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

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