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Blood, Vol. 113, Issue 12, 2732-2741, March 19, 2009
Capture and transfer of HIV-1 particles by mature dendritic cells converges with the exosome-dissemination pathway
Blood Izquierdo-Useros et al.
113: 2732
Supplemental materials for: Izquierdo-Useros et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 1.48 MB)
- Figure S1 (JPG, 52.9 KB)
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(A) Capture profile of increasing amounts of VLPs HIV-Gag-eGFP to iDCs and mDCs during 2 h at 37°C. Maturation of DCs resulted in increased VLP capture at 37°C compared to iDCs, and this capture was dose dependent. Histograms show a representative capture profile of iDCs (top) and mDCs (bottom) at 37°C. (B) Binding profile of carboxylated yellow fluorescent beads (comparable in size to HIV-1 particles; with approximately 100 nm of diameter) to iDCs and mDCs is similar at 4°C. However, maturation of DCs resulted in diminished bead capture at 37°C. Histograms show a representative binding and capture profile of iDCs and mDCs at 4°C and 37°C, respectively.
- Figure S2 (JPG, 126 KB)
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(A–C) Electron microscopy images of mDCs simultaneously pulsed with HIVNL43 and ExosomesDiI. Particles displaying viral morphology (with an electro-dense core; red arrows) or exosome morphology (with lighter core; yellow arrows) accumulated in the same area of the membrane (A) or within the same vesicles (B and C). (D–G) Confocal microscopy analysis of mDCs pulsed simultaneously with VLPHIV-Gag-eGFP and ExosomesDiI and then stained with DAPI. (D) Composition of a series of x-y sections of a mDC collected through part of the cell nucleus and projected onto a two-dimensional plane to show the x-z plane (bottom) and the y-z plane (right). VLPHIV-Gag-eGFP and ExosomesDiI accumulated within the same compartment. (E) Isosurface representation of the cell shown in (D), computing the nucleus and vesicle surfaces within a three-dimensional volumetric x-y-z data field. (F) Quantification of the percentage of VLPHIV-Gag-eGFP co-localizing with ExosomesDiI and vice versa, obtained analyzing 35 vesicles from mDCs of 3 different donors. The mean and S.D. of the correlation coefficients of Manders and Pearson (obtained considering all the images) were 0.84 ± 0.08 and 0.75 ± 0.1, respectively, indicating co-localization. (G) Confocal images exemplifying vesicles analyzed in (F), where the green, the red and the blue fluorescence are merged (top) or the vesicles are marked in regions of interest (squares) showing the mask of overlapping fluorescence signals (bottom).
- Figure S3 (JPG, 71.6 KB)
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(A) Fluorescent microscopy images of control mDCs compared to mDCs treated with 2.5 mM amantadine. Left panels show projection of stack images obtained by merging the red, blue and green fluorescence of mDCs exposed to VLPs HIV-Gag-eGFP for 2 h at 37°C and then labeled with DAPI and TRITC-wheat germ agglutinin (to stain the cell membrane). Right panels show projection of stack images obtained by merging only the blue and green fluorescence. (B) mDCs viability in the presence of increasing concentrations of amantadine was not compromised until cells were incubated with 5 mM amantadine (P=0.0175, one sample t test). Cells were labeled with propidium iodide and DIOC-6 to analyze the drug induction of necrosis and apoptosis with FACS. (C) Viability in the presence of increasing concentrations of chlorpromazine assessed as in panel (B). (D) Percentage of HIVNFN-SX captured by amantadine (dark bars) and chlorpromazine (clear bars) treated mDCs relative to untreated cells normalized to 100% of viral capture at 37°C. mDCs were pre-incubated with increasing concentrations of amantadine and chlorpromazine, exposed to the virus and lysed in triton to measure the cell associated p24Gag by an ELISA. Viral capture was inhibited in a dose dependent manner, reaching statistical significance at 2.5 mM of amantadine and 25 _M of chlorpromazine (P values on the graph, paired t test). Data show mean values and SEM from six donors and two independent experiments for each compound.
- Figure S4 (JPG, 145 KB)
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(A) Western blot of VLPHIV-Gag-eGFP co-transfected with an HIV-1 envelope glycoprotein plasmid to express gp120. Particles were treated or not with 400 µg/ml of pronase for 1 h at 4°C. Pronase treatment efficiently diminished gp120 detection levels compared to mock treated VLPs. mAb against p24Gag was used for detection of Gag-eGFP expression in both VLPs. (B) TCID50/ml of untreated and pronase-treated wild type HIVNL43 particles on TZM-BL cells containing the luciferase reporter gene. Upon pronase treatment there was an 18-fold reduction in infectivity (from a mean of 22,357 to 1,167). (C) Representative histograms of mDCs treated or not with 200 µg/ml of pronase for 30 min at 4°C and stained with an anti–DC-SIGN, CD4 and CD81 mAbs. Pronase treatment (light grey histogram) efficiently diminished DC¬SIGN, CD4 and CD81 expression levels or binding of trimeric HIV-1 envelope glycoprotein gp140 compared to mock treated cells (dark grey histogram) as seen by the reduction in geometric mean fluorescence intensity (MFI) (P values on the graphs, paired t test, n=6). Unlabeled cells are shown in blank dotted histograms. (D) When untreated (top panels) or pronase-treated mDCs (bottom panels) were pulsed at 37°C for 4 h (in the absence or in the presence of pronase, respectively) with VLPHIV-Gag-eGFP, ExosomesDiI or HIVNL43-vpr-eGFP, no differences in trafficking of the particles were observed by confocal microscopy, confirming our previous FACS results. (E) Binding of VLPHIV-Gag-eGFP at 16°C for 2 h was assessed by FACS employing mDCs previously exposed to 200 _g/ml of pronase or mock-treated. Noteworthy, by adding pronase we were not able to block viral binding when pulsing treated mDCs with VLPHIV-Gag-eGFP. (F) After pulse at 16°C, some cells were stained with DAPI and TRITC-labeled wheat germ agglutinin (to label the cell membrane), fixed with 2% paraformaldehyde and cytospun into glass-slides to analyze by deconvolution microscopy. Analysis of the projection of stack images revealed that at 16°C, most of the VLPs bound were not endocytosed but remained in the cell surface in both untreated (top panel) and pronase-treated mDCs (bottom panel). (G) After pulse at 37°C like in panel (D), pronase-treated mDCs were extensively washed, allowed to recover and then cocultured with cell tracker labeled Jurkat T cells. Confocal sections show VLPHIV-Gag-eGFP, ExosomesDiI and HIVNL43-vpr-eGFP polarization to the site of DC-T cell contact. Images depict the red and green fluorescence channels merged with DAPI and the bright field cellular shape.
- Figure S5 (JPG, 111 KB)
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(A–B) HEK-293T cell treatment with inhibitors of sphingolipid biosynthesis (50 µM of FB1 or 500 µM of NB-DNJ) for 4 days does not impact VLP release after HIV Gag-eGFP transfection, measured both by (A) p24Gag ELISA or (B) p24Gag western blot. (C) HEK-293T cell treatment with 500 µM of NB¬DNJ for 4 days does not impact HIVNL43 infectivity, while affecting virus produced from FB1-treated HEK-293T cells (P<0.0017, unpaired t test). TCID50 values were obtained with a TZM-BL reporter cell line and are normalized to the p24Gag levels released after transfection. Data show mean and SEM of viral stocks obtained in 5 different transfections. (D) At the viral dose employed to pulse mDCs (10 ng of p24Gag per 1 × 105 cells) HIVNL43 derived from FB1-treated HEK-293T cells only reduces its infectivity 20% ± 10% compared to untreated HIVNL43. Ghost CXCR4/CCR5 cells containing the GFP gene under the control of the HIV promoter were acquired with FACs 48 hours post-infection. Data show mean and SEM of viral stocks obtained in 3 different transfections. (E–F) Jurkat treatment with FB1 (50 µM) or NB-DNJ (500 µM) for 7 days does not impact exosome release after DiI labeling, measured both by (E) fluorescence assay or (F) protein quantification by Bradford assay. Dotted line in (E) indicates background levels of media assayed for RFUs.
- Video 1. Three-dimensional reconstruction of a mDC exposed to VLPHIV¬Gag-eGFP and ExossomesDiI and then stained with DAPI (MOV, 196 KB)
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A series of x-y sections collected through part of the cell nucleus shows the perinuclear vesicle in which VLPHIV-Gag-eGFP and ExossomesDiI accumulate.
- Video 2. Isosurface representation of the cell shown in Video 1, drawing the nucleus and vesicle within a three-dimensional volumetric x-y-z data field (MOV, 1.85 MB)
- Video 3. Three-dimensional reconstruction of a mDC exposed to VLPHIV¬Gag-eGFP and stained with DAPI (MOV, 66.3 KB)
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A series of x-y sections collected through part of the cell nucleus shows the perinuclear vesicle in which VLPHIV-Gag-eGFP accumulate.
- Video 4. Three-dimensional reconstruction of a series of x-y sections showing polarization of an internal vesicle accumulating VLPHIV-Gag-eGFP to the contact site of a mDC (stained with DAPI) with a Jurkat T cell (labeled with an orange cell tracker dye) (MOV, 87.6 KB)
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Bright field cellular shape corresponds to Fig. 5 B.
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