2. The Journal of Immunology
Early Recycling Compartment Trafficking of CD1a Is
Essential for Its Intersection and Presentation of
Lipid Antigens
Manuela Cernadas,*,1 Marco Cavallari,†,1 Gerald Watts,‡ Lucia Mori,† Gennaro De Libero,†,2
and Michael B. Brenner‡,2
A major step in understanding differences in the nature of Ag presentation was the realization that MHC class I samples peptides
transported to the endoplasmic reticulum from the cytosol, whereas MHC class II samples peptides from lysosomes. In contrast to
MHC class I and II molecules that present protein Ags, CD1 molecules present lipid Ags for recognition by specific T cells. Each of
the five members of the CD1 family (CD1a–e) localizes to a distinct subcompartment of endosomes. Accordingly, it has been widely
assumed that the distinct trafficking of CD1 isoforms must also have evolved to enable them to sample lipid Ags that traffic via
different routes. Among the CD1 isoforms, CD1a is unusual because it does not have a tyrosine-based cytoplasmic sorting motif
and uniquely localizes to the early endocytic recycling compartment. This led us to predict that CD1a might have evolved to focus
Downloaded from www.jimmunol.org on April 18, 2012
on lipids that localize to early endocytic/recycling compartments. Strikingly, we found that the glycolipid Ag sulfatide also
localized almost exclusively to early endocytic and recycling compartments. Consistent with colocalization of CD1a and sulfatide,
wild-type CD1a molecules efficiently presented sulfatide to CD1a-restricted, sulfatide-specific T cells. In contrast, CD1a:CD1b tail
chimeras, that retain the same Ag-binding capacity as CD1a but traffic based on the cytoplasmic tail of CD1b to lysosomes, failed
to present sulfatide efficiently. Thus, the intracellular trafficking route of CD1a is essential for efficient presentation of lipid Ags
that traffic through the early endocytic and recycling pathways. The Journal of Immunology, 2010, 184: 1235–1241.
T
he CD1 family of Ag-presenting molecules is unique in its d isoforms is determined by their unique cytoplasmic tail tyrosine-
ability to present lipid, glycolipid, and lipopeptide Ags to based sorting motif (2). The intracellular localization of the CD1b
CD1-restricted T cells. In contrast to MHC-restricted Ag isoform, for example, is almost exclusively in the lysosomal com-
presentation, CD1 molecules are functionally nonpolymorphic and partment. This CD1b localization is mediated by the adaptor protein
have been shown to present both exogenous microbial Ags as well as AP-3 through interactions with the cytoplasmic tyrosine-based
endogenous lipid ligands (1). Similar to MHC class I molecules, CD1 motif of CD1b (3).
molecules are noncovalently associated with b2-microglobulin. The CD1a isoform is almost exclusively expressed on pro-
CD1a, b, and c molecules are primarily expressed on professional fessional APC, including Langerhans cells (LCs) and other myeloid
APCs and have been implicated in adaptive immunity against mi- dendritic cell (DC) subsets (2). CD1a is distinct among the CD1
crobial lipids. In contrast, CD1d-restricted NK T cells are innate- isoforms in that it does not contain a tyrosine-based cytoplasmic
like lymphocytes that may bridge the innate and adaptive immune motif. CD1a has been shown to localize intracellularly to the en-
system. Each of the five members of the human CD1 family—CD1a, docytic recycling compartment (ERC) (4, 5). In contrast to CD1b,
b, c, d, and e—has a distinct cellular distribution and intracellular CD1a internalization into endosomes is independent of clathrin or
trafficking pattern. The intracellular localization of the CD1b, c, and dynamin. Once internalized, CD1a molecules follow a Rab22a- and
ADP ribosylation factor 6-dependent recycling pathway (5). Under
*Division of Pulmonary and Critical Care Medicine and ‡Division of Rheumatology, steady-state conditions, CD1a is not found to localize to late en-
Immunology and Allergy, Brigham and Women’s Hospital, Harvard Medical School, dosomes (LEs) or lysosomes (LYs) (4). Moreover, CD1a molecules
Boston, MA 02115; and †Experimental Immunology, Department of Biomedicine,
University Hospital Basel, Basel, Switzerland
have also been shown to localize in Birbeck granules, which are
1 intracellular compartments unique to LCs that have been demon-
M.C. and M.C. contributed equally to this work.
2
strated to be subdomains of the endocytic system (6).
G.D.L. and M.B.B. contributed equally to this work.
Several T cell Ags presented by CD1a have been described in-
Received for publication December 11, 2008. Accepted for publication November
17, 2009.
cluding didehydroxymycobactin, a lipopeptidic Ag isolated from
Mycobacterium tuberculosis, and sulfatide (7, 8). Sulfatide (39-
This work was supported by grants from the National Institutes of Health (AI 028973
to M.B.B.), the Swiss National Science Foundation (3100AO-109918), and Roche sulfated b1-D-galactosylceramide) is an endogenous glycolipid
Research Foundation (to G.D.L.). highly expressed in neuronal cells, kidney, and pancreas (9–11), and
Address correspondence and reprint requests to Dr. Michael B. Brenner, Division of its synthesis is upregulated in DCs upon bacterial infection (12).
Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Harvard Sulfatide can be presented by all group 1 CD1 molecules including
Medical School, Smith Building, Room 552, 1 Jimmy Fund Way, Boston, MA 02115.
E-mail address: mbrenner@rics.bwh.harvard.edu mouse and human CD1d (8, 13, and M. Cavallari and G. De Libero,
Abbreviations used in this paper: DC, dendritic cell; EEA-1, early endosome Ag-1; ERC, unpublished results). DC pulsed with sulfatide maintained the
endocytic recycling compartment; LAM, lipoarabinomannan; LAMP-1, lysosome- ability to stimulate CD1a-restricted T cells over a 3 d period. In
associated membrane protein 1; LC, Langerhans cell; LE, late endosome; LY, lysosome; contrast, the ability of the sulfatide-pulsed DC to stimulate CD1b-
Trf, transferrin; WT, wild-type.
and CD1c-restricted T cells was reduced by ∼75% over the same
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 time period. These findings suggest that CD1a has a unique ability
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0804140
3. 1236 CD1a TRAFFICKING IS ESSENTIAL FOR LIPID ANTIGEN PRESENTATION
to present sulfatide. However, these studies cannot distinguish be- Hercules, CA). Stable clones were isolated by limiting dilution under se-
tween higher affinity of CD1a binding for sulfatide versus more lection with 1 mg/ml G418 sulfate (Invitrogen) in DMEM complete media.
Transient transfections were performed with FuGENE 6 (Roche, Basel,
extensive colocalization of the Ag-presenting molecule with the Ag. Switzerland) as per manufacturer’s instructions. CD1a cell surface ex-
In this study, we show that the intracellular localization of the pression was confirmed by flow cytometric staining and intracellular traf-
CD1a isoform to the early endocytic system allows it to intersect with ficking by confocal microscopy as detailed below. Monocyte-derived DCs
lipids that are primarily or, in the case of sulfatide, almost exclusively, were generated as previously described (15). Briefly, CD14+ monocytes
localized in the early endocytic system. This colocalization was were isolated from buffy coats obtained from normal blood donors as per
institutional guidelines. Mononuclear cells were isolated with Ficoll-Paque
shown to be critical for the sustained ability of CD1a molecules to PLUS (GE Healthcare, Buckinghamshire, U.K.) and monocytes positively
stimulate CD1a-restricted T cells. When the same CD1a extracellular selected with CD14 MicroBeads (MACS; Miltenyi Biotec, Auburn, CA).
domain was fused to the CD1b tail, redirecting it to LYs, efficient and DCs were differentiated in RPMI 1640 complete media (Life Technologies,
prolonged presentation of CD1a lipid Ag was abrogated. This finding Carlsbad, CA), 10% heat-inactivated FCS (HyClone, Logan, UT), 2 mM L-
glutamine, 10 mM HEPES, and 100 U/ml penicillin-streptomycin (Life
has significant implications for the efficient and sustained pre- Technologies) supplemented with 300 U/ml GM-CSF (Immunex, Thousand
sentation of other CD1a-binding lipid Ags by DC in vivo. This work Oaks, CA) and 200 U/ml IL-4 (PeproTech, Rocky Hill, NJ). WT CD1a-
also highlights the importance of understanding lipid trafficking as (16), CD1aab- (14), or CD1abb-transfected T2 cells were used as APCs.
a critical factor in the study of CD1 Ag presentation and function. Briefly, stable transfectants were achieved by electroporation and selection
with G418 sulfate (0.8 mg/ml; Calbiochem, La Jolla, CA). Surface ex-
pression of CD1a was confirmed by flow cytometry, and intracellular lo-
Materials and Methods calization studies were performed by confocal microscopy. The CD1a-
Chimeric CD1a:CD1b constructs restricted, sulfatide-specific human T cell clone K34B9.1 was derived and
cultivated as previously described (8).
Three types of CD1a:CD1b hybrid constructs containing sequences
encoding the CD1a extracellular domain fused to three different CD1b Flow cytometry
chimeric tails were generated by PCR using human CD1a and CD1b cDNA
Downloaded from www.jimmunol.org on April 18, 2012
as the templates. The first CD1a:b tail chimeric construct contains the HeLa transfectants and monocyte-derived DCs were analyzed by flow
tyrosine-based motif of CD1b and two additional 39 amino acids from cytometry. In the case of the HeLa transfectants, the cells were detached by
CD1b wild-type (WT) sequence (highlighted in boldface) swapped with brief incubation with 0.5% trypsin-EDTA (Life Technologies/Invitrogen)
the terminal sequence (last three amino acids) of CD1a [i.e., WT CD1a (… and washed prior to staining. The cells were stained with PE-conjugated
FRKRCFC) was changed to (… FRKRRSYQNIP)]. The CD1a:CD1b anti-CD1a or isotype control mouse IgG1 mAb (BD Biosciences, San Jose,
chimeric tail construct was cloned between the BamHI and XhoI sites of CA) on ice. The cells were analyzed using a FACScan or Canto flow cy-
pcDNA3 vector (Invitrogen, Carlsbad, CA) and sequenced before trans- tometer (BD Biosciences).
fection. This construct is referred to in the text as CD1aab, indicating the
Lipid Ags
origin of the extracellular domain (first letter), transmembrane (second
letter), and cytoplasmic sequences (CD1b). The second construct is es- Bovine sulfatide and ceramide (C24) were obtained from Matreya (Pleasant
sentially identical to the first with the exception of one amino acid. In the Gap, PA) and lipoarabinomannan (LAM) from Colorado State University,
second CD1a:CD1b tail chimeric construct, the WT CD1a (…FRKRCFC) Fort Collins, CO.
tail was changed to (…FRRRSYQNIP) as previously described (14). This
construct, which was used to transfect the T2 cells, will also be referred to Confocal microscopy
CD1aab. The CD1aab constructs were independently generated, account-
ing for the one amino acid difference. Each laboratory included additional Laser confocal microscopy was performed on the HeLa transfectants and
amino acids from the transmembrane region of CD1b because replacement monocyte-derived DCs as previously described (3, 15). Briefly, HeLa trans-
of the tyrosine motif alone was not sufficient to fully redirect CD1a to the fectants were cultured on coverslips and DC plated on fibronectin-coated cov-
LY-associated membrane protein-1+ (LAMP-1+) lysosomal compartments. erlips overnight. After washing, the cells were fixed with paraformaldehyde,
In the third construct called CD1abb, the extracellular CD1a region was permeabilized with 0.2% saponin, and stained with mAb against CD1a (10H3,
joined to the transmembrane and cytoplasmic sequences of CD1b. The mouse IgG1), CD1b (BCD1, mouse IgG1), LAMP-1 (H4A3, mouse IgG1),
latter two sequences were cloned into pBluescript II KS+ (Stratagene, La sulfatide (04, mouse IgM supernatants, generously provided by Dr. Joan Boggs,
Jolla, CA), sequenced, and subcloned into the XhoI-NotI sites of the Hospital for Sick Children, Toronto, Ontario, Canada) (17), LAM (rabbit anti-
BCMGSNeo vector for transfection. All PCR primer sequences used to LAM antisera was generously provided by Dr. Daniel Clemens, University of
generate the constructs are available upon request. California, Los Angeles, Los Angeles, CA), Rab5 (BD Biosciences), early en-
dosome Ag-1 (EEA-1; BD Biosciences), and appropriate species and isotype
Cells controls for 60 min at room temperature. The purified Abs were used at 10 mg/ml
and the supernatants at 1:5 dilution. The cells were stained with the appropriate
HeLa cells (American Type Culture Collection, Manassas, VA) were secondary Ab, Alexa 546- or Alexa 488-conjugated anti-mouse IgM, Alexa 488-
transfected with WT CD1a (CD1aaa) and HeLa CD1aab chimeric con- conjugated F(ab9)2 fragment of goat anti-mouse IgG (Molecular Probes/In-
structs by electroporation (Bio-Rad Gene Pulser II, 280V, 960 mF; Bio-Rad, vitrogen), and FITC-conjugated F(ab9)2 fragment of donkey anti-rabbit IgG Abs
FIGURE 1. CD1a localization is redirected to LYs in the CD1aab tail chimera. HeLa CD1a WT, CD1b WT, and CD1aab tail chimera transfectants were
analyzed by confocal microscopy. A, The CD1 transfectants were stained with Abs directed at their extracellular domains. CD1a WT transfectants show cell
surface and perinuclear staining. The CD1b WT and CD1aab chimera have similar intracellular staining patterns. B, Colocalization studies with anti–
LAMP-1 mAbs demonstrated the localization of the CD1aab chimera to LAMP-1+ compartments similar to WT CD1b. There is no colocalization between
CD1a and LAMP-1. The fluorescent label used for each protein is indicated by color.
4. The Journal of Immunology 1237
(Jackson ImmunoResearch, West Grove, PA) for 60 min at room temperature. Sulfatide colocalizes to the early endocytic/recycling pathway
Additional staining was performed with Alexa 488-conjugated anti–LAMP-1
mAb (H4A3, BD Biosciences) and FITC-conjugated transferrin (Trf) (In- Despite extensive prior analyses of CD1a and CD1b trafficking, only
vitrogen) at 10 mg/ml. In the case of the Trf experiments only, the HeLa trans- rarely has lipid Ag trafficking been directly visualized, because tag-
fectants were serum-starved in DMEM media with 0.2% BSA for 30 min. The ging of lipid Ags often alters their trafficking, and few mAb against
cells were washed three times prior to fixation and confocal analysis. For the Ag lipids exist. No prior studies have examined the trafficking of CD1a-
loading studies, the cells were incubated for 30 min, unless clearly indicated
otherwise as in the time course analysis, at 37˚C with sulfatide at 10 mg/ml, after presented lipid Ags. We developed a protocol to determine the in-
which the cells were washed three times with PBS at room temperature prior to tracellular distribution of sulfatide using confocal microscopy and
confocal staining. In the case of the Trf experiments, 100 mg/ml sulfatide was the anti-sulfatide specific mAb (O4) in sulfatide-pulsed HeLa CD1
used. The slides were analyzed with an inverted microscope (TE2000, Nikon, transfectant cells. Sulfatide was sonicated in complete media at 37˚C
Melville, NY) with C1 confocal system and the Plan Apochromat 603 oil (NA
1.4) objective lens using EZ-C1 software (Nikon) at room temperature. Only
and then incubated with CD1a HeLa transfectants for the time points
representative images were acquired. indicated in Fig. 2A. After 30 min of incubation, sulfatide was taken
up by the cells as revealed by well-defined, punctuate staining with
Pulse and chase Ag presentation assays anti-sulfatide mAb (Fig. 2A), whereas minimal to no background
CD1a WT and CD1aab HeLa transfectants (1 3 106) were irradiated (5000
staining was observed with the anti-sulfatide mAb in the unpulsed
rads, [137Cs] source) and pulsed with 2 mg (pulse experiments) of sonicated cells. Strikingly, HeLa CD1a transfectants incubated with sulfatide
sulfatide for indicated times. CD1a WT-, CD1aab-, and CD1abb-transfected demonstrated an almost exclusive localization of sulfatide to the
T2 cells were pulsed for 3 h with 10 mg (chase experiments) of sonicated early endocytic and recycling pathways, identified using anti-Rab5
sulfatide. For the these experiments, purified synthetic C24:1 (nervonoyl-)
sulfatide (18) was used and was kindly provided by L. Panza (Universita `
del Piemonte Orientale, Novara, Italy). After pulse, the cells were washed
and chased or not for indicated times. At time zero, the cells for all the
conditions were collected, counted, and plated (2.5 3 104/well) in RPMI
Downloaded from www.jimmunol.org on April 18, 2012
1640 medium containing 10% FCS with or without the addition of freshly
sonicated Ag (at the same concentration as pulse). T cells (1 3 105/well)
were added in triplicate or quadruplicate. Supernatants were harvested
after 24 h, and released cytokines were measured by ELISA.
ELISAs
MaxiSorp ELISA plates (Nunc, Roskilde, Denmark) were coated overnight at
4˚C with 1 mg/ml 8D4-8 (anti-human IL-4, BD Biosciences), 1 mg/ml MAb1
(anti-human TNF-a, BD Biosciences), or 3 mg/ml HB-8700 (anti-human IFN-g,
American Type Culture Collection) mAb, blocked with PBS 0.05% Tween-20
and 10 mg/ml BSA, and incubated with the supernatants of the Ag presentation
assays. For detection, 1 mg/ml MP4-25D2 (anti-human IL-4 biotin labeled, BD
Biosciences), 0.5 mg/ml MAb11 (anti-human TNF-a biotin labeled, BD Bio-
sciences), or 0.72 mg/ml g69 (anti-human IFN-g biotin labeled) (19) mAb were
used. The plates were developed colorimetrically, and concentrations were de-
termined by comparison with standards of human IL-4 (human IL-4 secreting
X63, kind gift of U. Grawunder, 4-Antibody AG, Basel, Switzerland), re-
combinant human TNF-a (ImmunoKontact, Abingdon, U.K.), and recombinant
human IFN-g (Bender MedSystems, Burlingame, CA).
Results
Relocation of CD1a to LY by providing residues of CD1b
cytoplasmic tail
It has been previously shown that CD1a localizes to the ERC,
whereas CD1b localizes to the LE/LY. To investigate the importance
of tyrosine-based motifs in CD1 molecule trafficking, HeLa CD1a:
CD1b tail chimeric constructs containing the tyrosine-based motif of
CD1b were generated and intracellular localization of CD1 mole-
cules analyzed by confocal microscopy. The CD1aab tail chimera
showed a different localization from CD1a WT and an identical
staining pattern compared with WT CD1b (Fig. 1A). This was further
demonstrated by colocalization of the CD1aab chimera to LE/LY
with LAMP-1, as it was also demonstrated for WT CD1b. This is in
agreement with the known important role for the tyrosine-based
sorting motif of CD1b in binding AP-3 and directing trafficking to
the LY. In contrast, as expected, there was no colocalization between FIGURE 2. Sulfatide localizes to the early endocytic and recycling
WT CD1a and LAMP-1 (Fig. 1B). Both WT and chimeric proteins compartments. A, The anti-sulfatide mAb (O4) specifically labels sulfa-
were well expressed on the cell surface, as demonstrated by flow tide-pulsed HeLa CD1a transfectants. Numbers above panels indicate the
sulfatide pulsing times. IgM control staining was performed at 60 min. B,
cytometry. The CD1aab chimera transfectants consistently showed
Colocalization of sulfatide with the early endocytic markers Rab5 and
lower surface mean fluorescence intensity compared with WT CD1a
EEA-1 was performed in HeLa CD1a WT cells pulsed with sulfatide for
as determined by flow cytometry (data not shown). It should be 30 min. Individual Ab stainings and the merged images are presented. C,
noted that there was significant anti-CD1a mAb intracellular stain- Colocalization pattern of CD1a and Trf in FITC-Trf–pulsed HeLa CD1a
ing with anti-CD1a mAb of the CD1aab chimera, indicating con- WT transfectants (top panels) and sulfatide and Trf in sulfatide- and FITC-
formational stability of the CD1a extracellular domain in the late Trf–pulsed HeLa CD1a WT (bottom panels). Individual Ab stainings and
endocytic system (Fig. 1A). the merged images are displayed.
5. 1238 CD1a TRAFFICKING IS ESSENTIAL FOR LIPID ANTIGEN PRESENTATION
and EEA-1–specific mAb and fluorescently-labeled Trf. There was CD1aab tail chimera, there was a marked reduction in the co-
marked colocalization between sulfatide and the early endocytic localization of sulfatide with the CD1aab tail chimeric protein
markers Rab5 and EEA-1 (Fig. 2B). Sulfatide was also present in the (Fig. 3A). This suggests that the intracellular localization provided
ERC as demonstrated by its partial colocalization with Trf (Fig. 2C). by the CD1b tyrosine-based motif determines the degree of in-
CD1a and Trf also demonstrated partial overlap (Fig. 2C) consistent teraction of the CD1aab tail chimera with sulfatide. The distri-
with the previously published localization of CD1a in the ERC (4, 5). bution of sulfatide was very similar in cells expressing CD1a WT
However, there was no colocalization with LAMP-1 (Fig. 1B), and CD1aab tail chimera as demonstrated by the almost complete
a marker of the late endocytic system. colocalization of sulfatide and Rab5. Furthermore, there was no
redirection of sulfatide to the late endocytic system with the
Differential colocalization of CD1a and CD1b with sulfatide CD1aab tail chimera (Fig. 3B).
Next, the colocalization pattern of CD1a and CD1b and the CD1aab Time course studies of sulfatide distribution were also performed
chimera with sulfatide was examined. HeLa CD1a WT, CD1b WT, to investigate whether sulfatide traffics to LEs at late time points.
and CD1aab chimera transfectants were pulsed with sulfatide for The transfectants were incubated with sulfatide for various time
30 min and stained with anti-CD1a, anti-CD1b, and anti-sulfatide periods before fixation prior to confocal microscopy. These studies
mAb. Consistent with the above findings, there was marked show that there is no detectable colocalization of LAMP-1 with
colocalization of CD1a with sulfatide in the HeLa CD1a WT sulfatide up to 20 h after sulfatide pulse (Fig. 3B, 3C). This lack of
transfectant cells (Fig. 3A). On the other hand, although small colocalization was observed for both the CD1a WT and CD1aab
amounts of punctate colocalization of CD1b with sulfatide were tail chimera transfectants. Minor colocalization was observed for
observed in some cells, the majority of sulfatide did not colocalize sulfatide and CD1b at late time points (data not shown). At all time
with CD1b in the HeLa CD1b WT transfectants (Fig. 3A). For the points, the vast majority of sulfatide staining remained in the early
Downloaded from www.jimmunol.org on April 18, 2012
FIGURE 3. Marked intracellular
colocalization between CD1a and
sulfatide. HeLa CD1a WT, CD1b
WT, and CD1aab chimera trans-
fectants were pulsed with sulfatide
and analyzed by confocal micros-
copy. A, Colocalization between
each CD1 molecule and sulfatide
was determined for each trans-
fectant. The fluorescent labels used
for each protein are indicated by
color. B, Colocalization between
sulfatide and early (Rab5) and late
endocytic (LAMP-1) markers was
determined in sulfatide-pulsed
CD1a WT and CD1aab tail chimera
transfectants. C, The colocalization
pattern between CD1a and LAMP-1
was analyzed in CD1a WT trans-
fectants pulsed with sulfatide for 4 h
(top panel) and 20 h (bottom panel).
D, Confocal staining for CD1a (flu-
orescently labeled in red) and sul-
fatide (labeled in green) was
performed on CD1a WT trans-
fectants incubated with sulfatide
(top panels) and C24 ceramide
control (bottom panels) at increasing
concentrations for 30 min.
6. The Journal of Immunology 1239
endocytic and recycling compartments. The absence of sulfatide CD1a intracellular trafficking alters sulfatide Ag presentation
detection in LAMP-1+ compartments even at later time points is to specific T cells
likely due to its preferential sorting to the early endocytic and re- In order to determine the functional relevance of the intracellular
cycling compartments and to the degradation of sulfatide by en- trafficking of CD1a in Ag presentation, we next examined the
dogenous lysosomal arylsulfatases that are well described to be ability of CD1a WT and different CD1a:CD1b tail chimeras to
present in this compartment (20). Lastly, to rule out the possible activate CD1a-restricted sulfatide-specific T cells. First, to assess
contribution of sulfatide to changes in CD1a expression, in- the appearance of sulfatide:CD1a stimulatory complexes, we
tracellular distribution, or cell morphology, confocal microscopy performed pulse experiments using HeLa CD1a WT and CD1aab
was performed with increasing concentrations of sulfatide or cer- chimera transfectants with matched cell surface expression of
amide (C24) as an additional control. No changes were observed by CD1a as determined by flow cytometry. The transfectants were
confocal microscopy in the distribution or localization of CD1a or in pulsed with sulfatide for 0, 7, 20, 25, 45, and 60 min and washed
the transfectant morphology after culture with either lipid (Fig. 3D). before the addition of T cells. The ability of sulfatide-pulsed
CD1a but not CD1b colocalizes with sulfatide in human transfectants to stimulate the sulfatide-specific T cell clone
monocyte-derived DCs K34B9.1 increased with increasing pulse period independently of
the measured cytokine (IFN-g, TNF-a, or IL-4). CD1a WT
These findings were confirmed in primary cells, human monocyte- transfectants outperformed the CD1aab chimera at pulse periods
derived DCs that express all CD1 isoforms. Human monocyte- longer than 30 min (Fig. 5). CD1aab chimera transfectants even
derived DCs were pulsed with sulfatide as described above. showed a slight decrease in stimulatory capacity at pulse periods
Confocal microscopy of DCs demonstrated areas of strong longer than 30 min. Then, to assess the disappearance of sulfatide:
colocalization between sulfatide and CD1a molecules but not CD1a stimulatory complexes, we performed chase experiments
between sulfatide and CD1b (Fig. 4A). These findings are in using T2 CD1a WT and CD1a:CD1b chimeras instead of HeLa
Downloaded from www.jimmunol.org on April 18, 2012
agreement with the known localization of CD1a and CD1b mol- transfectants to avoid potential effects of trypsinization required
ecules in monocyte-derived DCs to the ERC or to LE/LY com- for the release of the HeLa transfectants. The T2 transfectants were
partments, respectively (21, 22). pulsed with sulfatide for 3 h, washed, and chased for 0, 24, 48, and
72 h before addition of T cells and cytokine release measurement.
Colocalization of CD1b with LAM in DC
At each time point, control groups were represented by trans-
Next, the colocalization of CD1a and CD1b with LAM, an Ag fectants incubated with sulfatide together with T cells to determine
presented by CD1b, was analyzed. Monocyte-derived DCs were the maximal Ag-presenting capacity. The ability of sulfatide-
incubated with LAM for 30 min and analyzed by confocal mi- pulsed transfectants to stimulate the sulfatide-specific clone
croscopy. Strikingly, in contrast to the specific colocalization of K34B9.1 decayed with increasing chase period independently of
CD1a with sulfatide in DC, LAM strictly colocalized with CD1b the measured cytokine. WT CD1a transfectants showed half
but not with CD1a (Fig. 4B). The localization of LAM with CD1b maximal stimulation at 40 h of chase, whereas CD1abb trans-
is consistent with the known interaction of LAM with mannose fectants showed half maximal stimulation at 20 h. CD1aab trans-
receptors that likely mediates the internalization and direct tar- fectants showed intermediate behavior, with half maximal
geting of LAM and similar Ags to the late endocytic system (23). stimulation at 30 h (Fig. 6). After 72 h, CD1a WT transfectants
These findings suggest that CD1 molecules select the lipid Ag to were still capable of stimulating sulfatide-specific T cells, whereas
be presented according to their capacity to bind and form stable both chimera transfectants were no longer stimulatory.
complexes and also according to the endosomal localization of
each lipid Ag and its intersection with the relevant CD1 molecule.
Discussion
Successful Ag presentation involves an orchestrated series of steps
that are critically dependent on the intersection of Ags with the
relevant Ag-presenting molecules. Lessons from the MHC class I
and II pathways have made it clear that access to the proper Ag
loading compartment is a key feature for those presentation systems
to succeed. The proteolytic environment present in LYs of DCs plays
a critical role in their ability to process protein Ags for prolonged Ag
presentation. APCs such as macrophages with high levels of lyso-
somal proteases rapidly degrade internalized proteins, reducing their
capacity for sustained Ag presentation compared with DCs (24).
Although the ability of MHC class I and II molecules to survey
different intracellular compartments and Ags is a hallmark of these
systems, the role of CD1 trafficking in the presentation of exoge-
nous and endogenous Ags has been examined in only a few cases.
The unique intracellular distribution of each CD1 isoform has
been appreciated for some time. It has been demonstrated for the
CD1b and CD1c isoforms that their intracellular trafficking is
directed by their respective tyrosine-based motifs using tail-deleted
FIGURE 4. Reciprocal intracellular localization patterns of CD1a and constructs (3, 25). Tail-deleted CD1b molecules have been dem-
sulfatide and CD1b and LAM. The pattern of CD1a and CD1b colocali- onstrated to have significant impairment in their Ag-presenting
zation with sulfatide (A) and LAM (B) was analyzed by confocal mi- abilities in vitro (3). Glucose monomycolate possessing a long
croscopy in human monocyte-derived DCs pulsed with sulfatide. The lipid tail has been shown to require the acidic environment found
fluorescent labels used for each protein are indicated by color. in the late endocytic system to be loaded efficiently (26, 27).
7. 1240 CD1a TRAFFICKING IS ESSENTIAL FOR LIPID ANTIGEN PRESENTATION
FIGURE 5. WT CD1a transfectants present sulfatide more efficiently than CD1aab chimeric transfectants. WT CD1a (white diamonds) HeLa trans-
fectants showed increased capacity to stimulate the CD1a-restricted sulfatide-specific T cell clone K34B9.1 as compared with CD1aab chimeric trans-
fectants (gray diamonds) for pulse periods longer than 30 min. Both transfectants were pulsed with nervonoyl sulfatide for indicated times, then washed and
coincubated with T cells. Supernatants were harvested after 24 h, and the release of human IL-4 (A), human IFN-g (B), and TNF-a (C) was measured by
ELISA. Error bars represent SD of triplicate measurements.
CD1a is the only CD1 isoform that does not have a tyrosine-based enzymatically or as a result of acidic pH in the late endocytic
motif and almost exclusively localizes to the early endocytic and system. This is supported by the enhanced ability of WT CD1a to
recycling pathways. In this study, we demonstrate that the locali- present sulfatide compared with the CD1aab and CD1abb tail
zation of CD1a to the early endosomal and recycling compartments chimeras and the sustained ability of sulfatide-pulsed DCs to ac-
is critical for the efficient and sustained presentation of specific Ags tivate CD1a- compared with CD1b- and CD1c-restricted sulfatide-
Downloaded from www.jimmunol.org on April 18, 2012
that also predominantly localize to the same endosomal sites. CD1a specific T cells. Additional interactions or pathways contributing to
and sulfatide are almost exclusively colocalized to early endocytic CD1a Ag presentation may also be contributory. For example, the
and recycling compartments. The redirection of CD1a to the LE by colocalization of CD1a with the unique LC-specific C-type lectin
the addition of the transmembrane and/or cytoplasmic CD1b ty- required for Birbeck granule formation, langerin, in this endosomal
rosine-based motif to the extracellular domain of CD1a resulted in subcompartment has been shown to augment CD1a-mediated Ag
loss of colocalization with sulfatide and a correspondingly signif- presentation (28). We cannot rule out the possibility that the
icant reduction in the ability of these transfectants to stimulate CD1aab–sulfatide complexes dissociate in the LY. We should also
CD1a-restricted sulfatide-specific T cells. Because the extracellular mention the possibility that the anti-sulfatide mAb may not be able
domains of the chimeric molecules are identical, the most likely to detect sulfatide in the LY secondary to changes to the antigenic
explanation for these findings is the difference in trafficking epitope recognized. However, similar sulfatide colocalization pat-
imparted by the chimeric cytoplasmic tails. The activity and lo- terns have been observed with other anti-sulfatide mAbs, making
calization of endogenous sulfatases suggests that even if some this less likely.
sulfatide is delivered to LYs, it would be rapidly degraded. There are The differential localization of CD1a to the early endocytic
two classes of sulfatases, catabolic and synthetic. The catabolic compartment may be important in vivo. The efficient and sustained
sulfatases are localized in LYs and exert their activity at acidic pH on presentation of Ags such as sulfatide by CD1a+ DC during their
a wide range of molecules, including sulfolipids and glyco- migration to regional lymph nodes is likely critical for maximiz-
soaminoglycans. Accumulation of these molecules occurs in the ing T cell activation and proliferation. This may be even of greater
setting of lysosomal sulfatase deficiency and leads to human disease, importance in vivo, where Ag concentrations are likely lower than
including several mucopolysaccharidoses and metachromatic leu- our studies where higher concentrations of sulfatides were added
kodystrophy (20). The prolonged ability of CD1a molecules to to allow for Ab detection and localization by microscopy.
present sulfolipids such as sulfatide may be critically dependent on The specific intracellular distribution of CD1 molecules and their
their localization to early endocytic compartments where the sul- Ags may also explain the evolutionary diversity of the CD1 iso-
fatide Ag is not degraded by sulfatases. The absence of a tyrosine- forms (29). It is interesting to speculate that the distribution of the
based motif allows CD1a to traffic and recycle through the early different CD1 isoforms throughout the endocytic system may have
endocytic system, where it can most efficiently present lipid Ags developed to allow for the presentation of differentially distributed
that preferentially localize there or that would be destroyed either Ags or Ags that are destroyed either enzymatically or by the acidic
FIGURE 6. CD1a:CD1b chimeras have shorter-lived ability to stimulate sulfatide-specific T cells than WT CD1a molecules. WT CD1a (white diamonds)
T2 transfectants showed prolonged capacity to stimulate the CD1a-restricted sulfatide-specific human T cell clone K34B9.1 as compared with CD1aab
(gray diamonds) or CD1abb (black diamonds). All transfectants were pulsed with nervonoyl sulfatide and chased for indicated times before addition of
T cells. In control wells, fresh Ag was added to chased T2 cells together with T cells. Supernatants were harvested after 24 h, and the release of human IL-4
(A), human IFN-g (B), and human TNF-a (C) was measured by ELISA and expressed as percent of control at time zero 6 SD (of triplicates or quad-
ruplicates) normalized at each time point for the maximal Ag-presenting capacity of the APC calculated as (chase/chase with fresh Ag)/(no chase/no chase
fresh Ag) 3 100.
8. The Journal of Immunology 1241
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Disclosures 24. Delamarre, L., M. Pack, H. Chang, I. Mellman, and E. S. Trombetta. 2005.
The authors have no financial conflicts of interest. Differential lysosomal proteolysis in antigen-presenting cells determines antigen
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