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

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

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

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

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

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

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

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

Screening strategy for selecting capture agents against anti-HIV antibodies 3D6 and 4B3.

<p>The flow chart represents the use of the A21 and A22 cyclic peptides as anchor ligands for separate in situ click screens against a large OBOC azide-presenting peptide library.</p