Volume 9, Issue 2, Pages 277-287 (August 1998) Intracellular Neutralization of HIV Transcytosis across Tight Epithelial Barriers by Anti- HIV Envelope Protein dIgA or IgM  Morgane Bomsel, Martine Heyman, Hakim Hocini, Sylvie Lagaye, Laurent Belec, Christophe Dupont, Claude Desgranges  Immunity  Volume 9, Issue 2, Pages 277-287 (August 1998) DOI: 10.1016/S1074-7613(00)80610-X

Figure 1 HIV Transcytosis Is Blocked by Specific Anti-HIV Polymeric IgA or IgM Loaded in the Cell via the pIg-R Basolateral-to-Apical Transcytosis Pathway (a) Characterization of the polyclonal dIgA and IgM anti-HIV envelope protein against the HIV envelope protein precursor gp160. Polymeric Igs (IgM): nonspecific (nonspecific dIgA [30 μg], anti-HIV envelope protein dIgA [30 μg], IgM [30 μg], or IgM, 1:10 [3 μg]) or with additional anti-SC serum (+ anti-SC) were basolaterally loaded in epithelial cells (25 or 15 min, respectively) before mucosal (apical) addition of HIV+ cells. Transcytosis was evaluated in the basolateral medium 150 min later by p24 antigenemia. Results are presented as percent of transcytosis in absence of Igs (standard). HIV transcytosis was induced from NDK-CEM T cell lines (b) or from HIV+ PBMCs (c). Immunity 1998 9, 277-287DOI: (10.1016/S1074-7613(00)80610-X)

Figure 3 HIV Neutralized Intracellularly Is Cleared from the Epithelial Cell Line Barrier Using the Last Leg of the pIg-R Pathway and Released in the Mucosal Environment as a Complex with dIgA or IgM HIV transcytosis was assayed as in Figure 1 with antibody-free medium in the basolateral chamber (lane 1 and 2) or after basolateral exposure to nonspecific polymeric Igs (lane 3) or to an anti-HIV envelope protein (dIgA, lane 4; IgM, lane 5). After a 2 hr pulse at 37°C, HIV+ cells were discarded and the epithelium chased for an additional hour in cell-free medium (lanes 1, 3, 4, and 5) or with polymeric IgA and M (lane 2). At the end of the chase period, both apical (Apical) and basolateral (Basolateral) media were collected and subjected to immunoprecipitation with anti-α or -μ chain antibodies and anti-SC serum. The precipitate was resolved by SDS-PAGE, electrotransfered, and blotted for HIV p24. As a control, an immune complex between HIV and polymeric Igs was formed and directly precipitated as the apical media (lane 6). Immunity 1998 9, 277-287DOI: (10.1016/S1074-7613(00)80610-X)

Figure 4 Inhibition of HIV Transcytosis by Various Monoclonal IgM Anti-HIV Envelope across a Human Epithelial Cell Line Barrier In a similar assay as described in Figure 1, the serosal pole of the epithelial cell line barrier was exposed to one of the various monoclonal IgM anti-HIV envelope proteins at the indicated concentration. HIV transcytosis was induced from HIV+ PBMCs. Transcytosis was evaluated in the basolateral medium 150 min later by p24 antigenemia. Results are presented as percent of transcytosis in the absence of Igs (standard). Values are means ± SEM of at least three independent experiments. Immunity 1998 9, 277-287DOI: (10.1016/S1074-7613(00)80610-X)

Figure 2 Neutralization of HIV Transcytosis by Specific Anti-HIV dIgA or IgM Occurs Intracellularly (a) After basolateral uptake of polymeric Igs (IgM), HIV transcytosis was induced by addition of HIV-infected cells (NDK-CEM cells) in the apical compartment and allowed to proceed for 90 min. Epithelial cells were then fixed (pulse; optical sections 1–15) or chased for an additional hour after extensive washing of HIV+ cells from the apical chamber before fixation (+ chase; optical sections 3, 7, 11, and 15). Indirect immunofluorescence allowed detection of HIV (green) and polymeric Igs (red) by two-color confocal microscopy. For each of the 24 optical sections (0.5 μM apart), the corresponding green and red fluorescence images were overlaid. Colocalization sites of the two markers appear as yellow spots. For the pulse condition, only the first 15 sections covering the apical half of the epithelial monolayer are shown, since the additional 9 basolateral sections were devoid of signal. For the chase condition, only 4 sections numbered as in pulse condition are shown. To emphasize the extent of overlapping, pixel intensity histograms taken for each chromophore along the white line drawn on focal plane 3 were generated. Thick- and fine-line histograms correspond to polymeric Igs and HIV pattern, respectively. It shows an extensive overlap for HIV by polymeric Igs. Scale bar = 5 μm. Immunity 1998 9, 277-287DOI: (10.1016/S1074-7613(00)80610-X)

Figure 5 The Conserved Sequence ELDKWA Is One Epitope Involved in the Intracellular Neutralization of HIV by Polymeric Igs (a) Reversion on monoclonal IgM and polyclonal D-IgA and IgM across a human epithelial cell line barrier. In a similar assay, described in Figure 1, the serosal pole of the epithelial cell line barrier was exposed to one of the various monoclonal IgM anti-HIV envelope prteins that had been previously preincubated (+) or not (−) with the ELDKWA peptide. HIV transcytosis was induced from HIV+ PBMCs. Transcytosis was evaluated in the basolateral medium 150 min later by p24 antigenemia. Results are presented as percent of transcytosis in absence of Igs (standard). Values are means ± SEM of at least three independent experiments. (b) Specific activity of the polymeric Igs for the ELDKWA peptide (aa 662–667), the aa 642–665 peptide recognized by the CA45C IgM, and the aa 630–59 peptide in ELISA. The dotted line represents the background level. Immunity 1998 9, 277-287DOI: (10.1016/S1074-7613(00)80610-X)

Figure 6 HIV Transcytosis Occurs through a Human Duodenal Biopsy The transcytosis assay was performed across human duodenal biopsies mounted as a tight functional barrier in Ussing chambers. (a) The viability of the biopsies was monitored by the constant recording of potential difference (PD) and short-circuit current (Isc), which remained stable throughout the time course of the experiments (n = 7 biopsies). (b and c) The integrity of the biopsies was controlled by the electrical resistance (R) and [14C]mannitol fluxes, which were maintained at the initial steady-state value. (d) Transcytosis was initiated by addition of HIV+ PBMCs in the mucosal compartment. Alternatively, cell-free HIV was used as the infectious agent. After 130 min, the serosal medium was collected and tested for its HIV infectivity. In some experiments, CA45C IgM was pulsed from the serosal side of the biopsy for 30 min before mucosal addition of HIV+ PBMCs. The serosal medium was analyzed for its HIV content after up to 2 weeks of coculture with uninfected PBMCs and quantitated by p24 antigenemia. Values are means ± SEM of at least six independent experiments. Immunity 1998 9, 277-287DOI: (10.1016/S1074-7613(00)80610-X)