Changes in gene expression in DC-induced latently infected CD4⁺ T cells.

<p>(<b>A</b>) Fold change scatterplot comparing gene expression between HIV T (+DC) (CD4⁺ T cells cultured with DC and HIV), and Mock T (+DC) (CD4⁺ T cells cultured with only DC) relative to their controls, HIV T (CD4⁺ T cells cultured with HIV) and Mock T (CD4⁺ T cells cultured in media alone) respectively. The solid line indicates absolute 2-fold change. (<b>B</b> and <b>C</b>) Top two gene interaction networks as ranked by Ingenuity Pathway Analysis. The networks were built from the list of differentially expressed genes induced by HIV T (+DC), relative to Mock T (+DC) after subtracting HIV T and Mock T from each group respectively. Genes highlighted in red were up-regulated and those in blue were down-regulated. The different node shapes indicate genes in different functional categories according to the legend. The interactions between the different nodes are shown as solid (direct interaction) or dashed (indirect interaction) lines (edges). (<b>D</b>) Fold change in gene expression values for selected genes from the Illumina BeadArrays plotted against Real-Time PCR (qPCR) deltaCt values for each target gene. PCR targets were mapped to BeadArray probes by matching the official gene symbols.</p

Soluble factors in DC-induced HIV latency.

<p>(<b>A</b>) Resting CD4⁺ T cells were cultured alone (light grey) or with sorted pDC (grey) or mDC (dark grey). At day 5 post-infection, cytokines and chemokines were quantified in culture supernatants using cytometric bead arrays, n = 3. *<i>P</i>&lt;0.05; **<i>P</i>&lt;0.01 (paired t-test). (<b>B</b>) Latent infection was quantified in eFluor670^hiEGFP⁻ resting CD4⁺ T cells that were cultured either alone or with sorted pDC in the presence of media alone (light grey), anti-IgG (grey) or anti-IFN-alpha (dark grey) following stimulation with anti-CD3/CD28 in the presence of L8, n = 5. (<b>C</b>) Resting CD4⁺ T cells were co-cultured with mDC with (grey) or without (light grey) the addition of equal numbers of pDC. Productive infection was determined at day 5 post-infection. Latent infection was determined in sorted eFluor670^hiEGFP⁻ CD4⁺ T cells following stimulation with anti-CD3/CD28 in the presence of L8, n = 5. (<b>D</b>) Latent infection was quantified in eFluor670^hiEGFP⁻ resting memory CD4⁺ T cells that were cultured either alone or with sorted mDC in the presence of media alone (light grey), anti-IgG (grey) or neutralising antibodies (dark grey) to IL-6, IL-10-receptor, CXCR3 or CCL19, n = 5. (<b>E</b>) eFluor670-labelled resting CD4⁺ T cells were cultured either alone (light grey) or with blood mDC (dark grey). Virus was added to (<b>i</b>) CD4⁺ T cells cultured alone; (<b>ii</b>) CD4⁺ T cells co-cultured with mDC; (<b>iii</b>) CD4⁺ T cells cultured with mDC in the presence of a 0.4 µm membrane transwell and latency determined at day 5 post-infection, n = 5. (<b>F</b>) eFluor670-labelled resting CD4⁺ T cells were cultured either (<b>i</b>) alone (light grey) or (<b>ii</b>) with blood mDC (dark grey) and infected. (<b>iii</b>) Following 24 hours, supernatant from infected mDC-T cell co-cultures was added to uninfected resting CD4⁺ T cells and these cells were then infected, n = 3. Columns represent the median of 3–5 donors and error bars indicate the interquartile range. *<i>P</i>&lt;0.05; **<i>P</i>&lt;0.01 (Wilcoxon signed-rank test).</p

Myeloid DC induce post-integration latency in non-proliferating memory CD4⁺ T cells.

<p>SNARF-labelled resting CD4⁺ T cells were cultured alone (light grey) or with syngeneic plasmacytoid (pDC; grey) or myeloid DC (mDC; dark grey). (<b>A</b>) Productive infection (EGFP⁺ cells) was determined by flow cytometry on day 5 post-infection. (<b>B</b>) Latent infection was quantified in SNARF^hiEGFP⁻ cells following either addition of PHA-activated PBMC, n = 5; or (<b>C</b>) direct activation with anti-CD3/CD28 in the presence or absence of the integrase inhibitor L8. (<b>D</b>) Integrated HIV DNA was quantified in the sorted SNARF^hiEGFP⁻ T cells by Alu-LTR real-time PCR, n = 3. (<b>E</b>) Productive and latent infection was determined in SNARF^hiEGFP⁻ CD4⁺ T cells from mDC-T cell co-cultures following infection with nef-deficient (-nef) or nef-competent EGFP HIV. (<b>F</b>) Latent infection was determined in sorted SNARF^hiEGFP^- CD4⁺ T cells, cultured alone or with mDC, following activation with PHA-PBMC. (<b>G</b>) Productive and latent infection was determined in SNARF^hiEGFP⁻ CD4⁺ T cells from mDC-T cell co-cultures with and without Staphylococcus Enterotoxin B (SEB), n = 4. (<b>H</b>) SNARF-labelled resting CD4⁺ T cells were cultured alone (light grey) or with syngeneic mDC (grey) at decreasing DC∶T cell ratios and latent infection quantified in sorted SNARF^hiEGFP⁻ T cells following addition of PHA-activated PBMC, n = 5. (<b>I</b>) Resting CD4⁺ T cells were cultured either alone or in the presence of mDC. At day 5 post-infection, SNARF^hiEGFP⁻ cells were sorted into naïve (light grey) or memory (grey) CD4⁺ T cells and latent infection quantified, n = 5. The lower limit of detection is represented by a dotted line. Columns represent the median of 3–7 donors and error bars indicate the interquartile range. *<i>P</i>&lt;0.05; **<i>P</i>&lt;0.01; ns, not significant (Wilcoxon signed-rank test).</p

DC-induced latency in resting CD4⁺ T cells.

<p>(<b>A</b>) Resting CD4⁺ T cells were isolated from the blood of healthy donors and labelled with the proliferation dye SNARF, which decreases in intensity following each round of cell division allowing identification of non-proliferating cells. SNARF-labelled resting CD4⁺ T cells were cultured either alone or with syngeneic blood DC. Following 24 hours of culture, cells were infected with NL(AD8)-nef/EGFP at an MOI of 0.5. All culture media was supplemented with IL-2 (2 U/mL). (<b>B</b>) At day 5 post-infection, the number of productively infected (EGFP⁺) cells was determined and the non-proliferating (SNARF^hi) cells that were not productively infected (EGFP⁻) were sorted. The sorted SNARF^hiEGFP⁻ cells were stimulated with PHA/IL-2 in the presence of PBMC and cultured for 5 days to amplify any replication competent latent infection. (<b>C</b>) Productive infection and (<b>D</b>) latent infection following infection of T cells cultured alone (light grey) or in the presence of DC (grey) is shown. (<b>E</b>) Latent infection in the presence of DC cultured with (grey) or without (light grey) 0.1 µM Indinavir. (<b>F</b>) Expression of the early (CD69; black) and late (HLA-DR; grey) surface activation markers and the intracellular proliferation marker Ki67 (light grey) was quantified by flow cytometry on sorted SNARF^hiEGFP⁺ CD4⁺ T cells following HIV infection of T cells cultured alone or in the presence of DC. The lower limit of detection of each assay is represented by a dotted line. Columns represent the median of 5 independent experiments and error bars indicate the interquartile range. *<i>P</i>&lt;0.05 (Wilcoxon signed-rank test).</p

MOESM4 of HIV integration and the establishment of latency in CCL19-treated resting CD4+ T cells require activation of NF-ÎşB

Additional file 4: Figure S4. Integration site selection and gene activation in chemokine treated cells. A, Gene expression was determined by Illumina bead array in unactivated, CCL19-treated or PHA-IL2 activated CD4+ T cells after 6 or 72 h. The ratio of expression of genes at the sites of integration was determined in each in vitro condition. B, Expression of individual genes at the site of HIV integration in CCL19-treated resting CD4+ T cells (x-axis) compared to unactivated (y-axis; upper panel) or PHA-IL2 activated CD4+ T cells (y-axis; lower panel). C, The distance of integration sites to specific genomic elements including LINE, H4K20me3 and H4R3me following HIV infection of unactivated, CCL19-treated and PHA-IL2 activated CD4+ T cells, or CD4+ T cells from HIV-infected patients on cART or randomly selected sites. Log distance is shown as box plots (median and quartiles) with violin plot of the kernel distribution. The means are shown as a red horizontal line

Graphic summary of the ability of each compound to activate HIV within each cell model: (A) primary CD4 T cell models and patient cell outgrowth assay (QVOA), and (B) J-Lat T cell line clones.

<p>Each compound and concentration tested is listed on the X-axis. In the primary CD4 cell models, each compound was tested using cells from 2, 3 or 4 different donors and in duplicate or triplicate with cells from each donor (See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003834#s4" target="_blank">Methods</a> Section for details). For the QVOA, results from the limiting dilution cultures from 3 patients were pooled to calculate one common IUPM (infectious units per million cells) value which was then normalized to that obtained with PHA. With the J-Lat clones, experiments were performed in triplicate. Asterisks represent “not done”.</p

List of stimuli used in this study and their corresponding signaling pathways.

<p>List of stimuli used in this study and their corresponding signaling pathways.</p

Heatmap visualization of the ability of each compound to activate HIV within each model when excluding (A) and including (B) data from the QVOA model.

<p>A reduced set of compounds was analyzed in (<b>B</b>) since not every compound was run at every concentration in the QVOA. The clustergram at the left of each heatmap reflects the relationships between compounds based on their ability to activate HIV across compounds. Since cells in all models responded to PHA with high strength, ranking was normalized within each model to the response to PHA at 10 µg/mL and, therefore, all models display in the heatmap the same relative responsiveness to this treatment. The clustergram at the top of each heatmap reflects the relationship between each model based on their response to compounds. Clustergrams were created by calculating Euclidean distances and then clustering distances using the average linkage method. The numbers at the nodes of clusters are AU p-values where 95% represents a <i>p</i>-value cut-off of 0.05 and only values 95% or greater are depicted. Red cells in the heatmaps reflect HIV activation whereas blue or blank cells indicate that the compound did not effectively activate HIV.</p