Figure S1. Western blot analysis of clathrin RNA interference in human DCs Human immature DCs were transfected with 100 nm Clathrin SMARTpool or

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Estella Pitts
5 years ago
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1 Figure S1. Western blot analysis of clathrin RNA interference in human DCs Human immature DCs were transfected with 100 nm Clathrin SMARTpool or control nontargeting sirnas. At 90 hr after transfection, clathrin levels were analyzed by Western blot by using an antibody against clathrin light chain (LC).

2 Figure S2. Routing of neck binding antibody is independent of Fc receptors Fc receptors on DCs were blocked by incubating cells with serum-free medium (none), medium with 2% human serum (HS) or medium with 10% human Fc receptor block (Myltenyi Biotec). Subsequently, DCs were incubated with the anti-neck antibody (DCN46, red) at 4 C, washed and incubated at 37 C for 5 hours. After acid strip, fixation and permeabilization, the early endosomal marker EEA1 was labeled (green) and the samples were analyzed by confocal microscopy. Cells are representative of several images from two independent experiments. Scale bar, 10 µm.

3 Figure S3. LPS does not affect the intracellular routing of anti-neck antibodies After incubating DCs with medium with 2% human serum to block aspecific binding of antibodies, DCs were incubated with the anti-neck antibody (DCN46, red) at 4 C, washed and incubated at 37 C for 5 hours in the absence or in the presence of 2 µg/ml LPS. After acid strip, fixation and permeabilization, the early endosomal marker EEA1 was labeled (green) and the samples were analyzed by confocal microscopy. Cells are representative of several images from two independent experiments. Scale bar, 10 µm.

4 Figure S4. DC-SIGN expression and routing in DC-SIGN transgenic mice A) Mouse bone marrow cells of wild-type and DC-SIGN transgenic mice were cultured for 7 days in the presence of GM-CSF to generate BMDCs. Subsequently, cells were stained using directly-labeled antibodies recognizing mouse CD11c and human DC-SIGN and analyzed by flow cytometry. In BMDC cultures of transgenic mice, 66% of the cells expressed CD11c, whereas human DC-SIGN was detected on 62% of the CD11c + cells. B) BMDCs were incubated with anti-neck antibody (DCN46, green) at 4 C, washed and either kept on ice (steady state) or shifted to 37 C for indicated time points to trigger endocytosis. After acid strip of cells incubated at 37 C, fixation and permeabilization, the lysosomal marker LAMP-1 was labeled (red), and the samples were analyzed by confocal microscopy. Cells are representative of several images from two independent experiments. Scale bar, 10 µm.

5 Figure S5. Presentation of antigen targeted to the DC-SIGN neck and CRD domain The efficacy by which neck-targeted and CRD-targeted antigens are presented via MHC I and II were compared using BMDCs from human DC-SIGN transgenic mice. Antigen presentation experiments were performed at the indicated antigen concentrations either in the absence (immature DCs) or presence (mature DCs) of a maturation stimulus. Experiments in the absence of a maturation stimulus were performed by incubating BMDCs with antibody/ova conjugates and (A) OT-1 or (B) OT-2 T cells for 3 days. In experiments with maturation stimulus, BMDCs were incubated with antibody/ova conjugates for 3 hours followed by removal of antigens and addition of LPS and OT-1 and OT-2 T cells. Data represent mean ± s.e.m. of two experiments performed in duplicate.

6 Mean cell fluorescence (% of saturation) anti-neck/ova anti-crd/ova Antibody/OVA (μg/ml) Figure S6. Differences in DC binding between the antibody/ova conjugates Human DCs were incubated at various concentrations of anti-neck/ova or anti-crd/ova (both containing approximately 3 OVA molecules per antibody, on average, as determined by ELISA measurements) at 4 C for 30 minutes. In addition, some DCs were incubated with isotype/ova constructs to determine non-specific binding. OVA-binding to cells was determined by staining cells using rabbit anti-ova and goat anti-rabbit Alexa488-labeled antibodies. OVA binding to DCs was saturated at 10 µg/ml of construct for both anti-neck/ova and anti CRD/OVA. Mean cell fluorescence is given as a percentage of maximum binding, after correcting for non-specific binding. Data represent mean ± s.e.m. of two experiments performed in duplicate.

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