A representative flow diagram of the suppression assay.

<p>A representative flow diagram of the suppression assay.</p

CD69 expression is upregulated following treatment with PKC-agonists.

<p>PBMCs from 8 HDs were treated with LRAs for 6 h, washed, then cultured for up to three days and examined for CD69 expression on CD8 + T cells. Mean expression ± standard error is indicated for each treatment at (A) 6 hours, (B) day 1, (C) day 2 and (D) day 3. Symbols directly above treatments indicate differences from the DMSO control. * p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Cells were either non treated (NT) or treated with DMSO (0.1%), bryostatin-1 at 10 or 1 nM (BRYO 10 and BRYO 1 respectively), prostratin at 0.3 uM (PRO), ingenol-B at 100nM (ING), PMA at 50ng/mL (PMA) and JQ1 at 1uM (JQ1). In some experiments the PKC-agonists were combined with JQ1 at the same concentrations (BRYO 10/JQ1, BRYO 1/JQ1, PRO/JQ1, ING/JQ1).</p

The effect of Ingenol-B on the suppressive capacity of elite suppressor HIV-specific CD8+ T cells

<div><p>Background</p><p>Some latency-reversing agents (LRAs) inhibit HIV-specific CD8+ T cell responses. In a prior study of protein kinase C (PKC) agonists, we found that bryostatin-1 inhibited elite controller/suppressor (ES) CD8+ T cell suppressive activity whereas prostratin had no effect. Ingenol-B is another PKC agonist with potent LRA activity both by itself and in combination with the bromodomain inhibitor JQ1; however its effect on CD8+ T cell mediated control of HIV-1 replication is unknown.</p><p>Methods</p><p>CD8+ T cells were isolated from ES and treated with bryostatin-1, prostratin, ingenol-B, and JQ1 as well as a combination of each PKC-agonist with JQ1. The cells were then tested in the viral suppression assay. To assess possible mechanisms of inhibition, CD8+ T cells were treated with the LRAs and analyzed for the expression of various immune cell markers.</p><p>Results</p><p>Ingenol-B had no effect on the ability of ES CD8+ T cells to suppress viral replication, however, the combination of ingenol-B and JQ1 caused a modest, but significant decrease in this suppressive capacity. The mechanism of the inhibitory effect of the JQ1 and ingenol-B combination relative to ingenol-B alone was unclear but the effect appeared to be dose dependent.</p><p>Conclusions</p><p>Ingenol-B does not inhibit HIV-specific CD8+ T cell responses in vitro. These responses are however modestly inhibited when 100 nMingenol-B is combined with JQ1. Since HIV-specific CD8+ T cell activity may be essential for the eradication of reactivated latently infected cells, the potency of latency-reversal activity of drug combinations must be balanced against the effects of the combinations on HIV-specific CD8+ T cell responses.</p></div

Surface CD8 expression is modestly downregulated by some LRAs.

<p>PBMCs from 8 HDs were treated with LRAs for 6 h before being washed and then cultured for up to three days and examined for CD8 expression. Mean expression ± standard error is indicated for each treatment at (A) 6 hours, (B) day 1, (C) day 2 and (D) day 3. Symbols directly above treatments indicate differences from the DMSO control. * p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Cells were either non treated (NT) or treated with DMSO (0.1%), bryostatin-1 at 10 or 1 nM (BRYO 10 and BRYO 1 respectively), prostratin at 0.3 uM (PRO), ingenol-B at 100nM (ING), PMA at 50ng/mL (PMA) and JQ1 at 1uM (JQ1). In some experiments the PKC-agonists were combined with JQ1 at the same concentrations (BRYO 10/JQ1, BRYO 1/JQ1, PRO/JQ1, ING/JQ1).</p

Elite suppressor CD8 + T cell responses are not inhibited by 10nMingenol-B in combination with JQ1.

<p>CD8 + T cells from 5ES were pre-incubated with 10nMingenol-B and 1uM JQ1 for six hours, washed, then added to autologous CD4 + T cells infected with a lab strain HIV-1 pseudovirus at a 1:1 effector:target ratio. Percent suppression of viral replication was determined after 3 days. Triplicates were performed and mean values are shown for each individual.</p

Surface CD3 expression is downregulated following treatment with PKC-agonists.

<p>PBMCs from 8 HDs were treated with LRAs for 6 h before being washed and then cultured for up to three days and examined for CD3 expression. Mean expression ± standard error is indicated for each treatment at (A) 6 hours, (B) day 1, (C) day 2 and (D) day 3. Symbols directly above treatments indicate differences from the DMSO control. * p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Cells were either non treated (NT) or treated with DMSO (0.1%), bryostatin-1 at 10 or 1 nM (BRYO 10 and BRYO 1 respectively), prostratin at 0.3 uM (PRO), ingenol-B at 100nM (ING), PMA at 50ng/mL (PMA) and JQ1 at 1uM (JQ1). In some experiments the PKC-agonists were combined with JQ1 at the same concentrations (BRYO 10/JQ1, BRYO 1/JQ1, PRO/JQ1, ING/JQ1).</p

Elite suppressor CD8 + T cell responses are not inhibited by ingenol-B.

<p>CD8 + T cells from 5ES were either not treated (NT) or pre-incubated with DMSO oringenol-B at 3 different doses for six hours, washed, then added to autologous CD4 + T cells infected with a lab strain HIV-1 pseudovirus at a 1:1 effector:target ratio. Percent suppression of viral replication was determined after 3 days. Triplicates were performed and mean values are shown for each individual.</p

Ingenol-B and ingenol-B/JQ1 combination causes modest levels of cell death in CD8+ T cells.

<p>PBMCs from eight HDs were isolated and treated with LRAs for 6 h, washed, then cultured for up to three days and examined for cell death as measured by percent annexin V expression. Mean expression ± standard error is indicated for each treatment at (A) 6 hours, (B) day 1, (C) day 2 and (D) day 3. Symbols directly above treatments indicate differences from the DMSO control. * p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Cells were either non treated (NT) or treated with DMSO (0.1%), bryostatin-1 at 10 or 1 nM (BRYO 10 and BRYO 1 respectively), prostratin at 0.3 uM (PRO), ingenol-B at 100nM (ING), PMA at 50ng/mL (PMA) and JQ1 at 1uM (JQ1). In some experiments the PKC-agonists were combined with JQ1 at the same concentrations (BRYO 10/JQ1, BRYO 1/JQ1, PRO/JQ1, ING/JQ1).</p

Clinical characteristics of ESused in the study.

<p>Clinical characteristics of ESused in the study.</p

Silencing of TLR2 expression attenuates the increased susceptibility to HIV.

<p>Expression of TLR2 increases at the transcription level in cells stimulated with <i>M. bovis</i> BCG when compared to cells stimulated with <i>M. smegmatis</i> (A). The expression levels of TLR4 and TLR9 do not show a significant difference in cells stimulated with different strains of mycobacteria as evidenced by RT-PCR. Comparison of HIV infectivity of CD4+ cells transfected with scrambled siRNA and subsequently stimulated with <i>M. bovis</i> BCG to CD4+ cells transfected with TLR2 siRNA and subsequently stimulated with <i>M. bovis</i> BCG (B) **p<0.005. TLR2 expression on CD4+ cells after stimulation with <i>M. bovis</i> BCG, <i>M. tuberculosis</i>, and <i>M. smegmatis</i> compared to TLR2 expression on CD4+ cells transfected with TLR2 siRNA and subsequently stimulated with <i>M. bovis</i> BCG (C). The expression showed similar trends for the four samples tested. Dot plots comparing the percentage of surface expression of TLR2 on unstimulated CD4+ cells and CD4+ cells stimulated with <i>M. bovis</i> BCG, <i>M. tuberculosis</i> (CDC1551), <i>M. smegmatis</i> (MC²155), and CD4+ cells transfected with TLR2 siRNA and subsequently stimulated with <i>M. bovis</i> BCG (D).</p