UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl)
UvA-DARE (Digital Academic Repository)
Molecular determinants of FVIII immunogenicity in hemophilia A
Wróblewska, A.
Publication date
2013
Link to publication
Citation for published version (APA):
Wróblewska, A. (2013). Molecular determinants of FVIII immunogenicity in hemophilia A.
General rights
It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).
Disclaimer/Complaints regulations
If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible.
5
Immune complexes enhance
uptake of blood coagulation factor
VIII by antigen presenting cells
Aleksandra Wroblewska, Eszter Herczenik, Jill W. C. Claassens,J. Sjef Verbeek, Anja ten Brinke and Jan Voorberg
FVIII-immune c omple xe s
5
Abstract
Development of antibodies directed against FVIII (referred to as FVIII inhibitors) is a common complication in hemophilia care. Immune tolerance induction (ITI) comprising frequent administration of high doses of FVIII is the treatment of choice for hemophilia A patients with inhibitors. Thus far, the molecular mechanisms contributing to tolerance induction have not been defined. Here we studied endocytosis of FVIII-containing immune complexes by antigen presenting cells. Bone marrow-derived dendritic cells (DC) were used as model antigen-presenting cells, as DCs are known for their ability to take up and process immune complexes via Fc gamma receptors (FcgRs). Bone marrow-derived murine DCs were able to efficiently take up FVIII pre-complexed with anti-FVIII antibodies (IC) in a dose-dependent manner. Endocytosis of FVIII-IC was 3-6 fold more efficient when compared to equimolar concentrations of soluble FVIII. Moreover, enhanced endocytosis led to stronger FVIII-specific T cell proliferation. Analysis of subclass-specific anti-FVIII IgG revealed that IgG2a/ IgG2b antibodies were most potent in enhancing FVIII endocytosis. Uptake of FVIII-IC, but not FVIII alone, could be inhibited with 2.4G2 antibody indicating functional involvement of FcgRII/III in this process. These results were confirmed using murine DCs isolated from FcgR-deficient mice. Uptake of FVIII-IC was similar to that of FVIII in DCs derived from mice lacking all four FcgRs. Genetic ablation of FcgRII or FcgRIII did not affect the ability of anti-FVIII IgG to promote the uptake of FVIII. Collectively, these data provide further insight into the modulation of FVIII endocytosis by anti-FVIII antibodies.
FVIII-immune c omple xe s
5
Introduction
Hemophilia A is an X-linked bleeding disorder caused by a deficiency or dysfunction of blood coagulation factor VIII (FVIII). Patients with severe hemophilia A frequently suffer from spontaneous joint and muscle bleedings whereas patients with mild hemophilia A usually bleed only after trauma of surgery. 1 Conventional treatment consists of frequent intravenous administration of FVIII. However, approximately 25% of patients with the severe form of hemophilia A develop an immune response resulting in the formation of antibodies that inhibit the procoagulant activity of FVIII. Anti-FVIII antibodies are primarily composed of IgG1 and IgG4 and have undergone affinity maturation through somatic hypermutation. 2-5 Both somatic hypermutation and class switching are dependent on CD4+ T cells which have been extensively characterized in both hemophilic mice 6-10 and hemophilia A patients. 11-15 Once inhibitor development occurs, infused FVIII can bind to circulating antibodies creating FVIII-specific immune complexes. Formation of immune complexes has been shown to modulate not only antigen half-life time in the circulation, but also immune responses in general. 16
Dendritic cells (DCs) are professional antigen-presenting cells specialized in induction of primary T cell response to foreign antigens in both MHC class I and II-dependent manner. 17 DCs can capture antigens either in soluble form or through the uptake of larger particles such as exosomes, apoptotic or necrotic cells and antigen-IgG complexes. 18 Typically, endogenous antigens are presented on MHC class I molecules for presentation to CD8+ T cells while peptides derived from exogenous, internalized antigens are loaded on MHC class II molecules and activate CD4+ T cells. Although macrophages and B cells also express MHC class II molecules and other proteins involved in MHC class II-dependent presentation, DCs have been exclusively shown to activate MHC class II-restricted CD4+ T cells
in vivo after injection of immune complexes. 19 Several studies have shown that IgG-complexed antigen is more efficiently cross-presented to MHC class I-restricted CD8+ T cells when compared to soluble antigens. 20-23
Here, we studied whether FVIII-containing immune complexes (FVIII-IC) are efficiently endocytosed by dendritic cells. We show, that endocytosis of equimolar concentrations of FVIII-IC is much more efficient than FVIII alone. The enhanced uptake of FVIII-IC was not observed in DCs derived from FcgRI-IV deficient mice indicating that FcgR promote the uptake of FVIII-IC. Genetic ablation of neither FcgRII nor FcgRIII alone reduced the uptake of FVIII-IC suggesting that these receptors do not exclusively promote endocytosis of FVIII-IC.
Methods
Materials
For culturing murine bone marrow-derived dendritic cells (BMDCs), mouse recombinant GM-CSF (R&D System, Minneapolis, MN, USA) was used. Penicillin/ streptomycin, RPMI-1640 and serum-free X-VIVO 15 medium were from Lonza
FVIII-immune c
omple
xe
s
5
(Walkersville, MD, USA. Fetal calf serum was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Culture flasks and 96-well microtiter plates were purchased from Nunc (Roskilde, Denmark). Ultrapure methanol-free paraformaldehyde was from Polysciences (Eppelheim, Germany). Antibodies used were: anti-murine CD83-APC, anti-murine CD86, anti-murine CD11b-FITC, rat IgG isotype control antibody conjugated with FITC, APC or biotin, streptavidin-APC, anti-mouse CD14-biotin, anti-mouse CD45R-biotin, anti-mouse Gr-1-biotin and anti-mouse CD8 (eBioscience, San Diego, CA, USA); monoclonal anti-FVIII antibodies ESH4 and ESH5 (Sekisui Diagnostics, Stamford, CT, USA). Anti-CD11c antibody producing cell line (clone HB-224) was purchased from ATCC (Manassas, VA, USA). Monoclonal antibodies CAgA, CAg9, CAg12 and CLB-CAg117 targeting different domains of FVIII have been described previously. 24-27
Fluorescent labeling of recombinant FVIII
Recombinant human FVIII was kindly provided by Dr. B.M. Reipert (Baxter Healthcare Corporation, Vienna, Austria). FVIII was labeled using the Microscale Alexa Fluor 488 protein-labeling kit (Invitrogen). Protein concentration and efficiency of labeling was spectrophotometrically determined at 280 and 495 nm. The degree of labeling was usually between 3 and 5 Alexa Fluor 488 molecules per 1 molecule of FVIII. The integrity of labeled FVIII (FVIII-488) was confirmed by SDS-PAGE (data not shown).
Mice
Hemophilic E17-KO mice, characterized by a targeted disruption of exon 17 of the FVIII gene 28 were backcrossed into the C57BL/6J background as described previously. 29 Genotype of hemophilic mice was confirmed by polymerase chain reaction analysis of genomic DNA extracted from ear clippings, as described previously. 6 FcgRIIb
B6─/─ 30, FcgRIII─/─31, FcgRI/II/III/IV─/─ and FcgRIIbB6flox/flox30 mice were bred and maintained in the Animal Facility at Leiden University Medical Center. Unless stated otherwise, mice used in this study were male and aged between 6 and 12 weeks at the beginning of the experiment.
Generation of BMDCs
Murine BMDCs were essentially prepared as described before. 32 Briefly, bone marrow cells were isolated by flushing femurs with PBS supplemented with 2% FCS. After erythrocyte lysis, cells were resuspended at 1x106 cells/ml containing 20 ng/ml mouse recombinant GM-CSF and cultured for 8-10 days in RPMI 1640 medium supplemented with 2.5 mM HEPES, 55 μM 2-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin, 5 mM glutamine and 10% FCS. Based on MHC class II and CD11c expression, over 95% of the cells generated by this method were DCs.
Pulse loading of DCs with FVIII or FVIII–IgG complexes
FVIII–IgG immune complexes (FVIII-IC) were prepared by incubating recombinant human FVIII (Baxter) or recombinant human FVIII conjugated with Alexa Fluor 488 (FVIII-488) with a mixture of monoclonal anti-FVIII antibodies (CLB-CAgA,
FVIII-immune c
omple
xe
s
5
CLB-CAg9, CLB-CAg12, CLB-CAg117, ESH4 and ESH5) for 30 min at 37 °C in a molar ratio 1:5. In uptake experiments, 0.2x106 DCs were incubated with various concentrations of FVIII-488 or FVIII-488 pre-complexed with antibodies at 37°C for 1 hour in serum-free CellGro (CellGenix). Uptake was analyzed by flow cytometry (LSRII flow cytometer; BD Biosciences). Histograms were processed using FlowJo Version 7.6.5 software (TreeStar).Administration of FVIII in hemophilic E17-KO mice
Recombinant human FVIII was diluted to 10 μg/ml in sterile PBS and 100 μl was administered intravenously in E17-KO mice.
CD4+ T cell proliferation assay
Spleens collected after 5 weekly injections of FVIII were processed into single-cell suspension. Erythrocytes were removed and CD8+ T cells, CD19+ B cells and CD138+ plasma cells were depleted by magnetic bead separation using anti-CD8, anti-CD19 and anti-CD139 beads. Remaining splenocytes were cultured in round-bottomed 96-well plates for 96 hours in X-VIVO 15 medium supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin and 55 μM 2-mercaptoethanol in the presence of increasing concentration of FVIII or FVIII-IC to measure antigen-specific T cell proliferation. Proliferation was assayed by the addition of 1 μCi/ well of [3H]thymidine for the last 18-20 hours. The results are expressed as the stimulation index defined as counts per minute (cpm) of cells incubated in medium supplemented with FVIII divided by cpm of cells with medium only.
Statistical analysis
Unless stated otherwise, data were analyzed by Student’s t test and differences were considered significant at P < 0.05: * P < 0.05, ** P < 0.01, *** P < 0.001, ns – not significant.
Results
Endocytosis of FVIII immune complexes is more efficient than soluble FVIII
Inhibitory antibodies develop in a subset of hemophilia A patients in response to replacement therapy. Circulating anti-FVIII antibodies readily bind to infused FVIII resulting in the formation of FVIII-specific immune complexes. It has been shown for the model antigen ovalbumin (OVA) that formation of immune complexes enhances endocytosis and leads to more efficient presentation and activation of specific T cells. In order to verify whether that phenomenon also holds true for FVIII, we evaluated internalization of FVIII and FVIII-IC in vitro using murine bone-marrow derived dendritic cells (BMDCs) as antigen-presenting cells. To enable efficient monitoring of endocytosis, recombinant FVIII was fluorescently labeled with Alexa Fluor 488. FVIII-specific immune complexes were formed using a panel of six mouse anti-FVIII monoclonal antibodies, directed against different domains of FVIII (Figure 1). A 5-fold molar excess of antibodies over FVIII wasusedto minimize the amount of non-complexed FVIII. As shown in Figure 2 (panel A and B), FVIII pre-complexed with antibodies (FVIII-IC) was efficiently
FVIII-immune c
omple
xe
s
5
taken up by BMDCs in a dose-dependent manner. Moreover, endocytosis of FVIII-IC was much more efficient when compared to equimolar concentrations of soluble FVIII.
FVIII-IC induce stronger FVIII-specific T cell proliferation when compared to non-complexed FVIII
Subsequently, we studied whether enhanced endocytosis of FVIII-IC as compared to soluble FVIII will result in increased ability to prime FVIII-specific T cells, as it has been shown previously for ovalbumin-containing immune complexes. 19 FVIII-deficient mice were injected five times weekly with 1 µg of recombinant FVIII to enrich for FVIII-specific T cells. One week after last injection mice were sacrificed and isolated splenocytes were restimulated in vitro with increasing concentrations of either recombinant FVIII or FVIII pre-complexed with antibodies. Increased proliferation of FVIII-specific CD4+ T cells was observed upon incubation with FVIII-IC, as shown in Figure 3. Altogether, these data suggests that anti-FVIII IgG promotes endocytosis of FVIII by dendritic cells resulting in more efficient FVIII-specific T cell responses.
Endocytosis of FVIII immune complexes proceeds via Fcg receptors
We subsequently investigated whether uptake of FVIII-IC is dependent on Fcg receptors. BMDCs were first incubated with monoclonal antibody 2.4G2, blocking FcgR-mediated uptake by shielding a common epitope present in the extracellular domains of FcgRII and FcgRIII. 33 Next, increasing doses of FVIII-IC were added to the cells and incubated for 1 hour at 37°C. Flow cytometry analysis revealed that addition of 10 µg/ml 2.4G2 reduced the uptake of FVIII-IC by 80-90% for the highest concentration of FVIII used (Figure 4A). Uptake of FVIII
A1
A2
A3
C1
C2
Antibody FVIII domain specificity Isotype
ESH-5 A1 IgG1
CLB-CAg9 A2 IgG1
CLB-CAgA A3 (1801-1823) IgG2a
CLB-CAg12 A3-C1 IgG2b
CLB-CAg117 C2 IgG2a
ESH-4 C2 (2303-2332); non-competitive with CLB-CAg117 IgG2a
Figure 1. Three-dimensional model of FVIII and characterization of anti-FVIII monoclonal antibodies used for preparation of immune complexes. Left panel provides a three-dimensional model of FVIII. The positions of
the A1, A2, A3, C1 and C2 domains are indicated next to the model. The table on the right provides an overview of monoclonal antibodies used for preparation of FVIII-immune complexes (name, FVIII domain specificity and isotype). Model was based on FVIII crystal structure (PDB code 3cdz) and prepared using PyMol imaging software.
FVIII-immune c
omple
xe
s
5
alone was not affected upon pre-incubation with the 2.4G2 antibody (Figure 4B and 4C). These findings show that Fcg receptors mediate the uptake of FVIII when complexed with IgG.
To date, four different classes of FcgRs, known as FcgRI, FcgRIIb, FcgRIII and FcgRIV, have been identified in mice. 34 Three of them, namely FcgRI, FcgRIII and FcgRIV are activating receptors, whereas FcgRIIb is the only inhibitory one. To investigate the role of individual Fcg receptors in endocytosis of FVIII-IC, we isolated bone marrow cells from various FcgR-deficient mice (Figure 5) and differentiate them into dendritic cells. FcgRIIbflox/flox mouse strain, expressing all functional Fcg receptors, was used as a source of wild-type cells. Increasing concentrations of either FVIII or FVIII-IC were incubated with BMDCs for 1 hour
0.0010 0.01 0.1 1 10 100 25 50 75 100 125 FVIII FVIII-IC FVIII (nM) % M FI FVIII FVIII-IC 104 0 104 0 100 200 300 0 104 0 100 200 300 104 0 104 0 100 200 300 0 104 0 100 200 300 104 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 104 0 104 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 104 0 104 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 104 0 104 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 104 0 104 0 100 200 300 0 104 0 100 200 300 4 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 10 0 20 0 30 0 0 104 0 10 0 20 0 30 0 0 104 0 10 0 20 0 30 0 0 104 0 10 0 20 0 30 0 0 104 0 10 0 20 0 30 0 0 104 0 10 0 20 0 30 0 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0 104 0 100 200 300 0.1 nM 1.0 nM 5.0 nM 10.0 nM 0.01 nM FVIII Ce ll c oun t B A
Figure 2. Endocytosis of FVIII immune complexes is more efficient when compared to non-complexed FVIII. A, B.
Increasing concentrations (0-10 nM) of either soluble FVIII or FVIII pre-complexed with antibodies (FVIII-IC) were added to bone-marrow derived dendritic cells (BMDCs) and incubated at 37°C for 1 hour. Representative histograms are shown in A: gray-filled histograms show untreated cells, open histograms represent cells treated either with FVIII (upper panel) or FVIII-IC (lower panel). Results are expressed as % of mean fluorescent intensity (MFI) in panel B, where 100% refers to MFI measured for 10 nM FVIII-IC. Results from 3 independent experiments (mean ± SD) are shown.
0.00 0.01 0.10 0.50 1.00 0 2 4 6 8 10 FVIII FVIII-IC FVIII (nM) Pr olif er at io n ( SI )
Figure 3. FVIII-IC induce stronger FVIII-specific T cell proliferation when compared to soluble FVIII.
Hemophilic E17-KO mice were injected intravenously 5 times weekly with 1 μg of recombinant FVIII. One week after the last injection mice were sacrificed, spleens were collected and processed into single-cell suspensions. Splenocytes were assayed in a thymidine
(3H) incorporation assay by in vitro restimulation
with increasing concentrations of either FVIII or FVIII-IC. Proliferation was measured after 72 hours and thymidine was added for the last 18-20 hours. Results are shown as stimulation index (SI) from triplicate wells (mean ± SD) and are representative for 2 independent experiments.
FVIII-immune c
omple
xe
s
5
at 37°C and subsequently endocytosis was measured by flow cytometry. Single knock-out of either FcgRII or FcgRIII did not abolish the uptake of FVIII-IC (Figure 5A-C); however, in the absence of all four FcgRs, no increase in mean fluorescence intensity was observed upon addition of FVIII-IC to the cells, indicating functional involvement of Fcg receptors in uptake of FVIII-IC (Figure 5D). Lack of the effect for single knock-out strains can potentially be explained by the redundancy of Fcg receptors as reported previously. 19
0.0010 0.01 0.1 1 10 100 20 40 60 80 100 FVIII-IC FVIII-IC + 2.4G2 mAb FVIII (nM) % M FI FVIII FVIII-IC 0 50 100 150 200 Medium 2.4G2 % M FI B A FVIII FVIII-IC 2.4G2 Medium MFI FITC Ce ll co un t 0 104 105 0 50 10 0 15 0 20 0 0 104 105 0 50 10 0 15 0 20 0 0 104 105 0 50 10 0 15 0 0 104 105 0 50 10 0 15 0 0 104 105 0 50 10 0 15 0 20 0 0 104 105 0 50 10 0 15 0 20 0 0 104 105 0 50 10 0 15 0 0 104 105 0 50 10 0 15 0 0 104 105 0 50 100 150 200 0 104 105 0 50 100 150 200 0 104 105 0 50 100 150 0 104 105 0 50 100 150 0 104 105 0 50 100 150 200 0 104 105 0 50 100 150 200 0 104 105 0 50 100 150 0 104 105 0 50 100 150 C
Figure 4. Endocytosis of FVIII immune complexes depends on Fcg receptors. A-C. Prior to addition of FVIII-IC, BMDCs
were incubated with either 2.4G2 monoclonal antibody (10 µg/ml) or medium control for 15 minutes at 4°C. Subsequently, increasing concentrations of FVIII-IC (A) or a fixed concentration (5 nM) of FVIII or FVIII-IC (B and C) were added to the cells for 1 hour at 37°C. Representative histograms are shown in B: gray-filled histograms show untreated cells, open histograms represent cells treated either with FVIII (upper panel) or FVIII-IC (lower panel). Results are expressed as % of mean fluorescent intensity (MFI): in panel A 100% MFI refers to MFI measured for 10 nM FVIII-IC; in panel C 100% MFI refers to MFI measured for either FVIII or FVIII-IC alone (no 2.4G2 mAb added). Results are expressed as mean ± SD from 3 independent experiments.
FcγRIIbflox/flox 0.001 0.010 0.1 1 10 100 2000 4000 6000 8000 FcγRIIb -/-0.001 0.010 0.1 1 10 100 2000 4000 6000 8000 FcγRI/II/III/IV -/-0.001 0.010 0.1 1 10 100 2000 4000 6000 8000 FVIII FVIII-IC FcγRIII -/-0.001 0.010 0.1 1 10 100 2000 4000 6000 8000 A B D C
Figure 5. Endocytosis of FVIII and FVIII-IC by BMDCs isolated from FcgR-deficient mice. A-D. BMDC derived of
FcgRIIbflox/flox (A), FcgRIIb-/- (B), FcgRIII
-/-(C) or FcgRI/II/III/IV-/- (D) mice were
used for these studies. Increasing concentrations (0-10 nM) of either soluble FVIII or FVIII pre-complexed with antibodies (FVIII-IC) were added to BMDCs and incubated at 37°C for 1 hour. Results are expressed as mean fluorescent intensity (MFI) and are representative of 2 independent experiments.
FVIII-immune c
omple
xe
s
5
IgG2a/2b antibodies are most efficient in promoting FVIII endocytosis
The complexity in the FcgR family is mirrored by the presence of four different IgG subclasses (IgG1, IgG2a, IgG2b and IgG3), which bind with varying affinity and specificity to different FcgR receptors. 35,36 FcgRI is the only high affinity receptor and binds mainly IgG2a; this isotype, together with IgG2b, is also bound by FcgRIII and IV, while IgG1 is exclusively recognized by FcgRIIb and FcgRIII. 34 To unravel which IgG isotype predominantly mediates endocytosis of FVIII-IC by BMDCs, anti-FVIII antibodies were separated based on their isotype and incubated with recombinant FVIII as described previously. Subsequently, soluble FVIII or isotype-specific FVIII-IC were added to the cells for 1 hour at 37°C. IgG2a appeared to be more effective in enhancing endocytosis when compared to IgG1 (Figure 6). FVIII-IC comprised of a combination of IgG2a and IgG2b were most efficient in promoting FVIII-IC endocytosis (Figure 6), while a combination of IgG1 and IgG2b was less effective (Figure 6). Since IgG2a and IgG2b can bind to multiple FcgRs this also suggests that multiple FcgRs play a role in FVIII-IC endocytosis by BMDCs.
Discussion
Endocytosis of ICs by dendritic cells results in efficient processing and loading of antigen-derived peptides on MHC class I and/or II molecules and promotes subsequent induction of antigen-specific T cells. 34 Immune tolerance induction (ITI) using high dosage infusion of FVIII is frequently used to eradicate inhibitory antibodies to FVIII in patients with hemophilia A. 37 Infusion of FVIII in patients with anti-FVIII antibodies will result in rapid formation of FVIII-immune complexes (FVIII-IC). As yet our knowledge on the biological activity of FVIII-IC is limited. In this study we investigated the endocytosis of FVIII-IC by antigen-presenting cells as well as subsequent presentation and priming of FVIII-specific T-cells. We showed that FVIII-IC are more efficiently endocytosed when compared to uncomplexed FVIII. Subclass analysis suggested that anti-FVIII IgG2a and 2b promotes FVIII endocytosis more efficiently when compared to IgG1. Finally, enhanced endocytosis of FVIII-IC led to robust in vitro priming of FVIII-specific T cells. Altogether, these data suggest that formation of FVIII-IC reinforces already pre-existing anti-FVIII immune responses.
FVIII IgG2a IgG2a+2b IgG1 IgG1+2b IgG1+2a+2b
0 100 200 300 400 % M FI
Figure 6. Anti-FVIII antibodies of subclass IgG2a and 2b are most efficient in promoting endocytosis of FVIII. Anti-FVIII monoclonal
antibodies were divided based on their isotype (IgG1: ESH5 and CLB-CAg9; IgG2a: ESH4, CLB-CAgA and CLB-CAg117; IgG2b: CLB-CAg12) and subsequently incubated in molar ratio 1:5 with recombinant FVIII (2 nM) for 30 minutes at 37°C to allow formation of FVIII-immune complexes. Next, FVIII or FVIII-IC were added to BMDCs for 1 hour at 37°C. Results are expressed as % MFI, where 100% MFI corresponds to mean fluorescent intensity measured for FVIII alone. Results from 3 independent experiments (mean ± SD) are shown.
FVIII-immune c
omple
xe
s
5
Endocytosis of FVIII-IC was FcgR-dependent as shown by blockage with monoclonal antibody 2.4G2, as well as employing bone marrow-derived DC derived from FcgRI-IV deficient mice. Genetic ablation of neither FcgRII nor FcgRIII alone affected the uptake of FVIII-IC, suggesting that these receptors do not exclusively promote FVIII-IC endocytosis. Both FcgRI and FcgRIII have been implicated in the enhanced uptake of ovalbumin-immune complexes. 21 FcgRI binds with high affinity to monomeric IgG2a and therefore is constantly saturated with ligand, which would suggest that it might not play a major role in the uptake of immune complexes. 34 Nevertheless, several recent experimental findings suggest that FcgRI is an important player in immunity. 38-40 Simultaneous knockout of both FcgRI and FcgRIII, but not each of these separately, was shown to abolish MHC class I-restricted antigen presentation of ovalbumin-ICs, suggesting that the high-affinity FcgRI can cooperate with low-affinity FcgRIII in mediating endocytosis of immune complexes. 21 The strong potentiating effect on FVIII uptake by IgG2a antibodies and the inhibitory effect of antibody 2.4G2 which neutralizes FcgRI via its Fc-tail is compatible with a possible role for FcgRI in the enhanced uptake of FVIII-IC. FcgRIV is also expressed on bone marrow derived DCs and binds with intermediate affinity to IgG2a and IgG2b. 36 Therefore, we cannot fully exclude that FcgRIV also contributes to the uptake of FVIII-IC. Extrapolating data obtained from mouse models into pathophysiological situations in patients is always challenging due to differences between mice and men. Based on the genomic localization and sequence similarity in the extracellular portion, mouse FcgRIII is most closely related to human FcgRIIa, while mouse FcgRIV seems to be the orthologue of human FcgRIIIa. 41 Moreover, the affinity of the human FcgRs for the different IgG subclasses is significantly lower compared with their mouse counterparts. 34 Anti-FVIII antibodies developing in hemophilia mice are mostly of IgG1, IgG2a and IgG2b isotype 42; IgG4 and IgG1 are the most abundant IgG subclasses in hemophilia patients with FVIII inhibitors. 5 Titers of anti-FVIII IgG4 antibodies have been suggested to correlate with a poor prognosis of ITI therapy. 4 IgG1 containing IC bind to all human FcgRs, while IgG4 containing IC bind primarily to human FcgRI, FcgRIIa/b/c and FcgRIIIa. 43 However, formation of immune complexes by incubation of recombinant FVIII with panel of either mouse of human anti-FVIII monoclonal antibodies and subsequent addition to human monocyte-derived dendritic cells (moDCs) did not result in enhanced FVIII endocytosis, but led to inhibition of this process (data not shown). This could be potentially explained by limited expression of FcgRs on these cells (only FcgRIIa and IIa are present, see supplemental Figure S1A), 44 whereas circulating conventional DCs, freshly isolated from peripheral blood also express high levels of activating FcgRI. 44,45 Monocyte-derived macrophages (MΦ) express a broader panel of FcgRs than moDCs, namely also FcgRI and FcgRIII (supplemental Figure S1A). Consequently, when MΦ were used to study endocytosis of FVIII-IC, enhanced uptake was observed for FVIII-IC as compared to soluble FVIII (supplemental Figure S1B). Blocking antibodies showed major involvement of FcgRII, but also suggested that multiple FcgRs are needed for efficient endocytosis of FVIII-IC. Macrophages are
FVIII-immune c
omple
xe
s
5
considered to mediate mostly clearance of ICs, while modulatory effects on antigen-specific immune response are thought to be strictly dependent on DCs. Therefore, additional studies employing human anti-FVIII antibodies and blood-derived conventional dendritic cells are needed to elucidate whether anti-FVIII IgG promotes the uptake of FVIII by human DCs.Nevertheless, despite the obvious difference between mice model systems and human, many of the basic principles and mechanisms underlying different IgG isotypes activities and role of FcgRs have been identified in mice and have been shown to hold true also in humans. Here, we showed that formation of immune complexes leads to more efficient delivery of FVIII to dendritic cells, and, as a consequence, more robust proliferation of FVIII-specific T cells. This data can provide further insight the modulation of immune responses to FVIII in hemophilia A.
FVIII-immune c omple xe s
5
References1. Mannucci PM, Tuddenham EG. The hemophilias ─ from royal genes to gene therapy. N Engl J Med. 2001;344(23):1773-1779.
2. van den Brink EN, Turenhout EA, Bank CM, Fijnvandraat K, Peters M, Voorberg J. Molecular analysis of human anti-factor VIII antibodies by V gene phage display identifies a new epitope in the acidic region following the A2 domain. Blood. 2000;96(2):540-545.
3. van den Brink EN, Turenhout EA, Davies J, et al. Human antibodies with specificity for the C2 domain of factor VIII are derived from VH1 germline genes. Blood. 2000;95(2):558-563.
4. van Helden PM, van den Berg HM, Gouw SC, et al. IgG subclasses of anti-FVIII antibodies during immune tolerance induction in patients with hemophilia A. Br J Haematol. 2008;142(4):644-652.
5. Whelan SF, Hofbauer CJ, Horling FM, et al. Distinct characteristics of antibody responses against factor VIII in healthy individuals and in different cohorts of hemophilia A patients. Blood. 2012;12:12.
6. Qian J, Collins M, Sharpe AH, Hoyer LW. Prevention and treatment of factor VIII inhibitors in murine hemophilia A. Blood. 2000;95(4):1324-1329.
7. Reipert BM, Sasgary M, Ahmad RU, Auer W, Turecek PL, Schwarz HP. Blockade of CD40/CD40 ligand interactions prevents induction of factor VIII inhibitors in hemophilic mice but does not induce lasting immune tolerance. Thromb Haemost. 2001;86(6):1345-1352.
8. Qian J, Burkly LC, Smith EP, et al. Role of CD154 in the secondary immune response: the reduction of pre-existing splenic germinal centers and anti-factor VIII inhibitor titer. Eur J Immunol. 2000;30(9):2548-2554.
9. Wu H, Reding M, Qian J, et al. Mechanism of the immune response to human factor VIII in murine hemophilia A. Thromb Haemost. 2001;85(1):125-133.
10. Steinitz KN, van Helden PM, Binder B, et al. CD4+ T-cell epitopes associated with antibody
responses after intravenously and subcutaneously applied human FVIII in humanized hemophilic E17 HLA-DRB1*1501 mice. Blood. 2012;119(17):4073-4082.
11. Ragni MV, Bontempo FA, Lewis JH. Disappearance of inhibitor to factor VIII in HIV-infected hemophiliacs with progression to AIDS or severe ARC. Transfusion. 1989;29(5):447-449.
12. Jacquemin MG, Desqueper BG, Benhida A, et al. Mechanism and kinetics of factor VIII inactivation: study with an IgG4 monoclonal antibody derived from a hemophilia A patient with inhibitor. Blood. 1998;92(2):496-506.
13. Jones TD, Phillips WJ, Smith BJ, et al. Identification and removal of a promiscuous CD4+ T cell
epitope from the C1 domain of factor VIII. J Thromb Haemost. 2005;3(5):991-1000.
14. James EA, Kwok WW, Ettinger RA, Thompson AR, Pratt KP. T-cell responses over time in a mild hemophilia A inhibitor subject: epitope identification and transient immunogenicity of the corresponding self-peptide. J Thromb Haemost. 2007;5(12):2399-2407.
15. James EA, van Haren SD, Ettinger RA, et al. T-cell responses in two unrelated hemophilia A inhibitor subjects include an epitope at the factor VIII R593C missense site. J Thromb Haemost. 2011;9(4):689-699.
16. Nimmerjahn F, Ravetch JV. Antibody-mediated modulation of immune responses. Immunol Rev. 2010;236:265-275.
17. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature. 1998;392(6673):245-252.
18. Heath WR, Carbone FR. Cross-presentation in viral immunity and self-tolerance. Nat Rev Immunol. 2001;1(2):126-134.
19. de Jong JM, Schuurhuis DH, Ioan-Facsinay A, et al. Dendritic cells, but not macrophages or B cells, activate major histocompatibility complex class II-restricted CD4+ T cells upon
immune-complex uptake in vivo. Immunology. 2006;119(4):499-506.
FVIII-immune c
omple
xe
s
5
cell maturation and major histocompatibility complex class I-restricted antigen presentation after immune complex internalization. J Exp Med. 1999;189(2):371-380.
21. Schuurhuis DH, van Montfoort N, Ioan-Facsinay A, et al. Immune complex-loaded dendritic cells are superior to soluble immune complexes as antitumor vaccine. J Immunol. 2006;176(8): 4573-4580.
22. Amigorena S, Bonnerot C. Fc receptors for IgG and antigen presentation on MHC class I and class II molecules. Semin Immunol. 1999;11(6):385-390.
23. Amigorena S, Bonnerot C. Fc receptor signaling and trafficking: a connection for antigen processing. Immunol Rev. 1999;172:279-284.
24. Leyte A, Mertens K, Distel B, et al. Inhibition of human coagulation factor VIII by monoclonal antibodies. Mapping of functional epitopes with the use of recombinant factor VIII fragments. Biochem J. 1989;263(1):187-194.
25. Donath MS, Lenting PJ, Van Mourik JA, Mertens K. The role of cleavage of the light chain at positions Arg1689 or Arg1721 in subunit interaction and activation of human blood coagulation factor VIII. J Biol Chem. 1995;270(8):3648-3655.
26. Lenting PJ, van de Loo JW, Donath MJ, van Mourik JA, Mertens K. The sequence Glu1811-Lys1818 of human blood coagulation factor VIII comprises a binding site for activated factor IX. J Biol Chem. 1996;271(4):1935-1940.
27. Fijnvandraat K, Celie PH, Turenhout EA, et al. A human alloantibody interferes with binding of factor IXa to the factor VIII light chain. Blood. 1998;91(7):2347-2352.
28. Bi L, Lawler AM, Antonarakis SE, High KA, Gearhart JD, Kazazian HH, Jr. Targeted disruption of the mouse factor VIII gene produces a model of haemophilia A. Nat Genet. 1995;10(1):119-121.
29. Reipert BM, Ahmad RU, Turecek PL, Schwarz HP. Characterization of antibodies induced by human factor VIII in a murine knockout model of hemophilia A. Thromb Haemost. 2000;84(5): 826-832.
30. Boross P, Arandhara VL, Martin-Ramirez J, et al. The inhibiting Fc receptor for IgG, FcgammaRIIB, is a modifier of autoimmune susceptibility. J Immunol. 2011;187(3):1304-1313.
31. Hazenbos WL, Gessner JE, Hofhuis FM, et al. Impaired IgG-dependent anaphylaxis and Arthus reaction in Fc gamma RIII (CD16) deficient mice. Immunity. 1996;5(2):181-188.
32. Lutz MB, Kukutsch N, Ogilvie AL, et al. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. JImmunolMethods. 1999;223(1):77-92.
33. Araujo-Jorge T, Rivera MT, el Bouhdidi A, Daeron M, Carlier Y. An Fc gamma RII-, Fc gamma RIII-specific monoclonal antibody (2.4G2) decreases acute Trypanosoma cruzi infection in mice. Infect Immun. 1993;61(11):4925-4928.
34. Nimmerjahn F, Ravetch JV. Fcgamma receptors as regulators of immune responses. Nat Rev Immunol. 2008;8(1):34-47.
35. Dijstelbloem HM, van de Winkel JG, Kallenberg CG. Inflammation in autoimmunity: receptors for IgG revisited. Trends Immunol. 2001;22(9):510-516.
36. Nimmerjahn F, Ravetch JV. Divergent immunoglobulin g subclass activity through selective Fc receptor binding. Science. 2005;310(5753):1510-1512.
37. DiMichele DM. Immune tolerance in haemophilia: the long journey to the fork in the road. Br J Haematol. 2012;159(2):123-134.
38. Barnes N, Gavin AL, Tan PS, Mottram P, Koentgen F, Hogarth PM. FcgammaRI-deficient mice show multiple alterations to inflammatory and immune responses. Immunity. 2002;16(3):379-389.
39. Baudino L, Nimmerjahn F, Azeredo da Silveira S, et al. Differential Contribution of Three Activating IgG Fc Receptors (FcgRI, FcgRIII, and FcgRIV) to IgG2a- and IgG2b-Induced Autoimmune Hemolytic Anemia in Mice. The Journal of Immunology. 2008;180(3):1948-1953.
40. Ioan-Facsinay A, de Kimpe SJ, Hellwig SM, et al. FcgammaRI (CD64) contributes substantially to severity of arthritis, hypersensitivity responses, and protection from bacterial infection.
FVIII-immune c omple xe s
5
Immunity. 2002;16(3):391-402.41. Nimmerjahn F, Ravetch JV. Fcgamma receptors: old friends and new family members. Immunity. 2006;24(1):19-28.
42. Qadura M, Waters B, Burnett E, et al. Immunoglobulin isotypes and functional anti-FVIII antibodies in response to FVIII treatment in Balb/c and C57BL/6 haemophilia A mice. Haemophilia. 2010;17(2):288-95.
43. Bruhns P, Iannascoli B, England P, et al. Specificity and affinity of human Fcgamma receptors and their polymorphic variants for human IgG subclasses. Blood. 2009;113(16):3716-3725.
44. Boruchov AM, Heller G, Veri MC, Bonvini E, Ravetch JV, Young JW. Activating and inhibitory IgG Fc receptors on human DCs mediate opposing functions. J Clin Invest. 2005;115(10): 2914-2923.
45. Fanger NA, Wardwell K, Shen L, Tedder TF, Guyre PM. Type I (CD64) and type II (CD32) Fc gamma receptor-mediated phagocytosis by human blood dendritic cells. J Immunol. 1996;157(2):541-548.
FVIII-immune c omple xe s
5
Supplemental data
MaterialsMouse monoclonal blocking antibodies anti-human CD16 (FcgRIII), CD32 (FcgRII) and CD64 (FcgRI) were from BD Biosciences (San Jose, CA, USA).
Generation of human monocyte-derived macrophages (MΦ)
For macrophage culture, monocytes isolated from blood of healthy donors were resuspended at 2.5x106 cells/well in 6-well plates in RPMI 1640 medium supplemented with 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin and 50 ng/ml recombinant human M-CSF. Blood of healthy individuals was drawn in accordance with Dutch regulations and following approval from Sanquin Ethical Advisory Board in accordance with the Declaration of Helsinki.
Blocking experiments
To block the uptake of FVIII, monoclonal antibody anti-CD16, anti-CD32 and/or anti-CD64 were first preincubated with MΦ at 4°C for 30 minutes. Subsequently, 20 nM of FVIII was added for 1 hour at 37°C. Cells were fixed and internalized FVIII was detected with monoclonal antibody CLB-CAg117-FITC in the presence of saponin. Mean fluorescence intensities were determined by flow cytometry.
FVIII FVIII-IC 0 100 200 300 400 * % M FI
Ctrl FcγRI FcγRII FcγRIII FcγRI/II/III
0 25 50 75 100 125 *** * *** * % M FI A B C Macrophages Dendritic cells MFI FITC Ce ll co un t 1 04 1 05 0 1 04 1 05 0 3 0 0 6 0 0 9 0 0 1 .2 K 1 04 1 05 0 1 04 1 05 0 5 0 0 1 .0 K 1 .5 K 1 04 1 05 0 1 04 1 05 0 3 0 0 6 0 0 9 0 0 1 .2 K 1 04 1 05 0 1 04 1 05 0 5 0 0 1 .0 K 1 .5 K 0 1 04 1 05 0 3 0 0 6 0 0 9 0 0 1 .2 K 0 1 04 1 05 0 3 0 0 6 0 0 9 0 0 1 .2 K 0 1 04 1 05 0 3 0 0 6 0 0 9 0 0 1 .2 K 0 1 04 1 05 0 5 0 0 1 .0 K 1 .5 K 0 1 04 1 05 0 5 0 0 1 .0 K 1 .5 K 0 1 04 1 05 0 5 0 0 1 .0 K 1 .5 K 0 1 04 1 05 0 3 0 0 6 0 0 9 0 0 1 .2 K 0 1 04 1 05 0 3 0 0 6 0 0 9 0 0 1 .2 K 0 1 04 1 05 0 3 0 0 6 0 0 9 0 0 1 .2 K 0 1 04 1 05 0 5 0 0 1 .0 K 1 .5 K 0 1 04 1 05 0 5 0 0 1 .0 K 1 .5 K 0 1 04 1 05 0 5 0 0 1 .0 K 1 .5 K 0 1 04 1 05 0 3 0 0 6 0 0 9 0 0 1 .2 K 0 1 04 1 05 0 3 0 0 6 0 0 9 0 0 1 .2 K 0 1 04 1 05 0 5 0 0 1 .0 K 1 .5 K 0 1 04 1 05 0 5 0 0 1 .0 K 1 .5 K 0 1 04 1 05 0 3 0 0 6 0 0 9 0 0 1 .2 K 0 1 04 1 05 0 3 0 0 6 0 0 9 0 0 1 .2 K 0 1 04 1 05 0 5 0 0 1 .0 K 1 .5 K 0 1 04 1 05 0 5 0 0 1 .0 K 1 .5 K FcγRII FcγRIII FcγRI
Figure S1. Endocytosis of FVIII-IC by macrophages is dependent on FcgRI and FcgRII. A. Expression of Fcg
receptors on human monocyte-derived macrophages (upper panel) and DCs (lower panel). B. 5 nM of either soluble FVIII or FVIII-IC was incubated with MФ for 1 hour at 37°C. C. Prior addition of FVIII-IC, cells were incubated with blocking antibodies directed to FcgRI, FcgRII and/or FcgRIII. Mean ± SD from 3 independent experiments are shown. Results are expressed as % MFI, where 100% corresponds to mean fluorescence intensity obtained with FVIII (A) or FVIII-IC (B).
