MOESM5 of The role of antigen presenting cells in the induction of HIV-1 latency in resting CD4+ T-cells

Additional file 5: Table S2. Comparison of gene expression between latency inducing and non-inducing antigen presenting cell subpopulations using microarray. Using the bioinformatics databases DAVID, GeneCards and GeneCodis, gene expression compartment and function was determined. Genes expressed on the antigen presenting cell (APC)-surface with the ability to signal to T-cells were shortlisted

High titres of R5-tropic HIV.EGFP reporter virus are needed to infect resting CD4+ T-cells.

<p>Resting CD4+ T-cells from 4 donors were labelled with eFluor670 proliferation dye and cultured without (<i>red</i>) or with autologous mDCs at 1 mDC:10 T-cell ratio (<i>blue</i>) for 24 hours. Cells were incubated with increasing TCID₅₀ per cell of HIV^{NL4.3(AD8)-EGFP} for 2 hours and cultured for 5 days in a low concentration of IL-2 (2 U/ml). 5 days post-infection, cultures were analysed for EGFP expression by flow cytometry to quantify productive infection (A). Non-proliferating eFluor670^hiEGFP- cells were also sorted and cultured for 3 days with anti-CD3/anti-CD28 plus IL-7 to induce EGFP expression from latent infection (B). The percentage of EGFP+ cells per 10⁴ cultured cells is shown. Columns represent the median of donors tested with each donor shown as a different symbol.</p

Latency is established with higher efficiency following co-culture of resting CD4+ T-cells with mDC.

<p>Resting CD4+ T-cells from 2 donors were labelled with eFluor670 cytoplasmic dye and cultured for 24 hours either untreated (<i>red</i>), with 100 nM CCL19 (<i>purple</i>) or with autologous mDCs at 1 mDC:10 T-cell ratio (<i>blue</i>). Cells were incubated with increasing TCID₅₀ per cell of X4-tropic HIV^NL4.3-EGFP. After 2 hours, cells were washed, cultured for 5 days in a low concentration of IL-2 (2 U/ml) and analysed for EGFP expression by flow cytometry to quantify productive infection (A). Additionally, non-proliferating eFluor670^hiEGFP- cells were sorted, cultured for 3 days with anti-CD3/anti-CD28 plus IL-7, in the presence of the integrase inhibitor L-870812 (L8), to induce EGFP expression from post-integration latent infection (B). As a comparative control, aliquots of sorted cells were also cultured for 3 days with L-870812 (L8) but no reactivation stimuli in order to measure background, spontaneous EGFP expression during 3 further days of culture (C). The true level of post-integrated latency in cultures was calculated by subtracting the percentage of EGFP+ cells in the spontaneous cultures from the percentage of EGFP+ cells in reactivated cultures (D). The percentage of EGFP+ cells per 10⁴ cultured cells is shown. Columns represent the median of donor pairs with each donor shown as a different symbol.</p

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