1 Unité INSERM U 396, Institut Biomédical des Cordeliers, 15 rue
de l'Ecole de Médecine, 75006 Paris, France
2 Department of Biological Sciences, University of Maryland, Baltimore, MD
21250, USA
3 Department of Immunology, Institute of Ophthalmology, University College
London, 11-43 Bath Street, London EC1V 9EL, UK
Author for correspondence (e-mail:
nuala.mooney{at}bhdc.jussieu.fr)
Accepted 4 March 2003
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Summary |
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Key words: Lipid rafts, MHC class II, PKC, Actin, Tumor cell
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Introduction |
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The PKC family of serine/threonine kinases is classified into three groups
of isoenzymes based upon their activation requirements: the classical PKCs
(, ßI/II and
) are activated by diacylglycerol (DAG),
phosphatidylserine (PS) and Ca2+; the novel PKCs (
,
,
and
) are activated by DAG and PS; and the atypical PKCs (
and
) only respond to PS. The intracellular tail of the MHC class II
ß chain is required for activation of the PKC-
and PKC-ßII
isoforms (Cambier et al., 1987
;
Harton et al., 1995
;
Rich et al., 1997
;
St-Pierre et al., 1989
). By
contrast, the ability of MHC class II to mediate PTK activation is unaffected
by either mutations or truncation of the intracellular part of the ß
chain in B cells (Harton et al.,
1995
; Rich et al.,
1997
).
A role for MHC class II proteins in the antitumoral immune response has
been clearly shown in an immunotherapy model wherein vaccination of mice with
sarcoma cells transfected with and ß chains of I-Ak
molecules (SaI/Ak cells) protects against subsequent challenge with
the highly tumorigenic MHC class II- wild-type sarcoma SaI
(Ostrand-Rosenberg et al.,
1990
). I-Ak expression in this sarcoma enables
SaI/Ak cells to present intracellularly expressed antigens directly
to CD4+ T cells (Armstrong et
al., 1998
). By contrast, sarcoma cells transfected with
C-terminal-truncated
and ß chains (SaI/Ak tr cells)
lose their immunogenic properties and remain equally as tumorigenic as the
wild-type sarcoma (Ostrand-Rosenberg et
al., 1991
). Furthermore, single amino acid mutations in the
intracellular part of the I-Ak ß chain led to distinct
antitumoral responses (Laufer et al.,
1997
).
Recent studies have demonstrated a key role for specific microdomains of
the plasma membrane in signal transduction via MHC class II molecules
(Anderson et al., 2000;
Huby et al., 1999
;
Setterblad et al., 2001
).
These membrane microdomains, highly enriched in cholesterol and
glycosphingolipids, are proposed to function as preformed platforms essential
for sustaining immunoreceptor signaling and membrane trafficking (reviewed by
Harder and Simons, 1997
;
Simons and Ikonen, 1997
). Such
detergent-insoluble glycolipid-enriched complexes (DIGs) are also known as
glycosphingolipid-enriched membrane microdomains (GEMs), detergent-resistant
membranes (DRMs) or lipid rafts. They can be isolated based on their
insolubility in non-ionic detergent and their low buoyant density on sucrose
gradients. They preferentially recruit and concentrate specific proteins
involved in cellular signaling such as doubly acylated Src-family tyrosine
kinases, glycosylphosphatidylinositol (GPI)-anchored proteins and proteins
with saturated fatty acyl chains, while excluding others such as CD45
(Rodgers and Rose, 1996
), thus
optimizing activation and subsequent downstream signaling. Aggregation of MHC
class II molecules in the THP-1 cell line induced their recruitment to lipid
rafts and was required for tyrosine kinase activation
(Huby et al., 1999
). Ligand
binding to HLA-DR in B cells led to HLA-DR and PKC-
recruitment to DIGs
(Setterblad et al., 2001
).
Anderson et al. implicated lipid raft microdomains in antigen presentation
under conditions of low peptide concentration
(Anderson et al., 2000
). By
contrast, Kropshofer et al. proposed that antigen presentation was not
dependent on the integrity of MHC class II-containing lipid rafts but rather
was dependent upon the MHC class II molecules complexed with tetraspanin
molecules (Kropshofer et al.,
2002
).
We have determined whether I-Ak expressed in a sarcoma transmits
signals and whether the intracytoplasmic tail of I-Ak is required.
We report that the intracytoplasmic tail of I-Ak is not required
for the recruitment of I-Ak to DIGs, whereas PKC- activation
and actin rearrangement, mediated via I-Ak, are abrogated by
truncation of I-Ak. Inhibition of PKC activation and of
cytoskeletal integrity revealed that recruitment of I-Ak to lipid
rafts did not require either PKC-
activation nor actin reorganization,
but rather that these events were initiated via I-Ak localized
within lipid rafts and were prevented by disruption of lipid rafts.
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Materials and Methods |
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Cell culture
The SaI sarcoma is a methylcholanthrene-induced murine sarcoma of A/J
(H-2KkAkDd) strain mice. The SaI cell line
was maintained in culture in DMEM (Gibco-BRL, Rockville, MD) supplemented with
10% decomplemented FCS, 100 µg/ml streptomycin, 100 U/ml penicillin and 2
mM L-glutamine at 37°C in 5% CO2.
SaI cells were tri-transfected with Ak and
Aßk MHC class II cDNA plus pSV2neo vector DNA by
the calcium phosphate method (called SaI/Ak transfectants)
(Ostrand-Rosenberg et al.,
1990
). I-Ak molecules, prematurely truncated for the
C-terminal 12 and 10 amino acids, respectively, are expressed in SaI cells
(SaI/Ak tr), as previously described
(Ostrand-Rosenberg et al.,
1991
). The transfected SaI/Ak and SaI/Ak tr
tumor cells were maintained in culture in IMDM Glutamax-I (Gibco-BRL,
Rockville, MD) supplemented as described for SaI and maintained under
selection by treatment with 400 µg/ml geneticin (Gibco-BRL, Rockville, MD)
for one week per month.
Sample preparation
Cell treatment and stimulation
Cells were stimulated with either 10 µg/ml goat anti-mouse Ab (GAM,
isotype control) or 10 µg/ml 10.2.16 mAb crosslinked with 10 µg/ml GAM
Ab for different times at 37°C. DIG disruption was carried out by treating
cells with the cholesterol-sequestering agent MßCD (10 mM MßCD for
15 minutes at 37°C) prior to Ab stimulation. Neither cell viability nor
surface MHC class II expression levels were affected by this treatment. Where
indicated, SaI/Ak were pretreated at 37°C with either the
broad-spectrum PKC inhibitor calphostin C (50 nM, 30 minutes), the actin
polymerization inhibitor cytochalasin D (10 µM, 60 minutes) or an
equivalent volume of the appropriate diluent.
Preparation of total cell lysates (TCLs)
Cells (5x106) were washed in cold PBS and then lysed in
lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM
Na3VO4, 1 mM EDTA, 1 mM PMSF, 10 mM NaF, 1 µg/ml
leupeptin, 1 µg/ml pepstatin A and 4 µg/ml aprotinin) on ice for 30
minutes. Following centrifugation at 14,400 g, 4°C for 20
minutes to pellet cellular debris, nuclei and large insoluble material, the
protein concentration of the supernatants was determined using the Bradford
assay (Bio-Rad Laboratories). TCLs were immunoblotted to detect total
I-Ak expression in SaI/Ak and SaI/Ak tr.
Preparation of DIGs
DIGs were isolated as described by Xavier et al.
(Xavier et al., 1998). Cells
(30x106) were washed twice and incubated in 350 µl of
MBS-T buffer [25 mM morpholinoethanesulfonic acid (MES), 150 mM NaCl pH 6.5, 1
mM Na3VO4, 1 mM PMSF, 10 mM NaF, 1 µg/ml leupeptin, 1
µg/ml pepstatin A, 4 µg/ml aprotinin and 0.5% Triton X-100] for 30
minutes on ice. Lysates were ultracentrifuged after mixing with an equal
volume of 85% sucrose (w/v) in MBS at the bottom of a polycarbonate
ultracentrifuge tube (Beckman Instruments, Palo Alto, CA), which was then
overlaid with 2 ml of 35% sucrose followed by 1 ml of 5% sucrose in MBS
containing 1 mM Na3VO4, 2 mM EDTA, 1 mM PMSF and 1
µg/ml aprotinin. After centrifugation at 200,000 g for 18
hours at 4°C in a SW55Ti rotor (Beckman Instruments), eight fractions of
500 µl were collected from the top to the bottom of the tube. Fractions 3
and 4 correspond to the DIG-containing 5/35% sucrose interface.
Western blotting
10-15 µg of TCLs or 15 µl of each sucrose fraction were boiled in
Laemmli sample buffer and proteins were separated on a 10% SDS-PAGE (15% for
GM1 detection). After electrophoresis, the proteins were transferred onto PVDF
(AP Biotech, Buckinghamshire, UK) for 1 hour at 100 V. The membranes were
blocked for 1 hour at room temperature in PBS/0.1% Tween 20 (PBS-T) containing
5% nonfat dry milk. The blots were then probed for 1 hour with primary
antibody diluted in blocking buffer. After two washes in PBS, the blots were
incubated for 1 hour with HRP-conjugated secondary Ab. Bands were visualized
using the ECL system (AP Biotech). Arbitrary quantification of the bands
corresponding to PKC- and phospho-PKC was carried out using the NIH
Image based Scion software (Scion Corporation, Frederick, ML).
SDS-stability assay
MHC class II heterodimers are stabilized by peptide binding and resist
dissociation after incubation in SDS-detergent at room temperature. Heating to
95°C dissociates the heterodimer into free and ß chains.
Samples were incubated either at room temperature for 30 minutes or heated at
95°C for 10 minutes under reducing conditions and loaded onto a 10%
SDS-PAGE. Membranes were blocked with PBS-T/5% milk for 1 hour, washed twice
in PBS-T and incubated with 10.2.16 mAb at 1 µg/ml in PBS-T/2% milk for 1
hour. After two washes in PBS, the blots were incubated for 1 hour with
HRP-conjugated anti-mouse secondary Ab. I-Ak was detected by
electrochemiluminescence (ECL).
Flow cytometry analysis
Cells were washed once in PBS, resuspended in PBS containing 1% FCS and
0.1% sodium azide and incubated with either control isotype (mouse
IgG2b) or the I-Ak-specific mAb (10.2.16) for 15
minutes. Cells were washed in PBS/SVF/azide and then incubated with Alexa
Fluor488-conjugated goat anti-mouse Ig for 15 minutes on ice in the dark.
After washing, cells were resuspended in PBS containing 5 µg/ml propidium
iodide and analyzed by flow cytometry on a FACScan (Becton Dickinson). Dead
cells were excluded based on FSC/SSC profile and propidium iodide staining.
Histograms were constructed based on analysis of 10,000 cells.
Preparation of antibody-coated latex beads
Latex beads were coated with either irrelevant mouse Igs or 10.2.16
antibody according to the manufacturer's recommendations. 100 µg of
purified Ig was allowed to bind to 0.2 ml beads pretreated with 0.1 M borate
buffer, pH 8.5. Beads were washed and resuspended in storage buffer (PBS pH
7.4, 10 mg/ml BSA, 0.1% sodium azide, 5% glycerol) at 4°C.
Confocal microscopy
Cells were stimulated with either 10.2.16-coated beads or irrelevant mouse
Ig-coated beads for 30 minutes at 37°C at a ratio of one bead per cell.
Where indicated, cells were pretreated with either MßCD (10 mM, 15
minutes), calphostin C (50 nM, 30 minutes) or cytochalasin D (10 µM, 60
minutes) prior to stimulation. The cells were then cytospun and air-dried for
20 minutes before fixation in cold methanol for the PKC- staining or in
2.5% paraformaldehyde (PFA)/PBS followed by permeabilization with 0.1% Triton
X-100 for the actin staining. Cells fixed with PFA were incubated with 50 mM
NH4Cl in PBS. Cells were washed in PBS and labeled with primary
antibody (polyclonal anti-PKC-
Ab) or with Alexa Fluor488-labeled
phalloidin for 1 hour. FITC-conjugated anti-rabbit Ab was used to detect
PKC-
. Slides were washed in PBS and mounted with Vectashield
(VectorLab, Burlingame, CA). Images were acquired with a Zeiss LSM-510 laser
scanning confocal microscope (Carl Zeiss, Oberkochen, Germany) equipped with a
Zeiss Axiovert 100M (plan Apochromat 63X 1.40NA oil immersion objective). The
Alexa Fluor488 and FITC fluorophores were excited at 488 nm. To avoid
selection bias, a minimum of 200 randomly selected cell-bead interactions were
examined per slide. The results are expressed as percentages of cells having
undergone striking relocalization of the target protein (such as shown in Figs
4,
6).
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Results |
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Characterization of DIGs
SaI/Ak cells were lysed and fractionated on a sucrose gradient.
The GM1, caveolin and ß-tubulin content in the eight collected fractions
were analyzed by western blotting to identify the DIG fraction. GM1
ganglioside is a glycolipid localizing in DIGs that binds the ß subunit
of cholera toxin. Caveolin has been described as a DIG-resident protein,
whereas ß-tubulin typically resides outside of DIGs
(Cheng et al., 1999;
Harder and Simons, 1997
).
Fig. 2 shows the expression of
GM1 and caveolin in fractions 3 and 4, corresponding to the 5/35% sucrose
interface and therefore identifies these low-density fractions as the
DIG-containing fractions (Xavier et al.,
1998
). By contrast, ß-tubulin was exclusively expressed in
fractions 7 and 8, which correspond to fractions containing high-density
detergent-soluble material. The distribution of DIG and non-DIG markers was
identical in SaI/Ak tr and was unchanged by
I-Ak-mediated stimulation of either cell type (data not shown).
|
I-Ak engagement induces MHC class II recruitment to DIGs
independently of the intracytoplasmic domains
In order to determine whether MHC class II molecules expressed in this
sarcoma were constitutively present in DIGs, and whether such a localization
could be modified by I-Ak engagement, we screened the sucrose
gradient fractions for I-Ak molecules both in nonstimulated and
I-Ak-stimulated SaI/Ak and SaI/Ak tr
cells.
I-Ak was weakly expressed in the DIG fraction in nonstimulated
SaI/Ak cells (Fig.
3A) under boiled or non-boiled conditions, and the percentage of
DIG-associated MHC class II was estimated as 10.7%
(Fig. 3C). MHC class II was
enriched in fraction 3 following I-Ak engagement, indicating
recruitment to DIGs via I-Ak stimulation
(Fig. 3A). The recruited MHC
class II molecules were present both as compact ß dimers (55-60
kDa) and HMW complexes (>100 kDa). In boiled samples, the increased
expression of the ß chain in fraction 3 confirmed the recruitment of MHC
class II molecules to DIGs via I-Ak
(Fig. 3A). The percentage of
DIG-associated MHC class II upon I-Ak engagement was estimated as
21% (Fig. 3C). Disruption of
DIG integrity by cholesterol depletion using MßCD abrogated the
recruitment of I-Ak to DIGs
(Fig. 3A) so that only 8% of
MHC class II localized to DIGs under these conditions
(Fig. 3C). The
detergent-insoluble sediment migrating to the bottom of the gradient contained
only trace amounts of I-Ak and therefore was not further analyzed
(data not shown).
|
SaI/Ak tr cells were analyzed under identical conditions (boiled
and non-boiled). I-Ak was weakly expressed (7.3% of total MHC class
II) in the DIG fractions of nonstimulated cells
(Fig. 3B, lane 3). Recruitment
of truncated I-Ak to DIGs via I-Ak stimulation was
observed and the increased expression in fraction 3 corresponded to 22% of the
total MHC class II (Fig. 3C),
demonstrating that the truncation of the intracytoplasmic domains of the MHC
class II molecules does not impair the recruitment of compact ß
dimers to DIGs.
Intracytoplasmic domains are required for I-Ak-mediated
DIG-dependent actin reorganization
Cytoskeletal proteins have been attributed a role in the formation and
maintenance of DIGs (Ebert et al.,
2000; St Pierre and Watts, 1991). We therefore examined the effect
of I-Ak-mediated signals on the actin cytoskeleton. We stimulated
SaI/Ak and SaI/Ak tr cells with beads coated with
anti-I-Ak mAb and directly examined polymerized actin (F-actin) by
staining with phalloidin. In SaI/Ak cells, we observed
rearrangements of the actin cytoskeleton revealed by the recruitment of actin
to the precise site of I-Ak engagement
(Fig. 4A, panel d; 62% of cells
with actin relocalization). By contrast, stimulation via truncated
I-Ak molecules failed to induce reorganization of the actin
cytoskeleton since the distribution of actin was unchanged compared with cells
that had been allowed to interact with beads coated with irrelevant mouse Igs
(Fig. 4A, panel h versus j; 5%
and 5.7% of cells with actin relocalization, respectively). The specificity of
the I-Ak-mediated signaling was further confirmed by the absence of
actin rearrangement when beads coated with irrelevant mouse Igs were allowed
to interact with SaI/Ak (Fig.
4A, panel b; 11% of cells with actin relocalization). In addition,
disruption of DIG integrity by MßCD pretreatment of SaI/Ak
cells prior to I-Ak stimulation impaired actin reorganization,
indicating that this event occurs in a DIG-dependent manner
(Fig. 4A, panel f; 15.3% of
cells with actin relocalization).
We next examined the impact of I-Ak signaling on the actin content of DIG fractions. Actin was constitutively present in the cholesterol-dependent lipid-rich fractions from SaI/Ak cells and was unchanged by stimulation via I-Ak (data not shown).
PKC- is recruited to DIGs in response to I-Ak
stimulation
The PKC isoenzyme family mediates signals via MHC class II
(Brick-Ghannam et al., 1991;
Cambier et al., 1987
;
Rich et al., 1997
).
SaI/Ak and SaI/Ak tr cells were screened for the
expression of four PKC isoforms (
, ßII,
, µ). In both
cell lines, PKC-
,
and µ were readily detected, whereas
PKC-ßII was absent (data not shown). We therefore studied the
distribution of the
,
and µ PKC isoenzymes in DIGs. We found
that only PKC-
was constitutively present in the DIG fractions from
SaI/Ak cells and was actively recruited to this fraction by
I-Ak stimulation (Fig.
5A). By contrast, while PKC-
was present in the DIG
fraction of SaI/Ak tr, stimulation via I-Ak tr molecules
did not lead to PKC-
recruitment. Neither PKC-
nor PKC-µ were
detected in the lipid-rich fractions of either SaI/Ak or
SaI/Ak tr (Fig. 5A). DIG disruption by cholesterol depletion with MßCD reduced the
constitutive expression and prevented the recruitment of PKC-
to DIGs
of SaI/Ak cells (Fig.
5B). These data demonstrate that I-Ak signaling
specifically relocalizes PKC-
to DIGs in SaI/Ak.
|
DIG-dependent PKC- translocation to the site of
I-Ak engagement requires the intracytoplasmic domains
Confocal microscopy was used to directly examine the impact of the
I-Ak signal on PKC- localization in SaI/Ak and
SaI/Ak tr cells. PKC-
was specifically relocalized to the
site of I-Ak engagement in comparison with the diffuse localization
observed in nonstimulated cells (Fig.
6A, panel b and d; 8.6% and 63.6% of cells with PKC-
translocation, respectively). By contrast, stimulation of SaI/Ak tr
did not modify the distribution of PKC-
in comparison with that
observed in nonstimulated cells (Fig.
6A, panel h and j; 3.4% and 5.5% of cells with PKC-
translocation, respectively), demonstrating that the intracytoplasmic tails of
the I-Ak molecule are required for PKC-
translocation to the
site of I-Ak stimulation. Disruption of DIG integrity by MßCD
pretreatment of SaI/Ak cells prior to I-Ak stimulation
impaired PKC-
translocation, indicating that this event is DIG
dependent (Fig. 6A, panel f;
11.4% of cells with PKC-
translocation).
DIG-recruited PKC- is activated in response to I-Ak
stimulation
Activation of PKC can be revealed by phosphorylation of a C-terminal
residue (Keranen et al.,
1995). We therefore used an Ab directed against PKC phosphorylated
at this site to detect PKC activation. Phosphorylated PKC in DIG fractions
from SaI/Ak and SaI/Ak tr cells corresponded to a single
band with a molecular weight identical to that of PKC-
, and was
increased in DIGs only from SaI/Ak cells as a consequence of
I-Ak stimulation (Fig.
7A).
|
A time course analysis of PKC- recruitment and PKC phosphorylation
was performed on DIG fractions from SaI/Ak cells. I-Ak
recruitment was characterized in parallel on the same extracts in order to
compare the time course of recruitment. Constitutively DIG-localized
PKC-
was enriched via I-Ak engagement from 1 minute up to at
least 30 minutes as confirmed by densitometry
(Fig. 7B). PKC phosphorylation
in the DIG fractions increased following the same kinetics as PKC-
recruitment to the DIGs. However, the accumulation of I-Ak in DIGs
was visible within 1 minute of stimulation and precedes both PKC-
accumulation and PKC activation (Fig.
7B). Phospho-PKC expression was examined in parallel in the
soluble fractions and was not increased. This demonstrates that the enhanced
expression of phospho-PKC in DIGs is not due to an overall increase in
phospho-PKC expression.
MHC class II partitioning to DIGs is independent of MHC class II
signaling and does not require integrity of the actin cytoskeleton
Some of the earliest events following MHC class II oligomerization include
PKC activation (Cambier et al.,
1987) and cytoskeletal reorganization. Disruption of the actin
network has been previously shown to inhibit such signaling (St Pierre and
Watts, 1991). We examined whether signal transduction was required for
recruitment of I-Ak to DIGs. Pretreatment of SaI/Ak
cells with the inhibitor of actin polymerization cytochalasin D, and the
broad-spectrum PKC inhibitor calphostin C, did not affect recruitment of
I-Ak to DIGs induced by I-Ak engagement
(Fig. 8A).
|
The disruption of the cytoskeletal integrity and the inhibition of PKC
activation was demonstrated by confocal microscopy. Actin recruitment and
PKC- translocation induced via I-Ak were severely impaired
by cytochalasin D and by calphostin C pretreatments, respectively
(Fig. 8B,C). Taken together
with the relocalization of I-Ak tr to DIGs following engagement by
anti-I-Ak mAb (Fig.
3), these data confirm that the recruitment of MHC class II
molecules to DIGs is independent of MHC class II-mediated signaling.
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Discussion |
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Expression of I-Ak in the murine SaI sarcoma was shown to be
necessary and sufficient to provoke protection against subsequent inoculation
with the wild-type tumor
(Ostrand-Rosenberg et al.,
1990). The arguments supporting a role for I-Ak
signaling in the tumor rejection response are as follows: (1) I-Ak
molecules truncated in the intracytoplasmic tail do not provide protection
against tumor inoculation
(Ostrand-Rosenberg et al.,
1991
); (2) full-length I-Ak is required for expression
of costimulatory molecules by SaI/Ak in vivo
(Ostrand-Rosenberg et al.,
1996
); and (3) SaI/Ak presents cytosolically expressed
antigens and therefore potentially presents cytosolic tumor antigens
(Armstrong et al., 1997
;
Armstrong et al., 1998
;
Qi et al., 2000
).
The present data demonstrate that, in addition to its described role in
antigen presentation (Armstrong et al.,
1997; Armstrong et al.,
1998
), the MHC class II molecule I-Ak in a murine
sarcoma acts as a signal-transducing molecule. I-Ak oligomerization
leads to recruitment of MHC class II molecules and PKC-
to DIGs, as
well as to PKC-
activation and reorganization of the actin cytoskeleton
in a DIG-dependent manner. The proportion of the total cellular protein that
localizes to lipid-rich microdomains has been estimated at
2.5%
[(Drevot et al., 2002
) and our
personal observations]. As such, the recruitment of a given protein to
lipid-rich microdomains actually represents a major partitioning to such
microdomains. We have quantified the proportion of I-Ak localizing
in lipid rafts post-ligand binding as 20%; this figure is consistent with
estimations made for diverse immunoreceptors
(Anderson et al., 2000
;
Chung et al., 2001
;
Drevot et al., 2002
). The
mechanism of limiting the abundance of a given protein within lipid-rich
microdomains has not yet been clarified.
Data from previous studies indicate that signal transduction and antigen
presentation via MHC class II molecules can be intimately associated. Nabavi
et al. reported that truncation of MHC class II molecules severely impaired
PKC activation in B cells and abrogated antigen presentation to certain T-cell
clones (Nabavi et al., 1989).
The role of the intracytoplasmic tail in signaling via MHC class II molecules
was revealed by several studies (Harton et
al., 1995
; Rich et al.,
1997
). Truncation of the intracytoplasmic domain of the ß
chain of I-Ab abrogated cAMP elevation and antigen-dependent Ig
production (Harton et al.,
1995
). Truncation of the ß chain of HLA-DR impaired
activation of PKC-
and -ßII
(Rich et al., 1997
). The
present study demonstrates for the first time that, although the
intracytoplasmic domains of the MHC class II molecules are not required for
this recruitment to DIGs, they are essential for further PKC-
recruitment to, and activation within, lipid rafts. Activated PKC recruitment
to cholesterol-dependent DIGs is believed to be due to the localization of
major PKC substrates within such microdomains. GAP 43, myristoylated C kinase
substrates (MARCKS) and CAP 28, collectively termed GMC, are membrane
associated by acylation (myristoylation or palmitoylation) and are enriched in
Triton X-100-insoluble lipid rafts (Laux
et al., 2000
). The data presented here shows that signaling is not
required for the initial recruitment of I-Ak to lipid microdomains
and that signaling commences within DIGs.
The cytoskeletal integrity of the APC has been shown to be important for
the efficiency of antigen presentation
(Barois et al., 1998;
St-Pierre and Watts, 1991
).
Treatment of the APC with cytochalasin A (which impairs elongation of actin
filaments and receptor capping) completely inhibited MHC class II-mediated
activation of certain T-cell hybridomas (St Pierre and Watts, 1991).
Cytoskeletal reorganization in the APC was therefore proposed to be important
for the activation of T cells requiring signals from accessory molecules. This
notion is strongly reinforced by the data presented in this study, which
demonstrate that the MHC class II-mediated signal specifically reorganizes the
APC actin cytoskeleton at the site of I-Ak engagement. Furthermore,
lipid raft disruption experiments clearly revealed the DIG dependence of the
actin cytoskeleton reorganization. This dynamic reorganization of F-actin at
the site of I-Ak stimulation reveals a qualitative modification
that was independent of the actin content of DIGs.
MHC class II molecules are generally considered as 60 kDa heterodimers that
can be observed under different conformations including: (1) HMW multimers
(Roucard et al., 1996); (2)
compact dimers, which have bound peptide of high affinity; (3) floppy dimers,
which are believed to have `loosely' bound peptide
(Sadegh-Nasseri and Germain,
1991
); and (4) empty MHC class II molecules
(Ericson et al., 1994
;
Roucard et al., 1996
;
Santambrogio et al., 1999
). It
was therefore important to determine the conformation of the MHC class II
molecules recruited to DIGs. The present study reveals that engagement of
either I-Ak or I-Ak tr resulted in recruitment of
SDS-stable MHC class II molecules to DIGs, both as HMW complexes and as
dimers. This model does not allow us to determine whether one or other
conformation of I-Ak is preferentially recruited to DIGs since the
10.2.16 Ab recognizes both. However, the difference in the signaling response
was not therefore due to the absence of one or other form from
SaI/Ak tr.
Anderson et al. reported that DIG disruption impaired antigen presentation
under conditions of limited peptide availability
(Anderson et al., 2000),
whereas Kropshofer et al. did not find that disruption of lipid-rich
microdomains perturbed antigen presentation
(Kropshofer et al., 2002
).
However, neither of the previous studies examined the impact of DIG
localization on the signaling function of MHC class II. A previous study that
did address this point focused on the recruitment and activation of tyrosine
kinases after Ab-mediated recruitment of MHC class II to lipid rafts in
myeloid APCs (Huby et al.,
1999
). Our data show that the immediate recruitment of MHC class
II to PKC-
-containing DIGs ensures that signaling can be readily
triggered. Inhibition of PKC activation completely abrogated recruitment of
PKC-
to the site of I-Ak engagement without perturbing
enrichment of I-Ak in the lipid rafts. Signaling is therefore not
required for the relocalization of MHC class II to lipid rafts, as our data
indicate that it is the localization of the MHC class II molecules in lipid
rafts that triggers signaling. This argument is supported by the lack of
impact of I-Ak truncation on the I-Ak recruitment to the
lipid raft fraction and is similar to the observation that signaling via the
B-cell receptor (BCR) originates within lipid microdomains rather than is a
prerequisite for BCR relocalization to DIGs
(Cheng et al., 2001
).
Distribution of MHC class II in lipid-rich microdomains has, as yet, only
been described in professional APCs including monocytes, B cells and dendritic
cells (Anderson et al., 2000;
Huby et al., 1999
;
Setterblad et al., 2001
). A
recent study reported the accumulation of MHC class II-peptide complexes in
the contact zone formed by APCT-cell interaction initiated by an
agonist MHC class II-peptide complex
(Wulfing et al., 2002
). The
accumulated complexes included non-agonist MHC class II-peptide complexes that
were entitled `accessory ligands' since they were shown to enhance specific
T-cell activation. In addition, lipid-rich microdomains were shown to localize
to the site of APCT-cell interaction
(Bi et al., 2001
). In the
present tumor cell model, enrichment of I-Ak molecules in DIGs
mediated by I-Ak engagement could reflect an accumulation of
`accessory ligands'. The initiation of intracellular signaling in the sarcoma
as a direct consequence of I-Ak recruitment to DIGs could therefore
actively contribute to the antitumoral immune response. Furthermore, MHC class
II mAbs are currently under study as therapeutic tools for the treatment of
lymphoid malignancies (Nagy et al.,
2002
). The data presented in the current study reveal that the MHC
class II antigens should also be considered as potential targets for mAb
therapy of solid tumors.
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References |
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