## Results: 6

1.

Both mature dendritic cell (mDC)–mediated trans-infection and cell-free infection are similarly inhibited by b12 antigen-binding fragment (Fab). The x-axis shows the amount of b12 immunoglobulin G (IgG) and b12 Fab used in log μg/mL, and the y-axis shows the percentage inhibition relative to infection without any inhibitor. Each point represents an average of at least 3 independent experiments performed in triplicate and is the mean percentage inhibition, with whiskers denoting standard errors of the mean. Nonlinear regression was used to estimate a fitted curve in GraphPad Prism 5.

2.

Target cells were exposed to similar amounts of infectious virus when incubated with mature dendritic cell (mDC)–laden human immunodeficiency virus type 1 (HIV-1) vs cell-free stocks. The x-axis identifies the box plots for the relative light units (RLUs) 2 d after TZM-bl infections among the various HIV-1 isolates in mDC (

*unfilled box*) vs cell-free (*gray boxes*) infections. The black line in each box represents the median value, boxes show the interquartile range, and whiskers show the maximum and minimum values. Values were generated from a minimum of 10 independent experiments with at least 2 different virus stocks for each isolate.3.

Anti-gp41 broadly neutralizing antibody 4E10 accumulates at the mDC–T cell synaptic junction in a human immunodeficiency virus (HIV)–independent manner. Mature DCs were incubated with 4E10 (

*A*and*C*) or 2G12 (*B*and*D*) prior to coculture with autologous CD4^{+}T cells prestained with a blue fluorescent dye. Mature DC–T cell synaptic junctions were visualized by staining with anti-CD81 monoclonal antibodies 4E10 (*red*) and 2G12 (*red*) (*A*and*B*; middle panels). Alternatively, mature DCs preloaded with Gag-eGFP virus-like particles (*green*) were incubated with 4E10 (*C; red*) or 2G12 (*D*;*red*) and cocultured with autologous CD4^{+}T cells. Representative deconvolution images of mDC–T cell cocultures (×100 magnification) are shown. Merged bright field images for each of the stains are shown in the right panels.4.

Neutralization of mature dendritic cell (mDC)–mediated human immunodeficiency virus trans-infection by anti-gp120–directed broadly neutralizing antibodies is attenuated compared with cell-free virus infection. Mature DC–associated (

*filled squares*) and cell-free (*hollow squares*) neutralization is compared for Q23 (*A*and*F*), YU-2 (*B*and*G*), JRCSF (*C*and*H*), REJO (*D*and*I*), and CH077 (*E*and*J*). The x-axis shows the amount of VRC01 (*A*–*E*) and PG16 (*F*–*J*) used in log μg/mL, and the y-axis shows the percentage inhibition relative to infection without any antibody. Each point represents an average of at least 3 independent experiments performed in triplicate and is the mean percentage inhibition, with whiskers denoting standard errors of the mean. Nonlinear regression was used to estimate a fitted curve in GraphPad Prism 5.5.

Mature dendritic cell (mDC)–mediated transfer and cell-free infection are equally susceptible to anti-gp41–directed broadly neutralizing antibodies. Neutralization for Lai (

*A*and*E*), Lai/Balenv (*B*and*F*), JRCSF (*C*), NL4-3 (*D*and*H*), and 89.6 (*G*) is compared among mDC-associated virus (*filled squares*), cell-free virus (*hollow squares*), and mDC-associated virus preincubated with FcR blocking reagent (*filled circles*). The x-axis shows the amount of 2F5 (*A*–*D*) and 4E10 (*E*–*H*) used in log μg/mL, and the y-axis shows the percentage inhibition relative to infection without any antibody. Each point represents an average of at least 3 independent experiments performed in triplicate and is the mean percentage inhibition, with whiskers denoting standard errors of the mean. Nonlinear regression was used to estimate a fitted curve in GraphPad Prism 5.6.

Anti-gp41–specific broadly neutralizing antibodies (bNAbs) bind the surface of mature dendritic cells (mDCs) in the absence of human immunodeficiency virus type 1 (HIV-1) particles. Mature DCs were incubated with 4E10, 2F5, 2G12, b12, PG16, VRC01, or phosphate-buffered saline alone and were then washed and stained for human immunoglobulin G (IgG), using Alexa 594–conjugated secondary antibody and for nucleus using DAPI. Representative deconvolution merged bright field images (×100 magnification) of mDC staining observed with 4E10 (

*A*) and 2G12 (*B*) bNAbs is shown. Arrowheads indicate IgG binding to the mDC surface.*C*, The y-axis shows the percentage of mDCs bound by various bNAbs, shown on the x-axis. The black line in each box represents the median value; boxes show the interquartile range, and whiskers show the maximum and minimum values. The background signals were calculated as a mean plus 2 times the standard deviation of the signals from cells without bNAb. Cells expressing a signal above the background on the cell membrane were counted as positive for IgG binding. Graphs were derived from counting at least 10 images from each sample, with a minimum of 100 cells in 3 independent experiments with cells derived from 3 different donors.