1. Uptake of blood coagulation factor VIII by dendritic cells is mediated via its C1 domain 
Eszter Herczenik, PhD, Simon D. van Haren, MSc, Aleksandra Wroblewska, MSc, Paul Kaijen, MSc, Maartje van den Biggelaar, PhD, Alexander B. Meijer, PhD, Luisa Martinez-Pomares, PhD, Anja ten Brinke, PhD, Jan Voorberg, PhD 
Journal of Allergy and Clinical Immunology 
Volume 129, Issue 2, Pages e5 (February 2012)
DOI: /j.jaci
Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2. Fig 1
FVIII uptake by MDDCs. A, FVIII (0-160 nM) was incubated with MDDCs. Cells were analyzed by fluorescence-activated cell sorting. B, Cell surface–bound versus internalized FVIII was compared in the absence or presence of saponin. C, Internalized FVIII was quantified from the cell lysate by using ELISA. D, Uptake of light- and heavy-chain FVIII was evaluated from cell lysates by using ELISA. Results represent 4 individual experiments. FITC, Fluorescein isothiocyanate; MFI, mean fluorescence intensity.
Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci )
Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3. Fig 2
FVIII uptake is mediated by the C1 domain. A, 10 nM of FVIII or 250 μg/mL of Lucifer yellow (LY) was preincubated with 80 nM of KM33 or VK34. B and C, KM33 or VK34 (0-160 nM) was added to 10 nM of FVIII. Uptake was analyzed by using fluorescence-activated cell sorting (Fig 2, B) or ELISA (Fig 2, C). D, 250 μg/mL of LY was preincubated with KM33 and VK34 before addition to MDDCs. Graphs represent data of 3 independent experiments. FITC, Fluorescein isothiocyanate; MFI, mean fluorescence intensity.
Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci )
Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4. Fig 3
Uptake of FVIII by MDDCs is independent of LRP, MR, and DC-SIGN. A, MDDCs were preincubated with α2m, RAP, mannan, or anti–DC-SIGN before 40 nM of FVIII was added to the cells. B, Receptor expression 72 hours after siRNA transfection was measured by using fluorescence-activated cell sorting. Grey histograms represent isotype controls, black lines indicate specific (LRP, MR and DC-SIGN) antibody stainings of cells with nontargeting (scramble) siRNA, dashed lines correspond to receptor staining after knockdown. C, 20 μg/mL of α2m-fluorescein isothiocyanate, β-d-galactose-3-sulfate-PAA-fluor, or blood-type B-PAA-fluor was used to monitor the endocytosis through targeted receptors. D, Internalized FVIII (0-80 nM of FVIII) was quantified by using fluorescence-activated cell sorting. Data represent 3 independent experiments. β-d-gal, β-d-galactose-3-sulfate-PAA-fluor; Btri, blood-type B-PAA-fluor; FITC, fluorescein isothiocyanate; MFI, mean fluorescence intensity.
Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci )
Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5. Fig 4
KM33 blocks the binding of FVIII to LRP and DC-SIGN but not to MR. A, C, E, LRP1 cluster II-Fc (Fig 4, A), MR CTLD1-3-Fc or CTLD4-7-Fc (Fig 4, C), or DC-SIGN-Fc (Fig 4, E) (0-10 μg/mL) were added to immobilized FVIII. B, D, F, LRP1 cluster II-Fc (Fig 4, B), MR-Fc CTLD4-7-Fc (Fig 4, D), or DC-SIGN-Fc (Fig 4, F) were preincubated with KM33 Fab2 (0-100 μg/mL). B, D, F, RAP (Fig 4, B) and mannan (Fig 4, D and F) were used as positive controls. Data represent 2 experiments. OD, Optical density.
Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci )
Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6. Fig 5
Uptake of FVIII by BMDCs. A, FVIII (0-160 nM) was incubated with BMDCs. B, 0 to 160 nM of KM33 or VK34 was preincubated with 20 nM of FVIII. C, Prior to the endocytosis of 10 nM of FVIII, BMDCs were preincubated with 1, 2.5, or 5 mg/mL of mannan, 100 nM of α2m, or 1 mg/mL of RAP D, Expression of LRP and MR was determined by using fluorescence-activated cell sorting. Results represent 3 individual experiments. FITC, Fluorescein isothiocyanate; MFI, mean fluorescence intensity.
Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci )
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7. Fig 6
KM33 modulates immune responses against FVIII in a murine model for hemophilia A. A and B, Hemophilic E17 mice were preadministered with 1 mg control antibody or KM33, followed by 3 (Fig 6, A) or 5 (Fig 6, B) weekly FVIII injections. Anti-FVIII antibody titers from the collected plasma samples were determined. C, Inhibitory capacity of FVIII antibodies was measured by using Bethesda assay. D, Presence of KM33 in blood was monitored. Data were analyzed by using the nonparametric Mann-Whitney U test.
Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci )
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8. Fig E1
vWF blocks the uptake of FVIII; phosphatidylserine is not involved in endocytosis. A, Preincubation of 20 nM of FVIII with 100 nM of vWF results in reduced uptake of FVIII by MDDCs. B, Prior to the uptake of 20 nM of FVIII, MDDCs were preincubated with 200 nM of lactadherin for 15 minutes at 37°C. FITC, Fluorescein isothiocyanate.
Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci )
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9. Fig E2
Simultaneous knockdown of LRP, MR, and DC-SIGN does not affect the uptake of FVIII.Uptake of various concentrations of FVIII by MDDCs transfected with nontargeting (scramble) LRP and MR (A) or LRP, MR, and DC-SIGN (B) targeting siRNA pools. C, Uptake after knockdown is expressed as percentages of 80 nM of FVIII endocytosed by cells transfected with nontargeting (scramble) siRNA pool. Data obtained from 2 or 3 individual experiments performed with cells from different donors. FITC, Fluorescein isothiocyanate; MFI, mean fluorescein intensity.
Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci )
Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10. Fig E3
Binding of MR CTLD4-7-Fc and LRP1 cluster II to immobilized FVIII. First, 12 nM of FVIII was immobilized to the CM5 sensor chip through mAb EL14. Then, LRP1 cluster II (A) or MR CTLD4-7-Fc (B) was passed over the chip at 200 nM (1), 100 nM (2), 50 nM (3), 25 nM (4), and 12.5 nM (5) concentrations of LRP1 cluster II and 500 nM (1), 200 nM (2), 100 nM (3), 50 nM (4), and 10 nM (5) concentrations of MR-CTLD4-7-Fc. Ymax values obtained from 1-phase exponential association alignments of LRP1 cluster II (C) and MR CTLD4-7-Fc (D) binding curves were used to calculate the apparent KD and Bmax values (E). Experiments were performed in duplicate.
Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci )
Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions