[Lola] Albright... [Oleg] Vidov

Additional file 3: Figure 2. Ingenols do not induce pro-inflammatory cytokine release in purified resting CD4+ T cell cultures. Concentrations of pro-inflammatory cytokines TNFα, IFNγ, IL-1β and IL-6 were not significantly elevated in the presence of ingenol-3,20-dibenzoate (ingenol DB) or ingenol B compared to media alone control at 72 h in purified resting CD4+ T cell cultures. Mean values and standard deviation of seven independent experiments using resting CD4+ T cells from aviremic ART-treated HIV positive participants are shown. Pro-inflammatory cytokine concentrations were significantly elevated in positive control cultures (T cell receptor stimulation via CD3 and CD28 antibodies). ** P value <0.01; *** P value <0.001

Reactivation of HIV by H37Ra, PIM6, and H37Rv lysate in a primary T_CM model of latency.

<p>Cultured T_CM cells following 72-hour incubation with test conditions or co-stimulation with αCD3/αCD28. (A) Levels of intracellular p24 Gag were measured by flow cytometry. The horizontal line within the box represents the median, the boundaries of the box represent the 25^th- and 75^th-percentile, and the whiskers represent the maximum and minimum values. Significance for intracellular p24 Gag was determined using a 2-tailed, paired Student’s t-test versus PBS (*p<0.05). (B) Relative luminescence was measured from supernatant of cultured T_CM cells following 72-hour incubation with conditions or co-stimulation with αCD3/αCD28. The horizontal line within the box represents the median, the boundaries of the box represent the 25^th- and 75^th-percentile, and the whiskers represent the maximum and minimum values. Significance was determined using a 2-tailed, paired Student’s t-test versus PBS (*p<0.05). Significance of individual test conditions are as follows: αCD3/αCD28 (p≤0.01), PIM6 (p<0.05), H37Ra (p≤0.01), and <i>M</i>. <i>smegmatis</i> (p≤0.01).</p

PIM6 and H37Rv lysate induce GFP expression through TLR-2.

<p>A) JLAT cells were incubated for 16 hours and GFP expression was measured by flow cytometry. Data represent mean ± SD of four independent experiments run in triplicate. *p<0.05 compared to PBS control. B) JLAT-TLR2 cells were incubated for 16 hours and GFP expression was measured by flow cytometry. Data represent mean ± SD of ten independent experiments run in triplicate. *p<0.05 compared to PBS control. <b>C</b>) JLAT-TLR2 cells were pre-incubated with the TLR-2 neutralizing antibody, PAb-hTLR2, for 30 minutes prior to addition of test conditions. Cells were subsequently incubated for 16 hours and GFP expression was measured by flow cytometry. Data represent mean ± SD. *p<0.05 test condition in the absence of TLR-2 neutralizing antibody compared to test condition in the presence of TLR-2 neutralizing antibody. D) JLAT-TLR2 cells pre-incubated with BAY 11–7082 for 30 minutes prior to addition of conditions. Cells were subsequently incubated for 16 hours and GFP expression was measured by flow cytometry. Data represent mean ± SD. *p<0.05 test condition in the absence of BAY 11–7082 compared to test condition in the presence of BAY 11–7082.</p

Transcriptomic Analysis Implicates the p53 Signaling Pathway in the Establishment of HIV-1 Latency in Central Memory CD4 T Cells in an In Vitro Model

<div><p>The search for an HIV-1 cure has been greatly hindered by the presence of a viral reservoir that persists despite antiretroviral therapy (ART). Studies of HIV-1 latency <i>in vivo</i> are also complicated by the low proportion of latently infected cells in HIV-1 infected individuals. A number of models of HIV-1 latency have been developed to examine the signaling pathways and viral determinants of latency and reactivation. A primary cell model of HIV-1 latency, which incorporates the generation of primary central memory CD4 T cells (T_CM), full-length virus infection (HIV_NL4-3) and ART to suppress virus replication, was used to investigate the establishment of HIV latency using RNA-Seq. Initially, an investigation of host and viral gene expression in the resting and activated states of this model indicated that the resting condition was reflective of a latent state. Then, a comparison of the host transcriptome between the uninfected and latently infected conditions of this model identified 826 differentially expressed genes, many of which were related to p53 signaling. Inhibition of the transcriptional activity of p53 by pifithrin-α during HIV-1 infection reduced the ability of HIV-1 to be reactivated from its latent state by an unknown mechanism. In conclusion, this model may be used to screen latency reversing agents utilized in shock and kill approaches to cure HIV, to search for cellular markers of latency, and to understand the mechanisms by which HIV-1 establishes latency.</p></div

Effect of pifithrin-α upon establishment of latency.

<p>(A) Time-line of experimental procedures for pifithrin-α treatment of the latently infected (LI) condition. For the majority of experiments pifithrin-α was maintained in culture from day 10 to 17. (B) CD4 and p24 Gag staining from a representative donor throughout the experiment. Numbers in dot-plots represent percentages. (C) p24 Gag positive CD4 negative cells from 5 independent donors at day 13 and day 17 in cells treated (purple symbols) or untreated (black symbols) with pifithrin-α. (D) p24 Gag positive CD4 negative cells at day 19 following reactivation of LI cells with αCD3/αCD28 beads at day 17 in cells previously treated (purple symbols) or untreated (black symbols) with pifithrin-α (day 10 to 17). As a negative control, p24 Gag positive CD4 negative cells were also assessed in unstimulated cells treated with pifithrin-α in a similar manner. (E) In a deviation from the time-line presented in (A), pifithrin-α was added only during the reactivation period from day 17 to 19. p24 positive CD4 negative cells were then determined at day 19 following reactivation of LI cells that were treated (salmon symbols) or untreated (black symbols) with pifithrin-α. As a negative control, p24 Gag positive CD4 negative cells were also assessed in unstimulated cells treated with pifithrin-α in a similar manner. (F) HIV-1 integration analysis in LI cells at day 17 using Alu-PCR with 250 ng of genomic DNA. As a negative control, HIV-1 integration was also assessed in uninfected cells. (G) Correlation of integrated HIV-1 with the percentage of p24 Gag positive CD4 negative cells after reactivation with αCD3/αCD28 beads. Correlation was determined using the Pearson correlation coefficient. Black symbols represent untreated samples and purple symbols represent cells treated with pifithrin-α from day 10 to 17. (H) Reactivation index calculated as the percentage of cells expressing p24 (day 19) after αCD3/αCD28 reactivation divided by the number of integrated copies before reactivation (day 17). Throughout this figure each donor is represented with a different symbol and significance was determined with a paired <i>t</i>-test (<i>p</i>-values provided).</p

Markers of CD4 T cell activation are modulated following αCD3/αCD28 stimulation.

<p>The fold change of a panel of host gene markers of CD4 T cell activation was assessed pre- and post-stimulation with αCD3/αCD28 beads by (A) RNA-Seq and (B) RT-qPCR. Fold changes were calculated for both uninfected (white bars) and latently infected (black bars) cells. Fold changes for all genes assessed by RNA-Seq were significant (FDR corrected <i>p</i>-value <0.05). Fold changes for <i>IL2</i> and <i>KLF2</i> assessed by RT-qPCR were significant in a paired <i>t</i>-test at the following levels: **<i>p</i><0.01 ***<i>p</i><0.001. Fold change is plotted on the log₂ scale with error bars representing standard deviation. The horizontal dotted line in each figure indicates a log₂ fold change cut off of 1 (i.e., actual fold change cut off of 2). Abbreviations are as follows: <i>UI</i>, uninfected; <i>UIA</i>, uninfected activated; <i>LI</i>, latently infected; <i>LIA</i>, latently infected activated.</p

Functional analysis of genes modulated in HIV-1 latency.

<p>(A) Protein interaction network (PIN) generated using differentially expressed genes (N = 826) after a log₂FC > 0.5 filter was applied (N = 64). Any single nodes not attached to a main hub were removed for clarity. Abbreviations for the type of interaction are as follows: <i>B</i>–Binding, <i>C</i>–Cleavage, <i>CM</i>–Covalent Modification, <i>GR</i>–Group Relation, <i>+P</i>–Phosphorylation, <i>TR</i>–Transcription Regulation, <i>T</i>–Transformation. Edge coloring of Green indicates activation, Red indicates inhibition, Black indicates unspecified, while Blue is utilized for a group relation. Arrows indicate the direction of interactions. The color gradient of log₂FC for (A) is included with red indicating upregulation and blue indicating downregulation. (B) RT-qPCR validation of a panel of p53 related genes modulated in HIV-1 latency. Fold change is plotted on the log₂ scale with error bars representing standard deviation. Significance for RT-qPCR results was determined with a paired <i>t</i>-test (*p<0.05, **p<0.01, ***p<0.001) and all RNA-Seq results were significant (EdgeR’s FDR corrected <i>p</i>-value < 0.05). (C) Dot plots of gene expression data (i.e., counts per million) from RNA-Seq results for <i>TNFRSF10B</i> (DR5) and <i>FAS</i> (CD95). Abbreviations: <i>UI</i>, uninfected; <i>LI</i>, latently infected. (D) Histograms of DR5 (<i>TNFRSF10B</i>) and CD95 (<i>FAS</i>) protein surface expression from a representative donor. Red line indicates UI and blue line indicates LI. Geometric mean fluorescence intensity (gMFI) is indicated at the right top corner of the histogram and the grey histogram represents unstained control. (E) Surface expression was measured by FACS for DR5 (6 donors) or CD95 (5 donors). Significance was determined with a paired <i>t</i>-test (<i>p</i>-values provided).</p

Generation of latently infected cultured T_CM.

<p>(A) Time-line of experimental procedure. A full description of the methodology is provided in Materials and Methods. (B) Analysis of infection in a representative donor. At the time points indicated, CD4 expression and HIV-1 p24 Gag expression was measured by flow cytometry as indicated in Materials and Methods. X-axis indicates HIV-1 p24 Gag staining and Y-axis CD4 staining. At day 17, cells were sorted based on surface CD4 expression. Staining of the cells pre- and post-sorting is indicated. Numbers in dot-plots represent percentages.</p

Genes responsive to p53 activity.

<p>Genes responsive to p53 activity.</p

HIV-1 splicing variants are upregulated following αCD3/αCD28 stimulation.

<p>(A) The abundance of HIV-1 unspliced (US), singly spliced (SS), and multiply spliced (MS) transcripts in the LI and LIA conditions was estimated by mapping reads over the two major splice sites D1 and D4 (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006026#sec010" target="_blank">Materials and Methods</a> for details). No more than 4 reads were identified to map to the HIV-1 genome in samples from the uninfected conditions (UI and UIA), which may reflect expression from endogenous retroviral elements in the human genome. The significance of increases in each class of HIV reads upon activation was assessed using a one-tailed paired t-test but only an increase in MS reads was identified as significant (<i>p</i> = 0.015). (B) Fold changes upon activation of US RNA (US-Gag), MS RNA (MS-Tat/Rev) and polyadenylated RNA were determined by RT-qPCR. Significance of fold changes was confirmed using a one-tailed paired t-test and all RT-qPCR measurements were identified as significant at the <i>p</i><0.001 level. Fold change is plotted on the log₂ scale with error bars representing standard deviation. (C) Surface CD4 and intracellular HIV-1 p24 Gag expression were measured by flow cytometry as indicated in Materials and Methods after isolation and after reactivation with αCD3/αCD28 beads for 48 hours.</p