Within one hour, HIV-1 uses viral synapse to enter efficiently the inner, but not outer, foreskin mucosa and engages Langerhans-T-cell conjugates
Yonatan Ganor, Zhicheng Zhou, Daniela Tudor, Alain Schmitt, Marie-Cécile Vacher-Lavenu, Laure Gibault, Nicolas Thiounn, Jacques Tomasini, Jean-Philippe Wolf7, Morgane Bomsel
Supplementary material legends:
Supplementary Figure 1.(a) Retention of LCs in the epidermis following exposure to low concentration of cell-free HIV-1. Representative flow cytometry histograms of cells in epidermal single-cell suspensions prepared from inner foreskin explants exposed for 1hr to low concentration of cell-free HIV-1 V29 (top left) or RPMI medium (bottom left). Cells were stained with either PE-conjugated anti-human langerin mAb (thick lines) or matched isotype control (thin lines). Numbers represent the percentage of positive cells in the M1 region. Images are representative of three independent experiments. The means±SEM fold decreases in the percentage of epidermal langerin+ cells (calculated as [% langerin+ cells following exposure to RPMI / % langerin+ cells following exposure to low concentration of cell-free HIV-1 V29]), in inner (light gray bar) and outer (dark grey bar) foreskin explants, are derived of three experiments (graph). (b) Calculated mean folds±SEM of LCs percentage in epidermal single-cell suspensions, following 1hr exposure of inner (light gray bar) and outer (dark grey bar) foreskin explants to RPMI medium (compared to high concentration of cell-free HIV-1 V29) derived of three experiments. (c) No changes in percentage of epidermal LC-T-cell conjugates following exposure to high concentration of cell-free HIV-1. Representative flow cytometry profiles of cells in epidermal single-cell suspensions prepared from inner foreskin explants exposed for 1hr to high concentration of cell-free HIV-1 V29 (top right) or RPMI (top left). Cells were double stained with CD3-APC and langerin-PE mAbs and gated on CD3+ cells. Numbers represent the percentage of CD3+ / langerin+ / high FSC LC-T-cell conjugates. Images are representative of three experiments. The calculated means±SEM fold increase in the percentage of epidermal LC-T-cell conjugates following 1hr exposure of inner (light gray bar) and outer (dark grey bar) foreskin explants to high concentration of cell-free HIV-1 V29 (normalized against RPMI) derived of three experiments are also shown (graph).
Supplementary Figure 2.(a) Electron microscopy images of reconstructed inner (left) and outer (right) foreskins. The white double-headed arrows emphasize the increased number / thickness of the keratinocyte layers in outer compared to inner foreskin reconstructions. Fb, fibroblasts; Ker, keratinocytes. Scale bars = 5m. (b) Fluorescence microscopy images of normal and reconstructed inner (left) and outer (right) foreskins, stained with mouse-anti-human CK10 mAb, followed by TRITC-conjugated anti-mouse Ab. Cell nuclei were counterstained with DAPI. (c) Immunohistochemistry of normal and reconstructed inner (left) and outer (right) foreskins, stained for the expression of involucrin (brown; DAB peroxidase substrate). All images are representative of at least three experiments performed with different normal or reconstructed foreskins for each morphological / structural characteristic. Scale bars = 10m.
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