Fine tuning by human CD1e of lipid-specific
Federica Facciottia, Marco Cavallaria, Catherine Angénieuxb, Luis F. Garcia-Allesc, François Signorino-Gelob,
Lena Angmana, Martine Gilleronc, Jacques Prandic, Germain Puzoc, Luigi Panzad, Chengfeng Xiae, Peng George Wangf,
Paolo Dellabonag, Giulia Casoratig, Steven A. Porcellih, Henri de la Salleb, Lucia Moria,i,1, and Gennaro De Liberoa,1
aExperimental Immunology, Department of Biomedicine, University Hospital Basel, 4031 Basel, Switzerland;bInstitut National de la Santé et de la Recherche
Médicale, Unité Mixte de Recherche S725, Biology of Human Dendritic Cells, Université de Strasbourg and Etablissement Français du Sang-Alsace, 67065
Strasbourg, France;cCentre National de la Recherche Scientifique, Institut de Pharmacologie et de Biologie Structurale, 31077 Toulouse, France;dDepartment
of Chemistry, Food, Pharmaceuticals, and Pharmacology, Università del Piemonte Orientale, 28100 Novara, Italy;eState Key Laboratory of Phytochemistry and
Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan 650204, China;fDepartment of Biochemistry
and Chemistry, The Ohio State University, Columbus, OH 43210;gExperimental Immunology Unit, Division of Immunology, Transplantation, and Infectious
Diseases, Dipartimento di Biotecnologie (DIBIT), San Raffaele Scientific Institute, 20132 Milano, Italy;hAlbert Einstein College of Medicine, Bronx, NY 10461;
andiSingapore Immunology Network, Agency for Science Technology and Research, Biopolis, Singapore 138648
Edited by Peter Cresswell, Yale University School of Medicine, New Haven, CT, and approved July 20, 2011 (received for review June 5, 2011)
CD1e is a member of the CD1 family that participates in lipid an-
tigen presentation without interacting with the T-cell receptor. It
binds lipids in lysosomes and facilitates processing of complex
glycolipids, thus promoting editing of lipid antigens. We find that
CD1e may positively or negatively affect lipid presentation by
CD1b, CD1c, and CD1d. This effect is caused by the capacity of
CD1e to facilitate rapid formation of CD1–lipid complexes, as
shown for CD1d, and also to accelerate their turnover. Similar
results were obtained with antigen-presenting cells from CD1e
transgenic mice in which lipid complexes are assembled more ef-
ficiently and show faster turnover than in WT antigen-presenting
cells. These effects maximize and temporally narrow CD1-re-
stricted responses, as shown by reactivity to Sphingomonas pau-
cimobilis-derived lipid antigens. CD1e is therefore an important
modulator of both group 1 and group 2 CD1-restricted responses
influencing the lipid antigen availability as well as the generation
and persistence of CD1–lipid complexes.
CD1 restriction|lipid transfer proteins|natural killer T cells
CD1 molecules, CD1e is associated with β2 microglobulin and
binds lipid antigens. CD1e is the only CD1 molecule that
becomes soluble and is never expressed on the plasma mem-
brane, thus it cannot behave as an antigen-presenting molecule.
Moreover, CD1e shows a very peculiar intracellular distribution,
which in dendritic cells (DCs) correlates with their maturation
stage (1). In immature DCs, CD1e prevalently accumulates in
the trans-Golgi network (TGN), whereas, in mature DCs, it
distributes in late endosomes and lysosomes (LYs) where it is
cleaved into an active soluble form (2). In LYs of mature DCs,
CD1e accumulates and persists because of increased protein
stability, which is associated with a progressive shortening of the
carbohydrate side chains (3).
Relatively little is known about the role of CD1e in immune
responses. CD1e assists processing by the lysosomal α-man-
nosidase of mycobacterial hexamannosylated phosphatidyl-myo-
inositols (PIM6), containing six α-D-Manp units, into dimanno-
sylated forms (PIM2), containing only two α-D-Manp units (4).
This form of PIM is stimulatory to specific CD1b-restricted
Whether CD1e also participates in presentation of lipid anti-
gens by other CD1 molecules is matter of investigation. After the
exit from the endoplasmic reticulum, CD1 molecules traffic to
the cell surface via the secretory pathway before being reinter-
nalized into the endosomal compartments. CD1a molecules
undergo cycles of internalization into early/sorting endosomes
followed by early/recycling endosomes (5, 6), whereas CD1c
umans and some other species, but not rodents of the family
Muridae (mice and rats), express CD1e. Similar to the other
molecules traffic to early recycling endosomes and, to a lesser
extent, to late endosomes and LYs (5, 7). In contrast, CD1b and
human CD1d molecules recycle in late endosome/LYs com-
partments where they can colocalize with CD1e (4).
Because of the colocalization of CD1e and CD1b in LYs,
where CD1c and CD1d molecules are also found, we in-
vestigated whether CD1e could also assist lipid antigen pre-
sentation by CD1 molecules other than CD1b. Our findings show
that the activity of CD1e is broader than the one already
reported and provide evidence that CD1e operates with multiple
mechanisms to modulate the immune response to lipid antigens.
CD1e Participates in the Presentation of CD1b- and CD1c-Restricted
Antigens. To study the possible functions of CD1e in CD1-re-
stricted presentation of lipid antigens, THP-1 cells stably ex-
pressing CD1b, CD1c, or CD1d with or without CD1e were used
as antigen-presenting cells (APCs) for human T-cell clones
specific for self- or nonself-antigens. In each case, transfectants
expressing equal levels of CD1 molecules were chosen (Fig. S1).
The effects of CD1e expression on CD1b- or CD1c-restricted
T-cell clones reactive to self-antigens differed depending on the
particular clone tested and the antigen specificity (Fig. 1 A–C).
When APCs expressed CD1e, the autoreactive response of the
CD1b-restricted and ganglioside GM1-specific T-cell clone
GG123b was inhibited (Fig. 1A) and that of the CD1c-autor-
eactive (unknown antigen) K34B27.f clone was unaffected (Fig.
1B), whereas the response of the CD1c autoreactive (unknown
antigen) DN4.99 clone was increased (Fig. 1C).
CD1e also influenced, in a variable manner, the T-cell re-
sponse to exogenously added antigens presented by CD1b. CD1e
impaired the response of the GG33a clone to ganglioside GM1
(Fig. 1D), whereas it did not modify the response of Z4B27 cells
to a mycobacterial diacylsulfoglycolipid synthetic analog (8) (Fig.
1E), and it facilitated the response of the DS1C9b clone to sul-
fatide (Fig. 1F). Thus, CD1e can modulate the response to lipid
antigens presented by CD1b or CD1c in a variable manner.
Author contributions: L.M. and G.D.L. designed research; F.F., M.C., C.A., L.F.G.-A., F.S.-G.,
L.A., H.d.l.S., and L.M. performed research; M.G., J.P., G.P., L.P., C.X., P.G.W., P.D., G.C.,
S.A.P., and H.d.l.S. contributed new reagents/analytic tools; F.F., M.C., H.d.l.S., L.M., and
G.D.L. analyzed data; and L.M. and G.D.L. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| August 23, 2011
| vol. 108
| no. 34www.pnas.org/cgi/doi/10.1073/pnas.1108809108
Type 1 and Type 2 Natural Killer T (NKT) Clones Respond Differently to
Self-Lipids in the Presence of CD1e. Autoreactive type 1 NKT
(iNKT) and type 2 NKT cell clones (identified as CD1d-re-
stricted, Vα24-Jα18−, and CD161+) were stimulated with APCs
expressing only CD1d or both CD1d and CD1e without addition
of exogenous lipid antigens. Different human iNKT clones were
selected according to their T-cell receptor β (TCRβ) CDR3
sequences. All self-reactive iNKT clones showed increased acti-
vation in the presence of CD1e (between 1.5 and 4 times higher
than the response in the absence of CD1e) and varied according
to individual clones (Fig. 2A and Fig. S2A). In contrast, type 2
NKT clones showed a more variable responsiveness (Fig. 2B and
Fig. S2 B and C). Some clones were positively influenced (1.8–38
times higher response in the presence of CD1e) (Fig. 2B),
whereas other clones were negatively influenced by CD1e (30–
60% inhibition) (Fig. 2B and Fig. S2C). One clone was not af-
fected (Fig. S2B). Similar findings were observed when IL-4 and
IFN-γ were investigated and when C1R CD1-transfected cells
were used as APCs (Fig. S2 D and E), thus confirming general
effects on T-cell activation.
In most cases, CD1e significantly influenced T-cell response,
indicating that the human type 2 NKT cells studied here prob-
ably recognize antigens loaded within late endosomes, where
CD1e is localized. Antigen loading in late endosomes was ex-
cluded when a single mouse type 2 NKT hybridoma was studied
(9), suggesting that different antigens may stimulate type 2 NKT
These results suggested that CD1e also regulates the response
of CD1d-restricted T cells. The observed positive, negative, or
neutral effects could be related to the nature and relative
abundance of individual antigens that determine the final net
effect of CD1e on CD1-restricted presentation.
CD1e Effects on Exogenous Glycolipid Antigen Presentation to iNKT
Cells. To study insight into the mechanisms of CD1e modulation
of lipid antigen presentation, we focused on iNKT cells. Nonself-
reactive human iNKT clones were chosen to avoid confusing
effects of self-antigens. The effects of CD1e were studied on
presentation of α-galactosylceramide (α-GalCer), a potent iNKT
TCR agonist; α-lactosylceramide (α-LacCer), an agonist with
intermediate potency (10); and α-galacturonosylceramide (GSL′),
a weaker agonist (11). In these experiments, the antigens were
present during the entire period of T-cell stimulation. Surpris-
ingly, the expression of CD1e did not change the response to all
three antigens when GM-CSF release was measured (Fig. 3 A, D,
and G), whereas it caused reduction of IL-4 (Fig. 3 B, E, and H)
and IFN-γ (Fig. 3 C, F, and I) release. Similar results were ob-
served when C1R transfectants were used as APCs (Fig. S2 F, G,
and H). This reduction was observed at all doses of α-GalCer and
at high doses of GSL′, whereas α-LacCer produced intermediate
results. These differential effects, according to type of released
cytokine and antigen dose, excluded the possibility that CD1E-
transfected cells are per se impaired in antigen presentation and
suggested that CD1e influenced both loading and unloading of
other CD1 molecules.
CD1e Affects the Kinetics of CD1d–Antigen Complex Formation and
Duration. CD1e might directly influence the number of CD1d–
antigen complexes available over time, which has been shown to
affect the type of cytokines released (12–14). To test this possi-
bility, antigen-pulse experiments were performed to compare the
time required for the generation of CD1d–antigen stimulatory
complexes. CD1e expression induced a fast IL-4 release at 4 h
after pulsing with α-GalCer (10 ng/mL). Without CD1e, cytokine
release was observed only after 8 h (Fig. 4A). Similar kinetics
were observed when releases of GM-CSF or IFN-γ were mea-
sured. These results suggest that CD1e assisted the immediate
presentation of lipid antigens by accelerating the formation of
CD1–antigen complexes. At later time points, when the antigen
was available to APCs for 8–24 h, the CD1e effects were no
longer detected, in agreement with the results described for GM-
CSF release in Fig. 3A. These results apparently differed from
those observed when IL-4 and IFN-γ were detected (Fig. 3 B and
C) and might be ascribed to important differences in the ex-
perimental setup. The data shown in Fig. 3 were obtained with
living APCs and with the antigen and T cells always present
during the assay. In this case, T-cell activation integrated the
response to available CD1d–antigen complexes over time. In-
stead, the data of Fig. 4A were obtained with fixed APCs with T
cells added after fixation at the end of the antigen pulse. In this
second experimental setup, T-cell activation reflected the re-
sponse to the complexes available only at the time point when
lipid antigens. (A–C) Self-antigens. (A) CD1b-restricted clone GG123b re-
sponse to THP-1 CD1b and CD1e (●) and THP-1 CD1b only (○). (B and C)
CD1c-restricted clones K34B27.f (B) and DN4.99 (C) response to THP-1 CD1c
and CD1e (●) and THP-1 CD1c only (○). (D–F) CD1b-dependent exogenous
antigens. GG33a clone response to GM1 (D), Z4B27 clone response to SGL12
(E), and DS1C9b clone response to sulfatide (F) presented by THP-1 CD1b and
CD1e (●) and THP-1 CD1b only (○). Human GM-CSF (hGM-CSF) release is
expressed as mean ng/mL ± SD (n = 3). *P < 0.05, **P < 0.01. Data shown are
representative of at least three experiments.
CD1e participates in the presentation of CD1b- and CD1c-restricted
(A) and type 2 (B) NKT cell clones were incubated with single-transfected
(CD1D only) and double-transfected (CD1E and CD1D) THP-1 cells. The dif-
ference in activation is shown as fold increase (ratio of cytokine release in
response to double- and single-transfected cells). In total, 5 × 104T cells were
stimulated with 105THP-1 cells. Released hGM-CSF is expressed as mean ng/
mL ± SD (n = 3). Data shown are representative of at least three experiments.
Autoreactive CD1d-restricted clones are influenced by CD1e. Type 1
Facciotti et al.PNAS
| August 23, 2011
| vol. 108
| no. 34
APCs were fixed. A second important difference was that fixed
APCs are less efficient than living APCs in T-cell stimulation.
To investigate whether CD1e also influenced the unloading of
CD1d and the persistence of the stimulatory complexes, pulse–
chase experiments were performed. APCs expressing CD1d only
or both CD1d and CD1e were pulsed with α-GalCer for 2 h,
extensively washed, and chased for different lengths of time
before fixation and use in T-cell activation assays. The 2-h time
point was chosen because it was sufficient to load enough antigen
capable of stimulating iNKT cells (when living APCs were used),
without allowing antigen overloading of APCs.
The presence of CD1e negatively affected T-cell responses,
which had already declined when CD1e+APCs were chased for
18 h, whereas the response to CD1e−APCs at this time point
was still maximal (Fig. 4B). The response declined in both APCs
at later points and dropped below 50% at 36 h. Similar ki-
netics were observed when releases of GM-CSF or of IFN-γ
CD1e Facilitates Loading and Unloading of Lipid Antigens on CD1d.
These data suggested that CD1e might facilitate antigen loading
and unloading onto other CD1 molecules, thus acting as a lipid
transfer protein (LTP). To investigate whether CD1e directly
facilitates lipid binding to CD1d, we performed plate-bound
CD1d-based lipid-presentation assays. Recombinant soluble hu-
man CD1d coated onto culture plates was loaded with fixed
amounts of α-GalCer in the presence or absence of recombinant
CD1e. Plates were washed, and the response of iNKT cells was
tested after 18 h. Recombinant soluble saposins A, B, or C were
used as controls (15, 16). CD1e significantly enhanced T-cell
activation and was more efficient than saposins B and C (Fig. 4C).
We tested whether CD1e also facilitates unloading of soluble
CD1d molecules previously loaded with α-GalCer. A second type
of plate-bound assay was established in which soluble CD1d was
coated to the plastic, optimally loaded with α-GalCer, then
washed and incubated with soluble CD1e or saposin A, B, or C
before addition of iNKT cells (Fig. 4D). CD1e significantly de-
presentation of exogenously added antigen. THP-1 cells transfected with the
CD1D gene (○) or with the CD1D and CD1E genes (●) were incubated for 2 h
with different concentrations of α-GalCer (A–C), α-LacCer (D–F), or GSL′ (G–I)
before addition of the human iNKT clone VM-D5. Released cytokines are
expressed as mean ng/mL ± SD (n = 3). Data shown are representative of at
least three experiments.
CD1e influences the type of cytokines secreted in response to the
promotes α-GalCer presentation after a short time pulse. APCs were pulsed
for different lengths of time with α-GalCer (10 ng/mL) and fixed before
addition of human iNKT cells. (B) CD1e shortens the half-life of CD1d–α-
GalCer complexes. APCs were pulsed for 2 h with α-GalCer (2 ng/mL) and
chased for different lengths of time before the addition of human iNKT cells.
(A and B) THP-1 cells transfected with the CD1D gene (○) or with the CD1D
and CD1E genes (●) were used. Mean release of IL-4 is shown (±SD) (n = 3).
(C) Plate-bound CD1d was pulsed with α-GalCer (1 μg/mL) in the presence of
different soluble LTPs, BSA, or CD1e or with PBS before addition of human
iNKT cells. hGM-CSF release in the absence of α-GalCer was below detection
limits of the assay. (D). Plate-bound CD1d was loaded with α-GalCer (1 μg/
mL), washed, then incubated overnight with different LTPs or PBS before
addition of human iNKT cells. (C and D) Bars show mean hGM-CSF release
(+SD) (n = 3). *P < 0.05, **P < 0.01. Data shown are representative of at least
three experiments. (E and F) CD1e mediates the transfer of anionic lipids to
CD1d (E) and lipid unloading from CD1d–GD3 complexes (F). (E) IEF gel of
the products after incubation of CD1d (10 μM) for 1 h at pH 5.0 with
liposomes incorporating the indicated anionic lipid (15% molar ratio, 200 μM
final concentration) in the presence or absence of CD1e (10 μM). The first
two lanes show a control experiment using liposomes devoid of anionic
lipids. (F) IEF gel of the products when purified CD1d–GD3 complexes were
incubated with or without CD1e and the indicated lipids (15% molar ratio in
liposomes). The lipids used were sulfatide (SLF), GD3, bis-(monoacylglycero)
phosphate (BMP), and phosphatidylserine (PS).
CD1e facilitates antigen loading and unloading on CD1d. (A) CD1e
| www.pnas.org/cgi/doi/10.1073/pnas.1108809108Facciotti et al.
creased GM-CSF release (>50%), whereas saposins had no ef-
fect in this in vitro assay. Even greater inhibition was observed
when releases of IL-4 or IFN-γ were measured.
The CD1e activity on CD1d loading and unloading was also
investigated by isoelectrofocusing (IEF). CD1e facilitated load-
ing of soluble CD1d with the anionic lipids sulfatide and gan-
glioside GD3 (GD3). On the contrary, no effect was observed
when bis-(monoacylglycero)phosphate and phosphatidylserine
were used (Fig. 4E). CD1e also facilitated unloading of pre-
formed CD1d–GD3 complexes in the presence of acceptor lip-
osomes (Fig. 4F, lanes 1 and 2). Addition of α-GalCer, sulfatide,
and bis-(monoacylglycero)phosphate increased the unloading
effect of CD1e, using these in vitro experimental conditions (Fig.
4F, lanes 3–8). Similar unloading effects were observed when
CD1d–GM1 or CD1d–sulfatide complexes were used. The ca-
pacity of CD1e to influence CD1d unloading was much more
evident in the iNKT cell activation assay that is closer to physi-
ological conditions, indicating that the IEF assay is less sensitive
and probably influenced by several in vitro conditions. Alto-
gether, these experiments support the hypothesis that CD1e
facilitates both antigen loading and unloading of CD1d.
Facilitated Presentation of Lipid Antigens in CD1e Transgenic (Tg)
Mice. To further dissect the mechanisms by which CD1e facili-
tates CD1d-restricted antigen presentation, we generated Tg
mice expressing the human CD1E cDNA under the H-2 Eα
promoter directing expression on APCs. In Tg mice, the ex-
pression of CD1e was found in B cells, peritoneal macrophages,
and bone marrow-derived DCs (BMDCs), but not in T lym-
phocytes (Fig. S3A) or thymocytes. Soluble CD1e molecules
were detected by pulse–chase labeling experiments followed by
immunoprecipitation in DCs of Tg mice but not non-Tg litter-
mates (Fig. S4) and partially colocalized with the Golgi marker
TGN-38 and with MHC class II compartment (Fig. S3B), thus
showing a distribution similar to the one occurring in human
cells (1). Number and phenotype of iNKT cells in thymus and
periphery as well as of other lymphocyte populations and mon-
ocytes were comparable in Tg and WT mice (Figs. S5–S7). Eα-
CD1e Tg mice therefore represent a unique animal model in
which to study the role of CD1e in CD1d-restricted antigen
To investigate the effects of CD1e on CD1d-restricted pre-
sentation of endogenous lipid antigens, DCs from Tg and WT
mice were used to stimulate human autoreactive iNKT cells (Fig.
S8A). In control experiments, WT or Tg thymocytes (which do
not express CD1e) were used to stimulate the same iNKT cells
(Fig. S8B). CD1e presence in DCs positively affected the re-
sponse of iNKT cells, whereas Tg and WT thymocytes were
equally stimulatory. Additional control experiments showed that
the presentation of α-GalCer was identical when using the same
setup described for the four APC types and an iNKT cell hy-
bridoma (Fig. S8 C and D). CD1e may thus affect the pre-
sentation of endogenous lipid antigens in mouse APCs also.
We next investigated whether CD1d–antigen stimulatory
complexes were formed with faster kinetics in Tg DCs than in WT
DCs, as previously found in the human system. The presence of
CD1e resulted in stronger iNKT responses after 30-min α-GalCer
pulse, and this effect remained at later time points (Fig. 5A).
We also tested whether CD1e reduced the persistence of
CD1d–antigen complexes. Tg or WT DCs were loaded with
α-GalCer and chased for different lengths of time before fixation
and presentation to iNKT hybridoma cells. Similar to the be-
havior observed with human APCs, 50% reduction of maximal
stimulation was observed after 24-h chase of CD1e Tg APCs and
after 30-h chase with WT APCs (Fig. 5B). Staining with the L363
mAb, which detects CD1d–α-GalCer complexes (14, 17), con-
firmed the increased appearance of complexes on the plasma
membrane at very early time points (Fig. 5C) and their reduced
persistence at late time points (24 h) (Fig. 5D) despite equal
levels of surface CD1d. This effect was dose-dependent and was
no longer observed at the highest α-GalCer dose (Fig. 5D).
Hence, CD1e participated in efficient lipid exchange from
CD1d in both mouse and human APCs, facilitating immediate
loading of lipid antigens but also reducing the half-life of stim-
ulatory CD1d–lipid antigen complexes.
CD1e Tunes the Response of iNKT Cells to Infection. We asked
whether CD1e plays a role in promoting iNKT cell response to
Sphingomonas paucimobilis, which encodes iNKT-stimulatory
antigens (18, 19). When WT and Tg DCs were pulsed with dif-
ferent numbers of heat-killed S. paucimobilis bacteria and used
to stimulate iNKT hybridoma cells for 24 h, both APCs showed
equal stimulatory capacity (Fig. 6A). However, when APCs were
incubated for different lengths of time and immediately fixed, the
presence of CD1e facilitated the generation of stimulatory CD1d
complexes, as previously observed with α-GalCer (Fig. 5E). Tg
DCs already stimulated iNKT hybridoma cells at 2 h after
pulsing and induced a persistently higher cytokine release (Fig.
6B). These findings show that APCs expressing or lacking CD1e
have similar presentation capacity when the antigen remains in
the culture and that CD1e promotes a faster response immedi-
ately after lipid antigens become available. The effects of CD1e
during infection were investigated by using freshly isolated
spleen cells stimulated with S. paucimobilis-infected Tg or WT
DCs. The presence of CD1e induced more efficient activation of
iNKT cells as shown by increased CD69 up-regulation (Fig. 6C)
and increased percentage of CD69+iNKT cells (Fig. 6D). A final
series of experiments assessed the capacity of infected DCs to
activate iNKT cells after different infection times. Tg or WT
DCs were infected for differing times, then treated with genta-
DCs from Tg mice (●) or non-Tg littermates (○) were incubated for different
lengths of time with α-GalCer (10 ng/mL) and then fixed before addition of
iNKT mouse hybridoma cells. (B) The half-life of CD1d–α-GalCer complexes is
reduced in Tg DCs. DCs from Tg mice (●) or non-Tg littermates (○) were
pulsed for 2 h with α-GalCer (2 ng/mL) and chased for different lengths of
time before addition of iNKT mouse hybridoma cells. (A and B) Released mIL-
2 (ng/mL ± SD) was determined in triplicate. (C) Detection of CD1d–α-GalCer
complexes by L363 antibody staining on the surface of Tg (●) or WT (○) DCs
incubated for 24 h with α-GalCer. (D) Detection of CD1d–α-GalCer complexes
by L363 antibody on Tg (●) or WT (○) DCs pulsed for 24 h with different
doses of α-GalCer and chased for 24 h before staining. Data shown are
representative of three independent experiments.
CD1e expressed in Tg DCs influences presentation of self-lipids. (A)
Facciotti et al.PNAS
| August 23, 2011
| vol. 108
| no. 34
micin, washed, and used to stimulate spleen cells for 24 h. Tg
DCs already induced maximal iNKT cell activation at 12 h after
infection, whereas WT DCs only induced maximal activation at
48 h after infection (Fig. 6E). In conclusion, CD1e also enhanced
iNKT cell response during S. paucimobilis infection.
We have found that CD1e has evolved a unique capacity to
complement the antigen-presentation functions of other CD1
family members. The presence of CD1e may influence pre-
sentation of lipid antigens by CD1b, CD1c, and CD1d, the three
molecules that recycle within late endosomes/LYs where soluble
CD1e is also localized. The effects on antigen presentation differ
according to the presented antigen and not to the restricting
CD1 molecule. In some instances, presentation is enhanced; in
others, it is reduced. This finding might reflect a preference of
CD1e for binding some lipids more efficiently than others. In
some other instances, CD1e presence did not influence antigen
presentation, suggesting that not all lipid antigens can bind CD1e
or be unloaded from other CD1 molecules. These findings sug-
gest a mechanism whereby a low binding affinity of a lipid an-
tigen to CD1e might facilitate antigen presentation by CD1
molecules, whereas a high binding affinity to CD1e might reduce
loading of CD1 molecules. This latter case could have two out-
comes: a detrimental one such as impaired immune response to
certain microbial lipid antigens or a favorable one upon se-
questration of self-lipid antigens involved in autoimmunity. This
function can be exerted in different cell types where CD1e is
expressed: thymocytes, DCs, and Langerhans cells, which also
express group 1 and group 2 CD1 molecules.
CD1e also influenced the presentation of self-antigens to type
1 (iNKT) and type 2 NKT cells. All autoreactive iNKT cells
tested were positively influenced by the presence of CD1e, sug-
gesting that CD1e facilitated formation of CD1d–lipid com-
plexes with the endogenous unknown lipid antigen(s) that
stimulate the autoreactive iNKT cells tested in this study.
A set of human autoreactive iNKT cell clones is activated by
tail-deleted CD1d molecules that do not recycle through LYs
(20), indirectly suggesting that the antigen stimulating these cells
is not loaded within LYs. Our clones might represent a different
type of autoreactive iNKT cells that recognize self-antigens
loaded within endosomal compartments where CD1e colocalizes
with CD1d. This possibility is supported by our findings that these
iNKT cells do not recognize soluble CD1d molecules, which are
secreted without trafficking through endosomal stations.
In contrast to the positive effects on autoreactive iNKT cells,
CD1e may have opposite effects on type 2 NKT cells, which are
also CD1d-restricted. We found that CD1e facilitated the acti-
vation of four type 2 NKT cell clones, inhibited the response of
three clones, and was irrelevant for one clone. Both enhancing
and inhibitory effects were very pronounced (1.8–30 times ob-
served enhancement and 30–60% inhibition). We interpret these
findings with the fact that type 2 autoreactive NKT cells may
recognize a set of self-lipids that are not identical to those
stimulating self-reactive iNKT cells. Thus, in this case, the type
of lipid antigens and not the type of CD1 restriction also
determines the effects of CD1e.
The expression of CD1e also influenced the cytokines released
by T cells because iNKT cells stimulated with CD1e-expressing
APCs and three different exogenous antigens showed a marked
reduction of IFN-γ, a moderate reduction of IL-4, and almost no
change in released GM-CSF. These effects were observed with
two different types of transfectants (THP-1 and C1R), excluding
unique behavior associated with the nature of APCs. The dif-
ferential influence on the type of released cytokines probably
reflects the reported different signal strength required for se-
cretion of GM-CSF versus IL-4 and IFN-γ (12). The presence of
CD1e might influence the number and nature of CD1–antigen
complexes, leading to a reduced TCR signal strength that might
affect IFN-γ production more readily than that of GM-CSF.
Indeed, the most pronounced inhibitory effects occurred at high
antigen doses, suggesting that CD1e modulated the lipid antigen
presentation capacity of APCs. The impact of CD1e on CD1-
restricted T-cell responses might be the outcome of a CD1e-
mediated unloading of the antigen from other CD1 molecules.
This hypothesis is supported by the effect of CD1e on loading
and unloading kinetics of CD1d–lipid complexes. CD1e already
facilitated the formation of stimulatory α-GalCer complexes with
CD1d at 4 h after antigen pulsing, but it also induced a faster
reduction of the stimulatory capacity at later time points, which
was confirmed by using two types of in vitro CD1d loading and
unloading assays (plate-bound T-cell activation and IEF). In
addition, CD1e behaved similarly in Tg mice and human cells.
The mouse model confirmed that CD1e accelerates the forma-
tion of CD1d–lipid complexes and their rapid turnover when the
antigen is limiting, as shown by iNKT cell activation experiments
paucimobilis. (A) DCs from Tg mice (●) or non-Tg littermates (○) were in-
cubated for 2 h with heat-killed S. paucimobilis at different bacteria:APC
ratios and then used to stimulate iNKT hybridoma cells. mIL-2 (ng/mL ± SD)
was determined by ELISA. (B) DCs from Tg (●) or non-Tg littermates (○) were
incubated for different lengths of time with heat-killed S. paucimobilis
(bacteria:APC 200:1), washed, and then fixed before addition of iNKT hy-
bridoma cells. After 24 h, mIL-2 (ng/mL ± SD) was determined. (C and D) DCs
from Tg or non-Tg littermates were infected at a multiplicity of infection
(MOI) of 1:1 for 2 h with S. paucimobilis or were not infected, then washed
and incubated with gentamicin for 1 h, and finally cocultured with freshly
isolated WT mouse splenocytes. CD69 mean fluorescence intensity (MFI) (C)
and percentage (D) of CD69+CD1d–α-GalCer dimer+iNKT cells as measured
by flow cytometry. *P < 0.05. (E) Tg (●) or WT (○) DCs were infected for
different lengths of time with S. paucimobilis at a low MOI (1:1), incubated
for 1 h with gentamicin, and then used to stimulate freshly isolated WT iNKT
cells. Up-regulation of CD69 on CD1d–α-GalCer dimer+iNKT cells was mea-
sured by flow cytometry. ■ indicates CD69 expression on iNKT cells in-
cubated with noninfected DCs. Data shown are representative of at least
DCs from Tg mice induce a better response after stimulation with S.
| www.pnas.org/cgi/doi/10.1073/pnas.1108809108Facciotti et al.
and by staining with a mAb specific for mouse CD1d–α-GalCer
complexes. The increased turnover of stimulatory ligands may
represent an efficient way to dampen T-cell responses of iNKT
and group 1 CD1-restricted T cells at later time points.
Finally, CD1e also changed the early response of iNKT cells to
bacterial-derived antigens, suggesting a modulation of iNKT cell
responsiveness during infection. A significant increase of acti-
vated iNKT cells was observed when infection lasted between 12
and 24 h, whereas the enhancing effect was no longer visible
after 48 h. Because this initial time frame is the most important
for an innate immune response to infectious agents, CD1e can be
considered to be a molecule actively participating in innate im-
mune functions of iNKT cells.
In conclusion, CD1e may fine-tune the response to lipid
antigens, influencing loading and unloading of other CD1 mol-
ecules as well as having major consequences on the temporal
availability of stimulatory CD1–lipid complexes. These unex-
pected functions of CD1e have disclosed unique mechanisms
whereby the immune response to lipid antigens is regulated.
Materials and Methods
Cells. Human CD1d-restricted type 1 and type 2 NKT, CD1b-restricted and
CD1c-restricted T-cell clones, and mouse iNKT hybridomas were derived as
described (21–23) and maintained according to standard procedures. Human
monocytic THP-1 and C1R B cells were transfected with human CD1B, CD1C,
and CD1D cDNAs alone or in combination with human CD1E cDNA, using the
BCMGS-Neo and BCMGS-Hygro vectors (4). Transfected cells expressing
similar levels of CD1 molecules were selected by cell sorting to avoid a biased
stimulation of responder cells.
Lipid Antigens. Mycobacterial lipids were purified as described (24). SGL12 (8),
α-GalCer (25), and α-LacCer (10) were synthesized as previously published.
GM1 and sulfatide were purchased from Sigma. GSL′ was provided by
P. Seeberger (The Max Planck Institute of Colloids and Interfaces, Berlin,
Flow Cytometry. Human CD1 molecules were detected with BCD1b3.1 (anti-
CD1b), 10C3 (anti-CD1c), and CD1d42 (anti-CD1d) mAbs, and human MHCI
was detected with W6/32 mAb. Intracellular CD1e was detected in fixed and
permeabilized cells with 20.6 mAb (4) and FITC-conjugated goat anti-mouse
IgG1 (Southern Biotech). Samples were acquired on a CyAn ADP flow
cytometer (Dako). Nonviable cells were excluded on the basis of light scatter,
and incorporation of propidium iodide, doublets were excluded by pulse-
width parameter. Live cells were analyzed with FloJo software (Tristar).
Antigen Presentation Assays. Autoreactive T cells (105cells per well) were
stimulated with increasing numbers of APCs without addition of exogenous
antigen. For exogenous antigen assessments, transfectants (0.5 × 105cells
per well), mouse thymocytes (5 × 105cells per well), or DCs (105cells per well)
were preincubated for 2 h at 37 °C with antigens before addition of human
T cells (105cells per well) or mouse iNKT FF13 hybridoma cells (0.5 × 105cells
per well). For pulse experiments, transfectants or DCs (2 × 106cells per well)
were pulsed with α-GalCer (10 ng/mL), washed, fixed as described (24), and
plated at 105cells per well in a 1:1 ratio with T cells. For chase experiments,
transfectants or DCs (5 × 105cells per well) were pulsed for 2 h with 2 ng/mL
of α-GalCer, washed, and chased before addition of human T cells or FF13.
CD1d Loading and Unloading Experiments. Plates were coated with 10 μg/mL
Bir1.4 mAb (26). Soluble recombinant human CD1d was purified by IEF and
added overnight at room temperature at twofold molar excess over Bir1.4.
In loading assays, α-GalCer was added with or without recombinant LTPs
(4 μg/mL) or recombinant CD1e (10 μg/mL). In unloading assays, α-GalCer was
added overnight, washed, and then CD1e or LTPs were added for an addi-
tional 24 h. After washing, T cells (1.5 × 105cells per well) were added. In
antigen presentation assays, released cytokines were measured in super-
natants taken after 24 h [GM-CSF, IL-4, and mouse IL-2 (mIL-2)] or 48 h
(human IFN-γ) by ELISA (BD Biosciences).
For IEF analysis of CD1e effects on CD1d loading and unloading, see SI
Materials and Methods.
Statistical Analysis. Data are expressed as mean ± SD and analyzed with the
unpaired Student’s t test with Welch’s correction. P ≤ 0.05 was con-
ACKNOWLEDGMENTS. We thank J. Mattner and P. Seeberger for S. pauci-
mobilis antigens, D. Nebenius for the generation of Tg mice, M. Lepore for
discussion, and P. Cullen for reading the manuscript. This work was sup-
ported by Swiss National Science Foundation Grant 3100A0 122464/1, Euro-
pean Union Seventh Framework Programme Tuberculosis Vaccine (TBVAC),
University Hospital Basel, French National Research Agency Grant ANR-05-
MIIM-006, and Etablissement Français du Sang-Alsace.
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Facciotti et al.PNAS
| August 23, 2011
| vol. 108
| no. 34