Figure 1.
Absence of FcγRI engagement is associated with increased antibody requirement for DENV neutralization.
(A) Flow cytometry data of both FcγRI and FcγRII (green histogram) in THP-1 and K562. Mock (red histogram) represents staining with secondary antibody only. (B) Neutralization profile of DENV using various concentrations of h3H5 antibody in THP-1 (Black) and K562 (Red) at 72 h post infection, quantified by plaque assay. Unless indicated, the mean value of neutralization is either 0% or 100%. (C) Percentage of internalized DiD-DENV (% DiD positive cells) in complex with h3H5 antibody is represented by black bar at concentrations that mediated complete neutralization in THP-1 (3.125 µg/mL) and K562 (25 µg/mL), DiD-DENV without antibody is represented in grey bar, as assessed by confocal microscopy at 30 mins post infection. (D) Subcellular localization of DiD-DENV opsonized at h3H5 antibody concentrations required for complete neutralization in THP-1(3.125 µg/mL) and K562 (25 µg/mL). LAMP-1 is in green, DiD-DENV is in blue and h3H5 antibody is in red. Scale bar is 10 µm. Data are represented as mean ± s.e.m. * p<0.01. Results presented are mean of three independent experiments, each with biological triplicates.
Figure 2.
FcγRIIB knockdown did not result in additional increase in antibody requirement for DENV neutralization.
(A) K562 transfected with either control siRNA or FcγRIIB siRNA. The reduction in FcγRIIB expression was detected in cell lysate by western blot. LAMP-1 served as a loading control. (B) Subcellular localization of neutralized DENV immune complexes in K562 treated with either control siRNA or FcγRIIB siRNA at 30 mins post infection. LAMP-1 is in green, DiD-DENV is in blue and h3H5 antibody is in red. Scale bar is 10 µm. (C) Percentage of DiD positive cells in K562 treated with either control siRNA or FcγRIIB siRNA with DiD-DENV using 25 µg/mL antibody (black bar) or without antibody (white bar) after 30 mins post infection, assessed by confocal microscopy (D) Neutralization profile of h3H5 against DENV in K562 treated with either siRNA control (red) or siRNA against FcγRIIB (blue) at 72 h post infection, assessed by plaque assay. Unless stated, the mean percent neutralization is either 0% or 100%. Data are represented as mean ± s.e.m. * p<0.01. Results presented are mean of three independent experiments, each with biological triplicates.
Figure 3.
Reduced antibody requirement for neutralization and lowered ADE with FcγRI engagement.
(A) Histogram showing surface expression of FcγRI in THP-1 cells when treated with mock transfection (green), control siRNA (blue), FcγRI siRNA (red) or untransfected (yellow). Unstained (grey) indicates negative control stained with secondary antibody only. (B) Histogram showing surface expression of FcγRII in THP-1 cells when treated with mock transfection (green), control siRNA (blue), FcγRI siRNA (red) or untransfected (yellow). Unstained (grey) indicates negative control stained with secondary antibody only. (C) Neutralization profile of untransfected THP-1 cells (yellow) with mock transfection (green), siRNA control (black), siRNA FcγRI (red) or siRNA FcγRIIA (blue), 72 h post infection, as assessed by plaque assay. (D) Virus yield from THP-1 treated with control siRNA, FcγRI siRNA and FcγRIIA siRNA at 72 h post infection, as assessed by plaque assay. Although knockdown efficiency may vary between experiments, we observed similar trends. * p<0.01. Graphs shown are mean ± s.d of biological triplicates of a single experiment from three independent experiments.
Figure 4.
Preferential engagement of FcγRI results in uptake of neutralized DENV immune complexes.
(A) Sorting of AF488-DENV infected cells using fluorescence activated cell sorter at 120 mins post infection after synchronization, in absence of antibody (virus only), with neutralizing (3.125 µg/mL) or sub-neutralizing (0.39 µg/mL) antibody concentrations. For mock infection, cells were exposed to h3H5 antibody only. Percentages of AF488-DENV positive cells (green histogram) are numerically indicated. (B) Cellular localization of AF488-DENV immune complexes at neutralizing or sub-neutralizing concentration of h3H5 after 120 mins post infection. LAMP-1 is in red. DENV is in green and FcγRI/FcγRII is in blue. White areas in the merged image indicate the presence of co-localization. Scale bar is 10 µm. (C) Percent co-localization of AF488-DENV opsonized with either neutralizing or sub-neutralizing levels of h3H5, with respect to FcγRI or FcγRII at 120 mins post infection using the confocal microscope, Zen 2009 Software. Images shown are representative of at least 2 separate experiments. Data are represented as mean ± s.e.m. * p<0.01.
Figure 5.
Clustering of FcγRI with neutralized DENV immune complexes.
(A) THP-1 infected with AF488-DENV opsonized with neutralizing h3H5 antibody (3.125 µg/mL) were sorted using fluorescence activated cell sorter after 15, 30, 60 and 120 mins post infection (p.i). For mock infection, cells were exposed to h3H5 antibody only. Percentages of AF488-DENV positive cells (green histogram) for different time points are numerically indicated. (B) Cellular localization of AF488-DENV immune complexes, with FcγRI or FcγRII at various time points post infection. LAMP-1 is in red, DENV is in green and FcγRI or FcγRII is in blue. Scale bar is 10 µm. (C) Intensity of FcγRI or FcγRII when co-localized with DENV obtained using the Zen 2009 Software, keeping the selected area (70.5 ± 0.28) µm2 consistent for all samples and fields. Statistical test using ANOVA shows a significant increase in intensity of FcγRI with increasing time (p<0.0001) as compared to FcγRII. Images shown are representative of 2 separate experiments. Data are represented as mean ± s.e.m. * p<0.01.