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. 2015 Mar 11;7(4):464–476. doi: 10.15252/emmm.201404719

Figure 2. 24F4A induces rapid internalization of BDCA2 resulting in trafficking to LAMP1+ compartments and sustained IFNα inhibition.

Figure 2

  • A Whole blood was treated with the indicated concentrations of 24F4A or the IgG1 isotype control antibody for 0, 1, 3, 6, 9, and 24 h. FACS analysis was performed to determine BDCA2 levels on pDCs. Fluorescence minus one (FMO) represents background staining of BDCA2. Shown is a representative plot of three independent experiments.
  • B Isolated pDCs were treated with 10 μg/ml 24F4A-AF647 at 4°C or at 37°C and analyzed at the indicated time points. Subcellular localization of BDCA2 (red) compared to LAMP1 (green) was assessed using confocal microscopy. Phalloidin was used to delineate the cell membrane. Yellow represents co-localization of BDCA2 and LAMP1. Shown is a representative image of four experiments conducted.
  • C, D Whole blood was either treated with 24F4A for 1 h at 37°C (pre-incubation) or left untreated. PBMC were isolated from each condition. PBMC from the untreated whole blood were subsequently treated with 10 μg/ml 24F4A, and cells from all conditions were stimulated with CpG-A for 16 h. Plot represents mean of duplicate wells. Flow cytometry was used to measure BDCA2 levels (MFI). Shown is a representative donor of eight donors tested.
  • E Whole blood was pre-treated for 0, 1, 3, 6, and 9 h with 10 μg/ml of 24F4 or the isotype control and then stimulated with CpG-A for additional 16 h. IFNα levels were measured by ELISA. Shown is a representative plot of two independent experiments.

Source data are available online for this figure.

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