Upload
others
View
5
Download
0
Embed Size (px)
Citation preview
Immunity
Article
Polarized Secretion of Lysosomesat the B Cell Synapse Couples Antigen Extractionto Processing and PresentationMaria-Isabel Yuseff,1,6,* Anne Reversat,1,6 Danielle Lankar,1 Jheimmy Diaz,1 Isabelle Fanget,2 Paolo Pierobon,1
Violaine Randrian,1 Nathanael Larochette,2 Fulvia Vascotto,1 Chantal Desdouets,3 Bertrand Jauffred,4
Yohanns Bellaiche,4 Stephane Gasman,5 Francois Darchen,2 Claire Desnos,2 and Ana-Maria Lennon-Dumenil1,*1Inserm U932, Institut Curie, 12 rue Lhomond, 75005 Paris, France2CNRS/Universite Paris Descartes UMR8192, 45 rue des Saints-Peres, 75006 Paris, France3CNRS/Universite Paris Descartes UMR8104, Institut Cochin, 22 rue Mechain, 75014 Paris, France4Inserm U934, CNRS/UMR3215, Institut Curie, 26 rue d’Ulm, 75005 Paris, France5CNRS/UPR3212, Institut des Neurosciences Cellulaires et Integratives, Universite Strasbourg, 5 rue Blaise Pascal,67084 Strasbourg, France6These authors contributed equally to this work
*Correspondence: [email protected] (M.-I.Y.), [email protected] (A.-M.L.-D.)
DOI 10.1016/j.immuni.2011.07.008
SUMMARY
Engagement of the B cell receptor (BCR) by surface-tethered antigens (Ag) leads to formation of a syn-apse that promotes Ag uptake for presentation ontomajor histocompatibility complex class II (MHCII)molecules. We have highlighted the membrane traf-ficking events and associated molecular mecha-nisms involved in Ag extraction and processing atthe B cell synapse. MHCII-containing lysosomes arerecruited to the synapse where they locally undergoexocytosis, allowing synapse acidification and theextracellular release of hydrolases that promote theextraction of the immobilized Ag. Lysosome recruit-ment and secretion results from the polarization ofthe microtubule-organizing center (MTOC), whichrelies on the cell division cycle (Cdc42)-downstreameffector, atypical protein kinase C (aPKCz). aPKCzis phosphorylated upon BCR engagement, associ-ates to lysosomal vesicles, and is required for theirpolarized secretion at the B cell synapse. Regulationof B lymphocyte polarity therefore emerges as acentral mechanism that couples Ag extraction to Agprocessing and presentation.
INTRODUCTION
The activation of B lymphocytes is critical for the onset of the
adaptive immune response. In lymph nodes, it is initiated by
the engagement of the B cell receptor (BCR) with antigens (Ag)
immobilized at the surface of macrophages or follicular or
migrating dendritic cells (Batista and Harwood, 2009). The
BCR includes a surface immunoglobulin (Ig) responsible for Ag
recognition and a signaling module composed of the Iga/Igb
dimer (Cambier et al., 1994; Reth and Wienands, 1997). BCR
Im
engagement induces a complex cascade of signaling events
that ultimately leads to cell proliferation and initiation of germinal
center (GC) development. To complete GC formation, activated
B lymphocytes must present BCR-uptaken Ag to primed CD4+
T cells (Mitchison, 2004). Productive Ag presentation relies on
the convergence of BCR-Ag complexes with lysosomal-like
compartments containing MHCII molecules, hydrolases in
charge of Ag degradation, and H2-DM molecules required for
peptide loading (Lankar et al., 2002).
In vivo, activation of B cells is mainly achieved by Ag retained
at the surface of neighboring cells (Carrasco and Batista, 2007;
Junt et al., 2007; Suzuki et al., 2009), leading to the formation
of an immune synapse (IS) that resembles the one originally
described in T cells (Grakoui et al., 1999; Kupfer et al., 1987).
Synapse formation allows sustained BCR signaling (Harwood
and Batista, 2008; Tolar et al., 2008) and further promotes the
efficient extraction of the immobilized Ag for processing and
presentation to T cells (Fleire et al., 2006). Ag extraction at the
IS occurs through a two-phase membrane spreading and cell
contraction response that is orchestrated by the local reorgani-
zation of the actin cytoskeleton and involves the small GTPases
Rac1 and Rac2 (Arana et al., 2008; Brezski and Monroe, 2007;
Fleire et al., 2006). Actin reorganization and activation of the
motor protein Myosin II are also required for convergence of
BCR-internalized Ag with MHCII-containing lysosomal compart-
ments (Le Roux et al., 2007; Vascotto et al., 2007).
In contrast to the actin cytoskeleton, the role of microtubules
(MTs) in formation of the B cell synapse and in Ag extraction
and processing remains unaddressed. Several studies support
their possible involvement: first, MHCII molecules use MTs to
move bidirectionally in melanoma cells (Wubbolts et al., 1999);
second, by ensuring the targeted delivery of effector molecules,
the polarization of the MT organizing center (MTOC) promotes
IS formation (Martın-Cofreces et al., 2008) and the delivery of
secretory granules in T cells (Stinchcombe and Griffiths, 2007).
These findings led to the proposal that recruitment of the
MTOC to a specific area of theplasmamembrane allows the local
regulation of exocytosis and endocytosis by focusing the
munity 35, 361–374, September 23, 2011 ª2011 Elsevier Inc. 361
A B
Anti-IgM
LAMP-1 H2-DM
IgG+ B lymphoma cells
MHCII-GFP LAMP-1 Merge Merge
I-Aβ-GFP-IgM+ spleen B cells
Anti-IgG Anti-IgM
Anti-IgG
0:00 0:06
0:11
E C
IgG+ B lymphoma cells
D
0:16
Num
ber o
f Lys
osom
es
Time (min) 10 30 60
35 30 25 20 15 10
5 0
Anti-IgG plates (30 min)
LAMP-1
60 120 0
Figure 1. BCR Engagement with Immobilized Ag Induces the Recruitment of MHCII-LAMP-1+ Lysosomes at the IS
(A and B) Spleen B cells fromMHCII-GFP mice (A) or mouse lymphoma IgG+ B cells (B) were incubated with anti-IgM- or anti-IgG-coated beads for 1 hr at 37�C.Cells were stained for LAMP-1 and GFP or for H2-DM and LAMP-1. Scale bars represent 3 mm. Dashed circles indicate bead position.
(C) Kinetics of LAMP-1+ recruitment to the IS in B lymphoma cells interacting with anti-IgG-coated beads. Time is shown in minutes. Scale bars represent 3 mm.
(D) TIRFM images of CatD-RFP+ vesicles in B lymphoma cells plated on anti-IgG-coated slides. The white arrow shows vesicle clustering. Time is shown in
seconds; scale bar represents 5 mm.
(E) Lysosome number in the subplasmalemmal region increased over time (bars represent mean number of lysosomes per cell ± SEM; n = 29, 82, and 60 cells at
10, 30, and 60 min, respectively). p values: 10 versus 30 min, p < 0.0001; 10 versus 60 min, p = 0.0002 (Mann-Whitney).
Immunity
Polarized Lysosome Secretion at the B Cell Synapse
required molecular machineries (Griffiths et al., 2010). Whether
polarized vesicle trafficking and secretion take place at the B
cell synapse and contribute to its function in extraction of
immobilized Ag has not been investigated so far.
We here show that B cells rapidly polarize their MTOC together
with MHCII+ and lysosomal-associated membrane protein
(LAMP-1)+ lysosomes toward the BCR-Ag interface, a process
that relies on the small GTPase Cdc42 and its downstream
effector, atypical protein kinase C zeta (aPKCz). Polarized lyso-
somes are locally secreted, allowing the acidification of the B
cell synapse and the extracellular release of lysosomal proteases
that promote the extraction of immobilized Ag. Accordingly,
protease inhibition or impairment of lysosome exocytosis via
Cdc42 or aPKC silencing compromises Ag processing and
presentation to T lymphocytes. Thus, MTOC-dependent exocy-
tosis of secretory lysosomes at the immune synapse is critical for
B lymphocytes to acquire their Ag presentation function.
RESULTS
BCR Engagement with Immobilized Ag Inducesthe Recruitment of MHCII-Containing Lysosomesat the Immune SynapseWe investigated the role of membrane trafficking in extraction
and processing of immobilized Ag at the B cell synapse. Latex
beads coated or not with specific BCR ligands were used to
mimic Ag presentation to B cells (Batista and Neuberger,
2000). Mouse spleen IgM+-IgD+ B cells incubated for 1 hr with
362 Immunity 35, 361–374, September 23, 2011 ª2011 Elsevier Inc.
anti-IgM-F(ab0)2-coated beads displayed a cluster of MHCII+,
H2-DM+, and LAMP-1+ vesicles at the cell-bead interface (Fig-
ure 1A). This cluster was not observed in B cells stimulated
with nonspecific BCR ligands (fibronectin or anti-IgG-F(ab0)2),indicating that BCR engagement is required to recruit MHCII+
lysosomes to the synapse. Similarly, stimulation of IgG+ mouse
B lymphoma cells with anti-IgG-F(ab0)2-coated beads triggered
the formation of a LAMP-1+ cluster that appeared after 30 min
and formed a ring around the Ag-coated bead, which became
more evident after 2 hr (Figures 1B and 1C). Because B lym-
phoma cells display very high surface MHCII expression, MHCII
intracellular distribution cannot be studied in these cells by
immunofluorescence. However, cryoimmunoelectron micros-
copy experiments showed that LAMP-1, MHCII, and the Ag
colocalize in these cells, suggesting that extraction did indeed
take place (see Figure S1A available online). Hence, MHCII+
lysosomes displaying all the characteristics of Ag processing
compartments are recruited to the synapse that forms at the
contact site between the BCR and Ag-coated beads in both
IgG+ and naive IgM+ spleen-derived B lymphocytes. This
synaptic structure will be referred to as the IS hereafter.
We next characterized lysosome dynamics at the IS by using
total internal reflection fluorescence microscopy (TIRFM) to
image the cell juxta-membrane region. IgG+ B lymphoma cells
expressing Cathepsin D-monomeric-red-fluorescent-protein
(CatD-RFP) were plated onto glass slides coated or not with
specific BCR ligands and 1 min duration movies were acquired.
Because mRFP is not pH sensitive, CatD-RFP can be used as
Immunity
Polarized Lysosome Secretion at the B Cell Synapse
a live-cell marker of MHCII+ and LAMP-1+ vesicles (Figures S1B
and S1C). B cells readily spread onto glass slides coated with
F(ab0)2 anti-IgG but not with F(ab0)2 anti-IgM (not shown), indi-
cating that such surface appropriately mimicked Ag-presenting
membranes. The number of CatD-RFP-labeled lysosomes de-
tected in the evanescent field increased between 10 and
30 min after B cell plating on Ag-coated slides (Figure 1E). Local
vesicle clustering and collectivemovements were observed (Fig-
ure 1D, white arrows; Movie S1). Although a minor fraction of
lysosomes moved rapidly, most of them displayed slow diffusive
movements or were almost immobile, suggesting that they were
attached to the plasma membrane. We conclude that engage-
ment of the BCR by surface-tethered Ag triggers the recruitment
of lysosomes and their immobilization at the IS.
MTOC Polarization Is Required for Recruitmentof Lysosomes to the Immune SynapseBecause MHCII+ vesicles have been shown to move on MTs
(Wubbolts et al., 1999), we investigated whether polarization of
the MT cytoskeleton accounts for the recruitment of MHCII+
lysosomes to the IS. Immunofluorescence experiments where
a-tubulin was labeled showed that the MHCII+-LAMP-1+ cluster
that formed at the cell-bead contact site was apposed to the
MTOC, suggesting that BCR engagement with immobilized Ag
induced MTOC polarization (Figures 2A and 2B). Labeling of
the MTOC with a g-tubulin Ab showed that it was indeed polar-
ized to the IS in activated primary and lymphoma B cells (Figures
2C and 2D). A similar observation was made when incubating
spleen B cells that express a hen egg lysozyme (HEL)-specific
BCR with HEL-coated beads (Figure 2C). MTOC polarization
was quantified by calculating a ‘‘polarity index’’ corresponding
to the distance between the MTOC and the bead center of
mass divided by the distance between both the cell and the
bead centers of mass (Figure 2E). Most of the cells incubated
with nonactivating beads displayed a polarity index similar or
higher than 1, indicating that their MTOC was located near the
cell center (Figures 2F and 2G). In contrast, a large proportion
of B cells treated with activating beads exhibited a polarity index
between 0.5 and 0.75, showing that their MTOC had been posi-
tioned toward the IS. Similar results were obtained when moni-
toring MTOC polarization toward anti-IgG-coated glass slides
by TIRFM: when we used a penetration depth of 300 nm, we
found that �70% of the cells had polarized their MTOC toward
the immobilized Ag within 30 min after plating (Figure S2).
Time-lapse analysis of B cells purified from transgenic mice
expressing the MTOCmarker Centrin fused to green fluorescent
protein (GFP) showed that BCR engagement triggered dynamic
oscillations of the MTOC (Figure 2H; Movie S2). The MTOC
moved back and forward, progressively positioning toward the
IS, while the polarity index shifted from 1 to 0.6 (Figure 2I). Similar
observations were made in B lymphoma cells (Figure 3A; Movie
S3). Cotransfection of Centrin-GFP and CatD-RFP in these cells
showed that MTOC and lysosome recruitment to the cell-bead
interface occurred concomitantly (Figures 3A and 3B). We
conclude that BCR engagement triggers the polarization of
both lysosomes and the MTOC toward the IS.
To determine whether polarization of the MTOC directs the
trafficking of lysosomes to the IS, we analyzed the impact of
MTOC disruption on lysosome distribution via two-photon laser
Im
ablation. Strikingly, CatD-RFP+ vesicles remained dispersed all
over the cell cortex and did not cluster at the IS in cells whose
MTOC had been ablated (Figure 3C). Indeed, whereas the
distance between the bead and lysosome centers of mass
decreased in time in nonablated cells, it did not vary in cells
whoseMTOC had been eliminated (Figure 3D). Importantly, lyso-
some recruitment and clustering at the cell-bead contact site
was not affected when ablating one lysosomal vesicle, demon-
strating that the impact of MTOC destruction on lysosome polar-
ization did not result from a bystander effect of laser ablation
(Figure 3C). Hence, polarization of the MTOC triggers the
directed trafficking of MHCII+-LAMP-1+ lysosomes to the
synapse that forms upon BCR engagement with immobilized Ag.
MTOC and Lysosome Polarization to the ImmuneSynapse Requires Cdc42To define the impact of BCR-induced polarity on IS function, we
searched for potential regulators. A likely candidate was the
small GTPase Cdc42, which is proposed to control the posi-
tioning of the MT network in many cell types (Iden and Collard,
2008). Pull-down experiments with p21-activated kinase
(PAK1) fused to gluthatione S-transferase (GST) protein revealed
that Cdc42 was activated upon BCR engagement with immobi-
lized Ag (Figure S3A). Transfection of the Cdc42-N17 dominant-
negative form in B lymphoma cells severely compromised the
polarization of the MTOC toward the IS (Figure 4A; Figure S3B),
whereas no effect was observed when transfecting the Cdc42-
V12 constitutive active form. Silencing of Cdc42 with an
shRNA-GFP construct that led to a�50% decrease in its protein
expression (Figure S3C) significantly impaired MTOC polariza-
tion to the cell-bead interface (Figures 4B and 4C). MTOC polar-
ization was rescued when introducing an shRNA-insensitive
Cdc42 construct in silenced cells, excluding off-target effects
(Figure S3D). Time-lapse analysis showed that although
Cdc42-depleted B lymphoma cells displayed important MTOC
oscillations upon contact with the activating bead, they did not
lead to its polarization to the IS (Figure S4). Equivalent conclu-
sions were reached in mouse splenic B cells transfected with
two different Cdc42-specific siRNAs (Figure S3E): neither their
MTOC nor their MHCII-GFP+-LAMP-1+ compartment was re-
cruited to the cell-bead synaptic interface (Figures 4D and 4E).
Quantification of both MTOC and lysosome polarization in single
B lymphoma cells showed that lysosomes from Cdc42-silenced
cells that displayed a nonpolarized MTOC were not recruited to
the IS, even after 2 hr of incubation (Figures 4F and 4G). In agree-
ment with this result, Cdc42 silencing significantly lowered the
density of CatD-mRFP+ vesicles detected by TIRFM in cells
plated on anti-IgG slides (Figure S6B). Together, our data show
that Cdc42 is activated upon BCR engagement, an event that
is required to polarize the MTOC and drive the recruitment of
lysosomes to the IS.
Nonpolarized Cdc42-Silenced BCells Show Impaired AgProcessing and PresentationHaving shown that B cell polarization needs Cdc42, we used
this finding to assess whether MTOC and lysosome recruitment
at the IS impacts on the processing of immobilized Ag. B
lymphoma cells were incubated with beads coated with specific
BCR ligands plus the LACK Ag from Leishmania major. Their
munity 35, 361–374, September 23, 2011 ª2011 Elsevier Inc. 363
A
Anti-IgM
B
Anti-IgM-bead
IgM+ spleen B cells
FN-beadHEL-beadC
E
a
b
b- Distance: CM-Center of Bead a- Distance: MTOC-Center of Bead
CM MTOC
D
0
10
20
30
40
50
60
70
80 FN (n=38)
anti-IgM (n=30) HEL (n=27)
Polarity index a/b
Cel
ls (%
)
0.25-0.5 0.5-0.75 0.75-1 >1.00
F
Anti-IgG-bead Anti-IgM-bead FN-bead
IgG+ B lymphoma cells
G
Cel
ls (%
)
Polarity index a/b
0
10
20
30
40
50
60
70
80
anti-IgG (n=30) anti-IgM (n=33) FN (n=29)
Anti-IgG
MHCII-GFP αTubulin Merge αTubulinLAMP-1 Merge
IgG+ B lymphoma cells I-Aβ-GFP-IgM+ spleen B cells
H I
Time (min)
Pola
rity
Inde
x
0 10 20 30 40 50 60 70 80 0
0.2 0.4 0.6 0.8
1 1.2 1.4 1.6
Δ=0.34
Centrin-GFP
0.25-0.5 0.5-0.75 0.75-1 >1.00
0 7:30 15:00 22:30 30:00
37:30 52:30 45:00 60:00 67:30
γTubulin γTubulin
Figure 2. B Cells Polarize Their MTOC toward the IS
(A and B) Spleen B cells (A) or mouse lymphoma B cells (B) were incubated with anti-IgM- or anti-IgG-coated beads for 1 hr. Merged images contain the 3D
projections of the a-tubulin staining.
(C and D) Cell-bead conjugates were stained for g-tubulin to determine the centrosome position.
(E) Scheme depicting the distance between (a) the MTOC or (b) the cell and the bead center of mass. Polarity indexes correspond to the a/b ratio.
(F and G) Distribution of MTOC polarity indexes in primary (F) and lymphoma (G) B cells upon interaction with Ag-coated beads for 1 hr, three independent
experiments. p values for primary B cells: anti-IgM versus FN = 2.9 3 10�5; HEL versus FN = 1.0 3 10�5; anti-IgM versus HEL = 0.7. p values for IgG+ B cells:
anti-IgG versus anti-IgM = 1.3 3 10�6; anti-IgG versus FN = 2.3 3 10�3; anti-IgM versus FN = 0.28 (Kolmogorov-Smirnov [KS] test).
(H) Sequential images of a GFP-centrin spleen B cell engaged with an anti-IgM-coated bead. Time is shown in min.
(I) MTOC polarity index is plotted in time.
Scale bars represent 3 mm.
Immunity
Polarized Lysosome Secretion at the B Cell Synapse
ability to present LACK-derived MHCII-peptide complexes to
a specific T cell hybridoma was then measured by monitoring
interleukin-2 (IL-2) secretion. Ag presentation was observed
only if LACK was coupled to beads coated with specific BCR
364 Immunity 35, 361–374, September 23, 2011 ª2011 Elsevier Inc.
ligands (anti-IgG+LACK), demonstrating that Ag targeting to
the BCR was required for presentation (Figure 5A). Noticeably,
expression of Cdc42-N17 or Cdc42 shRNA constructs signifi-
cantly decreased the presentation of bead-associated LACK to
C
D
MTOC ablation
Lysosome ablation
After ablation
No ablation
0:00 10:00 24:00 36:00 50:00 58:00
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 10 20 30 40 50 60
MTOC ablation No ablation
Lysosome ablation
Dis
tanc
e of
Lys
osom
es to
Bea
d C
M
Time (min)
60:00 0:00 12:00 36:00 50:00
0:00 12:00 36:00 52:00 60:00
After ablation
Dis
tanc
e to
Bea
d C
M (µ
m)
Time (min)
CM of CathepsinD-RFP vesicle population MTOC
0
2
4
6
8
0 10 20 30 40 50 60
B A Centrin-GFP
Cathepsin D-RFP
30:00 40:00 50:00 20:00
0 4:00 8:00 12:00
Centrin-GFPCathepsin D-RFP
Figure 3. MTOC Polarization Directs the Trafficking of Lysosomes toward the IS
(A) Sequential images of a B lymphoma cell expressing CatD-RFP and Centrin-GFP and engaged with an anti-IgG-coated bead (dashed circles). Scale bar
represents 3 mm.
(B) The distance between the bead center of mass (CM) and either the MTOC (green dots) or the CM of the CatD-RFP vesicle population (red dots) was plotted
in time.
(C) Sequential images of a B lymphoma cell expressing CatD-RFP and Centrin-GFP engaged with anti-IgG-coated beads of an untreated cell (top) and in a cell in
which a lysosome (bottom) or theMTOC (middle) was ablated with a 2-photon laser. Arrows show the position of theMTOC in control and lysosome-ablated cells
and white circles show ablated regions. Scale bars represent 3 mm.
(D) The relative distance between CatD-RFP lysosomes and the Ag-coated bead was calculated for each condition as in (B).
Immunity
Polarized Lysosome Secretion at the B Cell Synapse
Immunity 35, 361–374, September 23, 2011 ª2011 Elsevier Inc. 365
A
siControl
MHCII-GFPγTubulin
LAMP-1γTubulin
B
F
γTubulinLAMP-1
Time 0 shControl shCdc42 shControl shCdc42 shControl shCdc42
Time 1 hr Time 2 hr
D
0
10
20
30
40
50
60 shControl (n=45) shCdc42 (n=45)
Cells
(%)
0-0.5 0.5-0.75 0.75-1 >1.00
Polarity index a/b
Cdc42-N17 Cdc42-V12 Cdc42-WT
Cdc42-GFPγTubulinLAMP-1
0 2 4 6 8 10 12 Distance MTOC-bead (μm)
0 20 40
60 80
100 Time 1 hr
0 2 4 6 8 10 12 Distance MTOC-bead (μm)
0 20 40
60 80
100 Time 2 hr
0 2 4 6 8 10 12 Distance MTOC-bead (μm)
Lam
p-1(
%)
0 20 40
60 80
100 Time 0
siCdc42-A
C shCdc42 shControl
γTubulin LAMP-1
Merge
Merge
I-Aβ-GFP-IgM+ spleen B cells
Bead Center of Mass
1 2 3
G
shCdc42 shControl
MTOC
E
0
10
20
30
40
50
60
siControl (n=42) siCdc42-A (n=22)
Cel
ls (%
)
Polarity index a/b 0-0.5 0.5-0.75 0.75-1 >1.00
siCdc42-B (n=18) siCdc42-A+B (n=56)
M
Lam
p-1(
%)
Lam
p-1(
%)
Figure 4. MTOC and Lysosome Polarization to the IS Rely on Cdc42
(A) WT, constitutive active (V12), or dominant-negative (N17) Cdc42-GFP-expressing B lymphoma cells were incubated with anti-IgG-coated beads for 1 hr and
stained for g-tubulin and LAMP-1. Insets show magnification of the MTOC surrounded by Cdc42-GFP and LAMP-1+ vesicles.
(B and D) Control and Cdc42-silenced B lymphoma cells that coexpress GFP (B) and MHCII-GFP primary B control (siControl) or Cdc42-silenced (siCdc42) cells
(D) were activated as described above and stained for g-tubulin and LAMP-1.
(C and E) Distribution of MTOC polarity indexes observed in control and Cdc42-silenced B lymphoma cells and in IgM+ spleen B cells after 1 hr of incubation with
Ag-coated beads. p values: shControl versus shCdc42 = 7.633 10�6; siControl versus siCdc42-A = 3.673 10�2; siControl versus siCdc42-(A+B) = 1.0183 10�2
(KS test).
(F) Scheme depicting how MTOC and LAMP-1 polarization toward the bead were measured.
(G) shControl and shCdc42 GFP+ B lymphoma cells were incubated with anti-IgG-coated beads. Cell-bead conjugates containing GFP-positive cells (GFP not
shown) were stained for g-tubulin and LAMP-1. Graphs in the lower panel show the distance between the MTOC and the bead CM and the percent of LAMP-1
recruited to the bead (n R 20, 4 independent experiments).
Scale bars represent 3 mm.
Immunity
Polarized Lysosome Secretion at the B Cell Synapse
T cells (Figure 5A; Figure S5A). Equivalent results were obtained
when silencing Cdc42 in primary B cells via RNA interference
(Figure 5B). Cdc42 silencing had no effect on peptide presenta-
366 Immunity 35, 361–374, September 23, 2011 ª2011 Elsevier Inc.
tion (Figures 5A and 5B). To verify that Cdc42 is needed for
formation of MHCII-peptide complexes from bead-associated
Ag, we took advantage of the restricted mAb 2C44 that
B shControl shCdc42
I-Ad-LACK156-173LAMP-1
Merge
0
100
200
300
400
Peptide (μM)
Control shCdc42 B cells T cells
IL-2
(pg/
ml)
0 2 4 6 shControl
(n=33)shCdc42(n=35)
0
10
20
30
40
50
60
Mea
n Fl
uore
scen
ce In
tens
ity *** 70
D
700
0
200 300
500 600
400
100
siControl siCdc42-A
10 20 0 5 15
IL-2
(pg/
ml)
C A
si Cdc42-A
Anti-IgM+LACK
FN+LACK
si Control
si Control
0
100
200
300
600
500
400
*** 700
IL-2
(pg/
ml)
IL-2
(pg/
ml)
0 20 40 60 80
100 120 140 160 180 200
WT
**
Sh Control
sh Cdc42
Anti-IgG+LACK
Anti-IgM+LACK
G
Anti-IgM
Anti-IgG
Merge γ-Tubulin LAMP-1 γ-Tubulin
F
0 20 40 60 80
120
PLK4 Control
IL-2
(pg/
ml)
**
IL-2
(pg/
ml)
0 50
100 150 200 250 300 350
0 5 10 20
Control PLK4
15
E
100
140
Peptide (μM)
Peptide (μM)
Figure 5. MTOC and Lysosome Polarization to the IS Promotes Ag Presentation
(A and B) Upper panels show Ag (Lack) presentation assays with control and Cdc42-silenced B lymphoma cells (A) or control and Cdc42-silenced spleen B cells
(B). Lower panels show the peptide controls for cells used in the Ag presentation assays. Mean amounts of IL-2 ± SD are shown for a representative experiment.
p values: shControl versus shCdc42, p = 0.0120; siControl versus siCdc42, p < 0.0001 (t test).
(C) Confocal images of control or Cdc42-silenced B cell lines incubated with anti-IgG-Lack-beads for 4 hr at 37�C. Cells were fixed and stained for LAMP-1 and
I-Ad-LACK156-173 complexes. Scale bars represent 5 mm.
(D) Mean fluorescence intensity (MFI) of I-Ad-LACK156-173 complexes quantified per cell pooled from two independent experiments; p = 0.0002 (t test).
(E) Confocal images of B lymphoma cells overexpressing PLK4 interacting with anti-IgG- or anti-IgM-coated beads for 1 hr. 3D projections of g-tubulin and
LAMP-1 staining are shown; scale bars represent 5 mm. The inset shows a magnification of multiple MTOCs surrounded by LAMP-1+ vesicles at the Ag-bead
contact site.
(F) Ag presentation assay for control and PLK4-overexpressing cells. Mean amounts of IL-2 ± SD were obtained by pooling two independent experiments;
control versus PLK4, p = 0.0022 (Mann-Whitney test).
(G) Peptide controls for control and PLK4-overexpressing cells.
Immunity
Polarized Lysosome Secretion at the B Cell Synapse
Immunity 35, 361–374, September 23, 2011 ª2011 Elsevier Inc. 367
A
shControl
B
shCdc42
0 1 hr 2 hr
OVALAMP-1
OVA
on
bead
(%)
0 1 2 Time (hr)
** **
0
20
40
60
80
100
120
shControl: anti-IgM+OVA
shControl: anti-IgG+OVAshCdc42: anti-IgG+OVA
OVA
ext
ract
ion
(%)
0
20
40
60
80
10 μM DCG04: anti-IgG+OVA (n=114)
DMSO: anti-IgG+OVA (n=187)50 μM Chloroquine: anti-IgG+OVA (n=120)
E
shControl (n=72)shCdc42 (n=69)
G
Fanti-IgG-CypherCathepsin-D-RFP
anti-IgG-Cypher LysoTracker
H
100 μM Pepstatin: anti-IgG+OVA (n=72)10 μM GM: anti-IgG+OVA (n=120)All: anti-IgG+OVA (n=46)
***
Control 6:00 2:00
+ chloroquine 6:00 2:00
0:00 6:00 12:00 18:00 24:00
30:00 36:00 42:00 48:00 54:00
C
N° e
vent
s/ce
ll/m
in
0
0.04
0.08
0.12
0-5 5-10 10-15 >15
** **
ShControl ShCdc42
Sna
psho
t occ
urre
nce
Duration of immobilization (s)
D
Cathepsin-D-mRFP-pHluorin
mRFP pHluorin
0.0
0.5
1.0
1.5
2.0
2.5
3.0
ShControl (n=24)ShCdc42 (n=28)
**
00:00 00:00
30:00
40:00
Cel
ls (%
)
0 20 40 60 80
100
Cypher- Cypher+
Figure 6. Exocytosis of Lysosomes Takes Place at the IS and Is Required for Efficient Extraction of Immobilized Ag
(A) Control and Cdc42-silenced GFP+ lymphoma B cells (GFP not shown) were incubated with anti-IgG+OVA or anti-IgM+OVA beads for different time points.
Fixed cell-bead conjugates were stained for OVA and LAMP-1. Scale bars represent 3 mm.
(B) TheOVA amount on beads interacting withGFP+ shControl and shCdc42 cells was calculated (see Experimental Procedures). Each bar represents themean ±
SEM percent of OVA relative to time 0 and was obtained from four independent experiments (n > 25). p values: shControl versus shCdc42 at t = 1 hr, p = 0.0015;
and at t = 2 hr, p = 0.00054 (t test).
Immunity
Polarized Lysosome Secretion at the B Cell Synapse
368 Immunity 35, 361–374, September 23, 2011 ª2011 Elsevier Inc.
Immunity
Polarized Lysosome Secretion at the B Cell Synapse
specifically recognizes I-Ad-LACK156-173 complexes but does
not react with its individual components. I-Ad-LACK156-173
complexes were detected in LAMP-1+ compartment clusters
after incubating control cells for 4–6 hr with activating LACK-
coated beads but were significantly decreased in Cdc42-
silenced cell (Figures 5C and 5D). Hence, Cdc42 is required for
processing and presentation of immobilized Ag recognized
through the BCR.
To strengthen the link between MTOC polarization and Ag
processing by B cells, we evaluated how artificial MTOC ampli-
fication impacts Ag presentation. For this, we expressed in B
lymphoma cells the protein Polo-like kinase, PLK4, which
controls centriole duplication and—when overexpressed—
leads to MTOC amplification (Basto et al., 2008). Transient
transfection of PLK4 led to the appearance of extra MTOCs
surrounded by numerous lysosomes in 10%–20% of B cells
(Figure S5B). Interestingly, these cells were larger than control
cells and their MTOCs were dispersed when incubated with
nonactivating particles but clustered toward the beads when
these engaged their BCR (Figure 5E). Strikingly, MTOC amplifi-
cation in 10%–20% of B cells led to a �30% increased in
Ag presentation (Figure 5F), suggesting that the presence of
multiple MTOCs promotes Ag presentation. No effect of
MTOC amplification on peptide presentation was observed,
indicating that the increase in Ag presentation did not merely
result from enhanced cell size or surface MHCII expression
but rather from improved Ag processing (Figure 5G). Together,
these data suggest that the ability of B cells to process and
present immobilized Ag is linked to their capacity to polarize in
response to BCR stimulation.
B Cell Polarization Leads to Lysosome Secretionat the Immune SynapseWe next investigated whether MTOC and lysosome polarization
to the IS promotes Ag processing by facilitating the extraction
of immobilized Ag. Ag extraction was evaluated by monitoring
the disappearance of ovalbumin (OVA) coupled to activating or
nonactivated beads by immunofluorescence. A substantial
decrease in the amount of bead-associated OVA was observed
at 1–2 hr after incubation with control cells (Figures 6A and 6B,
anti-IgG+OVA-beads). This decrease was not observed when
coupling OVA to nonactivating beads, indicating that OVA
extraction from the bead surface needed BCR engagement
(Figures 6A and 6B, anti-IgM+OVA-beads). Remarkably, OVA
(C) Lysosomes containing CatD-RFP-pHluorin are red (top left) and their green
exocytosis as shown in the bottom panel (enlarged view of the area framed in top p
indicated in seconds. Scale bar represents 5 mm. The graph shows the mean nu
(D) Lysosomes were tracked (>6000 trajectories/condition) to detect duration of
bilization > 10 s). Cdc42 silencing reduced the snapshot occurrence of lysosom
trajectory, a lysosome experienced a docking period of a given duration (snapsh
10–15 s, p = 0.0059; <15 s, p = 0.0057 (Mann-Whitney).
(E) Sequential images of a CatD-RFP-expressing B cell engaged with a Cypher5-
the appearance of fluorescence on the bead as CatD-RFP+ vesicles are recruite
(F) Quantification of the percent of anti-IgG-Cypher5 beads associated to shCon
values after 90 min at 37�C (three independent experiments). p value = 8.6 3 10
(G) Sequential images of Lysotracker-Red-loaded IgG+ B cells engaged with anti-
addition of 50 mM chloroquine (bottom). Scale bars represent 3 mm.
(H) Role of proteases in Ag extraction. Control cells (DMSO) and cells pretreated
beads for 2 hr. Bead-associated OVA was measured as described in (A), where ea
(one-way ANOVA test).
Im
disappearance was significantly reduced in Cdc42-silenced
cells. In contrast, BCR-mediated endocytosis of soluble multiva-
lent ligands was not affected in these cells (Figure S6H), showing
that the decrease in Ag extraction of Cdc42-silenced cells was
not due to a global defect in endocytosis. Given that BCR endo-
cytosis relies on cortical actin (Onabajo et al., 2008; Stoddart
et al., 2005), this implies that (1) Cdc42 silencing does not disturb
the actin cortex and (2) impaired Ag extraction in Cdc42-silenced
cells does not result from defective Ag internalization because of
altered actin organization.
We next investigated the mechanisms by which Cdc42-
dependent cell polarization regulates the extraction of immobi-
lized Ag.We postulated that lysosomesmay undergo exocytosis
at the IS, resulting in the local release of proteases that would
facilitate Ag extraction. To test this hypothesis, we fused the
pH-insensitive CatD-mRFP construct to a super ecliptic pHluorin
domain, whose fluorescence is low at acidic pH and steadily
increases upon pH elevation (pKa = 7.1; Figure S6C; Puthen-
veedu et al., 2010; Sankaranarayanan et al., 2000). As expected,
lysosomes from B lymphoma cells expressing CatD-mRFP-
pHluorin displayed green fluorescence only when exposed to
pH-neutralizing drugs (Figure S6D). When these cells were
plated on anti-IgG-coated slides and analyzed by TIRFM, their
lysosomes were red but displayed low green fluorescence, indi-
cating that their luminal pH had remained acidic (Figure 6C).
Noticeably, monitoring the dynamics of these vesicles showed
the occasional appearance of green lysosomes, showing that
their acidic content had been neutralized most probably upon
exocytosis (Figure 6C, see arrows). Green fluorescent signals
were transient (�2 s), suggesting that fluorescent CatD diffused
after secretion. Signal intensity was rather low, but they were
reproducibly observed (�2.4 events/cell/min in control cells; Fig-
ure 6C). Not surprisingly, this number decreased to �0.9 in
Cdc42-silenced cells, showing that lysosome exocytosis at the
IS relies on Cdc42. Interestingly, lysosome mobility was altered
in Cdc42-silenced cells: calculation of diffusion coefficients
(Dxy) along lysosome trajectories indicated that the probability
of lysosome docking (defined as an immobilization period longer
than 10 s, see Supplemental Experimental Procedures; Huet
et al., 2006; Desnos et al., 2007) was significantly decreased in
Cdc42-silenced cells (Figure 6D). Altogether our data suggest
that Cdc42 regulates lysosome exocytosis at the IS by pro-
moting their recruitment and by facilitating their local immobiliza-
tion and fusion with the plasma membrane.
fluorescence (top right) is dim because of their low pH but increases upon
anel). Imageswere taken every 150ms; one frame out of three is shown. Time is
mber of events/cell/min ± SEM, p value = 0.0008 (Mann-Whitney).
immobilization periods, an index of docking at the plasma membrane (immo-
e docking. Bars represent the probability that, at any given time point of its
ot occurrence) ± SEM; n = 59 cells; four independent experiments; p values:
conjugated anti-IgG-coated bead. Scale bar represents 3 mm. Insets highlight
d.
trol and shCdc42 cells that showed a Cypher5 MFI > 10% above background�3 (KS test).
IgG-Cypher beads. Images show a control cell (top) and a cell before and after
for 20 min with different protease inhibitors were incubated with anti-IgG+OVA
ch bar represents the mean ± SEM percent of OVA relative to time 0. p < 0.001
munity 35, 361–374, September 23, 2011 ª2011 Elsevier Inc. 369
G
B
D
C A
shControl
shCdc42
aPKC-P LAMP-1 aPKC-P LAMP-1 Merge
IL-2
(pg/
ml)
Peptide (µM)0 5 10 15 20
0
100
200
300
400
500
600
sh-aPKCζ-B sh-aPKCζ-A shControl
H I
sh-aPKCζ shControl
γTubulin LAMP-1
8 9
10
0 1 2 3 4 5 6 7
aPK
C-P
Int
ensi
ty (A
.U)
ShControl ShCdc42
anti-IgG anti-IgG anti-IgM
***
Cel
ls (%
)
0 20 40 60 80
100
Cypher- Cypher+
shControl (n=46) sh-aPKCζ (n=69)
E
F Centrin-GFP
Cathepsin-D-RFP
0 1 2 3 4 5 6 7 8 9
10 ***
anti-IgG anti-IgG anti-IgM
Control + aPKCζ Inhibit.20μM
Fold
of a
PKC
-P I
nten
sity
(A.U
)Anti-IgG
Anti-IgM
2 hr
Merge aPKC-P LAMP-1 LAMP-1 aPKC-P
1 hr
1 hr
Anti-IgGaPKC-P
Actin
+ aPKCζ Inhibitor
anti-I
gG-B
ead
anti-I
gM-B
ead
anti-I
gG-B
ead
20μM 0 0
75kDa
37kDa
IL-2
(pg/
ml)
20 40 60 80
100 120 140 160
0
Anti-IgG+Lack
Anti-IgM+Lack
sh Control
sh aPKCζ-
A
sh aPKCζ-
B
sh Control
***
Polarity Index a/b
Cel
ls (%
)
0.5-0.75 0.75-1 >1.00 0-0.5
60
50 40
30
20
10
0
shControl (n=103) sh-aPKCζ (n=74)
time (min)
sh-aPKCζ 20:00
40:00
60:00
20:00 shControl
40:00
60:00
Lam
p-1(
%)
0
20
40
60
80
100 ***
shControl sh-aPKCζ
(n=169) (n=44) (n=43)
(n=70) (n=37) (n=65)
Figure 7. Cdc42-Dependent Activation of aPKCz Controls MTOC Polarization and MHCII-LAMP-1+ Secretion at the IS
(A) Mouse B lymphoma cells were incubated with anti-IgG- or anti-IgM-coated beads for 1 hr at 37�C ± 20 mM of the aPKCz pseudo-substrate. Lysates were
analyzed by immunoblot for phospho-aPKCz (aPKC-P) and actin expression.
(B) B lymphoma cells were incubated with anti-IgG or anti-IgM beads for 1 hr at 37�C and stained for aPKC-P and LAMP-1.
(C) Quantification of aPKC-P integrated fluorescence intensity (FI) in cells interacting with anti-IgM beads or anti-IgG beads ± 20 mMof aPKCz pseudo-substrate.
The FI for each cell was normalized to the mean FI of the negative control (anti-IgM beads; MFI = 1); three independent experiments, p < 0.0001
(Mann-Whitney test).
(D) Ag presentation assay with control and aPKCz-silenced B cells. Mean amounts of IL-2 ± SD were obtained by pooling three independent experiments.
p values: shControl versus sh-aPKCz-A, p < 0.0001; shControl versus sh-aPKCz-B, p = 0.0009 (t test). The lower panel shows the peptide control for B cells used
in a representative experiment.
Immunity
Polarized Lysosome Secretion at the B Cell Synapse
370 Immunity 35, 361–374, September 23, 2011 ª2011 Elsevier Inc.
Immunity
Polarized Lysosome Secretion at the B Cell Synapse
Lysosome Secretion at the Immune SynapseIs Critical for Efficient Extraction and Processingof Immobilized AgHaving shown that lysosomes are secreted at the IS, we inves-
tigated whether the local release of their proteolytic content
contributes to the extraction of the immobilized Ag. To this
mean, we added protease inhibitors and/or the alkalinizing
agent chloroquine to the extracellular milieu prior to B cell
activation and evaluated their impact on Ag extraction, as
measured by OVA disappearance from activating beads. Chlo-
roquine as well as all protease inhibitors decreased the ability of
B cells to extract the Ag, the more effective being the cysteine
protease inhibitor DCG-04 (Figure 6H). DCG-04 is non-cell-
permeable (Lennon-Dumenil et al., 2002) and in B cells was
not targeted to endolysosomal compartments by fluid phase
endocytosis, as indicated by the fact that it could not be de-
tected in cellular lysates from cells incubated with 10 mM
DCG-04 for 1 hr (Figure S6E). Thus, the activity of extracellular
proteases is required for efficient extraction of immobilized Ag
by B cells.
To verify that the Ag was indeed being proteolyzed extracellu-
larly, we coupled it to Cypher5, a dye whose fluorescence
increases at acidic pH (Milasta et al., 2005). We reasoned that,
if internalized prior to degradation, Cypher5-labeled Ag should
be observed in polarized lysosomes. On the contrary, the lack
of fluorescence in lysosomes would indicate that bead-associ-
ated Ag are proteolyzed at the IS at first. Strikingly, Ag-coated
beads acquired a strong fluorescent signal approximately
30 min upon cell contact (Figure 6E), suggesting that the
synaptic pH had become acidic. Accordingly, we observed a
rapid disappearance of the green fluorescent signal after
addition of chloroquine (Figure 6G). Bead fluorescence was sig-
nificantly decreased upon Cdc42 silencing (Figure 6F), consis-
tent with inhibition of lysosome secretion. Importantly, local
acidification of the extracellular space resulting from lysosome
exocytosis might account for the relatively low pH-phluorin
signals observed by TIRFM. Labeling of cell-associated acti-
vating beads without cell permeabilization showed that bead-
coupled Ag rapidly became partially or not accessible to Abs
because of membrane spreading (Figures S6F and S6G). These
findings suggest that the cell-bead interface is a sealed zone that
can prevent proton diffusion and allow local pH acidification.
Importantly, Cypher5-labeled anti-IgG was not detected in
intracellular vesicles, suggesting that it had been proteolyzed
at least in part prior to internalization (Figure 6E). We conclude
that polarized secretion of lysosomes at the IS allows the local
release of hydrolases that contribute to efficient extraction of
immobilized Ag having engaged the BCR.
(E) Control and aPKCz-silenced B lymphoma cells were incubated with anti-IgG be
distribution of MTOC polarity indexes. shControl versus sh-aPKCz, p value = 1.9
recruited to the Ag-coated bead (three independent experiments); shControl ver
(F) Sequential images of a control and aPKCz-silenced B lymphoma cell cotransfe
Arrows show the MTOC surrounded by lysosomes.
(G) Percent of anti-IgG-Cypher beads engaged to shControl and sh-aPKCz cells
(H) shControl and shCdc42 GFP+ cells were incubated with anti-IgG beads for 1
(I) Quantification of aPKC-P FI in cells interacting either with anti-IgG or anti-IgM
experiments).
Scale bars represent 3 mm.
Im
Lysosome Secretion at the Immune Synapse Relies onthe Activation of Atypical PKCz Downstream of Cdc42We next searched for Cdc42 effector molecules involved in
polarized secretion of lysosomes at the B cell IS. Atypical protein
kinase C (aPKC) was a good candidate because, together with
partitioning defective proteins Par6 and Par3, it forms the Par
complex known to control cell polarity downstream of Cdc42
in multiple cell types (Goldstein and Macara, 2007). aPKC auto-
phosporylates upon interaction with Par6-bound Cdc42-GTP,
resulting in Par3 phosphorylation and MTOC polarization (Iden
and Collard, 2008). Interestingly, it was shown that aPKC is acti-
vated upon BCR stimulation and mice genetically ablated for
aPKCz—the aPKC form expressed in lymphocytes—fail to
mount an efficient humoral immune response (Guo et al., 2009;
Martin et al., 2002). Immunoblot analysis showed that expres-
sion of phospho-aPKCz strongly increased upon B cell incuba-
tion with activating beads (Figure 7A). Phospho-aPKCz was
not observed when incubating B cells with nonactivating beads
or when inhibiting its kinase activity, showing the specificity of
the phospho-aPKC Ab (Figures 7A–7C). Silencing of aPKCz
with two different shRNA constructs strongly impaired the
presentation of bead-associated LACK Ag to T cells (Figure 7D;
Figure S7A). Equivalent results were obtained when silencing
aPKCz expression in purified primarymouseB cells (Figure S7B).
Silencing of aPKCz did not affect peptide presentation, showing
that it regulates the processing of immobilized Ag but does not
impact B-T lymphocyte interactions (Figure 7D).
Having shown that aPKCz is activated upon BCR engagement
and required for Ag processing, we investigated its role in polar-
ized secretion of lysosomes at the IS. Strikingly, phospho-aPKCz
was found in close association with lysosomes while they
accumulated at the IS (Figure 7B). Phospho-aPKCz was barely
detected when silencing Cdc42 or expressing its dominant-
negative form, demonstrating the requirement of Cdc42 for
aPKCz activation downstream of the BCR (Figures 7H and 7I;
Figures S7G and S7H). Consistently, phospho-aPKC was found
to bind to Cdc42-GTP in pull-down experiments (Figure S7F).
MTOC and lysosome polarization to the IS were impaired in
aPKCz-silenced cells, as shown by a significant increase in
polarity indexes as well as in the recruitment of the LAMP-1+
vesicles at the cell-bead interface (Figure 7E; Figure S7C).
Similar results were obtained when treating the cells with an
aPKC inhibitor (Figure S7D). Time-lapse analysis showed that
in aPKCz-silenced cells, the MTOC and lysosomes had started
to polarize toward the IS but that polarity was lost after
�30 min after BCR engagement (Figure 7F). Remarkably,
synapse acidification was not observed in these individual cells,
as shown by the lack of fluorescent signal from beads coated
ads for 1 hr and stained for LAMP-1 and g-tubulin. Themiddle panel shows the
3 10�10 (KS test). The graph in the lower panel shows the percent of LAMP-1
sus sh-aPKCz, p < 0.0001 (t test).
cted with CatD-RFP and Centrin-GFP engaged with an anti-IgG-coated bead.
quantified as described in Figure 6F; p = 0.084 (KS test; one experiment).
hr and stained for aPKC-P and LAMP-1.
beads, as described in (C); p < 0.0001 (Mann-Whitney test; three independent
munity 35, 361–374, September 23, 2011 ª2011 Elsevier Inc. 371
Immunity
Polarized Lysosome Secretion at the B Cell Synapse
with Cypher5-coupled Ag (Figure 7G; Figure S7E), indicating that
lysosome secretion at the synapse is compromised in the
absence of the enzyme. We conclude that the Cdc42 effector
protein aPKCz is required for MTOC polarization and exocytosis
of lysosomes at the B cell synapse.
DISCUSSION
Establishment of cell polarity is crucial for many cellular pro-
cesses, including asymmetric cell division, directed cell migra-
tion, and tissue development. We here bring evidence for B
lymphocyte polarization in response to Ag stimulation and
provide insights on how they use polarity to promote their Ag
presentation function. Our results support a role forMTOC polar-
ization in directing the trafficking of MHCII+-LAMP-1+ vesicles
toward the synapse, thereby facilitating their local recruitment
and secretion to couple Ag extraction to processing. Cell polarity
thus emerges as an essential mechanism for B cells to acquire
their Ag presentation function.
BCR-mediated uptake of membrane-tethered Ag relies on the
formation of an IS that allows Ag gathering and extraction for
presentation to T cells (Batista et al., 2001; Fleire et al., 2006).
The precise mechanism used by B cells to extract and process
immobilized Ag remains unclear. Our results show that exocy-
tosis of lysosomes at the IS provides an acidic and protease-
rich environment that facilitates Ag extraction prior to internaliza-
tion. This finding is reminiscent of a recent observation made in
macrophages where lysosome secretion provides a sealed
acidic extracellular zone to favor digestion of aggregated lipo-
proteins by lipases and proteases (Haka et al., 2009). The model
Ag used in our study showed an important role for secreted
proteases in Ag extraction, but lysosomal lipases may equally
contribute to extraction when synapses form between the BCR
and membrane-bound Ag.
In addition to hydrolase secretion, polarized trafficking of
lysosomes facilitates the local concentration of MHCII and
accessory molecules required for Ag processing. Thus, polar-
ized lysosome secretion couples Ag extraction to formation of
the Ag processing compartment, allowing the spatio-temporal
coordination of these two events ultimately required for T cell-
B cell cooperation. Different nonexclusive scenarios can be en-
visioned for the nature of the compartment where Ag processing
takes place. BCR-bound large Ag chopped by secreted prote-
ases may be directly loaded onto surface MHCII molecules
(Moss et al., 2007). Alternatively, lysosome exocytosis might
be associated to compensatory endocytic events, allowing the
rapid uptake of predigested BCR-Ag complexes, which would
be further degraded and loaded onto MHCII molecules intracel-
lularly. This second scenario is consistent with data obtained in
T cells, where secreted LAMP-1+ vesicles are rapidly retrieved
by endocytosis at the IS (Liu et al., 2009). One could speculate
that the nature and amount of hydrolases secreted at the B cell
synapse define whether the Ag is processed extra- and/or intra-
cellularly, determining the size of the peptide Ag to be presented
to T cells and the fate of the immune response.
We found that MTOC and lysosome polarization rely on BCR-
dependent activation of the small GTPase Cdc42 and its effector
protein aPKC. This finding is consistent with observations made
inothercell types—fromthebuddingyeast to activatedT lympho-
372 Immunity 35, 361–374, September 23, 2011 ª2011 Elsevier Inc.
cytes—where Cdc42 plays a major role in cell polarity (Iden and
Collard, 2008). Importantly, genetic evidence for the involvement
of both Cdc42 and aPKC in B cell function has been provided:
Cdc42 genetic ablation impairs B cell development and aPKCz-
null mice show altered humoral immune responses (Guo et al.,
2009; Martin et al., 2002). aPKCz is activated downstream of
Cdc42 and is found in association to lysosomes. Interestingly,
although Cdc42- and aPKCz-silenced B cells display impaired
MTOC and lysosome polarization upon BCR engagement, time-
lapse analysis showed that they intervene at different stages:
although silencing of Cdc42 compromises MTOC and lysosome
polarization, their polarization occurs in the absence of aPKCz
but is rapidly lost. These results suggest that Cdc42-GTP inter-
acts with (1) unknown effector(s) that generate the driving force
needed for MTOC polarization and (2) aPKC that allows the
stabilization of polarized MTOC and lysosomes at the synapse.
This aPKC-dependent step of polarity stabilization is required
for the docking and secretion of lysosomes at the IS to promote
Ag extraction and processing. This model shows that Cdc42
couples lysosome recruitment at the synapse to local exocytosis
and is consistentwith the role proposed for the IS asaplatform for
regulated events of polarized trafficking.
What are the Cdc42 effectors regulating MTOC polarization to
the IS? Dynein regulates MTOC polarization in migrating fibro-
blasts downstreamofCdc42and in concertwithmyosin II (Gomes
et al., 2005). We found that myosin II is activated upon BCR
engagement and controls lysosome polarization at the IS (M.-I.Y.
and A.-M.L.-D., unpublished data). Time-lapse imaging showed
that MTOC polarization occurred concomitantly with phases of
actomyosin contraction that stopped once polarity was stably
achieved. In addition, Cdc42-silenced B cells displayed uncon-
trolled cortical contractility, suggesting that myosin II activity is
altered in Cdc42-silenced cells. Thus, MTOC polarization to the
ISmight rely on the concertedaction of dynein andmyosin IImotor
proteinsactingdownstreamofCdc42.Focusingonmolecules that
regulate the MT-actin crosstalk shall provide insights on how Ag
extraction and processing are regulated at the B cell synapse.
EXPERIMENTAL PROCEDURES
Mice and Cells
I-Abb-GFP genetically targeted mice (MHCII-GFP) were provided by H. Ploegh
(Whitehead Institute, MIT, MA) (Boes et al., 2002). The transgenic mouse lines,
centrin-GFP, and MD4 have been described (Goodnow et al., 1988; Pulecio
et al., 2010). The mouse lymphoma cell line IIA1.6 (Lankar et al., 2002) and
the LMR7.5 T cell hybridoma that recognizes I-Ad-LACK156–173 complexes
were cultured as reported (Le Roux et al., 2007; Vascotto et al., 2007). Resting
mature spleen IgM+-IgD+ B cells were purified as described (Vascotto et al.,
2007). All mice were housed at the Institut Curie in an accredited specific-
pathogen-free colony and were sacrificed in accordance with the guidelines
and regulations of the French Veterinary Department.
Preparation of Ag-Coated Beads
43 107 3 mm latex NH2-beads (Polyscience) were activated with 8% glutaral-
dehyde for 2 hr at room temperature (RT). Beads were washed with PBS and
incubated overnight (O/N) with different ligands: 10 mg/ml of either F(ab0)2 goatanti-mouse-IgM or F(ab0)2 goat anti-mouse-IgG (MP Biomedicals) or hen egg
white lysozyme (HEL) (Sigma) or 1 mg/ml of Fibronectin (FN) (Sigma).
Stimulation of B Cells and Immunofluorescence Analysis
Cells were activated with Ag-coated NH2-beads at a 1:1 ratio and plated on
poly-L-lysine-coated glass coverslips for different time points at 37�C, fixed
Immunity
Polarized Lysosome Secretion at the B Cell Synapse
in 3%paraformaldehyde (PFA) for 10min at RT, and quenched in 1mMglycine
for 10 min. Fixed cells were incubated with antibodies in PBS-0.2% BSA-
0.05% saponin. For detection of I-Ad-LACK156–173 complexes, biotinylated
2C44 and streptavidin-546 (Tyramide kit, Invitrogen) were used as previously
described (Vascotto et al., 2007). Immunofluorescence images were acquired
on a confocal microscope (LSM Axiovert 720; Carl Zeiss MicroImaging, Inc.)
with a 633 1.4 NA oil immersion objective.
Time-Lapse Analysis
A Nikon Eclipse TE2000-U microscope equipped with a 1003 1.45NA, oil
immersion objective, a PIFOC Objective stepper, a Yokogawa CSU22
confocal unit, and a Roper HQ2 CCD camera was used. B cells expressing
the plasmids of interest were attached on a poly-L-lysine-coated 35 mm
dishes (Fluorodish) at 37�C and images were acquired with Metamorph
software.
Centrosome Ablation
Cells were plated on poly-L-lysine-coated 35 mm dishes and imaged with
a confocal microscope (Zeiss 710 NLO) equipped with an oil immersion lens
(633-1.4 OIL DICII PL APO). Stacks of images were taken every 2 min for
1 hr at 37�C and 5% CO2. After acquiring the first image, laser ablation was
performed with a pulsed laser (Mai-Tai HP, Spectra Physics) controlled by
the Zen2009 software. The laser beam was focused to a region around the
centrosome or lysosome, marked by centrin-GFP or CatD-RFP and 2 itera-
tions of 800 nm laser pulse, corresponding to 40 mW were applied. Ablation
was monitored by the absence of recovery of the RFP or GFP signal.
Ag Presentation
Lack was coupled to glutaraldehyde-activated NH2 beads together with
F(ab0)2 anti-mouse-IgM or anti-mouse-IgG fragments in equal concentrations.
Spleen B cells from BALB/c WT mice or IIA1.6 cells (I-Ad) were incubated with
Lack-coated beads or with Lack Peptide for 4 hr at 37�C. B cells were washed
and incubated with 0.75 3 105 Lack T cell hybridoma for 4 hr. Supernatants
from each condition were obtained and IL-2 was measured (BD Bioscience).
Ag-Extraction Assay
OVA and F(ab0 )2 anti-mouse-IgM or anti-mouse-IgG fragments were coupled
to NH2-beads in equal concentrations. Cells incubated in a 1:1 ratio with
Ag-coated beads were plated on poly-L-lysine slides for different time points
at 37�C, fixed, and stained for OVA with a polyclonal Ab. The amount of OVA
present on the beads after each time point was calculated by establishing
a fixed area around beads in contact with cells and the fluorescence intensity
was measured on 3D projections obtained from the sum of each plane
(Metamorph).
TIRFM
Cells expressing shRNAs were transfected with plasmids encoding Centrin-
GFP, CatD-RFP, or CatD-RFP-pHluorin 48 hr before imaging by TIRFM and
plated on coverslides (MatTek) coated with anti-IgG-F(ab0)2 or anti-IgM-
F(ab0)2 30min before imaging. TIRF imaging was performed on a custom setup
as described previously (Huet et al., 2006) with a penetration depth of
150–300 nm. One pixel corresponds to 107.5 nm. Frames were acquired at
7–10 Hz with an exposure time of 100–150 ms. Image segmentation and
single-particle tracking were done with multidimensional image analysis soft-
ware (MIA) as described (Desnos et al., 2007). The diffusion coefficientDxywas
calculated along trajectories, immobilization periods were defined as portions
of trajectories during which Dxy < 3 3 10�4mm2s�1, and their snapshot occur-
rence was computed (Desnos et al., 2007).
Lysosome Exocytosis Visualized by TIRFM
Cells expressing shRNAs and CatD-RFP-pHluorin were plated for 30 min on
coverslides coated with anti-IgG-F(ab0)2 and imaged by TIRFM. Cells were
selected in the red channel (excitation 514 nm, emission 605–655 nm) and
imaged in the green channel (excitation 488 nm, emission 522–548 nm) to
detect the transient increases in pHluorin fluorescence that follow exocy-
tosis-induced lysosome alkalinization. Stacks of 1200 frames were acquired
at �7 Hz with an exposure time of 150 ms. A denoising algorithm was applied
(Boulanger et al., 2010) before counting the number of exocytic events.
Im
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
seven figures, and five movies and can be found with this article online at
doi:10.1016/j.immuni.2011.07.008.
ACKNOWLEDGMENTS
The authors thank H. Ploegh for providing I-Abb-GFP genetically targetedmice
as well as S. Amigorena, C. Hivroz, and G. Dumenil for critical reading of the
manuscript. The authors greatly acknowledge the Nikon Imaging Centre at
Institut Curie-CNRS, especially V. Fraisier, V. Calco (CNRS, UPR3212, INCI,
Strasbourg, France), O. Leroy, and O. Renaud for technical assistance,
G. Raposo for support with electron microscopy experiments, the Institut
Curie animal facility, R. Basto and V. Marthiens (UMR144, Institut Curie, Paris,
France) for PLK4 constructs and discussion, andM. Piel andM. Heuze for their
precious help with image analysis. A.R. especially thanks F.-X. Gobert for
technical support, notably in lentiviral production. M.-I.Y. especially thanks
A. Sanchez-Squella for help with statistical analysis. M.-I.Y. had been sup-
ported by fellowships from the Curie Institute, Inserm and Fondation pour la
Recherche Medicale. A.R. and B.J. were supported by fellowships from the
Ministere Francais de la Recherche. This work was funded by grants from
the Fondation pour la Recherche Medicale (help for platform creation,
DPT20081214431), the Agence Nationale pour la Recherche to F.D. (ANR-
06-BLAN-0211-02) and A.-M.L.-D. (ANR-06-JCJC-0054), the Young Investi-
gator Program from the City of Paris to A.-M.L.-D., and the European Research
Council to Y.B. (CePoDro 209718) and A.-M.L.-D. (Strapacemi 243103).
Received: March 11, 2010
Revised: April 30, 2011
Accepted: July 8, 2011
Published online: August 4, 2011
REFERENCES
Arana, E., Vehlow, A., Harwood, N.E., Vigorito, E., Henderson, R., Turner, M.,
Tybulewicz, V.L., and Batista, F.D. (2008). Activation of the small GTPase Rac2
via the B cell receptor regulates B cell adhesion and immunological-synapse
formation. Immunity 28, 88–99.
Basto, R., Brunk, K., Vinadogrova, T., Peel, N., Franz, A., Khodjakov, A., and
Raff, J.W. (2008). Centrosome amplification can initiate tumorigenesis in flies.
Cell 133, 1032–1042.
Batista, F.D., and Harwood, N.E. (2009). The who, how and where of antigen
presentation to B cells. Nat. Rev. Immunol. 9, 15–27.
Batista, F.D., and Neuberger, M.S. (2000). B cells extract and present immobi-
lized antigen: Implications for affinity discrimination. EMBO J. 19, 513–520.
Batista, F.D., Iber, D., and Neuberger, M.S. (2001). B cells acquire antigen from
target cells after synapse formation. Nature 411, 489–494.
Boes, M., Cerny, J., Massol, R., Op den Brouw, M., Kirchhausen, T., Chen, J.,
and Ploegh, H.L. (2002). T-cell engagement of dendritic cells rapidly rear-
ranges MHC class II transport. Nature 418, 983–988.
Boulanger, J., Kervrann, C., Bouthemy, P., Elbau, P., Sibarita, J.B., and
Salamero, J. (2010). Patch-based nonlocal functional for denoising fluores-
cence microscopy image sequences. IEEE Trans. Med. Imaging 29, 442–454.
Brezski, R.J., and Monroe, J.G. (2007). B cell antigen receptor-induced Rac1
activation and Rac1-dependent spreading are impaired in transitional
immature B cells due to levels of membrane cholesterol. J. Immunol. 179,
4464–4472.
Cambier, J.C., Pleiman, C.M., and Clark, M.R. (1994). Signal transduction by
the B cell antigen receptor and its coreceptors. Annu. Rev. Immunol. 12,
457–486.
Carrasco, Y.R., and Batista, F.D. (2007). B cells acquire particulate antigen in
a macrophage-rich area at the boundary between the follicle and the subcap-
sular sinus of the lymph node. Immunity 27, 160–171.
munity 35, 361–374, September 23, 2011 ª2011 Elsevier Inc. 373
Immunity
Polarized Lysosome Secretion at the B Cell Synapse
Desnos, C., Huet, S., Fanget, I., Chapuis, C., Bottiger, C., Racine, V., Sibarita,
J.B., Henry, J.P., and Darchen, F. (2007). Myosin va mediates docking of
secretory granules at the plasma membrane. J. Neurosci. 27, 10636–10645.
Fleire, S.J., Goldman, J.P., Carrasco, Y.R., Weber, M., Bray, D., and Batista,
F.D. (2006). B cell ligand discrimination through a spreading and contraction
response. Science 312, 738–741.
Goldstein, B., andMacara, I.G. (2007). The PAR proteins: Fundamental players
in animal cell polarization. Dev. Cell 13, 609–622.
Gomes, E.R., Jani, S., and Gundersen, G.G. (2005). Nuclear movement regu-
lated by Cdc42, MRCK, myosin, and actin flow establishes MTOC polarization
in migrating cells. Cell 121, 451–463.
Goodnow, C.C., Crosbie, J., Adelstein, S., Lavoie, T.B., Smith-Gill, S.J., Brink,
R.A., Pritchard-Briscoe, H., Wotherspoon, J.S., Loblay, R.H., Raphael, K.,
et al. (1988). Altered immunoglobulin expression and functional silencing of
self-reactive B lymphocytes in transgenic mice. Nature 334, 676–682.
Grakoui, A., Bromley, S.K., Sumen, C., Davis, M.M., Shaw, A.S., Allen, P.M.,
and Dustin, M.L. (1999). The immunological synapse: A molecular machine
controlling T cell activation. Science 285, 221–227.
Griffiths, G.M., Tsun, A., and Stinchcombe, J.C. (2010). The immunological
synapse: A focal point for endocytosis and exocytosis. J. Cell Biol. 189,
399–406.
Guo, F., Velu, C.S., Grimes, H.L., and Zheng, Y. (2009). Rho GTPase Cdc42 is
essential for B-lymphocyte development and activation. Blood 114, 2909–
2916.
Haka, A.S., Grosheva, I., Chiang, E., Buxbaum, A.R., Baird, B.A., Pierini, L.M.,
and Maxfield, F.R. (2009). Macrophages create an acidic extracellular hydro-
lytic compartment to digest aggregated lipoproteins. Mol. Biol. Cell 20,
4932–4940.
Harwood, N.E., and Batista, F.D. (2008). New insights into the early molecular
events underlying B cell activation. Immunity 28, 609–619.
Huet, S., Karatekin, E., Tran, V.S., Fanget, I., Cribier, S., and Henry, J.P. (2006).
Analysis of transient behavior in complex trajectories: Application to secretory
vesicle dynamics. Biophys. J. 91, 3542–3559.
Iden, S., and Collard, J.G. (2008). Crosstalk between small GTPases and
polarity proteins in cell polarization. Nat. Rev. Mol. Cell Biol. 9, 846–859.
Junt, T., Moseman, E.A., Iannacone, M., Massberg, S., Lang, P.A., Boes, M.,
Fink, K., Henrickson, S.E., Shayakhmetov, D.M., Di Paolo, N.C., et al. (2007).
Subcapsular sinus macrophages in lymph nodes clear lymph-borne viruses
and present them to antiviral B cells. Nature 450, 110–114.
Kupfer, A., Swain, S.L., and Singer, S.J. (1987). The specific direct interaction
of helper T cells and antigen-presenting B cells. II. Reorientation of the micro-
tubule organizing center and reorganization of the membrane-associated
cytoskeleton inside the bound helper T cells. J. Exp. Med. 165, 1565–1580.
Lankar, D., Vincent-Schneider, H., Briken, V., Yokozeki, T., Raposo, G., and
Bonnerot, C. (2002). Dynamics of major histocompatibility complex class II
compartments during B cell receptor-mediated cell activation. J. Exp. Med.
195, 461–472.
Le Roux, D., Lankar, D., Yuseff, M.I., Vascotto, F., Yokozeki, T., Faure-Andre,
G., Mougneau, E., Glaichenhaus, N., Manoury, B., Bonnerot, C., and Lennon-
Dumenil, A.M. (2007). Syk-dependent actin dynamics regulate endocytic
trafficking and processing of antigens internalized through the B-cell receptor.
Mol. Biol. Cell 18, 3451–3462.
Lennon-Dumenil, A.M., Bakker, A.H., Maehr, R., Fiebiger, E., Overkleeft, H.S.,
Rosemblatt, M., Ploegh, H.L., and Lagaudriere-Gesbert, C. (2002). Analysis of
protease activity in live antigen-presenting cells shows regulation of the phag-
osomal proteolytic contents during dendritic cell activation. J. Exp. Med. 196,
529–540.
374 Immunity 35, 361–374, September 23, 2011 ª2011 Elsevier Inc.
Liu, D., Bryceson, Y.T., Meckel, T., Vasiliver-Shamis, G., Dustin, M.L., and
Long, E.O. (2009). Integrin-dependent organization and bidirectional vesicular
traffic at cytotoxic immune synapses. Immunity 31, 99–109.
Martin, P., Duran, A., Minguet, S., Gaspar, M.L., Diaz-Meco, M.T., Rennert, P.,
Leitges, M., and Moscat, J. (2002). Role of zeta PKC in B-cell signaling and
function. EMBO J. 21, 4049–4057.
Martın-Cofreces, N.B., Robles-Valero, J., Cabrero, J.R., Mittelbrunn, M.,
Gordon-Alonso, M., Sung, C.H., Alarcon, B., Vazquez, J., and Sanchez-
Madrid, F. (2008). MTOC translocation modulates IS formation and controls
sustained T cell signaling. J. Cell Biol. 182, 951–962.
Milasta, S., Evans, N.A., Ormiston, L., Wilson, S., Lefkowitz, R.J., and Milligan,
G. (2005). The sustainability of interactions between the orexin-1 receptor and
beta-arrestin-2 is defined by a single C-terminal cluster of hydroxy amino
acids and modulates the kinetics of ERK MAPK regulation. Biochem. J. 387,
573–584.
Mitchison, N.A. (2004). T-cell-B-cell cooperation. Nat. Rev. Immunol. 4,
308–312.
Moss, C.X., Tree, T.I., and Watts, C. (2007). Reconstruction of a pathway of
antigen processing and class II MHC peptide capture. EMBO J. 26, 2137–
2147.
Onabajo, O.O., Seeley, M.K., Kale, A., Qualmann, B., Kessels, M., Han, J., Tan,
T.H., and Song, W. (2008). Actin-binding protein 1 regulates B cell receptor-
mediated antigen processing and presentation in response to B cell receptor
activation. J. Immunol. 180, 6685–6695.
Pulecio, J., Petrovic, J., Prete, F., Chiaruttini, G., Lennon-Dumenil, A.M.,
Desdouets, C., Gasman, S., Burrone, O.R., and Benvenuti, F. (2010). Cdc42-
mediated MTOC polarization in dendritic cells controls targeted delivery of
cytokines at the immune synapse. J. Exp. Med. 207, 2719–2732.
Puthenveedu, M.A., Lauffer, B., Temkin, P., Vistein, R., Carlton, P., Thorn, K.,
Taunton, J., Weiner, O.D., Parton, R.G., and von Zastrow, M. (2010).
Sequence-dependent sorting of recycling proteins by actin-stabilized endoso-
mal microdomains. Cell 143, 761–773.
Reth, M., andWienands, J. (1997). Initiation and processing of signals from the
B cell antigen receptor. Annu. Rev. Immunol. 15, 453–479.
Sankaranarayanan, S., De Angelis, D., Rothman, J.E., and Ryan, T.A. (2000).
The use of pHluorins for optical measurements of presynaptic activity.
Biophys. J. 79, 2199–2208.
Stinchcombe, J.C., and Griffiths, G.M. (2007). Secretory mechanisms in cell-
mediated cytotoxicity. Annu. Rev. Cell Dev. Biol. 23, 495–517.
Stoddart, A., Jackson, A.P., and Brodsky, F.M. (2005). Plasticity of B cell
receptor internalization upon conditional depletion of clathrin. Mol. Biol. Cell
16, 2339–2348.
Suzuki, K., Grigorova, I., Phan, T.G., Kelly, L.M., and Cyster, J.G. (2009).
Visualizing B cell capture of cognate antigen from follicular dendritic cells.
J. Exp. Med. 206, 1485–1493.
Tolar, P., Sohn, H.W., and Pierce, S.K. (2008). Viewing the antigen-induced
initiation of B-cell activation in living cells. Immunol. Rev. 221, 64–76.
Vascotto, F., Lankar, D., Faure-Andre, G., Vargas, P., Diaz, J., Le Roux, D.,
Yuseff, M.I., Sibarita, J.B., Boes, M., Raposo, G., et al. (2007). The actin-based
motor protein myosin II regulates MHC class II trafficking and BCR-driven
antigen presentation. J. Cell Biol. 176, 1007–1019.
Wubbolts, R., Fernandez-Borja, M., Jordens, I., Reits, E., Dusseljee, S.,
Echeverri, C., Vallee, R.B., and Neefjes, J. (1999). Opposing motor activities
of dynein and kinesin determine retention and transport of MHC class II-con-
taining compartments. J. Cell Sci. 112, 785–795.