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* Lymphocyte Biology Section, Division of Rheumatology, Immunology, and Allergy, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115;
Netherlands Cancer Institute, Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands; and
Department of Environmental Health, Harvard School of Public Health, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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for maturation, we observed that none of the CD1 isoforms underwent
changes in intracellular trafficking that mimicked MHC class II
molecules during DC maturation. In contrast to the striking increase in
surface expression of MHC class II on mature DCs, the surface
expression of CD1 molecules was either increased only slightly (for
CD1b and CD1c) or decreased (for CD1a). In addition, unlike MHC class
II, DC maturation-associated transport from lysosomes to the plasma
membrane was not readily detected for CD1b despite the fact that both
molecules were prominently expressed in the same MIIC lysosomal
compartments before maturation. Consistent with this, DCs efficiently
presented CD1b-restricted lipid Ags to specific T cells similarly in
immature and mature DCs. Thus, DC maturation-independent pathways for
lipid Ag presentation by CD1 may play a crucial role in host defense,
even before DCs are able to induce maximum activation of peptide
Ag-specific T cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
subunits are translocated into the endoplasmic reticulum, where
they associate with the invariant chain (2). After
transport to the trans-Golgi network, these :invariant
chain complexes are mainly sorted to endocytic pathways directly or
transported to the cell surface and then internalized to the endocytic
compartments, ultimately localizing to a specialized lysosomal
compartment called the MHC class II compartment (MIIC)
(3). In DCs exposed to Ag and maturational stimuli, such
as LPS and TNF-, peptide loading is thought to occur in the MIIC,
followed by transport of Ag-bound MHC class II molecules to the cell
surface (4, 5, 6, 7). Concurrently, endocytosis is
down-regulated, resulting in the accumulation of MHC class II/peptide
complexes on the cell surface (8, 9). These DC
maturation-associated changes in MHC class II trafficking are critical
for peptide Ag presentation.
CD1 molecules constitute a separate lineage of MHC-like molecules that
function to bind and present lipid Ags to T cells (10, 11). Human CD1a, -b, -c, and -d are predominantly expressed on
professional APCs such as DC and B cells. The CD1a, -b, -c, and -d
isoforms have been shown to traffic and localize in distinct
subcompartments of the endocytic system. Following assembly with
2-microglobulin in the endoplasmic reticulum,
CD1b heavy chains are transported to the cell surface and then
internalized via specific interaction of their cytoplasmic tail
tyrosine-based sequence with the AP-2 clathrin coat-associated adaptor
protein complex (12, 13, 14). Subsequently, CD1b molecules are
further transported deeply into the endocytic system and reach
lysosomes, including the MIIC, as a result of selective binding of the
AP-3 complex that is known to interact with certain lysosome-resident
proteins, such as lysosome-associated membrane protein-1 (lamp-1)
(15). CD1c and CD1d also contain similar cytoplasmic tail
tyrosine-based sorting motifs, but their failure to bind the AP-3
complex results in their substantial distribution to the early
endocytic system, although a fraction of them can reach lysosomes
through an undefined pathway (15, 16, 17). Unlike other CD1
molecules, CD1a lacks the cytoplasmic tail tyrosine-based sorting
sequence, and, within the endocytic system, is expressed solely in
early endosomes of the recycling pathway (18). Together,
these observations underscore the capacity of CD1 to broadly survey the
endocytic system for sampling lipid Ags that may localize in various
endocytic subcompartments (19).
These studies, however, only determined the intracellular trafficking
and localization of CD1 molecules in immature DCs as well as non-DC
lineages of cells transfected with CD1. The distinct intracellular
trafficking of CD1 and the distinct chemical nature of Ags presented by
CD1 as well as the different subsets of T cells they activate raised
the possibility that the CD1 and MHC trafficking and function might be
differentially controlled during DC maturation. In the present study
using monocyte-derived immature DCs and those stimulated with TNF-
for maturation, we found that none of the CD1 isoforms was up-regulated
on the cell surface after DC maturation to levels comparable to those
of MHC class II. Whereas immature DCs contained a large pool of MHC
class II and CD1b molecules, both localized in lysosomes, the lysosomes
of mature DCs were almost devoid of MHC class II, but still contained
the majority of the CD1b molecules. This suggests that unlike MHC class
II, CD1b molecules continue to survey lysosomes in a manner that may
not be dramatically altered upon DC maturation. Further, CD1b-mediated
presentation of lipid Ags occurred efficiently even without
maturational stimuli. These studies detecting the differential pathways
for Ag presentation by CD1 and MHC molecules during DC maturation
support the concept that CD1-based Ag presentation may occur earlier in
the immune response than MHC class II-based Ag presentation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The tissue culture medium used throughout this study was RPMI
1640 complete medium (20 mM HEPES, 2 mM L-glutamine, 1 mM
sodium pyruvate, 100 µg/ml penicillin-streptomycin, 55 µM 2-ME (all
from Life Technologies, Gaithersburg, MD), and 10% heat-inactivated
FCS (HyClone, Logan, UT)). To generate immature DCs, human peripheral
blood monocytes were isolated as described previously (20)
and cultured in RPMI 1640 complete medium containing 300 U/ml rGM-CSF
(Immunex, Seattle, WA) and 200 U/ml rIL-4 (PeproTech, Rocky Hill, NJ)
for 3 days. For induction of DC maturation the cells were stimulated
with either 50 ng/ml TNF- (Pierce, Rockford, IL) or 1 µg/ml LPS
(Sigma, St. Louis, MO) for an additional 1 or 2 days in the presence of
the same concentrations of GM-CSF and IL-4. The glucose monomycolate
(GMM)-specific, CD1b-restricted T cell line, LDN5 (21),
and the tetanus toxoid (TT)-specific, HLA-DR-restricted T cell line,
SPF3 (22), were cultured as previously described.
Flow cytometry
Flow cytometry was performed as previously described (23), using the following mAbs: P3 (control) (23), W6/32 (anti-MHC class I) (24), L243 (anti-HLA-DR) (25), 10H3.9.3 (anti-CD1a) (26), BCD1b3.1 (anti-CD1b) (27), F10/21A3.1 (anti-CD1c) (28), CD1d42.1.1 (anti-CD1d) (29), anti-CD83 (Ancell, Bayport, MN), anti-CD86, and anti-CD14 (both from BD PharMingen, San Diego, CA). These mouse Abs were followed by FITC-conjugated goat F(ab')2 Abs against mouse IgG and IgM (BioSource International, Camarillo, CA). For double staining, cells were double labeled with FITC-conjugated mouse mAbs against MHC class II (Ancell), CD1a (BD PharMingen), CD1b (BD PharMingen), and CD1c (Ancell) and PE-conjugated mouse mAbs against CD83 (BD PharMingen).
Endocytosis assay
The endocytic capacity of DCs was examined as previously
described (5). Briefly, GM-CSF/IL-4-stimulated immature
DCs and TNF--treated mature DCs (5 x
105/ml) were incubated with 1 mg/ml of
FITC-conjugated dextran (Sigma) in RPMI complete medium at 37°C for
the indicated periods of time. At each time point, the cells were
quickly chilled and washed three times in cold PBS containing 1% FCS.
The accumulation of FITC-conjugated dextran within the cells was
measured by flow cytometry.
Confocal immunofluorescence microscopy
DCs were adhered on glass slides by a cytospin procedure and labeled with anti-CD83 Ab, followed by incubation with Texas Red-conjugated donkey F(ab')2 anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). The incubation was performed on ice for 30 min. The cells were then fixed with 2% formaldehyde in PBS for 10 min at room temperature, permeabilized with 0.2% saponin, and double labeled with either FITC-conjugate anti-HLA-DR (LB3.1) (30) or Alexa488-conjugated anti-CD1b (BCD1b3.1). For detection of CD1a and CD1c, biotinylated Abs (10H3.9.3 and F10/21A3.1, respectively) were used, followed by incubation with FITC-conjugated streptavidin. For colocalization studies with lamp-1, DCs were first fixed and permeabilized as described above, and double labeling with rabbit anti-lamp-1 (31) and LB3.1, 10H3.9.3, BCD1b3.1 or F10/21A3.1 was performed, followed by incubation with Texas Red-conjugated donkey anti-rabbit IgG and FITC-conjugated donkey anti-mouse IgG (both from Jackson ImmunoResearch Laboratories). The labeled cells were examined using a Leica TCS-NT confocal laser scanning microscope (Deerfield, IL) fitted with krypton and argon lasers as previously described (32).
T cell proliferation assay
CD83- immature DCs were prepared, using
GM-CSF/IL-4-stimulated monocytes and anti-CD83-coupled magnetic
beads for depletion. The CD83- cells were pulsed
with TT (for SPF3) or with purified long chain
(C80) GMM (for LDN5) for 10 min at 37°C. The
cells were then chilled on ice, washed three times with cold PBS, and
resuspended in RPMI 1640 complete medium supplemented with GM-CSF and
IL-4 either with or without 50 ng/ml of TNF-. After 24 h, DCs
(5 x 104/well) were fixed with 0.04%
glutaraldehyde and incubated with either SPF3 or LDN5 (1 x
105/well) for 3 days. T cell proliferation was
assessed by measuring the uptake of
[3H]thymidine (1 µCi/well) for the last
6 h of culture.
Electron microscopy
Monocytes grown in GM-CSF/IL-4-containing medium either with or
without TNF- were fixed using 2% paraformaldehyde and 0.2%
glutaraldehyde, and processed for cryosectioning as previously
described (33). Ultrathin sections were cut at -120°C
and immunogold labeled at room temperature using mouse anti-CD1b Ab
(BCD1b2.1) (27) and rabbit anti-MHC class II Ab
(34). Bound anti-CD1b Ab was detected using rabbit
antiserum against mouse IgG (DAKO, Glostrup, Denmark) and protein A
conjugated to 15 nm gold (EM Laboratory, Utrecht University, The
Netherlands), whereas bound anti-MHC class II Ab was directly
detected using protein A conjugated to 10 nm gold. Immunogold-labeled
sections were viewed using a CM10 transmission electron microscope
(Philips Electronic Instruments, Mahway, NJ).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To study Ag presentation by CD1 and MHC class II during DC
maturation we first established an in vitro method to drive DC
maturation. Human peripheral blood monocytes were cultured in the
presence of GM-CSF and IL-4 for 3 days to induce their differentiation
into immature DCs. The cells were then stimulated to mature by
incubation for an additional 2 days with TNF- in the continued
presence of GM-CSF and IL-4. During the first 3 days of culture in
GM-CSF and IL-4, most cells developed fine projections and lost
adherence to the surface of tissue culture plates. These cells acquired
an even more dendritic appearance after an additional 2-day stimulation
with TNF- (data not shown). This morphological alteration was
associated with phenotypic changes as determined by flow cytometry
(Fig. 1
A). Fresh monocytes
(Fig. 1A, upper panels, day 0)
prominently expressed MHC class I and class II molecules as well as
CD86 and CD14, but almost completely lacked the expression of CD1a, -b,
and -c molecules. After exposure to GM-CSF and IL-4 for 5 days without
TNF- (day 5, -TNF-), the surface expression of CD1a, -b, and -c
molecules was markedly induced (Fig. 1A, middle
panels). Consistent with an immature DC phenotype, a majority of
these cells lacked expression of CD83, a marker of mature DCs
(35). In contrast, when monocytes were incubated with
GM-CSF and IL-4 for the first 3 days and then further stimulated with
TNF- in the continued presence of GM-CSF and IL-4 for an additional
2 days (day 5, +TNF-), marked changes associated with DC maturation
were observed. The TNF--stimulated DCs prominently expressed CD83 as
well as high levels of MHC class II and CD86 and largely lost the
expression of CD14. Furthermore, as shown in Fig. 2, the endocytic activity of
TNF--treated DCs (+TNF-/37°C), assessed by uptake of
extracellular FITC-conjugated dextran, was significantly reduced
compared with that of DCs that were not exposed to TNF-
(-TNF-/37°C). Together, these phenotypic and functional analyses
confirmed that the cells obtained after stimulation of monocyte-derived
immature DCs with TNF- were mature DCs. These immature and mature
DCs were used in the present study, addressing the impact of DC
maturation on CD1 and MHC class II-dependent Ag presentation.
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In contrast to MHC class II, whose surface expression on DCs was
prominently up-regulated upon TNF- stimulation, changes in surface
expression levels of CD1a, -b, and -c molecules were not as apparent
(Fig. 1
A, compare day 5, -TNF- with day 5, +TNF-). We
considered the possibility that TNF--stimulated DCs might be
heterogeneous in the degree of maturation. Indeed, a small, but
significant, fraction of TNF--stimulated DCs remained negative for
CD83 (Fig. 1A) and thus were not mature DCs. Conversely, a
small fraction of TNF--unstimulated DCs expressed low levels of CD83
(Fig. 1A). To better analyze the surface expression of CD1
molecules on immature DCs and mature DCs in these heterogeneous
cultures, we performed double labeling with mAbs to CD83 (PE) and
either MHC class II or CD1 molecules (FITC), and the major DC
subpopulations in each culture, namely CD83-negative DCs without
TNF- stimulation and CD83-positive DCs after TNF- stimulation,
were compared for MHC class II and CD1 expression (Fig. 1B).
As expected, the surface levels of MHC class II increased markedly
after TNF- stimulation on CD83+ DC (mean
fluorescence intensity (MFI) of 481 on CD83- DC
before TNF- stimulation to MFI of 2577 on
CD83+ DC after TNF- stimulation). In contrast
to the striking increase in surface expression of MHC class II, the
cell surface expression of CD1 molecules was either increased only
slightly (for CD1b and CD1c) or decreased (for CD1a). In the
representative experiment depicted in Fig. 1B, the surface
expression of CD1b was increased with MFI from 62 (on
CD83- DC without TNF- stimulation) to 158 (on
CD83+ DC after TNF- stimulation), and CD1c
expression was increased with MFI from 110 to 192, whereas the surface
expression of CD1a decreased from MFI 436 to 221. The level of CD1d
surface expression remained essentially unchanged. To illustrate the
changes in surface expression of Ag-presenting molecules during DC
maturation, we calculated a ratio of MFI of mature DCs (CD83-positive
population) to that of immature DCs (CD83-negative population) for each
Ag-presenting molecule in 11 independent experiments and plotted the
results (Fig. 1C). After TNF- stimulation, surface
expression of CD1a was decreased by half, whereas that of CD1b and CD1c
was increased 
1- to 3-fold. These small increases contrasted the
larger up to 5-fold increase in the surface expression of MHC class II
molecules (Fig. 1C). These results suggested that
trafficking of CD1 and MHC class II molecules might be controlled
differently during DC maturation.
CD1 and MHC class II molecules followed distinct intracellular pathways during DC maturation
We applied confocal immunofluorescence microscopy to study the
intracellular distribution of CD1 and MHC class II molecules during DC
maturation. To distinguish mature DCs from a small faction of immature
DCs that might be present even after TNF- stimulation, cells were
labeled first with anti-CD83 mAb before permeabilization and then
permeabilized for detection of intracellular CD1 and MHC class II
molecules. The monocyte-derived immature DCs lacked the expression of
CD83 and contained a pool of intracellular MHC class II molecules in
peripherally distributed vesicles that corresponded to the MIIC (Fig. 3A, top panels,
indicated with arrowheads). After TNF- stimulation, most cells
gained expression of CD83 on the cell surface and displayed a striking
shift of MHC class II from the intracellular pool to the plasma
membrane (tailed arrow), whereas a small fraction of cells that did not
express CD83 still contained intracellular vesicles expressing MHC
class II (Fig. 3A, middle panels, arrowhead). The
redistribution of MHC class II to the plasma membrane in
TNF--stimulated DCs was comparable to that seen in LPS-stimulated
DCs (Fig. 3A, bottom panels).
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A, upper panels), CD1b was prominent in
peripherally distributed vesicles (Fig. 3B, upper
panels, arrowheads). After TNF- stimulation, however, little
shift from lysosomes to the plasma membrane occurred for CD1b compared
with that which occurred for MHC class II (Fig. 3B,
lower panels). Instead, CD1b-containing vesicles remained
intracellularly and appeared clustered (Fig. 3B, lower
panels, arrows). These results underscored the possibility that
CD1b and MHC class II resided in the same lysosomes in immature DCs,
but might follow distinct intracellular pathways during DC maturation,
as discussed in more detail below.
Unlike CD1b, CD1a molecules are prominently expressed on the surface of
immature DCs, with a small fraction localized to early recycling
endosomes (18). Thus, labeling of immature DCs with
anti-CD1a mAb revealed strong cell surface staining (arrowhead) as
well as a few CD1a+ intracellular vesicles (Fig. 3C, upper panels). After TNF- stimulation, the
surface CD1a staining became appreciably less bright, and the
proportion of intracellular CD1a (arrow) to surface CD1a (arrowhead)
was increased (Fig. 3C, lower panels). Thus,
consistent with the flow cytometric results presented in Fig. 1C, the surface expression of CD1a was down-regulated upon
DC maturation. Labeling of immature DCs with anti-CD1c mAb revealed
its expression both on the cell surface (Fig. 3D,
upper panels, arrow) and in intracellular vesicles
(arrowheads), and its cellular localization was not noticeably altered
after TNF- stimulation (Fig. 3D, lower
panels). Thus, the flow cytometric and confocal immunofluorescence
microscopic studies suggested that CD1a, -b, and -c molecules
trafficked separately from MHC class II during DC maturation.
CD1b and MHC class II molecules differentially trafficked out of the lysosome during DC maturation
Both CD1b and MHC class II molecules are expressed prominently in
the same lysosomal compartments in immature DCs. As demonstrated above,
however, striking alterations in intracellular localization were
detected for both molecules after TNF- stimulation. Whereas most MHC
class II molecules redistributed to the plasma membrane after TNF-
stimulation, a majority of CD1b molecules remained in intracellular
vesicles. To gain further insight into pathways for their intracellular
transport during DC maturation, localization of CD1b and MHC class II
molecules by confocal fluorescence microscopy was compared with that of
lamp-1, a marker protein for late endosomes and lysosomes. Immature and
mature DCs were double labeled with Abs to lamp-1 and either CD1 or MHC
class II molecules. As expected from the results presented above, MHC
class II molecules were expressed prominently in peripherally
distributed vesicles containing lamp-1 in immature DCs (Fig. 4A, upper panels,
arrowheads). After TNF- stimulation, the majority of MHC class II
molecules trafficked out of the lysosomes and reached the plasma
membrane (Fig. 4A, lower panels, solid-tailed
arrows), while lamp-1+ lysosomes became clustered
mainly at the perinuclear region (open-tailed arrows).
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B, upper panels,
arrowheads), but unlike MHC class II, only a marginal increase in
surface expression was observed for CD1b after TNF- stimulation.
Instead, a majority of CD1b molecules remained intracellularly and were
expressed in a large cluster of vesicles at the perinuclear region
(Fig. 4B, lower panels, tailed arrows). These
CD1b-containing vesicles were most likely lysosomes, since a similar
distribution was observed for lamp-1, but because of their highly
clustered distribution of these vesicles it was difficult to determine
their identity at this level of resolution. To overcome this problem,
we conducted immunogold-labeled transmission electron microscopy. In
immature DCs both MHC class II (small gold particles, indicated with
small arrows) and CD1b molecules (large gold particles, indicated with
large arrowheads) were expressed in electron-dense multilamellar
compartments, confirming their prominent localization in lysosomes
(Fig. 5A). After 48-h
maturation with TNF-, the electron-dense lysosomes appeared smaller
in diameter, but remained as separate compartments (Fig. 5B). These lysosomal compartments still contained CD1b
molecules (large gold particles indicated with large arrowheads) and
lamp-1 (not shown), while MHC class II molecules largely redistributed
from the lysosomes to the plasma membrane (small arrows). These
observations suggested that lysosomes underwent dynamic changes during
DC maturation in their morphology, their localization, and possibly
their function, resulting in a redistribution to the plasma membrane
only of MHC class II and not a majority of CD1b molecules.
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It has been established that remarkable transport of Ag-loaded MHC
class II molecules from lysosomes to the cell surface occurs during DC
maturation, resulting in efficient activation of Ag-specific T cells
(1). Given the substantial difference, detected above,
between MHC class II and CD1b molecules in their net transport from
lysosomes to the cell surface during DC maturation, we reasoned that,
unlike MHC class II, CD1b might function independently of DC
maturation. To address this hypothesis, the ability of immature DCs to
present endocytosed protein and lipid Ags to specific T cells was
evaluated in the presence or the absence of TNF-. Peripheral blood
monocytes were differentiated into immature DCs by a 3-day incubation
with GM-CSF and IL-4. Following complete removal of a small fraction
(1–10%) of contaminating CD83+ mature DCs using
anti-CD83 Ab and magnetic beads, the remaining
CD83- immature DCs were pulsed for 10 min with
various concentrations of either TT or GMM, and then washed extensively
to remove free Ags. Subsequently, the Ag-pulsed DCs were incubated for
24 h in the presence or the absence of TNF- stimulation. At the
end of the culture the TT- and GMM-pulsed DCs were fixed with
glutaraldehyde, and their ability to stimulate the TT-specific,
HLA-DR-restricted T cell line (SPF3) and the GMM-specific,
CD1b-restricted T cell line (LDN5), respectively, was assessed by
measuring [3H]thymidine uptake by the T
cells. When TT-pulsed immature DCs were stimulated with TNF- to
undergo maturation, they activated the SPF3 T cells more efficiently
(by 2- to 3-fold at every Ag concentration tested) compared with
those DCs cultured without TNF- (Fig. 6, upper panel). In contrast,
little effect of TNF- stimulation was detected on CD1b-mediated
presentation of GMM to the LDN5 cells (Fig. 6, lower panel).
Thus, unlike MHC class II that required DC maturation to mediate
efficient protein Ag presentation, CD1b-mediated presentation of lipid
Ags functioned independently of DC maturation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Although
immature DCs maintained a large lysosomal pool of both MHC class II and
CD1b molecules, lysosomes of mature DCs were almost devoid of MHC class
II, but still contained a large fraction of the CD1b molecules,
suggesting a much more pronounced net transport from lysosomes to the
plasma membrane for MHC class II than for CD1b during DC maturation
(Figs. 4 and 5). Immature DCs are highly phagocytic (Fig. 2) and appear
to efficiently endocytose both protein and lipid Ags of foreign origin.
However, MHC class II-mediated presentation of protein Ags, but
not CD1b-mediated presentation of lipid Ags, depended substantially on
DC maturation (Fig. 6). Taken together, these data underscored the
distinct regulation of MHC and CD1 function during DC maturation. The ability of immature DCs to efficiently mediate CD1-dependent lipid Ag presentation even in the absence of maturational stimuli may be of physiological importance. During microbial infection, immature DCs sample microbe-derived protein Ags, but are unable to present them to T cells until they undergo substantial maturation. To initiate maturation, immature DCs use their Toll-like receptors and detect microbial components, such as LPS from Gram-negative bacilli, lipoteichoic acids from Gram-positive bacilli, and lipoarabinomannan from mycobacteria (36, 37). Once activated by proper stimuli, protein Ag-loaded DCs undergo maturation while migrating into draining lymph nodes, where fully matured DCs efficiently activate peptide Ag-specific naive T cells in a CD28-dependent manner (1, 38). Although this type of acquired immunity is essential for adaptive, peptide Ag-specific T cell responses against microbes, it takes days to occur. In contrast, the results obtained in the present study indicate that presentation of microbial lipids does not require maturation of DCs. The ability of immature DCs to present lipid Ags without maturational stimuli may provide a valuable opportunity for the immune system to mediate prompt CD1-specific T cell responses, even before peptide Ag-specific T cells are fully differentiated and expand. It should be noted that virtually all microbial lipid Ag-specific, CD1-restricted T cells analyzed to date lack the expression of CD28 (27), and thus, unlike most peptide Ag-specific T cells, activation of lipid Ag-specific T cells does not appear to require interaction with costimulatory ligands, such as CD80 and CD86, that are expressed at high levels on DCs only after maturation.
DC maturation-associated reorganization of lysosomes has been noted
previously in a murine DC line and may account for efficient transport
of MHC class II molecules from lysosomes to the plasma membrane during
DC maturation (39). In human monocyte-derived DCs analyzed
in the present study we also observed that lysosomes are deprived of
MHC class II after maturation and loose their tightly packed membranes
(Fig. 5). In contrast to MHC class II, a large pool of CD1b molecules
appeared to be maintained in lamp-1+ lysosomes
throughout DC maturation, and no evidence for the net transport of CD1b
from lysosomes to the plasma membrane was noted (Figs. 4 and 5).
Delivery of lipid Ag-bound CD1b molecules from lysosomes to the plasma
membrane must occur in both Ag-pulsed immature and mature DCs, as
suggested by functional experiments (Fig. 6). Three pathways of
delivery from lysosomes to the plasma membrane have been described.
Tubulation and vesicle formation of the lysosome were proposed to
account for delivery of MHC class II molecules to the plasma membrane
during DC maturation (39). Previously, a specialized CIIV
vesicle was identified in murine DC and was also proposed as a delivery
route for MHC class II during DC maturation (7). For
CD1-based Ag presentation, however, it is also necessary to account for
the ability of immature DCs to deliver Ag-loaded complexes from
lysosomes to the plasma membrane. One potential mechanism that may
account for this is the exosome-dependent pathway previously noted to
occur in murine immature and mature DCs as well as in human B cells
(40, 41). In this pathway the limiting membrane of
lysosomes fuses with the plasma membrane, resulting in delivery of the
limiting membrane-bound proteins to the plasma membrane while
exocytosing the inner membranes. Consistent with this
possibility, it should be noted that a majority of CD1b molecules
reside on the limiting membrane of lysosomes, whereas most MHC class II
molecules are detected on the inner membranes (13).
Once DC maturation has occurred, both molecules may reach the plasma membrane during the unraveling of lysosomes that occurs during DC maturation, but CD1b may continue to recycle and be delivered back to the clustered, postmaturation lysosomes, while MHC class II remains on the cell surface with a prolonged half-life (8). The current data cannot distinguish the relative contributions of differences in transport from lysosomes to the cell surface vs differences in rates of internalization from the plasma membrane and delivery back to lysosomes as the major pathway that accounts for the profound differences in steady state localization of CD1b and MHC class II after DC maturation.
Although both MHC and CD1 molecules mediate Ag-specific T cell responses of acquired immunity, recent studies suggest that CD1d molecules (42) and CD1a, -b, and -c molecules (43, 44) (M. Vincent, D. Leslie, and M. B. Brenner, unpublished observations) have the capacity for foreign Ag-independent T cell recognition that suggests a role for some CD1-reactive T cells at early points in the immune response. This type of self-reactive, CD1-specific recognition may provide an interface between innate and acquired immunities. The functional experiments presented here that reveal the distinct regulation of MHC and CD1 during DC maturation provide further support for the possibility that CD- restricted, foreign Ag recognition by T cells may occur early in immunological responses even before MHC class II molecules are fully functional.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael B. Brenner, Division of Rheumatology, Immunology, and Allergy, Brigham and Women’s Hospital, Smith Building Room 552, One Jimmy Fund Way, Boston, MA 02115. E-mail address: mbrenner{at}rics.bwh.harvard.edu
3 Abbreviations used in this paper: DC, dendritic cell; GMM, glucose monomycolate; MFI, mean fluorescence intensity; MIIC, MHC class II compartment; lamp-1, lysosome-associated membrane protein-1; TT, tetanus toxoid.
Received for publication June 13, 2002. Accepted for publication August 27, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 subset in human mycobacteria-responsive T cells suggests germline gene encoded recognition. J. Immunol. 147:3360.[Abstract]
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  D. L. Hava, N. van der Wel, N. Cohen, C. C. Dascher, D. Houben, L. Leon, S. Agarwal, M. Sugita, M. van Zon, S. C. Kent, et al. Evasion of peptide, but not lipid antigen presentation, through pathogen-induced dendritic cell maturation PNAS, August 12, 2008; 105(32): 11281 - 11286. [Abstract] [Full Text] [PDF]
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 A. Smed-Sorensen, M. Moll, T.-Y. Cheng, K. Lore, A.-C. Norlin, L. Perbeck, D. B. Moody, A.-L. Spetz, and J. K. Sandberg IgG regulates the CD1 expression profile and lipid antigen-presenting function in human dendritic cells via Fc{gamma}RIIa Blood, May 15, 2008; 111(10): 5037 - 5046. [Abstract] [Full Text] [PDF]
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 M. J. Raftery, M. Hitzler, F. Winau, T. Giese, B. Plachter, S. H. E. Kaufmann, and G. Schonrich Inhibition of CD1 Antigen Presentation by Human Cytomegalovirus J. Virol., May 1, 2008; 82(9): 4308 - 4319. [Abstract] [Full Text] [PDF]
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 I. Sloma, M.-T. Zilber, T. Vasselon, N. Setterblad, M. Cavallari, L. Mori, G. De Libero, D. Charron, N. Mooney, and C. Gelin Regulation of CD1a Surface Expression and Antigen Presentation by Invariant Chain and Lipid Rafts J. Immunol., January 15, 2008; 180(2): 980 - 987. [Abstract] [Full Text] [PDF]
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P. Gogolak, B. Rethi, I. Szatmari, A. Lanyi, B. Dezso, L. Nagy, and E. Rajnavolgyi Differentiation of CD1a- and CD1a+ monocyte-derived dendritic cells is biased by lipid environment and PPAR{gamma} Blood, January 15, 2007; 109(2): 643 - 652. [Abstract] [Full Text] [PDF]
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I. Potolicchio, S. Chitta, X. Xu, D. Fonseca, G. Crisi, V. Horejsi, J. L. Strominger, L. J. Stern, G. Raposo, and L. Santambrogio Conformational Variation of Surface Class II MHC Proteins during Myeloid Dendritic Cell Differentiation Accompanies Structural Changes in Lysosomal MIIC J. Immunol., October 15, 2005; 175(8): 4935 - 4947. [Abstract] [Full Text] [PDF]
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S. Della Bella, S. Nicola, A. Riva, M. Biasin, M. Clerici, and M. L. Villa Functional repertoire of dendritic cells generated in granulocyte macrophage-colony stimulating factor and interferon-{alpha} J. Leukoc. Biol., January 1, 2004; 75(1): 106 - 116. [Abstract] [Full Text] [PDF]
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M. Cernadas, M. Sugita, N. van der Wel, X. Cao, J. E. Gumperz, S. Maltsev, G. S. Besra, S. M. Behar, P. J. Peters, and M. B. Brenner Lysosomal Localization of Murine CD1d Mediated by AP-3 Is Necessary for NK T Cell Development J. Immunol., October 15, 2003; 171(8): 4149 - 4155. [Abstract] [Full Text] [PDF]
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and •) and untreated (
and 
) DCs were incubated
with FITC-conjugated dextran for the indicated periods of time at
37°C (
