moDC-to-PBMC infection is insensitive to TFV.

<p>(<b>A</b>) FACS plots of DC-free T cell or moDC-to-PBMC infection in the absence of presence of 5 μM of TFV. (<b>B</b>) Drug insensitivity measured by transmission index (T_x) of DC-free culture or moDC-T cell coculture. TFV at 0 <b>μ</b>M, 5 μM, 10 μM (wedges). (<b>C</b>) Fold difference between the T_x values of moDC-PBMC and DC-free PBMC infections. Each symbol represents one donor. Mean ± s.e.m (η = 3 donors). Data is representative of two independent experiments (<b>A</b>-<b>C</b>).</p

HIV transmission between moDCs and PBMCs or isolated CD4⁺ T cells is efficient and insensitive to RAL.

<p>(<b>A, D</b>) The moDCs were cocultured with autologous PBMCs (A) or CD4⁺ T cells (D) at ratios of 1:1, 1:8, and 1:32. The cultures were infected with NL4-3. The frequency of infected p24⁺ T cells was measured using flow cytometry. (<b>B, E</b>) The moDC-PBMC coculture (B) or moDC-CD4⁺ T cell coculture (E) or DC-free cultures were infected with NL4-3 in the presence or absence of 10 μM of TFV or 10 μM of RAL and drug insensitivity (T_x) was measured. (<b>C, F</b>) Fold differences between the T_x values of moDC-PBMC coculture (C) or moDC-CD4⁺ T cell coculture (F) and DC-free culture were measured. Mean ± s.e.m (η = 3 technical replicates). *, p <0.05; **, p <0.01, ***, p<0.005 (student’s T-test). n.s., p >0.05. Data is representative of one donor from two independent experiments (<b>A</b>-<b>F</b>).</p

Dendritic cells efficiently transmit HIV to T Cells in a tenofovir and raltegravir insensitive manner

<div><p>Dendritic cell (DC)-to-T cell transmission is an example of infection in <i>trans</i>, in which the cell transmitting the virus is itself uninfected. During this mode of DC-to-T cell transmission, uninfected DCs concentrate infectious virions, contact T cells and transmit these virions to target cells. Here, we investigated the efficiency of DC-to-T cell transmission on the number of cells infected and the sensitivity of this type of transmission to the antiretroviral drugs tenofovir (TFV) and raltegravir (RAL). We observed activated monocyte-derived and myeloid DCs amplified T cell infection, which resulted in drug insensitivity. This drug insensitivity was dependent on cell-to-cell contact and ratio of DCs to T cells in coculture. DC-mediated amplification of HIV-1 infection was efficient regardless of virus tropism or origin. The DC-to-T cell transmission of the T/F strain CH077.t/2627 was relatively insensitive to TFV compared to DC-free T cell infection. The input of virus modulated the drug sensitivity of DC-to-T cell infection, but not T cell infection by cell-free virus. At high viral inputs, DC-to-T cell transmission reduced the sensitivity of infection to TFV. Transmission of HIV by DCs in trans may have important implications for viral persistence <i>in vivo</i> in environments, where residual replication may persist in the face of antiretroviral therapy.</p></div

moDC-to-PBMC drug resistance depends on physical contact between cells.

<p>(<b>A</b>) FACS plots of DC-free PBMC and moDC-to-PBMC infection with (<i>bottom</i>) or without (<i>top</i>) a transwell system, in which DCs are physically separated from PBMCs by a transwell membrane. Infection occurs in the absence of presence of 10 μM of TFV. (<b>B</b>) Drug insensitivity (T_x) of DC-free or moDC-to-PBMC infection with or without 10 μM of TFV and in the absence of presence of a transwell system. Mean ± s.e.m (η = 3 donors). *, p <0.05 (student’s T-test). n.s., p >0.05. Each symbol represents a donor. Data is representative of two (<b>A</b>, <b>B</b>) independent experiments.</p

moDCs significantly amplify PBMC infection.

<p>(<b>A</b>, <b>B</b>) moDCs were treated with LPS and analyzed by flow cytometry. FACS histogram plots showing expression of DC-SIGN and CD14. Number in the top left gate indicates percentage of CD14^-DC-SIGN⁺cells (<b>A</b>). FACS histogram plots showing surface expression of activation markers CD86 and HLA-DR (<b>B</b>). (<b>C</b>) Infection of PBMCs without (<i>left</i>) or with DCs (<i>center</i>) was analyzed by flow cytometry. Uninfected moDC-PBMC coculture (<i>right</i>) was included as a negative control. Number in the top right gate indicates percentage of infected T cells. (<b>D</b>) Frequency of infected p24⁺ T cells or moDCs was measured in DC-free culture or moDC-PBMC cell coculture. Each symbol represents one donor. Mean ± s.e.m (η = 6 donors). ***, p <0.0001 (student’s T-test). Data is representative of two independent (<b>A</b>, <b>B</b>), six independent (<b>C</b>) or pooled from six independent experiments (<b>D</b>).</p

moDC-to-PBMC drug resistance depends on physical contact between cells.

<p>(<b>A</b>) FACS plots of DC-free PBMC and moDC-to-PBMC infection with (<i>bottom</i>) or without (<i>top</i>) a transwell system, in which DCs are physically separated from PBMCs by a transwell membrane. Infection occurs in the absence of presence of 10 μM of TFV. (<b>B</b>) Drug insensitivity (T_x) of DC-free or moDC-to-PBMC infection with or without 10 μM of TFV and in the absence of presence of a transwell system. Mean ± s.e.m (η = 3 donors). *, p <0.05 (student’s T-test). n.s., p >0.05. Each symbol represents a donor. Data is representative of two (<b>A</b>, <b>B</b>) independent experiments.</p

HIV transmission between primary myeloid DCs and CD4⁺ T cells is capable of drug-insensitivity.

<p>Primary myeloid DCs (mDCs) were cocultured with autologous CD4⁺ T cells at ratios of 1:4 and 1:8. (<b>A</b>) The mDC-CD4⁺ T cell cocultures and DC-free CD4⁺ T cell cultures were infected with NL4-3 and the frequency of infected p24⁺ T cells was measured using flow cytometry. (<b>B</b>) The mDC-CD4⁺ T cell cocultures and DC-free CD4⁺ T cell cultures were infected with NL4-3 in the presence or absence of 10 μM of TFV and drug insensitivity (T_x) was measured. (<b>C</b>) Fold difference between the T_x values of mDC-CD4⁺ T cell coculture and DC-free culture were measured. Mean ± s.e.m (η = 3 technical replicates). *, p <0.05 (student’s T-test); n.s., p >0.05. Data is representative of one donor from two independent experiments (<b>A</b>-<b>C</b>).</p

DC amplification of T cell infection with different HIV-1 isolates.

<p>(<b>A</b>) Infection of PBMCs with or without moDCs by CXCR4-tropic, dual-tropic, CCR5-tropic and T/F isolates, which were normalized to similar amounts of p24, were analyzed by flow cytometry. Frequency of p24⁺ T cells was measured by flow cytometry. (<b>B, C</b>) PBMCs with or without moDCs were infected with or without 10 μM of TFV and drug insensitivity (T_x) (B) and fold difference between the T_x values were measured (C). Mean ± s.e.m (η = 2 donors). Data is representative of three independent experiments (<b>A-C</b>).</p

TFV insensitivity of moDC-to-PBMC transmission is dependent on infectious dose of NL4-3.

<p>(<b>A</b>) PBMCs with or without moDCs were infected with varying doses of NL4-3. Frequency of p24⁺ T cells was measured by flow cytometry. (<b>B, C</b>) PBMCs with or without moDCs were infected with varying doses of NL4-3 in the presence or absence of 10 μM of TFV and drug insensitivity (T_x) (B) and fold difference between the T_x values were measured (C). Mean ± s.e.m (η = 2 donors). Data is representative of two independent experiments (<b>A-C</b>).</p

Schematic overview of dynamic proteomics for exploring cell cycle dependent changes in level and localization.

<p>(A) CD tagging was used to insert YFP as an exon into the introns of genes on the chromosome of a human cell line clone (H1299), resulting in a full length protein fused to YFP expressed from its endogenous locus. (B) A panel of 6 representative clones with different tagged proteins from the LARC library (C) Time-lapse microscopy and automated image analysis allow capturing proteins levels and localizations in individual cells over time. Yellow arrow indicates a cell in mitosis, green arrow indicates cells post mitosis. (D) Fluorescence traces of individual cells over a 40 hours movie (tagged protein is DDX5). Sharp decreases are at division events (E) In silico synchronization is done by plotting cell dynamics on a time axis which indicates time from previous or next division. Time is divided by mean cell cycle duration, to provide fraction of cell cycle elapsed. G, S and G2 phases are estimated from Sigal <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048722#pone.0048722-Sigal2" target="_blank">[15]</a>. Grey lines- cells with two mitosis events in the movie, blue, red lines: cells with one mitotic event. The fluorescence level is normalized to the maximal level before cell division. (F) In silico synchronized dynamics are used to examine cell-cycle dependence on levels and localizations. On the top panel, Protein profile (blue) that is significantly different from the average profile (black) is considered cell cycle dependent. On the bottom panel : nuclear protein shows a nuclear ratio (nuc/total) profile close to 1 most of the cell cycle, while cytoplasmic protein, shows a nuclear ratio close to 0 most of the cell cycle (besides during the mitosis, where the nucleus and cytoplasm are hard to segment apart). Protein that change its localization from the cytoplasm to the nucleus in a cell cycle dependent manner, present a nuclear ratio that is variable across the cell cycle.</p