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INTRODUCTION |
Binding of antigens
(Ag)1 to the B cell receptor
(BCR) not only triggers B cell activation but also facilitates Ag
presentation. BCR-mediated Ag uptake allows presentation at much lower
Ag concentrations than fluid phase endocytosis (1). This is because of
targeting of BCR-bound Ag to specialized endocytic compartments wherein Ag processing can take place (for review see Ref. 2). BCR-Ag complexes
first enter early endosomes and are targeted to late endocytic compartments where they are degraded in peptides (3, 4).
These peptides are then loaded onto MHC class II and the complexes are
displayed at the surface of Ag presenting cells (2, 5, 6).
Peptide loading occurs mostly in late endocytic compartments, called
the MHC class II-enriched compartment, an endocytic multivesicular
and/or multilaminar structure, which bears markers of late endosome
(7-9). We have recently shown in murine B cells that BCR engagement
dynamically induces the formation of an Ag-processing compartment
presenting with these characteristics (10).
Several studies have shown that BCR-dependent Ag
presentation is signaling dependent (11, 12). BCR is a multimeric
receptor complex in which an Ag-recognition subunit, membrane-bound Ig, is noncovalently associated with one heterodimer of Ig
and Ig
(13), which assures its signaling capacity (14). Accelerated Ag
targeting to Ag-processing compartments by the BCR seems to be
dependent on Ig
/Ig
because mutants of Ig that do not bind to
Ig
/Ig
were unable to target Ag for MHC class II presentation (15,
16). Moreover we have shown that Ig
and Ig
are both able to
increase the efficiency of Ag presentation (17) although they differ in
their endocytic traffic (18).
Signaling pathways induced after BCR triggering have been widely
investigated, however, the distinct contribution of these pathways to
BCR-induced Ag presentation are still poorly characterized. One of the
pathways activated after BCR activation involves phosphoinositide 3-kinase (PI3K) (19). PI3K activation by the BCR leads to activation of
the serine/threonine kinase Akt (20, 21) and regulates membrane
association and function of the Bruton's tyrosine kinase (22). Recent
studies have shown the key role of PI3K in B cells, disruption of its
expression by genetic deletion results in impaired B cell
development (23). In other cellular models, PI3K has been shown to
control vesicular transport of several proteins in early and late
endocytic compartments (24, 25), endosomes fusion (26), and
morphogenesis of multivesicular bodies (MVB) (27, 28).
We herein report that PI3K inhibitors block Ag presentation induced by
the BCR, Ig
but not Ig
and that this blockage is linked to
inhibition of de novo formation of a multivesicular Ag
processing compartment induced by BCR and Ig
triggering.
Furthermore, using fluorescent probes for PI3K products, we demonstrate
that PI3K is activated along the Ag endocytic journey and that Ig
is
the chain that couples BCR to this PI3K activation. Altogether these
data strongly suggest that Ig
-dependent PI3K activation creates the lipidic environment necessary for neogenesis of an endocytic compartment where Ag processing can efficiently take place.
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MATERIALS AND METHODS |
Chemicals and Antibodies--
RPMI 1640 with glutamax, PBS,
penicillin, streptomycin, sodium pyruvate, 2-mercaptoethanol, Tris,
glycine, and G418 were purchased from Invitrogen; LY 294002 from
Calbiochem; and Pronase from Roche Molecular Biochemicals. Other
chemicals were from Sigma.
The following Abs were used: 2.4G2 (rat IgG) an anti-mouse FcR mAb; a
polyclonal rabbit anti-phospho-Ser-AKT Ab and a polyclonal mouse
anti-phospho-mitogen-activated protein kinase Ab (Cell Signaling); a
polyclonal goat anti-ERK2 (Santa Cruz Biotechnology); a polyclonal rabbit anti-HRP Ab (ICN); 1D4B an anti-mouse Lamp1 mAb, native and biotinylated rat anti-mouse IL-2 mAb (BD Pharmingen),
F(ab')2 fragments of fluorescein isothiocyanate-coupled
donkey anti-rat IgG Ab and donkey anti-rabbit (Immunotech); a
biotinylated rat anti-H2-M (a kind gift from L. Karlsson); a
polyclonal rabbit anti-mouse IgG F(ab')2 (ICN) was
biotinylated following the manufacturer's protocol (Biotin, Pierce);
and streptavidin Alexa 594 was from Molecular Probes.
Cell Culture--
The following cell lines have been used and
previously described (11). IIA1.6, a Fc
R-defective variant of the
A20 B lymphoma cells has been transfected with constructs encoding the
cytoplasmic domain of the Ig
or Ig
subunits linked to the
extracellular and transmembrane domains of the mouse Fc
RII,
respectively, Fc
R-Ig
and Fc
R-Ig
, as described in Ref. 29.
Anti-TNP A20 cells were obtained by transfection of genomic clones
encoding the light and heavy µ chain of the BCR specific for TNP
(11). CD4+ T cell hybridoma 24.4 recognizes the 12-26
peptide derived from the
C1 repressor complexed to I-Ad
class II molecules (11). All cell lines were grown in RPMI glutamax,
10% fetal calf serum (Dutcher), 5 mM sodium pyruvate, 50 µM 2-mercaptoethanol, 100 units/ml penicillin, 100 µg/ml streptomycin.
Internalization and Degradation--
Anti-TNP A20 cells (2 × 106 cells/point) were washed in internalization buffer
(RPMI, 5% fetal calf serum, 20 mM Hepes, pH 7.4), treated
20 min at 37 °C with wortmannin or Me2SO (vehicle), incubated on ice for 1 h with 4 µg/ml iodinated TNP-BSA, washed, and incubated for various time at 37 °C in the presence of
wortmannin. Cells were either submitted to trichloroacetic acid
precipitation (20% final) to study degradation, or incubated in
internalization buffer containing 10 mg/ml Pronase at 4 °C overnight
to remove surface bound radioactivity. Radioactivity present in half of the supernatant was counted and percentage of internalized or degraded
Ag was calculated. Degradation assay by Fc
R-Ig
-expressing cells was performed with the same protocol, except that cells were
incubated on ice 2 h with 10 µg/ml iodinated TNP-BSA
precomplexed with 20 µg/ml anti-DNP Abs.
Internalization assay by chimera was performed as previously described
(17). Fc
R-Ig
cells were washed in internalization buffer, treated
20 min at 37 °C with wortmannin or Me2SO, incubated on
ice for 1 h with preformed immune complexes against HRP (IC-HRP, 10 µg/ml HRP and 400 µg/ml anti-HRP rabbit polyclonal), washed, and
incubated for various times at 37 °C. Internalization was stopped by
addition of cold PBS. After washing, half of the cells were lysed with
PBS, 1% Triton X-100 for 5 min at room temperature, and transferred on
ice, the other half was kept on ice in PBS. Cell surface and total HRP
activity were measured by addition of cold OPD substrate buffer.
Antigen Presentation--
Ag presentation was assessed as
previously described (11) with the following modifications. A fragment
of the recombinant C1
repressor (from amino acid 1 to 102) was
produced, it was either complexed with mAbs anti-C1
repressor 22D
and 51F for studies on Fc
R-chimera, or TNP-coupled for studies on
BCR, as previously described (11). B cells were incubated 20 min at 37 °C with 200 nM wortmannin, 10 µM
LY294002 or Me2SO before addition of Ags. After different
times of incubation with the Ags, B cells were fixed 5 min on ice with
0.05% glutaraldehyde. T cell hybridoma were then incubated for 20 h with the Ag-pulsed B lymphoma and IL-2 production in the supernatant
was measured by enzyme-linked immunosorbent assay, according to the
manufacturer's protocol (BD Pharmingen). TMB substrate (BD Pharmingen)
was used for revelation. All experiments were performed in triplicates.
Western Blot Analysis--
Cells were washed in culture medium
without fetal calf serum, and incubated with wortmannin, LY294002, or
Me2SO for 20 min prior to Ag stimulation. Ag used for
stimulation was either TNP coupled to ovalbumin or immune complexes
against C1 (IC-C1) preformed at 37 °C. Cells were harvested and
lysed in lysis buffer (20 mM Tris, pH 7.4, 140 mM NaCl, 0.5% Nonidet P-40, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 100 µM
Na3VO4, 1% aprotinin, 2 µg/ml antipain,
pepstatin, leupeptin) for 20 min at 4 °C. Equal amounts of
postnuclear cell lysates were analyzed under reducing conditions by
SDS-PAGE and electroblotted on Immobilon P membrane (Millipore). The
antibody-Ag complexes were visualized by an enhanced chemiluminescence
detection system according to the manufacturer's instructions (ECL,
Amersham Biosciences).
Immunofluorescence and Confocal Microscopy--
To analyze PI3K
products, cells were transiently transfected with a plasmid encoding a
chimera of the Akt PH domain coupled to GFP (kind gift from T. Balla) and used for immunofluorescence 24 h after. Cells
were washed in PBS and put on 0.2% poly-L-lysine-coated glass
coverslips. After washing in internalization buffer, cells were
pretreated with PI3K inhibitors for 20 min. Anti-TNP A20 B cells were
incubated on ice for 1 h with OVA coupled to Cy3 and TNP.
Fc
R-chimera were incubated on ice for 1 h with preformed IC
containing HRP-rhodamine (10 µg/ml) and anti-HRP rabbit polyclonal Ig
(400 µg/ml) or biotinylated rabbit anti-mouse IgG F(ab')2
(ICN) precomplexed with streptavidin Alexa 594 (Molecular Probes).
Cells were washed, incubated at 37 °C for 30 min, washed in cold
PBS, fixed in 3% paraformaldehyde for 30 min, and washed with PBS, 20 mM glycine. After permeabilization with 0.05% saponin in
PBS supplemented with 0.2% BSA, the cells were incubated with 2.5 µg/ml anti-CD107a Ab for 45 min, washed, and incubated with 2.5 µg/ml F(ab')2 fragments of fluorescein isothiocyanate-coupled donkey
anti-rat IgG Ab for 45 min. Double immunofluorescence analysis were
performed using a Leica TCS SP2 confocal laser scanning microscope.
Immunoelectron Microscopy--
Cells were pretreated with
wortmannin or Me2SO, coated 1 h on ice with ×10
preformed IC-HRP, washed, and then incubated for 30 min at 37 °C.
Cells were fixed for 2 h at room temperature with 2%
paraformaldehyde in 0.2 M phosphate buffer, pH 7.4. Fixed cells were processed for ultrathin cryosectioning and immunogold labeling as described previously (30). For single MHC II labeling, sections were indirectly immunogold-labeled with the rat mAb anti-I-A M5114, followed by a rabbit anti-rat Ab (Dako, Denmark), for double MHC
II and Ii labeling, sections were then labeled with a rabbit serum
anti-Ii chain (IiNH2, a kind gift from J. Davoust). Double labeling for Lamp1 and MHC II was performed by incubating ultrathin cryosections with a rat monoclonal anti-Lamp1 Ab and a
biotinylated-M5114. These Abs were followed by a rabbit anti-rat Ab and
a rabbit anti-biotin Ab (Biovalley, Marne la Vallée,
France), respectively. Bound antibodies were detected with protein A
coupled to 10- and 15-nm gold particles (purchased from J. W. Slot, Utrecht University, Utrecht, The Netherlands). The sections were
contrasted, embedded in a mixture of 2% methylcellulose and 4% uranyl
acetate, and viewed with a CM120 Twin Phillips electron microscope.
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RESULTS |
Inhibitors of PI3K Block BCR-mediated Ag Presentation--
We
first verified that BCR triggering induces PI3K activation in our
model. Anti-TNP A20 B lymphoma cells were treated with two PI3K
inhibitors, wortmannin and LY294002, for 20 min, and activated with
TNP-coupled ovalbumin. BCR stimulation induced PI3K activation, as
shown by the phosphorylation of Akt on serine 473 (Fig.
1A). Both inhibitors
abolished Akt phosphorylation (31), they also impaired
BCR- induced mitogen-activated protein kinase activation as
previously shown (32). The inhibitors used in this study were carefully
titrated to ensure that they neither affected the surface expression of
BCR and MHC class II molecules, nor induced apoptosis of A20 B cells
(as shown by annexin V labeling, data not shown). For this reason the
concentrations of wortmannin and LY294002 used did not completely
inhibit Akt phosphorylation after 2 h of treatment.

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Fig. 1.
BCR-mediated Ag presentation depends on
PI3K. Anti-TNP A20 B cells were preincubated for 20 min at
37 °C with 0.1% Me2SO (dmso)
(control), 10 µM LY294002, or 200 nM wortmannin. A, Western blot analysis showing
phospho-Akt in total lysates from B cells stimulated with 10 µg/ml
TNP-OVA. Equal amounts of proteins were present in each lane as shown
with anti-ERK2 Abs. B and C, anti-TNP A20 B cells
were incubated with various concentrations of TNP-C1 (B) 6 µg/ml (C), or 10 µg/ml C1 peptide 12-26. Kinetic
studies (C) were performed by adding the Ag sequentially
every 30 min. After 2 h at 37 °C, cells were fixed and
incubated with the T cell hybridoma 24.4. Secreted IL-2 was measured by
enzyme-linked immunosorbent assay. D, Ag presentation was
assessed using the same protocol with uncoupled C1. Data are
representative of three independent experiments.
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We then studied the effect of PI3K inhibitors on BCR-induced Ag
presentation by TNP A20 B cells. Cells were pretreated for 20 min with
200 nM wortmannin, 10 µM LY294002, or vehicle
(Me2SO) alone and incubated for 2 h with different
concentrations of the 1-102 fragment of the
c1 repressor coupled to
TNP. B cells were then fixed, washed thoroughly, and incubated with the
24.4 T cell hybridoma, which recognizes peptide 12-26 from the
c1
repressor. IL-2 production by the T cell hybridoma was quantified in
the supernatants. As shown in Fig. 1B, TNP A20 B cells
incubated in the presence of either wortmannin or LY294002 displayed a
50% reduction in IL-2 production at all concentrations of Ag tested. A
kinetic analysis of BCR-mediated Ag presentation confirmed that PI3K
inhibitors diminished Ag presentation at all time points tested
(Fig. 1C).
Remarkably, this inhibition of Ag presentation by PI3K
inhibitors was selective for BCR-internalized Ag. Indeed, PI3K
inhibitors did not inhibit presentation of synthetic peptides 12-26 of
the
c1 repressor (Fig. 1, B and C). Moreover
LY294002 (Fig. 1D) and wortmannin (data not shown) did not
inhibit the fluid phase-mediated Ag presentation of the 1-102 fragment
of
c1 repressor when not coupled to TNP, notably this presentation
required 10-fold more
c1 repressor than the BCR-mediated Ag
presentation. We conclude that PI3K is specifically required for
presentation of Ag internalized via the BCR.
PI3K Controls Ig
- and Not Ig
-mediated Ag
Presentation--
Our results suggest that PI3K activity controls
BCR-mediated Ag processing. We and others have demonstrated that Ig
and Ig
have distinct signaling functions inside the BCR complex (29, 33). However, both are able to induce Ag presentation (17). We
therefore decided to examine the role of PI3K in Ig
- and
Ig
-mediated Ag presentation. To address this question we used an A20
IIA1.6-derived B cell line that expresses chimeric receptors consisting
of the extracellular and transmembrane domains of the mouse Fc
RII
coupled to the intracytoplasmic domain of Ig
or Ig
(29). We first assessed PI3K activation of both chimeras by measuring Akt
phosphorylation on serine 473. Chimera-expressing cells were incubated
with ICs consisting of
c1 repressor precomplexed with two
mAbs against the
c1 repressor. As shown in Fig.
2A, Akt was constitutively phosphorylated in cells expressing Fc
R-Ig
, this basal
phosphorylation was inhibited by pretreatment of the cells with
LY294002 or wortmannin. Cross-linking of Ig
did not induce any
increase of Akt phosphorylation, however, it induced mitogen-activated
protein kinase phosphorylation (Fig. 2A) showing the
functionality of this chimera. In contrast, cross-linking of the
Fc
R-Ig
chimera induced PI3K activity, as reflected by an
increased phosphorylation of Akt, which was inhibited by pretreatment
with LY294002 or wortmannin (Fig. 2B). This is the first
demonstration that PI3K is specifically activated by the Ig
chain.

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Fig. 2.
Ig -mediated Ag
presentation is PI3K-dependent,
Ig -mediated Ag presentation is not. Cells
were treated with Me2SO (dmso) (control),
LY294002, or wortmannin. A and B, Fc R-Ig
(A) and Fc R-Ig (B) expressing cells were
stimulated with pre-formed immune complexes of C1 for different times.
Cell lysates were assessed for phospho-AKT, phospho-mitogen-activated
protein kinase, and ERK2. C-F, Fc R-Ig (C
and D) or Fc R-Ig (E and F)
expressing cells were incubated with pre-formed IC of C1 at various
concentrations (C and E) and 1.2 µg/ml C1
complexed to IgG (D and F), or 10 µg/ml C1
peptide 12-26. Kinetic studies (D and F) were
performed by adding pre-formed IC sequentially every 30 min. Cells were
fixed, incubated with T cell hybridoma 24.4, and secreted IL-2 was
measured. Data are representative of three independent
experiments.
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We then studied the effect of PI3K inhibitors on Ig
- and
Ig
-induced Ag presentation. B cells expressing Fc
R-Ig
and
Ig
chimera were pretreated for 20 min with 10 µM
LY294002, 200 nM wortmannin, or vehicle (Me2SO)
alone. Ag presentation was measured by incubating the
chimera-expressing cells with several doses of
c1 repressor
precomplexed with two mAbs directed against the
c1 repressor. B
cells were then fixed, washed, incubated with the 24.4 T cell
hybridoma, and IL-2 production quantified in the supernatants. As shown
in Fig. 2C, PI3K inhibitors did not inhibit
Fc
R-Ig
-induced Ag presentation. A kinetic analysis of the
Ig
-mediated Ag presentation confirmed that PI3K did not play a
significant role in Ig
-mediated Ag presentation (Fig. 2E). In contrast, both PI3K inhibitors inhibited by 50%
Fc
R-Ig
-mediated Ag presentation (Fig. 2D). The role of
PI3K was confirmed in a kinetic analysis of Ig
-mediated Ag
presentation (Fig. 2F for LY294002, and data not shown for
wortmannin). Presentation of synthetic peptides 12-26 of the
c1
repressor by this B cell line was not significantly modified by
incubation with either LY294002 or wortmannin (Figs. 2, D
and F). We also verified that both inhibitors did not affect
the surface expression of chimera and MHC class II molecules (data not
shown). Altogether, these results demonstrate that PI3K is activated
specifically by Ig
and that Ig
-mediated Ag processing depends on
this PI3K activation.
PI3K Is Not Required for BCR-mediated Ag Internalization or
Degradation--
At which step of antigen presentation is PI3K
required? To address this question we monitored the ability of B cells
to internalize Ag in the presence or absence of PI3K inhibitors.
BCR-induced Ag internalization was followed in anti-TNP A20 cells using
iodinated TNP-coupled BSA. Cells were incubated at 4 °C with the
labeled Ag, washed, and further incubated at 37 °C for different
times. The internalized fraction was then determined. As shown in Fig. 3A, preincubation with the
different PI3K inhibitors did not alter Ag internalization. Equivalent
results were obtained when analyzing the internalization of pre-formed
ICs of HRP/anti-HRP Abs in Fc
R-Ig
expressing B cells (Fig.
3C). We conclude that PI3K activity is not required for
BCR-mediated Ag internalization.

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Fig. 3.
PI3K activity is not required for Ag
internalization or degradation. Cells were pretreated with 0.01%
Me2SO (dmso) (control) or 100 nM wortmannin for 20 min. A, anti-TNP A20 cells
were incubated with 125I-labeled TNP-BSA for 1 h at
4 °C, washed, and incubated at 37 °C. The internalized fraction
was calculated after overnight Pronase treatment of the cells.
B, Fc R-Ig chimera-expressing cells were coated with
IC-HRP Abs for 1 h on ice, washed, and incubated at
37 °C. Total and surface HRP activity was measured. C and
D, anti-TNP A20 B cells were treated with
125I-labeled TNP-BSA as in A, and Fc R-Ig
chimera-expressing cells with 125I-labeled TNP-BSA
precomplexed with anti-DNP Abs. Undegraded Ag was precipitated with
trichloroacetic acid. Radioactivity of degraded Ag in the supernatant
and trichloroacetic acid-precipitable undegraded Ag in the pellet was
determined. Data are representative of two or more independent
experiments.
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Do PI3K inhibitors alter Ag degradation? Internalized Ag appeared to be
degraded at normal rates in TNP A20 B cells treated by the PI3K
inhibitors, as determined by measuring the release of trichloroacetic
acid-soluble 125I into the medium after uptake of iodinated
TNP-coupled BSA (Fig. 3B). For Fc
R-Ig
expressing B
cell lines we incubated the cells with iodinated TNP-coupled BSA
precomplexed with anti-DNP Abs. Preincubation of the Fc
R-Ig
expressing cells with wortmannin inhibited Ag degradation at early
points (30 and 60 min), however, at 2 h this inhibition was barely
detectable although as shown previously in Fig. 2, Ag presentation was
still inhibited by 50%. BCR-induced Ag degradation therefore occurs
independently of PI3K activity.
PI3K Is Required for Ag Access to Lamp1+
Compartments--
Recent data from our group (10) and others (34, 35)
have shown that Ag processing compartments are dynamically modified upon BCR activation. BCR stimulation induced the transient accumulation of MHC class II molecules in newly formed Lamp1+ MVBs that
contain the Ag, and to which H2-M is recruited (10). Is PI3K activity
required for formation of such compartments? To address this question
Ag-stimulated cells were analyzed by immunofluorescence in the presence
and absence of PI3K inhibitors. Unstimulated TNP A20 cells and
Fc
R-Ig
expressing cells showed multiple small Lamp1+
vesicles (Fig. 4A,
green) distributed throughout the cytoplasm. Following
ligation of the BCR with TNP-OVA these vesicles were remarkably
redistributed and by 30 min formed a single large perinuclear aggregate
in most of the cells. Equivalent results were obtained after Ig
ligation, using preformed ICs of rhodamine-coupled-HRP/anti-HRP Abs
(Fig. 4). Endocytosed Ag fully co-distributed with Lamp1 in these
aggregates in 82% of TNP A20 cells and 70% of Fc
R-Ig
-expressing cells. As shown in Fig. 4C, Ig
-internalized Ags were also
found in H2-M positive compartments. Of note, redistribution of
Lamp1+ vesicles and targeting of Ag to the Lamp1
compartment were rarely observed when activating
Fc
R-Ig
-expressing cells (Ag fully co-distributed with Lamp1 only
in 15% cells, data not shown).

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Fig. 4.
PI3K plays a role in Ag access to
Lamp1+compartments. Anti-TNP A20 B cells
(A) and Fc R-Ig (B and
C)-expressing cells on polylysine-coated coverslips were
treated with 0.01% Me2SO (control) or 200 nM wortmannin, incubated 1 h on ice with TNP-OVA
coupled to Cy3 (A) or with rhodamine-coupled HRP complexed
with anti-HRP Abs (B and C). After washing cells
were incubated for 30 min at 37 °C, fixed, and labeled with a rat
anti-Lamp1 mAb followed by fluorescein isothiocyanate-coupled anti-rat
Abs (A and B). Left panel shows the
distribution of Lamp1 in untreated and unstimulated cells. Fc R-Ig
chimera-expressing cells were labeled with biotinylated anti-H2-M Abs
followed by Alexa 488-coupled streptavidin (C).
Me2SO and wortmannin-treated cells (+) are shown,
respectively, in the upper and lower rows.
Typical results were obtained from three independent experiments or
more.
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Pretreatment of B cells with 200 nM wortmannin prevented
both BCR and Fc
R-Ig
-induced redistribution of Lamp1+
vesicles in large perinuclear aggregates. This pretreatment also reduced the co-distribution of BCR or Fc
R-Ig
internalized Ag with
Lamp1 (Fig. 4, panels A and B) and H2-M (data not
shown and panel C). After 30 min, the co-distribution of Ag
with Lamp1 was observed only in 24% of TNP A20 cells and was not
observed in Fc
R-Ig
-expressing cells. As expected, no effect of
wortmannin was detected when analyzing Fc
R-Ig
-expressing cells
(data not shown).
Wortmannin Modifies the Formation of Ag Processing
Compartment--
Our data obtained by confocal microscopy strongly
suggest that BCR ligation through the Ig
chain generates a
compartment wherein Ag loading onto MHC class II molecules takes place
and that this is dependent on PI3K activity. To confirm this
hypothesis, we performed immunogold labeling on ultrathin cryosections,
to analyze the morphology of MHC class II-enriched compartments and the
distribution of MHC class II molecules. As shown in Fig.
5A, Fc
R-Ig
-expressing B
cells stimulated for 30 min with pre-formed ICs of HRP/anti-HRP Abs
displayed endosomal structures with multivesicular morphology, which
have been previously characterized upon BCR cross-linking (10). These
structures were scarcely observed in nonactivated cells (data not
shown). In these compartments, labeling for MHC class II was observed
in the internal membrane vesicles, which also contained Lamp1 (Fig.
5A), and Ag (data not shown) witnessing their Ag processing
function. In wortmannin-treated cells, pre-formed ICs did not induce
the formation of MVBs, instead endocytic compartments mostly devoid of
internal vesicles were seen (Fig. 5B). In a small proportion
of cells, wortmannin had an intermediate effect, classical
multivesicular Ag processing compartments were observed closely apposed
to electron luscent compartments (data not shown). This may explain the
partial inhibition of Ag presentation by wortmannin. However, in most
of the cells, only the electron luscent endocytic compartments devoid
of internal membranes were observed, their limiting membranes were
labeled with anti-class II and anti-Lamp1 antibodies (Fig.
5D), and they also
contain Ag (data not shown). In addition, in wortmannin-pretreated cells, labeling for MHC II was observed also in tubulovesicular structures closely apposed to the enlarged compartments (Fig. 5B). These endocytic compartments also contained the
invariant chain Ii (Fig. 5C). We previously showed that BCR
stimulation increases the intracellular MHC class II molecules by a
factor 3 after 30 min (10), a quantitative analysis of the class II present in the electron luscent compartment and the tubulovesicular network demonstrated that a similar increase was observed in
wortmannin-treated cells (data not shown).

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Fig. 5.
Wortmannin modifies the formation of the Ag
processing compartment. Ultrathin cryosections of
Fc R-Ig -expressing cells were single or double immunogold labeled
for MHC class II (M5114) (B), MHC class II and Ii
(C), or MHC class II and Lamp1 (A and
D). A, in cells stimulated for 30 min with
pre-formed IC-HRP, MHC class II protein A gold, 15 nm (PAG 15) are
present in multivesicular endosomes that also label for Lamp1 protein A
gold, 10 nm (PAG 10). MHC class II molecules are also detected
in small vesicles. B, cells pretreated with 200 nM wortmannin MHC class II (PAG 10) are visualized in
enlarged compartments mostly devoid of internal membranes. Occasionally
small membrane vesicles are observed in close apposition to the lumenal
side of limiting membrane. Note the small tubular vesicular structures
highly labeled for MHC class II surrounding the enlarged compartments.
C, in wortmannin-treated cells Ii (PAG 15) codistributed
with MHC class II (PAG 10) at the limiting membrane of electron luscent
compartments. D, in wortmannin-treated cells some electron
luscent compartments contained mostly class II (PAG 15) and others
contained class II and Lamp1 (PAG 10). Bars = 100 nm.
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Fig. 6.
PI3K is activated along the Ag endocytic
pathway. A, Fc R-Ig -expressing cells were
transiently transfected with a plasmid encoding the PH-Akt domain fused
to GFP. Cells on polylysine-coated coverslips were incubated 1 h
on ice with IC-HRP-rhodamine. After washing cells were incubated for 5 (upper panel) or 30 min (lower panel) at
37 °C, and fixed. Left panel shows the distribution of
PH-Akt-GFP in resting cells. B and C,
Fc R-Ig -expressing cells were treated as in A, except
that they were incubated with biotinylated anti-IgG F(ab')2
precomplexed with streptavidin-Alexa 594 to trigger the BCR
(C). Results are typical from three independent
experiments.
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Interestingly, in a large proportion of the wortmannin-treated cells we
observed juxtaposed electron luscent compartments differentially
enriched, at their limiting membranes, in MHC class II molecules or
Lamp1 (Fig. 5D). Altogether, these results strongly suggest that PI3K activity is implicated in de novo
formation of a multivesicular Ag processing compartment, which is
induced by BCR activation in an Ig
dependent fashion.
PI3K Is Activated Along the Ag Endocytic Pathway--
We have
shown that PI3K is implicated in de novo formation of a
multivesicular antigen-processing compartment and that PI3K is
activated by BCR and Ig
but not by Ig
(Fig. 2A). We
thus wondered if PI3K is activated in the endocytic compartments
wherein the Ag is transported. The PH domain of Akt has been shown to bind to phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (36-38) and can be used as a probe to
detect these PI3K products. We thus transfected Fc
R-Ig
and -Ig
chimera-expressing B cells with a PH-Akt domain coupled to GFP and
performed confocal microscopy experiments. In resting cells, PH-Akt-GFP
labeling was mainly observed at the plasma membrane, with some diffuse labeling in the cytoplasm and the nucleus. Five minutes after ligation
of Ig
chimera with pre-formed ICs of rhodamine-coupled HRP/anti-HRP
Abs, PH-Akt-GFP was detected in membrane ruffles surrounding the Ag
(Fig. 6A, upper panel). After 30 min, the
endocytic compartments containing the Ag were strongly labeled with
PH-Akt-GFP (Fig. 6A, lower panel). Closer
examination of these endocytic compartments often showed a large
compartment delimited by PH-Akt-GFP labeling enclosing smaller vesicles
containing Ag surrounded by PH-Akt-GFP (see inset, Fig. 6).
Notably, transfection of PH-Akt-GFP did not modify Ag targeting to
Lamp1+ compartments (data not shown). In B cells pretreated
with 200 nM wortmannin, PH-Akt-GFP did not label the plasma
membrane and labeling was more diffuse than in untreated
cells (data not shown). Wortmannin pretreatment also prevented
Fc
R-Ig
-induced redistribution of PH-Akt-GFP in membrane
ruffles at early time points as well as vesicular labeling at 30 min
(Fig. 6A).
Distribution of PH-Akt-GFP was similar in resting Fc
R-Ig
- and
Fc
R-Ig
-expressing cells. In contrast, ligation of ICs to Fc
R-Ig
rarely induced co-distribution of PH-Akt-GFP with the Ag
(Fig. 6B). These results confirmed the data presented above, i.e. Ig
stimulation does not induce PI3K activation (Fig.
2). Strikingly, in these very cells that express both Fc
R-Ig
chimera and IgG, BCR cross-linking did induce PH-Akt-GFP concentration at the site of contact (Fig. 6C, upper panel),
and on the Ag-containing endocytic vesicles (Fig. 6C,
lower panel). These results showed that the absence of PI3K
activation by Ig
is not because of a defect intrinsic of the
Fc
R-Ig
chimera-expressing cells.
BCR cross-linking with TNP-OVA-Cy3 in anti-TNP A20 B cells induced
PH-Akt-GFP translocation to Ag containing vesicle as well (data not
shown). Altogether, these results demonstrate that PI3K is
activated in the intracellular compartments wherein the Ag has been
endocytosed by the BCR and that this activation is mediated by
Ig
.
 |
DISCUSSION |
BCR is a multimeric receptor complex composed of an Ig
noncovalently associated with an heterodimer of Ig
and Ig
, which mediates BCR-induced signaling. BCR-mediated Ag uptake increases its
processing efficiency by targeting the Ag to compartments competent for
Ag degradation and peptide loading onto MHC class II molecules (39,
40). We and others have previously shown that signaling through the BCR
regulates Ag presentation (11, 12), however, the precise mechanisms
involved are still unknown. Here, we show that BCR-mediated PI3K
activation is instrumental for Ag presentation because it controls the
neogenesis of the multivesicular Ag processing compartment, which
contains MHC class II, Ii, H2-M, Lamp1, and the Ag. Moreover, we
demonstrate for the first time that PI3K activation accompanies the Ag
endocytic journey in an Ig
-dependent manner. These data
strongly suggest that modification by PI3K of the lipidic environment
of the endosomes wherein the BCR-bound Ag is targeted is required for
the dynamic formation of the Ag processing compartment.
Several groups have already suggested a role for PI3K in Ag
presentation (41, 42). However, the precise mechanisms implied were not
unraveled. In one of these studies, the authors showed that
Fc
Rc-induced Ag presentation was inhibited by LY294002 and wortmannin and that cross-linking of Fc
Rcs induced activation of
PI3K in peptide loading endocytic compartments (42). These results
obtained with another immunoreceptor are in accordance with ours and
probably reflects the fact that activation of Fc
Rc also induces
de novo formation of an Ag processing compartments.
Although we demonstrate that BCR-induced PI3K activation plays a
crucial role in Ag presentation, we do not know which PI3K species is
implicated. There is increasing evidence for an important role for
class IA PI3Ks in the regulation of the immune system (43). It has
recently been shown that p110
, which is highly expressed in
lymphocytes, is required for BCR signaling (23), it may thus be a key
player of BCR-induced Ag presentation. Alternatively, the murine
analogue of Vps34, which phosphorylates phosphatidylinositol at the 3'
position of the inositol ring, may be implicated. Indeed, Vps34 is
required for internal vesicle formation within multivesicular endosomes
(28). A previous study has shown in a melanoma cell line that PI3K is
implicated in MHC class II-enriched compartment formation (27). Our
study shows unambiguously that PI3K is required for neogenesis of an Ag
processing compartment with important consequences on Ag presentation.
Ig
and Ig
intracytoplasmic tails induce differential Ag
presentation (11) and differ in their signaling abilities (29, 33).
Herein, we show for the first time that Ig
triggering induces PI3K
activation whereas Ig
does not and that PI3K is activated
along the endocytic pathway of BCR- and Ig
-transported Ag. We
have previously shown that Ig
and Ig
differ both in their endocytic traffic (18) and in their ability to present Ag (17). Our
results strongly suggest that PI3K activation by Ig
is causal to
these differences. A recent study showed that Ag presentation requires
the recruitment of B cell linker protein by the Ig
BCR subunit (44).
Thus Ig
and Ig
by activating, respectively, a B cell linker
protein or PI3K-dependent pathways may differentially contribute to Ag presentation. We indeed observed that activation of
Fc
R-Ig
expressing B cells by ICs, which induces Ag presentation, does not induce de novo formation of MVBs in contrast to the
activation of Fc
R-Ig
expressing B
cells.2
Ligation of many tyrosine kinase receptors induces activation of their
kinase activity, autophosphorylation of their intracytoplasmic domains,
and recruitment of signaling proteins; this has been thoroughly
documented for the epidermal growth factor receptor. Signaling is
accompanied by enhanced endocytosis of receptor-ligand complexes.
Ligated receptors are associated with signaling proteins along the
endocytic pathway suggesting that intracellular signaling can take
place in endocytic compartments. By analogy, recruitment and/or
transport of signaling proteins by the BCR toward endosomal compartments may modify their composition. Herein, we point to one of
these effectors: PI3K. BCR triggering induces activation of PI3K, which
may then be transported together with the BCR along the endocytic
pathway. Alternatively PI3K could be specifically recruited to specific
intracellular compartments as suggested by the fact that membranes of
the Ag containing endocytic compartment are enriched in PI3K products
(Fig. 6). There, products generated by PI3K could recruit and/or
activate the molecular machinery necessary for the formation of the
multivesicular Ag processing bodies. In other models, it has
been shown that MVBs are enriched in phosphatidylinositol 3-phosphate
(45), a PI3K product and that inward vesiculation within MVBs is
inhibited by wortmannin (27, 28).
In this study we show that PI3K inhibition does not preclude
internalization of BCR-bound Ag and strikingly does not prevent degradation of BCR-internalized Ags despite inhibition of MVB formation. Alternative interpretations of this result can be proposed (1). In the absence of PI3K activation, Ag degradation takes place in
different endocytic compartments, implying that the generated antigenic
peptides will not be loaded onto MHC II molecules (2). Ag presentation
does not occur because Ag are not being degraded in a productive
fashion. They could be either degraded by a different set of proteases,
or completely destroyed. Our assay for Ag degradation does not allow us
to distinguish between these hypothesis. A definitive conclusion will
need further investigation.
Absence of Ag presentation may be because of inhibition of fusion of
endocytic compartments by wortmannin, indeed we observed a
non-negligible number of electron luscent compartments expressing only
MHC class II or only Lamp1 at their limiting membranes (Fig. 5D). This inhibition of fusion could preclude gathering of
several molecules that are essential for Ag processing. For example,
H2-M, which has been shown to play an important role in loading of
peptide on MHC class II molecules (46) and to be targeted to MVBs by BCR triggering (10), is not observed in Ag containing compartments in
wortmannin-treated cells (Fig. 4).
A lot remains to learn about the intracellular compartments responsible
for Ag processing and peptide loading as well as about the control of
their formation. Signaling induced by Ag binding to its receptor will
directly influence the environment of endosomes and thereby control
formation of Ag processing compartments. This will ultimately lead to
presentation of differently processed Ags. Identification of the
cytosolic effectors used by Ag receptors will allow to better
understand the molecular mechanisms that control Ag processing.