Supplementary Figure Captions
Supplementary Figure 1. Kinetics of IVIg cell-penetration in human firoblasts. Cells
were incubated with 1.6 mg/ml of IVIg (Sandoglobulin) for various time points ( 0, 2, 30,
60, 120 and 240 min) at 37oC. Confocal sections of IVIg staining (green) with the use of
anti-human IgG (H- and L-chain–specific)-Alexa488 conjugate as detection antibody
(green), and of TO-PRO-3 iodide (blue) for nucleus labeling. Images were captured with
a 63x HCX PLApo objective lens.
Supplementary Figure 2. In vitro cell penetration of healthy donor IgG. NIH-3T3 cells
were incubated with 1.6 mg/ml of an IVIg preparation (Sandoglobulin), and of IgG from
five healthy donors, or CM (control) for 2 h at 37oC. Confocal sections of IVIg staining
(green) with the use of anti-human IgG (H- and L-chain–specific)-Alexa488 conjugate as
detection antibody (green), and of TO-PRO-3 iodide (blue) for nucleus labeling. Images
were captured with a 63x HCX PLApo objective lens. Results are representative of at
least three independent experiments.
Supplementary Figure 3. In vitro cell penetration of IVIg in the presence of
cytochalasin D. Cells were pretreated for 30 min with 1 μg/ml cytochalasin D or CM and
then incubated with 1.6 mg/ml of IVIg (Sandoglobulin) or CM (control) for 2 h at 37oC in
the presence or absence of cytochalasin D, respectively. Maximum projections of
confocal sections of IVIg staining (green) in NIH-3T3 cells. TO-PRO-3 iodide (blue)
used for nucleus labeling and rhodamine–phalloidin-TRITC (tetramethylrhodamine; red)
(1:100 dilution; Molecular Probes) for labeling the actin-filaments. Confocal images
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captured with a 63x HCX PLApo objective lens. Results are representative of three
independent experiments.
Supplementary Figure 4. Semi-quantitative analysis of in vitro cell-penetration.
Confocal sections of NIH-3T3 cells after incubation for 2 h at 37oC with the three
affinity-purified antibody fractions (0.2 mg/ml), whole IVIg (1.6 mg/ml), effluent(s)
derived from IVIg passaging through each or all three IADs (1.6 mg/ml) or medium
(control). IVIg staining (green) and TO-PRO-3 iodide nucleus labeling (blue) observed
using a 63x HCX PLApo objective lens. Measurements of fluorescent intensity in
arbitrary units (a.u.) were performed at confocal images of the maximum projection,
obtained from at least 100 cells/sample (captured with the same settings), by the use of
Intensity Correlation Analysis software ImagePro 5 (Media Cybernetics, Maryland,
USA). Results are representative of three independent experiments (*p ≤ 0.05**p ≤
0.01,***p ≤ 0.001).
Supplementary Figure 5. In vivo cell penetration of IVIg in spleen cells. BALB/c mice
received a single intravenous injection of 2 g/kg of Intraglobin F-IVIg [n = 3, (∆)] and
controls 2 g/kg of effluent or 200 μl of physiological saline [n = 3, (○)]. Three hours later,
spleen and lymph nodes were prepared as cell suspensions and trypsinized. (A) Confocal
sections of cell suspensions (spleen and lymph nodes). IVIg staining (green) and TOPRO-3 iodide nucleus labeling (blue) observed using a 63x HCX PLApo objective lens.
Results are representative of three independent experiments. (B) Percentage of positive
cells stained for IVIg from spleen and lymph nodes suspensions after FACS analysis;
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horizontal lines indicating the means. Results obtained from three independent
experiments (***p ≤ 0.001). (C) Control experiment for the specific detection of
intracellular IVIg in spleen cells in vivo. Fluorescent intensity of IVIg (Kiovig) staining
in cell suspensions (gated granulocytes and monocytes) after fixed with PFA and cellpermeabilization (black line) or not (dotted line), in comparison to that of control cells
(permeabilized; gray area) and mean fluorescent intensity (MFI) by FACS analysis.
Supplementary Figure 6. In vivo cell penetration of Kiovig-IVIg 3 h postadministration. BALB/c mice received a single intravenous injection of 2 g/kg of KiovigIVIg [n = 6, (∆)] or physiological saline [n = 3, (○)]. Three hours later, various organs
were either snap frozen and subjected to cryostat sectioning, or prepared as cell
suspensions and trypsinized. (A) Maximum projection of confocal sections from organs
processed with cryostat (liver, kidneys, lungs, brain, ileum and heart). (B) Confocal
sections of cell suspensions (spleen and lymph nodes). IVIg staining (green) and TOPRO-3 iodide nucleus labeling (blue) observed using a 20x HC PlanApo and 63x HCX
PLApo objective lenses; scale bar corresponds either to 80 or 20 μm. Results are
representative of three independent experiments. (C) Percentage of IVIg-positive cells
from spleen and lymph nodes suspensions after FACS analysis; horizontal lines
indicating the means. Results obtained from three independent experiments (***p ≤
0.001).
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