
presented by Tx-100-extractable CD1d molecules and thus predicted
to be a Th2-biasing agonist. In contrast, agonists 5?21 were presented
by detergent-resistant (i.e., raft-localized) CD1d and were therefore
identified as potential strong IFNγ inducers and Th1-biasing agonists.
dx.doi.org/10.1021/ja200070u |J. Am. Chem. Soc. 2011, 133, 5198–5201
Journal of the American Chemical Society
In summary, we have described a new in vitro method for rapid
functionality. Our technique is simple and robust, and its use can
substantially reduce the time and materials required for initial
characterization of biological activity. The use of this straightfor-
structure?activity relationships for RGC analogues and thus
facilitate the discovery of iNKT cell agonists for a wide variety of
immunotherapeutic applications. Our findings also provide further
response. Further studies are underway to explore whether the
approach described here can be applied to predict functional
responses induced by glycolipid antigens in humans.
mentary information. This material is available free of charge via
the Internet at http://pubs.acs.org.
Complete ref 13 and supple-
rINSERM Unit 851 Facult? e de M? edecine, CHU Lyon-Sud,
OMcCormick and Company, Inc., Technical Innovation Center,
204 Wright Ave., Hunt Valley, MD 21031.
A.P. Flow cytometry resources were supported by the Einstein
Cancer Center (NIH/NCI CA013330) and the Einstein Center
for AIDS Research (NIH AI51519). G.S.B. acknowledges
support in the form of a Personal Research Chair from Mr.
James Badrick, Royal Society Wolfson Research Merit Award, as
a former Lister Institute-Jenner Research Fellow, the Medical
Council, and The Wellcome Trust (084923/B/08/7). S.-K.C.
and MOEHRD (KRF-2005-070-C-00078). A.R.H. was sup-
ported by NIH/NIGMS Grant GM087136.
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Figure 4. In vivo serum cytokine responses of selected RGC agonists.
(A) Serum IL-4 and IFNγ at the indicated times after glycolipid
injection were analyzed by ELISA. Results for 4 were compared with
those for the seven other agonists using one-way analysis of variance
(ANOVA) with Dunnet correction. *, **, and *** indicate p < 0.05, 0.01,
and 0.001, respectively. (B) Ratios of IFNγ observed 24 h after
glycolipid injection to that for peak IL-4 at the 2 h time point.
Figure 5. (A) Effect ofsaturated fatty acyl chain length onthe extent of
the fattyacylchainlengthoftheRGCagonist. Thisshowsaswitchfrom
lipid raft exclusion to lipid raft localization at a chain length of 16
carbons. (B) Comparison of the extent oflipid raft localization ofCD1d
loaded with 13 different Th1/Th0-biasing agonists vs Th2-biasing
agonists 2 and 4. The value for 2 was compared with the values for
the other agonists using one-way ANOVA with Dunnet correction.