Involvement of Secretory and Endosomal Compartments in
Presentation of an Exogenous Self-Glycolipid to Type II
Keshab Chandra Roy,* Igor Maricic,* Archana Khurana,†Trevor R. F. Smith,*
Ramesh C. Halder,* and Vipin Kumar2*
Natural Killer T (NKT) cells recognize both self and foreign lipid Ags presented by CD1 molecules. Although presentation
of the marine sponge-derived lipid ?GalCer to type I NKT cells has been well studied, little is known about self-glycolipid
presentation to either type I or type II NKT cells. Here we have investigated presentation of the self-glycolipid sulfatide to
a type II NKT cell that specifically recognizes a single species of sulfatide, namely lyso-sulfatide but not other sulfatides
containing additional acyl chains. In comparison to other sulfatides or ?GalCer, lyso-sulfatide binds with lower affinity to
CD1d. Although plate-bound CD1d is inefficient in presenting lyso-sulfatide at neutral pH, it is efficiently presented at acidic
pH and in the presence of saposin C. The lysosomal trafficking of mCD1d is required for ?GalCer presentation to type I NKT
cells, it is not important for presentation of lyso-sulfatide to type II NKT cells. Consistently, APCs deficient in a lysosomal
lipid-transfer protein effectively present lyso-sulfatide. Presentation of lyso-sulfatide is inhibited in the presence of prima-
quine, concanamycin A, monensin, cycloheximide, and an inhibitor of microsomal triglyceride transfer protein but remains
unchanged following treatment with brefeldin A. Wortmannin-mediated inhibition of lipid presentation indicates an im-
portant role for the PI-3kinase in mCD1d trafficking. Our data collectively suggest that weak CD1d-binding self-glycolipid
ligands such as lyso-sulfatide can be presented via the secretory and endosomal compartments. Thus this study provides
important insights into the exogenous self-glycolipid presentation to CD1d-restricted T cells.
2008, 180: 2942–2950.
sociation with ?2-microglobulin (?2m)3and can be categorized
into two groups: group I (CD1a, CD1b, CD1c and CD1e (being
intermediate) in humans) and group II (CD1d in humans and
mice) based on sequence homology (2). Unlike MHC molecules
that present peptides, CD1 proteins bind and display a wide
array of lipids, glycolipids, and lipopeptides (either foreign or
self) to T lymphocytes expressing ?- and ?-chains of the TCR
(3). CD1d presents lipids and glycolipids to at least two distinct
populations of NKT cells. One of these is referred to as invari-
ant (iNKT) or type I NKT cells, which use a single invariant
?-chain (mouse V?14-J?18 and the homologous human V?24-
J?18) in combination with a restricted number of ?-chains
(mouse V?8.2, V?7, and V?2, and human V?11) of TCR (4, 5).
The Journal of Immunology,
he CD1 proteins are Ag-presenting molecules that have
many characteristics similar to MHC molecules (1). Five
different isotypes of CD1 proteins are expressed in as-
Type I NKT cells can recognize the foreign Ag ?GalCer, a lipid
found in the marine sponge Agelas mauritianus (6), in response
to which they rapidly produce Th1 and Th2 cytokines (7–11).
These cells also recognize bacterial lipids as well as a self-
glycolipid, isoglobotrihexosyl ceramide iGB3 (4, 5). Other au-
toreactive CD1d-restricted T cells, which do not use the invari-
ant TCR ?-chain and use a wide variety of TCR ?-chains, are
referred to as type II NKT cells. A subset of the type II NKT
cells is able to recognize the self-glycolipid sulfatide in both
humans and in mice (5).
Like proteins, glycolipids also need processing to yield im-
munogenic complexes with CD1 molecules for successful pre-
sentation. APCs have evolved an Ag processing machinery and
intracellular trafficking of Ag presenting molecules including
CD1 to survey and bind with appropriate ligands for presenta-
tion to T cells. Recently adaptor proteins, several lysosomal
enzymes, lipid transfer molecules, the biophysical environment
inside the cell, and lipid carrier proteins have been shown to be
involved in surveillance as well as in the processing and loading
of lipid Ags onto CD1 molecules (12). It has been shown that
a high affinity ligand to CD1d such as ?GalCer does not require
processing for presentation to iNKT cells (13). However its
presentation to iNKT is inhibited in the absence of saposins,
MTP, and adaptor protein AP-3 (14–16). Sulfatide is a promis-
cuous ligand which binds to human CD1a, CD1b, CD1c, and
CD1d and does not require further processing because it forms
a stable immunogenic complex with CD1 in vitro (17). We have
previously reported that the murine non-invariant T cell hybrid-
oma 19.3 recognizes sulfatide presented by CD1d (18). Bovine
brain-derived sulfatide contains several species, including cis-
tetracosenoyl-, tetracosenoyl-, palmitoyl-, and lyso-sulfatide.
*Laboratory of Autoimmunity, Torrey Pines Institute for Molecular Studies and†La
Jolla Institute for Allergy and Immunology, San Diego, CA 92121
Received for publication February 13, 2007. Accepted for publication December
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 is supported by grants from the National Institutes of Health to V.K.
2Address correspondence and reprint requests to Dr. Vipin Kumar, Laboratory of
Autoimmunity, Torrey Pines Institute for Molecular Studies, 3550 General Atomics
Court, San Diego, CA 92121. E-mail address: Vkumar@tpims.org
3Abbreviations used in this paper: ?2m, ?2-microglobulin; WT, wild type; TD, tail
deleted; IEF, isoelectric focusing; CMA, concanamycin A; ER, endoplasmic reticu-
lum; EE, early endosome; RE, recycling endosome; iNKT, invariant NKT.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
Recently we have published a three dimensional structure of
cis-tetracosenoyl-sulfatide complexed with murine CD1d at 1.9
Å (19) showing binding of this natural self-glycolipid ligand.
Here we further define the Ag-fine specificity of the type II
NKT cell hybridoma 19.3. We show that its TCR is extremely
restrictive in that it recognizes lyso-sulfatide but not other gly-
colipids, including three other major species of sulfatide. Lyso-
sulfatide binds with a weak affinity to mouse CD1d. Sulfatide
bound mCD1d traffics into the endoplasmic reticulum (ER),
early endosome (EE), and recycling endosome (RE) for its pre-
sentation at the cell surface. Also we report for the first time
that trafficking of mouse CD1d and its Ag presentation ability is
regulated by phosphatidyl inositol 3-kinase. Although the oc-
currence of cross-presentation of lipid Ags in vivo is inferred
from earlier studies (20, 21), the present data further elucidate
the intracellular pathways involved in the presentation of ex-
ogenous self-glycolipids bound by CD1d to type II NK T cells.
Materials and Methods
Mice and reagents
Female C57BL/6 and AP-3 mice were purchased from The Jackson Lab-
oratory. They were maintained in specific pathogen-free conditions in vi-
varium at TPIMS. A20 cells transfected with the wild-type (WT) or tail-
deleted (TD) mouse CD1d (mCD1d), V?14?1.2 NKT cell hybridoma,
Hy-1.2 were kindly provided by Dr. M. Kronenberg (La Jolla Institute of
Allergy and Immunology, La Jolla, CA) and were cultured in RPMI sup-
plemented with 10% FBS and 2 mM L-glutamine. Sulfatide-reactive
V?14– hybridoma has earlier been described (18). Brefeldin A, prima-
quine, monensin, wortmannin, concanamycin A (CMA), glutaraldehyde,
L-lysine, asialo-GM1 and Tween 20 were purchased from Sigma-Aldrich.
Cycloheximide and lactacystin were purchased from A.G. Scientific. MTP
inhibitor (iMTP, NUP-LAB687) was gifted by Drs. Gary Ksander and
David Chao (Novartis Institute for Biomedical Research). Dr. Kronenberg
kindly provided wild type, prosaposin, and cathepsin L knock out mice
originally developed in the laboratories of Drs. Grabowski and A Ruden-
ski, respectively. All Abs and conjugates used for FACS analysis were
purchased from BD Biosciences. Recombinant mouse saposin C was pro-
vided by Dr. Masao Hiraiwa (Department of Neurosciences, University of
California San Diego). Semisynthetic lyso-sulfatide, palmitoyl-sulfatide,
cis-tetracosenoyl and tetracosenoyl sulfatide were purchased from Matreya
Inc. Synthetic ? GalCer was provided by Pharmaceutical Division of the
Kirin Brewery Co. All lipids were dissolved in vehicle (0.5% polysorbate
20 (Tween 20) and 0.9% NaCl solution). Enhanced protein-binding 96-
well ELISA plates were purchased from NUNC.
Staining for flow cytometry was performed as described previously (18). In
brief, splenocytes or A20 cells transfected with WT- or TD-mCD1d were
treated either in the presence or absence of inhibitors. Cells were washed
twice with PBS and suspended in FACS buffer (PBS, 0.01% NaN3, and
0.5% BSA). Resuspended cells were first blocked with Abs against FcR-?
(2.4G2) and then labeled with the indicated Abs. Finally cells were
fixed with 1% paraformaldehyde. Flow cytometric analysis was per-
formed on a FACSCalibur instrument using CellQuest software (BD
Biosciences). PE-labeled mCD1d–? GalCer tetrameric complexes were
made in a baculovirus expression system as described previously (22,
23). “Unloaded” mCD1d tetramers were prepared by preincubating bi-
otinylated mCD1d protein with vehicle only. All experiments were re-
peated at least two times.
In vitro Ag presentation assay using APCs
Initial Ag presentation assays were performed to define the fine specificity
of sulfatide-reactive hybridoma, Hy-19.3 in the presence different lipids.
5 ? 104of T cell hybridoma (Hy-19.3) were cocultured with irradiated
splenocytes (4 ? 105/well in triplicate) from 8 to 12 wk old C57BL/6 mice
in the presence of graded concentrations of glycolipids (10?2ng-1 ?g for
?GalCer and 0.05 ?g-25 ?g for other lipids). Supernatants from these 16-h
cultures were used in sandwich ELISA to the measure IL-2 production.
In some lipid presentation assay, APCs including splenocytes collected
from different WT and knock out mice or WT or TD mCD1d-transfected
A20 cells (4 ? 105/well) pulsed with lyso-sulfatide or ?GalCer for 4h
(irradiated but unfixed APC) or 2h (unirradiated, fixed APC). APCs 4 ?
105/well were cocultured with 5 ? 104/well T cell hybridoma, Hy-19.3 or
Hy-1.2 for lyso-sulfatide or ?GalCer, respectively in triplicate at 37°C in
RPMI 1640 medium containing 10% FCS. Supernatants were harvested for
IL-2 release assay as above. Data are expressed as mean ng/ml ? SD of
triplicates. All experiments were repeated at least two times.
To study the lipid Ag pathway, WT mCD1d-transfected A20 cells (2–
4 ? 106/ml) were incubated with a panel of Ag presentation pathway
inhibitors at their optimum concentrations, including 300 mM primaquine,
100 nM CMA, 8–10 ?g/ml brefeldin A, 50 mM monensin, 1.0 mM wort-
mannin for 2 h, 100 ?g/ml cycloheximide for 5 h and 10 mM LAB-687
(iMTP) for 72 h before the addition of lipid Ags (lyso-sulfatide: 10 ?g/ml
and ?GalCer: 200 ng/ml). Inhibitory reagents were titrated and class I-re-
stricted OT-I T cells were used as controls for nonspecific cytotoxicity.
Cells were further incubated for 2–4h at 37°C in continued presence of
inhibitor, washed and fixed and used to stimulate T cells.
Fixation of APCs
WT mCD1d-transfected A20 cells treated either in presence or absence of
inhibitor followed by washing twice and suspended in PBS (10 ? 106/ml)
containing 0.05% glutaraldehyde for 30 s at room temperature. Additional
fixation was blocked by incubating with 0.2 M of L-lysine for 2 min. It was
ascertained that the concentrations of the inhibitors and the fixation pro-
cedure used in the assay did not result in significant loss of cell membrane
integrity of the APC as determined by trypan blue exclusion.
Ag presentation assay using plate-bound CD1d
Flat bottom 96-well plates were coated for 18 h with soluble CD1d recov-
ered from baculovirus expression system (19, 22) at the concentration of
9.0, 3.0, 1.0 and 0.3 ?g/well at 4°C in PBS (pH 7.4). Plates were washed
three times with PBS and then incubated for another 4h at 37°C with
indicated concentrations of lyso-sulfatide or ?GalCer. After washing three
times with PBS, 5 ? 104/well T cell hybridomas (Hy-19.3 or Hy-1.2) were
added in triplicate. Supernatants were collected after 17 h to measure IL-2
To study the effect of pH on lyso-sulfatide loading and presentation, 1
?g of soluble mouse CD1d was coated on flat-bottom 96-well plates for
18 h at 4°C in 50 mM Na-acetate buffer (pH 5.0, 6.0 and 7.0). Plates were
washed three times with same buffer and then incubated for another 4h at
37°C with lyso-sulfatide 1.25 ?g/ml or ?GalCer 500 ng/ml in 100 ml of
buffer. Plates were washed thrice with PBS (pH 7.4) before addition of 5 ?
104/well T cell hybridoma in triplicate. Supernatants were collected after
17 h to measure IL-2 release.
To investigate the role of saposin in lyso-sulfatide loading and presen-
tation 1 ?g of soluble mouse CD1d was coated on flat-bottom 96-well
plates for 18 h at 4°C in 50 mM Na-acetate buffer (pH 5.0). Plates were
washed three times with same buffer. 5 mg/ml lyso-sulfatide in 100 ml of
50 mM Na-acetate buffer (pH 5.0) in the presence of different concentra-
tions of saposin C as indicated was added and plates were incubated for
another 6 h at 37°C. Plates were washed three times with PBS (pH 7.4)
before addition of 5 ? 104/well T cell hybridoma, Hy-19.3 in triplicate.
Supernatants were collected after 17 h to measure IL-2 release.
To compare the binding affinity of each four major species of sulfatide
(cis-tetracosenoyl, tetracosenoyl, palmitoyl, and lyso) to CD1d the com-
petition assay is designed as follows. One microgram of soluble mouse
CD1d was coated on flat-bottom 96-well plate for 18 h at 4°C in PBS (PBS,
pH 7.4). Plates were washed three times with PBS and then incubated for
another 2 h at 37°C with a mixture of indicated concentrations of individual
species of sulfatide with a fixed concentration of ?GalCer (250 ng/ml) in
100 ml volume. Plates were washed thrice with PBS before addition of 5 ?
104/well T cell hybridoma, Hy-1.2 in triplicate. Supernatants were col-
lected after 17 h to measure IL-2 release.
Isoelectric focusing gel electrophoresis of sulfatide/CD1d
The protocol is same as described earlier (19). In brief, recombinant mouse
CD1d (9 ?g/18 ml in PBS) was mixed with 4 ?g (2.0 ml) synthetic cis-
tetracosenoyl sulfatide, bovine brain-derived tetracosenoyl-, palmitoyl-,
and lyso-sulfatide and incubated at 37°C for 6 h followed by washing with
500 ml of PBS and concentrated using 10 kDa m.w. cut-off centrifugal
concentrators (Millipore). After adjusting the remaining volume, equal
amounts of protein were subjected to isoelectric focusing (IEF) gel elec-
trophoresis to monitor the loading efficiency of each species of sulfatide
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2950 PRESENTATION OF EXOGENOUS SULFATIDE VIA CD1D PATHWAY