Soluble HIV-1 Tat protein inhibits IL-7 induced Bcl-2 expression in CD4 T-cells.

<p>Purified CD4 T-cells from healthy HIV-negative volunteers were pre-incubated in medium alone or in medium containing Tat protein (10 µg/ml) for 48 hours. Cells were then stimulated with IL-7 (10 ng/ml) for an additional 72 hours and then fixed, stained for Bcl-2 expression and analyzed by flow cytometry. (A) Representative flow cytometry histogram showing Bcl-2 expression in untreated CD4 T-cells (grey fill), following IL-7 stimulation (black line), and following IL-7 stimulation of Tat pre-treated cells (grey line). (B, C) Bcl-2 expression as measured by mean channel fluorescence (MCF) in (B) CD4 T cells treated with IL-7 compared to medium controls, and (C) cells pre-incubated in medium or with Tat prior to IL-7 stimulation (n = 5).</p

Tat-induced CD127 down regulation is not due to CD4 T-cell activation.

<p>Purified CD4 T-cells from healthy HIV-negative volunteers (n = 4) were incubated in medium alone or with Tat protein (10 µg/ml) for up to 48 hours and then analyzed by flow cytometry. The changes in surface protein expression relative to medium controls are shown (p>0.19 for all proteins other than CD127 where p<0.04 at 48 hours). Graph represents mean values ± SEM. Representative flow histograms show CD4 T-cells from one individual incubated in media (grey fill) or media plus Tat (black line) at 48 hours.</p

Tat does not affect other CD4 T-cell surface proteins.

<p>Purified CD4 T-cells from healthy HIV-negative volunteers (n = 4) were incubated in medium alone or with Tat protein (10 µg/ml) for up to 48 hours and then analyzed by flow cytometry. (A) Changes in positive staining for the indicated surface proteins relative to medium controls (p>0.09 for all proteins other than CD127 where p<0.04). (B) Change in CD132+ CD4 T-cells relative to medium controls (p>0.12 at 48 hours). Graph represents mean values ± SEM. Representative flow histograms show CD4 T-cells from one individual incubated in media (grey fill) or media plus Tat (black line) at 48 hours.</p

Tat-induced CD127 down regulation is blocked by heparin and anti-Tat antibodies.

<p>Purified Tat protein (2 µg) was pre-incubated with 20 USP heparin or 20 µg anti-Tat polyclonal antibodies (equimolar ratio with Tat) for 30 minutes before being added to CD4 T-cells (n = 4). Representative flow cytometry histograms show CD127 surface expression on CD4 T-cells after 24 hours incubation with (A) 10 µg/ml Tat (grey line), Tat plus heparin (black line) or in medium alone (grey fill); and (B) 10 µg/ml Tat (grey line), Tat plus anti-Tat antibodies (black line) or in medium alone (grey fill). (C) Composite data (n = 5) showing CD127 expression relative to medium controls for Tat, Tat plus heparin, and Tat plus anti-Tat antibodies after 24 hours incubation. * denotes p value <0.05 compared to media. The relative changes are mean values ± SEM.</p

de novo protein synthesis is required for recovery of CD127 surface expression on CD4 T-cells following Tat-induced down regulation.

<p>Purified CD4 T-cells from healthy HIV-negative volunteers (n = 4) were incubated in medium alone or with Tat protein (10 µg/ml). (A) After 24 hours, cells incubated with Tat were washed twice and resuspended in fresh medium alone (Tat W 24) or in medium plus 100 µM CHX (Tat W 24 CHX). Cells were analyzed for CD127 surface expression by flow cytometry at the times indicated. (B) Cells were pre-incubated with and without proteasome inhibitors (5 µM MG132, 10 µM Lactacystin) and lysosome inhibitors (10 µM Leupeptin, 10 µg/ml Pepstatin) for 1 hour followed by incubation with 10 µg/ml Tat protein. After 24 hours, CD127 surface expression was measured by flow cytometry. The changes relative to medium controls are shown (mean values ± SEM). * denotes p value less than 0.05 compared to Tat treated control.</p

HIV-1 Tat protein down regulates CD127 surface expression on CD4 T-cells.

<p>Purified CD4 T-cells from healthy HIV-negative volunteers were incubated with Tat protein or in medium alone, stained for CD127 and analyzed by flow cytometry. (A) Percent change in CD127+ CD4 T-cells cultured with Tat (10 µg/ml) compared with media controls (n = 17; p<0.01). (B) Change in mean channel fluorescence relative to media controls for the same cultures (p<0.01). (C) Representative flow cytometry histograms from one individual showing progressive down regulation of CD127 surface expression at 48 hours with increasing amounts of Tat protein (black line) compared to medium (grey fill). (D) Composite data (n = 6; p<0.02) showing time and dose response to Tat in percent change in CD127+ CD4 T cells compared to media controls. (E) Purified CD4 and CD8 T-cells from the same healthy HIV-negative volunteers (n = 5) were incubated for up to 72 hours with HIV-1 Tat protein (10 µg/ml) or in medium alone. Percent changes in surface CD127 expression compared to medium controls are shown (p<0.01 for Tat-treated samples; p>0.5 between T-cell subsets). Graphs show mean values ± standard error of the mean (SEM).</p

CD127 surface expression recovers on CD4 T-cells after Tat is removed from the culture medium.

<p>Purified CD4 T-cells from healthy HIV-negative volunteers (n = 4) were incubated in medium alone or with Tat protein (10 µg/ml). At 24 (Tat W 24) and 48 (Tat W 48) hours the cells were washed twice in PBS and resuspended in fresh medium in the absence of Tat protein. Cells were analyzed for CD127 surface expression by flow cytometry at the times indicated. The changes relative to medium controls are shown (mean values ± SEM).</p

Tat protein does not affect the viability of purified CD4 T-cells.

<p>Purified CD4 T-cells from healthy HIV-negative volunteers (n = 4) were incubated in medium alone or with Tat protein (10 µg/ml) for up to 72 hours and then stained with propidium iodide or annexin V-FITC and analyzed by flow cytometry. Analyses were performed on the total ungated population as well as on only live cells based on forward and side scatter. (A) Propidium iodide exclusion showed no significant difference in viability between cells cultured in Tat or medium alone. (B) Apoptosis as indicated by positive annexin V staining averaged <10% in the gated population at 72 hours and was no different comparing cells cultured in medium alone or with Tat protein. Camptothecin (10 µM) induces apoptosis and was used as a positive control. * denotes p value <0.05 compared to media. Graphs show mean values ± SEM.</p

de novo protein synthesis is not required for Tat-induced down regulation of CD127.

<p>Purified CD4 T-cells from healthy HIV-negative volunteers were incubated in medium alone, with cyclohexamide (100 µM; CHX), Tat protein (10 µg/ml) or Tat plus CHX for 24 hours and then analyzed by flow cytometry. (A) Representative histogram showing CD127 surface expression on CD4 T-cells cultured in the presence of CHX alone (black line), Tat alone (dashed line) and CHX plus Tat (grey line) compared to medium control (grey fill). (B) Composite data from n = 4 demonstrating inhibition of protein synthesis with CHX does not prevent Tat-induced CD127 down regulation at the cell surface. CHX alone had no significant effect on CD127 expression (p = 0.07). There was no significant variance in surface CD127 expression between Tat and Tat + CHX treatments (p = 0.38) whereas both treatments showed significant down regulation of CD127 relative to medium (* denotes p<0.009). The percent changes relative to medium controls are shown (mean values ± SEM).</p

Pyrosequencing Dried Blood Spots Reveals Differences in HIV Drug Resistance between Treatment Naïve and Experienced Patients

<div><p>Dried blood spots (DBS) are an alternative specimen collection format for HIV-1 genotyping. DBS produce HIV genotyping results that are robust and equivalent to plasma when using conventional sequencing methods. However, using tagged, pooled pyrosequencing, we demonstrate that concordance between plasma and DBS is not absolute and varies according to viral load (VL), duration of HIV infection and antiretroviral therapy (ART) status. The plasma/DBS concordance is the highest when VL is ≥5,000 copies/ml and/or the patient has no ART exposure and/or when the duration of HIV infection is ≤2 years. Stepwise regression analysis revealed that VL is most important independent predictor for concordance of DBS with plasma genotypes. This is the first study to use next generation sequencing to identify discordance between DBS and plasma genotypes. Consideration should be given to VL, duration of infection, and ART exposure when interpreting DBS genotypes produced using next generation sequencing. These findings are of particular significance when DBS are to be used for clinical monitoring purposes.</p> </div