Identification of the molecular recognition domain present in gangliosides essential for mDC capture.

<p>(A) Comparative mDC capture of distinct LUV_HIV-tRed containing Cer, GM1a, or GM1 lacking sialic acid (Asialo GM1). A total of 2×10⁵ DCs were pulsed for 4 h at 37°C with 100 µM of LUVs, washed with PBS, and assessed by FACS to obtain the percentage of tRed-positive cells. Data show mean values and SEM from three independent experiments, including cells from nine donors. mDCs capture significantly higher amounts of GM1a-containing LUV_HIV-tRed than Asialo GM1-containing LUV_HIV-tRed (<i>p</i><0.0001, paired <i>t</i> test). Schematic of the gangliosides present in the LUVs of these experiments is shown underneath. (B) Comparative mDC capture of GM3-containing LUV_HIV-tRed and VLP_HIV-Gag-eGFP treated or not with neuraminidase to remove sialic acid. A total of 2×10⁵ DCs were pulsed for 2 h at 37°C with 25 µM of LUVs and 75 ng of VLP_HIV-Gag-eGFP Gag treated or not with <i>C. perfringens</i> neuraminidase for 12 h, washed with PBS, and assessed by FACS to obtain the percentage of tRed- and eGFP-positive cells. Data show mean values and SEM from two independent experiments including cells from five donors. mDCs capture significantly higher amounts of untreated particles than neuraminidase-treated particles (<i>p-</i>values on the graph, paired <i>t</i> test). (C) Comparative mDC capture of distinct LUV_HIV-tRed containing GalCer, GM4, GM3, or GM1a. A total of 2×10⁵ DCs were pulsed for 4 h at 37°C with 100 µM of LUVs, washed, and assessed by FACS to obtain the percentage of tRed-positive cells. Data show mean values and SEM from three independent experiments including cells from nine donors. mDCs capture significantly higher amounts of GM1a-containing LUV_HIV-tRed than GalCer or GM4-containing LUV_HIV-tRed (<i>p</i><0.0001, paired <i>t</i> test). Schematic of the molecules present in the LUVs for these experiments is shown underneath. (D) Graph representing the relative capture of GM3-containing LUV_HIV-tRed and VLP_HIV-Gag-eGFP by mDCs that had been pre-incubated with 10 mM of soluble lactose or with 5–10 mM of GM3 carbohydrate polar head group, normalized to the level of LUV/VLP capture by mock-treated mDCs (set at 100%). mDCs captured fewer particles upon treatment with GM3 polar head group (<i>p-</i>values on the graph, paired <i>t</i> test). Data show mean values and SEM from three independent experiments including cells from at least nine donors.