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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

mailto:[email protected]

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http://dx.doi.org/10.1016/j.immuni.2011.07.008

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

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LAMP-1

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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).

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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

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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


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

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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


G

Cel

ls (%

)

Polarity index a/b

0

10

20

30

40

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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.

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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


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

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MTOC ablation No ablation

Lysosome ablation

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d C

M

Time (min)

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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).

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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

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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 20 40

60 80

100 Time 2 hr


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).


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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-


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

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60

Mea

n Fl

uore

scen

ce In

tens

ity *** 70

D

700

0

200 300

500 600

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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.

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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



Immunity


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


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.

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Immunity


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).


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


Immunity


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-


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


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

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