Splenic marginal zone antigen-presenting cells are critical for the primary allo-immune response to...

ORIGINAL ARTICLE

Splenic marginal zone antigen-presenting cells are critical forthe primary allo-immune response to therapeutic factor VIII inhemophilia A

A. NAVARRET E ,*�� S . DASGUPTA ,*�� S . DEL IGNAT ,*�� G. C AL I G I UR I ,*�� O. D . CHR ISTOPHE ,§

J . BAYR Y ,*�� A . N I COLETT I , *�� S . V . KA VER I *�� and S . LACROIX-DESMAZE S*��*Centre de Recherche des Cordeliers, Universite Pierre et Marie Curie-Paris6, UMRS 872, Paris; �Universite Paris Descartes, UMRS872, Paris;

�INSERM, U872, Paris; and §INSERM U770, Universite Paris-Sud, Faculte de medecine Paris-Sud, IFR93, Le Kremlin-Bicetre, France

To cite this article: Navarrete A, Dasgupta S, Delignat S, Caligiuri G, Christophe OD, Bayry J, Nicoletti A, Kaveri SV, Lacroix-Desmazes S. Splenic

marginal zone antigen-presenting cells are critical for the primary allo-immune response to therapeutic factor VIII in hemophilia A. J Thromb

Haemost 2009; 7: 1816–23.

Summary. Background: Alloimmune responses to intrave-

nously administered protein therapeutics are the most common

cause of failure of replacement therapy in patientswith defective

levels of endogenous proteins. Sucha situation is encountered in

some patients with hemophilia A, who develop inhibitory anti-

factor (F)VIII alloantibodies after administration of FVIII to

treat hemorrhages. Objectives: The nature of the secondary

lymphoid organs involved in the initiation of immune responses

to human therapeutic has not been studied. We therefore

investigated this in the case of FVIII, a self-derived exogenous

protein therapeutic.Methods:The distribution of intravenously

administered FVIII was followed after FVIII-deficient mice

were injected with radiolabeled FVIII and using immunohisto-

chemistry. The role of the spleen and antigen-presenting cells

(APC) in the onset of the anti-FVIII immune response was

analyzed upon splenectomy or treatment of themicewithAPC-

depleting compounds. Results: FVIII preferentially accumu-

lated in the spleen at the level of metallophilic macrophages in

the marginal zone (MZ). Surgical removal of the spleen or

selective in vivo depletion of macrophages and CD11c-positive

CD8a-negative dendritic cells resulted in a drastic reduction in

anti-FVIII immune responses. Conclusions: Using FVIII-

deficient mice as a model for patients with hemophilia A, and

human pro-coagulant FVIII as a model for immunogenic self-

derived protein therapeutics, our results highlight the impor-

tance of the spleen and MZ APCs in the initiation of immune

responses to protein therapeutics. Identification of the receptors

implicated in retention of protein therapeutics in the MZ may

pave the way towards novel strategies aimed at reducing their

immunogenicity.

Keywords: allo-immunization, factor VIII, FVIII inhibitors,

hemophilia A, protein therapeutics, spleen.

Introduction

Replacement therapy is the treatment of choice for patients

with endogenous deficiencies of self-proteins. In several

instances, patients develop antibodies that inhibit the admin-

istered therapeutics [1]. Such a situation is encountered in

patients with hemophilia A, a X-linked bleeding disorder

consecutive to the lack of functional endogenous factor

(F)VIII: in 10%–30% of the patients, administration of FVIII

to treat hemorrhages results in induction of inhibitory anti-

FVIII IgG [2]. The development of allo-immune responses to

therapeutic FVIII suggests that exogenous FVIII is not solely

attracted to bleeding sites and trapped by catabolic organs, but

also reaches lymphoid organs where it is processed by antigen-

presenting cells (APCs) and presented to immune effectors.

The spleen plays a primary role in filtration of blood by

removing particulate components such as immune complexes,

bacteria or colloidal particles, and supports the development of

immunity, especially towards blood-borne antigens [3]. The

spleen contains three major types of professional APCs:

macrophages, B lymphocytes and dendritic cells (DCs) [4].

Blood-borne antigens reach the spleen through the splenic

artery which branches either towards the red pulp, and interacts

with red pulp macrophages, or towards the marginal zone

(MZ) of the spleen where B cells, metallophilic macrophages

and MZ macrophages reside [3]. Initiation of the immune

responses requires the antigens to reach the white pulp of the

spleen [5]. Depending on their size, solubility and concentra-

tion, blood-borne molecules diffuse directly to the white pulp

from the MZ [6] or are delivered to the follicles by APCs [7].

Correspondence: Sebastien Lacroix-Desmazes, INSERM UMR 872

Equipe 16, Centre de Recherche des Cordeliers, Paris F-75006, France.

Tel.:+33 1 55 42 82 65; fax: +33 1 55 42 82 62.

E-mail: [email protected]

AMN, SuDa and SaDe contributed equally to the work.

Received 4 June 2009, accepted 13 July 2009

Journal of Thrombosis and Haemostasis, 7: 1816–1823 DOI: 10.1111/j.1538-7836.2009.03571.x

� 2009 International Society on Thrombosis and Haemostasis

The localization and identification of the type of APCs

implicated in immune responses have been studied in the case

of exogenous model antigens [8], often as a prerequisite to

therapeutic targeting of defined APCs in vaccination strate-

gies [9]. To our knowledge, the identification of the lymphoid

organs and APCs implicated in immune responses to blood-

borne therapeutic self-proteins, a prerequisite to the selective

prevention of deleterious iatrogenic allo-immune responses,

has not been addressed. In the present study, we hypothe-

sized that intravenously administered therapeutic FVIII

accumulates in the spleen. Using FVIII-deficient mice, a

mouse model of hemophilia A, we demonstrate the relevance

of the spleen and of splenic phagocytes for the onset of the

naıve anti-FVIII allo-immune response. We further seek to

identify the populations of endocyting cells implicated in the

capture of FVIII.

Materials and methods

Animals

Mice used in the experiments were 7- to 12-week-old 129/B6

(H-2Db background) exon 16 FVIII-deficientmales (a kind gift

from Professor H.H. Kazazian, Department of Genetics,

University of Pennsylvania School of Medicine, Philadelphia,

USA). Animals were handled in agreement with local ethical

authorities (Comite regional d�ethique p3/2005/002).

Coupling of iodine125 to FVIII

Coupling of FVIII to iodine125 (I125) was done by the IODO-

GEN� (Pierce Chemical Co, Rockford, IL, USA) method.

Briefly, recombinant human FVIII (Kogenate�; Bayer

Healthcare, Lille, France; or Helixate�; CSL-Behring, Paris,

France; Kogenate� and Helixate� are the same molecule) was

solubilized in water and dialyzed against 100 mM Borate

buffer for 4 h. Dialyzed FVIII was incubated at room

temperature for 6 min with iodine125 (Amersham GE Health-

care, Buckinghamshire, UK) at a ratio of 10 lCi lg)1 of

FVIII. Radio-labeled FVIII was eluted using a PD-10 column

(Amersham GE Healthcare). FVIII-containing fractions were

eluted and passed on a gamma-counter. The specific activity

of coupling was 1065 cpm fmol)1 of FVIII. Comparison of

the binding of unlabeled and radiolabeled FVIII to von

Willebrand factor (VWF) and to a monoclonal anti-C2 IgG

(Bo2C11, a kind gift from Professor JM Saint-Remy, Leuven,

Belgium) in radio-immuno assays, indicated that labeled

FVIII retained 65%–75% of its capacity to bind VWF.

Organ distribution of I125-FVIII

FVIII-deficient mice were injected intravenously with I125-

FVIII (1 lg per mouse in 200 lL PBS). Mice were sacrificed

at different time points after injection. Organs were collected,

weighed and incorporated radioactivity was measured with a

gamma-counter. Five mice per time point were utilized.

Kinetics of I125-FVIII clearance were computed using a

double-exponential model: Q = Q1e�k1t þQ2e

�k2t, where Q

represents the quantity of FVIII (cpm lg)1 tissue) at a given

time point, k1 and k2 are the rate constants corresponding to

fast and slow phases of FVIII elimination in min)1, and Q1

and Q2 are quantities of FVIII removed during the fast and

slow phases of clearance, respectively. In VWF-deficient mice,

FVIII is eliminated in < 10 min. The 35%–25% of iodinated

FVIII molecules that do not bind to VWF will thus be

eliminated very rapidly after intravenous injection, and,

hence, will not bias our experimental results on FVIII half-

life.

Administration of FVIII to splenectomized mice

Anesthetized mice were splenectomized or sham-operated. One

day after surgery, mice were injected intravenously or subcu-

taneously with FVIII. In the case of intravenous injections,

recombinant human FVIII was administered (0.2 lg per

mouse) once a week for 4 weeks. For subcutaneous injection,

FVIII (20 lg per mouse) in the presence of Freund�s adjuvantwas injected three times at 1-week intervals. Blood samples

were collected 1 week after the last injection. Serumwas kept at

)20 �C until use.

Titration of anti-FVIII IgG and of inhibitors was performed

by ELISA and chromogenic assay respectively, as described

[10].

Administration of FVIII to clodronate-containing liposomes-

treated mice

Clodronate was a gift of Roche Diagnostics GmbH, Mann-

heim, Germany. It was encapsulated in liposomes as described

earlier [11]. FVIII-deficient mice were administered intrave-

nously with clodronate- or phosphate-buffered saline (PBS)-

containing liposomes (200 lL per mouse) twice with a weekly

interval. FVIII (0.2 lg per mouse) was injected intravenously

48 h after the first and second administrations of liposomes,

and two additional times at weekly intervals. Under such

conditions, clodronate-containing liposomes eliminate phago-

cytes [11]. Repopulation of macrophages starts 9 days after

treatment in the red pulp and 15 days in the MZ [12]. Blood

samples were collected 1 week after the last injection of FVIII.

Serum was kept at )20 �C until use. In some cases, mice were

sacrificed 48 h after the first administration of liposomes.

Spleens were collected and analyzed by immunohistochemistry

and flow cytometry to assess the extent of cell depletion.

Analysis of splenocytes by multi-color flow cytometry

Mice were sacrificed and spleens collected. Spleens were

digested with a solution containing 0.42 lg mL)1 Liberase

and 0.1 lg mL)1 DNAse (Roche, Indianapolis, IN, USA).

Thirty minutes later, single cell suspensions were prepared.

Splenocytes were washed, counted and incubated with anti-

mouse CD11c-PE, anti-mouse CD8a-pacific blue, anti-mouse

Allo-immunization to therapeutic FVIII 1817


B220 PE-TexRed (Pharmingen, BD bioscience, CA, USA)

and anti-mouse F4/80-APC (EBioscience, San Diego, CA,

USA) monoclonal rat antibodies, and analyzed using a

LSRII flow cytometer (BD bioscience). Dead cells were gated

using positive annexin V-FITC (Immunotech, Marseille,

France) staining and excluded from the analysis. More than

400 000 events were acquired in the annexin V-FITC negative

gate. Analysis used the FACSDIVA software v5.0.1 (BD

bioscience).

Analysis of splenocytes by immuno-histochemistry

Mice were sacrificed and spleens snap-frozen in liquid nitrogen.

Spleen sections (10 lm) were fixed in acetone at 4 �C for

10 min. Endogenous peroxidase and avidin-biotin activity were

eliminated (Dako Cytomation, Glostrup, Denmark). Metallo-

philic macrophages and macrophage-associated receptor with

collagenous structure (MARCO)-positive MZ macrophages

were identified with monoclonal rat anti-sialic acid-binding

lectin-1 (MOMA-1) and anti-MARCO antibodies (Abd Sero-

tec, Oxford, UK), followed by a rabbit anti-rat biotinylated

antibody (Dako). DCs were detected with a monoclonal

hamster anti-CD11c antibody (Pharmingen, BD Biosciences,

CA, USA), followed by a goat anti-hamster biotinylated

antibody. Red pulp macrophages were labeled with a mono-

clonal rat anti-F4/80 biotinylated antibody (eBiosciences) and

revealed with streptavidine-peroxydase from the ABC kit

(DAKO) and DAB substrate (Vector laboratories, CA, USA).

Analysis of FVIII distribution in the spleen by

immuno-fluorescence

Mice were injected with 3 lg of human recombinant FVIII.

Thirty minutes post-injection, the mice were sacrificed and

spleens collected and snap-frozen in liquid nitrogen. Serial

cryosections (10 lm)were air-dried and fixed in acetone. FVIII

was detected with a sheep polyclonal anti-human FVIII IgG

(Kordia, Leiden, the Netherlands) followed by a donkey anti-

sheep IgG conjugated to FITC (Abcam, Cambridge, UK) or to

Alexa Fluor 647 (Invitrogen, Orlando, FL, USA). Metallo-

philic macrophages, MZ macrophages and red pulp macro-

phages were stained using monoclonal rat anti-MOMA-1

FITC, rat anti-MARCO FITC (AbD Serotec, Oxford, UK)

and biotinylated anti-F4/80 (eBiosciences) antibodies followed

by Avidin-Alexa Fluor 555 (Invitrogen), respectively. DCs

were identified with a monoclonal hamster anti-CD11c,

followed by biotinylated goat anti-hamster polyclonal antibody

and Avidin-Alexa Fluor 555. Nuclei were counterstained with

0.1 lg mL)1 Hoechst 33342 and tissue sections were mounted

with ProLong� Gold antifade reagent (Invitrogen). Images

were acquired using an Axiovert� M200 microscope (Zeiss)

equipped with Apoptome� and four filters (Dapi, FITC,

Rhodamine, APC) connected to a monochromatique CCD

camera. Digital images were captured with AxioVision�

software and analyzed with Adobe Photoshop V.CS3 (Adobe,

Dublin, Ireland).

Results

FVIII accumulates in the spleen of FVIII-deficient mice after

intravenous administration

We investigated the distribution of intravenously administered

FVIII in the liver, spleen, blood, kidneys, lymph nodes and

lungs of FVIII-deficient mice after 15, 30, 60, 120, 240 and

480 min. FVIII declined from the circulation whereas it

accumulated in the spleen and liver (Fig. 1). FVIII density in

lungs, kidneys and lymph nodes was marginal. FVIII clearance

from blood, liver and spleen required a double exponential

model to fit the data (Table 1), with a fast phase and a slow

phase of FVIII elimination.

During the first 30 min after injection, the FVIII concentra-

tiondecreasedby7.6% ± 11.0%inthecaseofthespleenandby

11.0% ± 8.8% in the case of blood. It dropped by

48.2% ± 12.0% in the case of the liver (P < 0.01 as compared

with the spleen and blood using Mann–Whitney U-test).

Accordingly, the rate constant for the fast phase of FVIII

elimination tended to be significantly higher in the case of the

liver (0.078 ± 0.037 min)1) than the spleen (0.025 ± 0.025

min)1, P = 0.076). Together, the data indicate a longer

residence time and a higher density of FVIII in the spleen as

compared with other organs, including liver and lymph nodes.

The spleen is essential for mounting an allo-immune response

to therapeutic FVIII

In order to determine the importance of the spleen in a specific

immune response to therapeutic FVIII, FVIII-deficient mice

1600

Lymph nodesBloodLiverLungSpleenKidney

1200

800

400

010 100 1000

Time (min)

FV

III (

cpm

per

mg

tissu

e)

Fig. 1. Tissue distribution of factor (F)VIII after intravenous adminis-

tration. I125-FVIII (1 lg) was injected intravenously to FVIII-deficient

mice. At the indicated time points, mice were sacrificed. Lymph nodes (full

circles), blood (empty circles), liver (empty squares), lung (X), spleen (full

squares) and kidney (+) were collected, weighed and the incorporated

radioactivity was measured. Results depict the density of FVIII (cpm per

lg of tissue). Data represent mean values from five mice at each time point

and are representative of two independent experiments.

1818 A. Navarrete et al


were splenectomized and injected with FVIII. After the fourth

injection of FVIII, the anti-FVIII IgG titer was 4.4-fold lower

in splenectomized mice (81.3 ± 46.8 lg mL)1 mAb6-equiva-

lent) as compared with sham-operated animals (359.9 ±

204.4 lg mL)1 mAb6-equivalent, P < 0.05, Fig. 2A). Fur-

ther, the inhibitory activity in the serum of sham-operated mice

(494 ± 304 BU mL)1) was 4.3-fold higher than in the serum

of splenectomized animals (115 ± 104 BU mL)1, P < 0.05)

(Fig. 2B). Interestingly, total IgG titers were similar in both

groups of mice (Fig. 2C), indicating that removal of the spleen

does not alter the levels of circulating IgG over a period of

4 weeks after surgery. Furthermore, sham-operated and sple-

nectomized mice developed similar titers of anti-FVIII IgG

after subcutaneous immunization with FVIII in Freund�sadjuvant (data not shown), indicating that removal of the

spleen does not hamper the capacity of the mice to mount a

specific immune response to an antigen that is targeted to the

lymph nodes. Together, these results demonstrate that the

spleen is the major secondary lymphoid organ for the

development of an anti-FVIII immune response.

Accumulation of therapeutic FVIII at the level of macrophages

in the spleen

In order to identify the cells co-localizing with FVIII in the

spleen, we injected intravenously near to therapeutic doses of

exogenous FVIII (3 lg) to naıve FVIII-deficient mice. Fig-

ure 3A documents the accumulation of FVIII in the splenic

MZ after intravenous injection of FVIII (3 lg). Immunohis-

tochemistry experiments demonstrated preferential FVIII co-

localization with metallophilic macrophages (MOMA-1-posi-

tive, Fig. 3B) and, to a lesser extent, MARCO-positive MZ

macrophages (Fig. 3C). Occasionally FVIII was co-localized

with CD11c-positive DCs in theMZ (Fig. 3E). FVIII was only

sporadically detected with red pulp F4/80-positive macrophag-

es (Fig. 3D). FVIII was not detected at the level of B

lymphocytes in naıve mice (data not shown).

Depletion of macrophages hampers the development of the

anti-FVIII immune response

To determine the importance of macrophages in an anti-FVIII

immune response, mice were injected with clodronate-contain-

ing liposomes. Immuno-histochemistry confirmed depletion of

bothF4/80-positive redpulpmacrophagesandMZ(MOMA-1-

andMARCO-positive)macrophages from clodronate-contain-

ing liposome-treated mice (Fig. 4A). Numbers of red pulp F4/

80-positive macrophages and CD11c-positive DCs were

reduced by 3.1 (from 7.8% ± 1.1% to 2.6% ± 0.4% of the

total splenocyte population) and 1.3-fold (from 9.8% ± 0.5%

to 7.6% ± 0.8%) as compared with control mice, respectively

(P < 0.05, Fig. 4B). Changes in B lymphocytes numbers were

marginal (from 57.4% ± 0.5% to 50.9% ± 0.1%).

Table 1 Clearance of 125I-FVIII from the blood, liver and spleen of FVIII-

deficient mice

Organ

Q1 Q2 k1 k2

cpm lg)1 min)1

Blood 0.48 ± 0.25*� 0.55 ± 0.08* 0.059 ± 0.049 0.005 ± 0.001

Liver 2.87 ± 1.92* 0.36 ± 0.12* 0.078 ± 0.037� 0.006 ± 0.002

Spleen 2.16 ± 1.10� 0.81 ± 0.48 0.025 ± 0.025� 0.006 ± 0.005

The values (mean ± SD) of the kinetic rate constants k1 and k2, that

correspond to the fast and slow phases of FVIII clearance, and the

amount of FVIII removed during these two phases (Q1 and Q2,

respectively, expressed as cpm lg)1 of tissue) were determined by fit-

ting the clearance data depicted in Fig. 1 to the double exponential

decay curve: Q = Q1e�k1t þQ2e

�k2t, where t is the time in minutes.

Differences were statistically compared using the Mann–Whitney U-

test (*, blood vs. liver; �, blood vs. spleen; �, liver vs. spleen).

Underscored letters indicate tendency towards significance (P < 0.1),

otherwise significance (P < 0.05).

P < 0.05 P < 0.051000

800

600

400

Inhi

bito

ry a

ctiv

ity (

BU

mL–

1 )

200

0

SplnxSham

SplnxSham

1.6A B C

1.2

0.8

0.4

00.0001 0.001 0.01 0.1

Plasma dilution

IgG

bin

ding

to F

VIII

(opt

ical

den

sity

, 492

nm

)

ns800

600

400

Tota

l IgG

(A

U)

200

0SplnxSham

Fig. 2. Importance of the spleen in the immune response to factor (F)VIII in FVIII-deficient mice. FVIII-deficient mice were splenectomized (Splnx, eight

mice) or sham operated (five mice) and injected with FVIII (0.2 lg per mouse) intravenously four times at weekly intervals. Panel A. Levels of anti-FVIII

IgG in the serum of the mice weremeasured by (mean ± SEM). Panel B. Inhibitory titers (BUmL)1) in mouse serumwere measured using a chromogenic

assay. Panel C. Titers of total IgG were measured by ELISA in the same samples and are expressed as arbitrary units (AU). The data are representative of

two independent experiments. Statistical significance was assessed using the Mann–Whitney U-test (ns, non significant).



T lymphocytes numbers were not affected by clodronate-

containing liposome treatment (data not shown). Among

CD11c-positive DCs, CD8a-negative cells were significantly

reduced in numbers (from 6.3% ± 2.1% to 4.0% ± 0.4% of

the total splenocyte population, P < 0.05, Fig. 4C).

Clodronate-containing liposome-treated mice also demon-

strated significantly reduced titers of anti-FVIII IgG

(28.8 ± 20.0 vs. 573.7 ± 154.5 lg mL)1 mAb6-equivalent,

P < 0.01) and of inhibitory activity towards FVIII, as

compared with control mice (Fig. 5A and B). Clodronate-

containing liposomes treatment had no effect on the total levels

of circulating IgG over a period of 4 weeks after the initial

liposome treatment (Fig. 5C).

Discussion

Pharmacokinetics of FVIII have been studied in different

animal models and in human [13,14]. Under physiological

A

B

C

D

E

20 µm RP

RP

RP

RP

RP

RP

MZ

MZ

MZ

MZ

WP

WP

WP

WP

WP

Fig. 3. In situ localization of factor (F)VIII in the spleen of FVIII-deficient mice. FVIII-deficient mice were injected with FVIII or phosphate-buffered

saline (PBS), and sacrificed 30 min later. Panel A. FVIII (green) was detected on histological spleen sections in the case of FVIII- (left panel) and

PBS-treated mice (right panel). Panels B, C, D and E. Histological sections of spleens of FVIII-treated mice were labeled with anti-FVIII antibodies

(Green), and with MOMA-1, anti-MARCO, anti-F4/80 or anti-CD11c (panels B, C, D and E, respectively) antibodies (Red). Left panels show FVIII

staining alone, middle panels showMOMA-1, MARCO, F4/80 or CD11c staining alone and right panels showmerged images. Data are representative of

3–4 mice in each group. MZ, marginal zone, WP, white pulp, RP, red pulp, as identified using MOMA-1 and anti-F4/80 antibodies. Magnification 40·.



conditions, human FVIII is produced mainly in the liver and in

the lungs [15,16]. Recent evidence obtained in wild-type mice

(i.e. FVIII-sufficient animals) suggest that the liver is also the

site of FVIII elimination; disruption of the expression of

hepatic low-density lipoprotein-related protein receptor (LRP

or CD91) in conditional knock-out mice, or saturation of the

liver-expressed asialoglycoprotein receptor (ASGPR) using the

ASGPR antagonist asialo-orosomucoid, result in increased

FVIII residence time in the blood [17]. Here, we analyzed the

fate of exogenous FVIII in FVIII-deficient mice, a context

similar to that of patients with severe hemophilia A where the

endogenous molecule is absent. As reported [18], FVIII

administered at replacement doses was found at an elevated

density in the blood, the liver and the spleen. Conversely, low

FVIII density was detected in other organs.

The rate of exogenous FVIII elimination from the blood of

FVIII-deficientmice in the fast phase (0.059 ± 0.04 min)1)was

greater than previously reported (0.020 ± 0.003 min)1) [19].

Liposomecontent

Clodronate

PBS

Cel

l cou

ntC

D11

c10

510

410

310

2–1

0215

0010

0050

00

2000

1500

1000

500

0

0

105

104

103

102

–102

0

105

7.8 ± 1.1

F4/80

PBS-containingliposomes

Clodronate-containingliposomes

B220

9.8 ± 0.5 7.6 ± 0.8

2.6 ± 0.4

50.9 ± 0.157.4 ± 0.5

104103102–1020

105104103–103 0 105104103–103 0

105104103102–1020

MOMA

A

C

B

MARCO F4/80 CD11c

9*

6

3

0

% o

f spl

enoc

ytes

CD11c+CD8+

CD11c+CD8–

Fig. 4. In vivo depletion of macrophages and dendritic cells (DCs) by clodronate-containing liposomes. FVIII-deficient mice were injected with PBS- or

clodronate-containing liposomes. After 48 h, mice were sacrificed and spleens collected. Panel A. Spleen sections were analyzed by immuno-histochemistry

using MOMA-1, anti-MARCO, anti-F4/80 and anti-CD11c antibodies. Panel B. Splenocytes were purified, labeled with anti-F4/80, anti-CD11c, anti-

B220 and anti-CD8a antibodies, and analyzed by flow cytometry. The percentages of DCs (CD11c+), macrophages (F4/80+ CD11c)) and B lym-

phocytes (B220+ CD11c)) were calculated among total splenocytes. Panel C. The graphs depicts percentages of CD11c-positive cells that are positive or

negative for CD8a in the case of mice treated with PBS- (full bars) or clodronate-containing (empty bars) liposomes. Data represent the mean ± SD of

four mice per group. Statistical significance was assessed using the Mann–Whitney U-test (*P < 0.05).

1.2

1

0.8

0.6

0.4

0.2

00.0001 0.001 0.01 0.1

Plasma dilution

P < 0.05P < 0.01

ns800

600

400

IgG

tite

r (A

U)

200

0

200

150

100

Inhi

bito

ry a

ctiv

ity (

BU

mL–

1 )

50

0Control Clodronate Control Clodronate

Clodronate-containingliposomes

PBS-containingliposomes

IgG

bin

ding

to F

VIII

(opt

ical

den

sity

, 492

nm

)

A B C

Fig. 5. Importance of macrophages and dendritic cells (DCs) in the immune response to factor (F)VIII in FVIII-deficient mice. FVIII-deficient mice were

treated with phosphate-buffered saline (PBS)- (six mice) or clodronate-containing (eight mice) liposomes.Mice were injected with FVIII (0.2 lg per mouse)

intravenously four times at weekly intervals. The anti-FVIII immune response was assessed 7 days after the fourth injection. Panel A. Levels of anti-FVIII

IgG in mouse serum were measured by ELISA (mean ± SEM). Panel B. Inhibitory titers (BU mL)1) in the serum of the mice were measured using a

chromogenic assay. Panel C. Titers of total IgG were measured by ELISA in the same samples and are expressed as arbitrary units (AU). The data are

representative of two independent experiments. Statistical significance were assessed using the Mann–Whitney U-test (ns, non significant).



Similar rates were observed in wild-type mice under the same

experimental conditions (data not shown). Both the liver and

spleen demonstrated accumulation of exogenous FVIII early

after injection, and a biphasic elimination pattern of FVIII. The

two organs displayed different behaviors as far as FVIII

elimination is concerned.While the amount of FVIII in the liver

rapidly decreased during the first 30 min after administration, it

remained stable in the case of the spleen. The data may reflect

the different capacities of the two organs to catabolize FVIII, as

well as their differences in size. Thus, during the first 30 min

after intravenous administration, the catabolism of FVIII in the

spleen is balanced by its constant influx from the blood which

maintains a constant level of FVIII.

Blood-borne antigens have been shown to reach theMZ and

red pulp of the spleen and to be captured by resident APCs

[6,20]. However, such studies employedwild-type animals using

model antigens such as albumin. In contrast, here we have used

human pro-coagulant FVIII as a model for immunogenic self-

derived protein therapeutics administered to FVIII-deficient

mice, a suitable model for patients with the X-linked hemor-

rhagic disorder hemophilia A. In addition, the amount of

FVIII injected in our model was close to therapeutic doses, i.e.

‡ 30-fold lower than that used in the case of albumin.

During the first 30 min after intravenous administration,

accumulation of FVIII in the spleen may be of importance to

its immunogenicity. Indeed, the spleen screens the blood and

provides the immunological environment for initiation of

adaptive immune responses against blood-borne antigens [3].

Using FVIII-deficient mice, we show that the onset of the anti-

FVIII immune response occurs in the spleen. Removal of the

spleen resulted in a significant reduction in the amplitude of the

anti-FVIII humoral response. Our results are in line with

previous observations wherein disruption of splenic germinal

centers by intravenous injection of anti-CD154 antibodies was

followed by a reduction in anti-FVIII antibody titers and

abolition of T-cell responses to FVIII [21]. Interestingly,

development of a detectable anti-FVIII immune response to

therapeutic FVIII was observed in splenectomized animals,

indicating that alternative secondary organs, the lymph nodes

or possibly the bone marrow, may be involved in the immune

response to blood-borne protein therapeutics as well [22].

Whether such secondary lymphoid organs are also at play

when the spleen is present, remains to be validated.

Our data suggest a role for residentmacrophages andDCs in

trappingFVIII in themarginal zone:FVIII clustered at the level

of MZ macrophages, preferentially MOMA-1-positive cells

and, toa lesser extent,withMARCO-positivemacrophages and

CD11c-positive DCs in the marginal zone. Localization of

exogenously administered proteins at the level of MZ macro-

phages hasbeendocumented in the caseof the administrationof

elevated amounts of ovalbumin (OVA) [6]. In contrast, intra-

venously administered VWFwas recently found to accumulate

at the level of red pulp macrophages and to be absent from the

MZ [18].

MZ macrophages play a role in both T-dependent and

T-independent immune responses [12,23]. Here, depletion of

macrophages from the spleen of naıve FVIII-deficient mice

with clodronate-containing liposomes, prior to administration

of therapeutic doses of FVIII, resulted in close to complete

abrogation of the humoral anti-FVIII immune response.

Elimination of MZ macrophages by clodronate-containing

liposomes may have diverse side effects. Indeed, splenic MZ

macrophages have important alternative roles in the dynamic

equilibrium of the immune system. They secrete cytokines and

chemokines that determine the fate and intensity of immune

responses [24]. Further, they have been proposed to transport

and deliver antigens to immune effectors in the follicles [25].

Treatment of mice with clodronate-containing liposomes

also reduced the CD11c+CD8 a-negative DC population, as

described previously [26]. Targeting of CD8a-negative DCs

using ovalbumin-coupled 33D1-specific antibodies drives anti-

gen presentation through the MHC II pathway [8]. Our

observations of a drastically reduced immune response to

FVIII in clodronate-containing liposome-treated animals may

thus also result from the reduction in FVIII internalization by

CD11c+CD8a-negative DCs and presentation to T cells in

splenic germinal centers [27].

Several endocytic receptors specific for different FVIII

moieties have been characterized. Members of the low-density

lipoproteinreceptor(LDLR)familyrecognizeproteinstructures

in the heavy and light chains of FVIII [19,28,29]. ASGPRbinds

to galactose-ending glycans of the B domain of FVIII [17].

Recently,wehaveshownthatthemacrophagemannosereceptor

(MMR/CD206) interacts with mannose-ending glycans on the

A1 and C1 domains of the molecule [30]. The constitutive

expressionofMARCOandMOMA-1byMZmacrophageshas

been implicated in the capture of blood-borne antigens [31–33].

Whether the latter receptorsmaybe implicated in recognition of

FVIII by macrophages is being currently investigated.

Addendum

Designed research: S. Dasgupta, J. Bayry, A. Navarrete, S.V.

Kaveri, S. Lacroix-Desmazes; performed research: A. Navar-

rete, S.Dasgupta,S.Delignat,G.Caligiuri,O.D.Christophe,A.

Navarrete, S. Lacroix-Desmazes; analyzed data: A. Navarrete,

S. Dasgupta, S. Delignat, G. Caligiuri, O.D. Christophe, A.

Navarrete, S. Lacroix-Desmazes; wrote the paper: A. Navar-

rete, S. Delignat, J. Bayry, S.V. Kaveri, S. Lacroix-Desmazes.

Acknowledgements

This work was supported by INSERM, by CNRS, by UPMC-

Paris6, and by grants from Agence Nationale de la Recherche

(ANR-05-MRAR-030-1, ANR-07-JCJC-0100-01, ANR-07-

RIB-002-02) and Grifols (Barcelona, Spain). SuD and AMN

were recipients of fellowships from Fondation de la Recherche

Medicale and Region Ile-de-France (Paris, France).

We thank A. Rice (Mater Medical Research Institute and

University of Queensland, Australia) for giving us anti-CD11c

antibodies. Kogenate�, Helixate� and Advate� were kind gifts

from Bayer Healthcare (Lille, France), from CSL-Behring



(Paris, France) and from Baxter (Maurepas, France), respec-

tively. We thank C. Klein for advice with immunohistochem-

istry (�Cellular imaging and cytometry� facility, CRC,

INSERM U872, Paris).

Disclosure of Conflict of Interests

The authors state that they have no conflict of interest.

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