Increased expression of lymphoid tissue homing markers on circulating pDC of HIV-1 positive versus negative individuals.

<p>Panel A: representative flow cytometry plots showing expression of nine homing markers grouped by tissue specificity: CD62L and CCR7 (lymph nodes); CCR4 (skin); CD18 and CD29 (inflamed tissues); and CD49d, CD103, CCR9 and integrin β7 (GALT). Histogram plots were obtained by gating onto the live cell population as assessed by forward and side light scatter profile (not shown), and then onto the pDC population as identified by staining with anti-BDCA4 and anti-CD123 antibodies (rectangles within density plots). Top density plot and gray area histogram: healthy control; bottom density plot and black line histogram: HIV-1 patient. Panel B: summary results of CCR7, CD62L and CD103 expression on pDC of HIV-1 positive (<i>n</i> = 17) and negative individuals (<i>n</i> = 11). We assessed the Geometric Mean Fluorescence Intensity (GMFI) for each marker by flow cytometry (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011110#s2" target="_blank">Materials and Methods</a>). The panels show the Mean and SEM of the GMFI values determined for all individuals in the two study groups. Black bars: healthy controls; white bar: HIV-1 patients. Panel C: correlation analyses between CCR7 expression and frequency of pDC in PBMC of HIV-1 patients (left) and HIV-1 viremia (right). The line summarizing the data uses a linear model fitted on the logarithmic scale that expresses HIV-1 viremia. Closed circles (•): viremic patients; closed diamonds (⧫): aviremic patients.</p

Demographic and clinical parameters of lymph node donors.

<p>All data are Median (Interquartile Range) except for % pDC in total LNMC, which is Mean ± Standard Error of Means; n.a.  = not available; N/A = not applicable; LNMC = lymph node mononuclear cells; ART = Antiretroviral therapy.</p

Features of lymph node-homed pDC.

<p>Data are Mean ± Standard Error of Means of the Geometric Mean Fluorescence Intensity (GMFI) values determined for all individuals in each study group. For Annexin V, data are Mean ± Standard Error of the percent Annexin V positive values determined for all individuals in each study group.</p

Increased IFNα expression by pDC in lymph nodes of HIV-1 patients.

<p>Panel A: flow cytometry plot showing expression of IFNα by lymph node-homed pDC from a representative healthy control and an HIV-1 patient. Histogram plot was obtained by gating onto the live cell population as assessed by forward and side light scatter profile (not shown), and then onto the pDC population as identified by staining with anti-BDCA2 and anti-CD123 antibodies. Top density plot and gray area histogram: healthy control; bottom density plot and black line histogram: HIV-1 patient. Panel B: summary results for expression levels of IFNα by lymph node-homed pDC of healthy controls (black bar; <i>n</i> = 11) and HIV-1 patients (white bar; <i>n</i> = 18). We assessed the Geometric Mean Fluorescence Intensity (GMFI) value with cells from all individuals in the two study groups (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011110#s2" target="_blank">Materials and Methods</a>). Panel shows the Mean and SEM of the GMFI values.</p

Higher rates of cell death by pDC in lymph nodes of HIV-1 patients.

<p>Panel A: flow cytometry plot showing Annexin V staining of lymph node-homed pDC from a representative HIV-1 patient and control individual. Histogram plots were obtained by gating onto the live cell population as assessed by forward and side light scatter profile (not shown), and then onto the pDC population as identified by staining with anti-BDCA2 and anti-CD123 antibodies. Top density plot and gray area histogram: healthy control; bottom density plot and black line histogram: HIV-1 patient. Panel B: summary results for expression levels of Annexin V by lymph node-homed pDC of healthy controls (black bar; <i>n</i> = 11) and HIV-1 patients (white bar; <i>n</i> = 18). We determined the percentage of Annexin V positive pDC from all individuals in the two study groups (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011110#s2" target="_blank">Materials and Methods</a>). Panel shows the Mean and SEM values of the percent Annexin V positive pDC.</p

Activated but immature phenotype of lymph node-homed pDC from HIV-1 positive versus negative individuals.

<p>Panel A: identification of pDC populations in LNMC of a representative control (top panel) and HIV-1 positive (bottom panel) individual. LNMC were stained with anti-BDCA2 and anti-CD123 antibodies; analyses were carried out by gating on the live cell population as determined by forward and side scatter light profiles. Panels B, D, F and H: flow cytometry plots showing expression levels of activation (CD40 and BDCA2) and maturation (CD83 and CD86) markers on lymph node-homed pDC from a representative HIV-1 patient and control individual. Histogram plots were obtained by gating onto the live cell population as assessed by forward and side light scatter profile (not shown), and then onto the pDC population as identified by staining with anti-BDCA2 and anti-CD123 antibodies (as shown in density plots). Gray area: healthy control; black line: HIV-1 patient. Panels C, E, G, and I: expression levels of CD40, BDCA2, CD83 and CD86 on lymph node-homed pDC of control individuals (black bars; <i>n</i> = 11) and HIV-1 patients (white bars; <i>n</i> = 18). We assessed the Geometric Mean Fluorescence Intensity (GMFI) values for each marker (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011110#s2" target="_blank">Materials and Methods</a>). Panels show the Mean and SEM of the GMFI values determined with cells from all individuals in the two study groups.</p

Accumulation of pDC in lymph nodes of HIV-1 patients.

<p>Panel A: flow cytometry plots showing the pDC population in lymph node mononuclear cells (LNMC) of a representative control individual (top) and HIV-1 patient (bottom). We identified pDC by staining whole LNMC with anti-BDCA2 and anti-CD123 antibodies (rectangles within density plots) and by gating onto the live cell population as assessed by forward and side light scatter profile (not shown). Panel B: summary results for the frequency of pDC in LNMC of healthy controls (black bar, <i>n</i> = 11) and HIV-1 patients (white bar: total patients, <i>n</i> = 18; downward diagonal bar: patients with <4.5 log₁₀ copies/ml HIV-1 RNA, <i>n</i> = 9; upward diagonal bar: patients with >4.5 log₁₀ copies/ml HIV-1 RNA, <i>n</i> = 9). The panel shows the Mean and SEM values of pDC frequencies for each study group.</p

Demographic and clinical parameters of peripheral blood donors.

<p>All data are Median (Interquartile Range) except for % pDC in total PBMC, which is Mean ± Standard Error of Means; n.a.  = not available; N/A = not applicable; PBMC = peripheral blood mononuclear cells; ART = Antiretroviral therapy.</p

Influence of FPR cut-offs on PPV and number of patients elegible for MVC treatment.

<p>Values were calculated using all available RNA (n = 87) and DNA (n = 60) samples; ESTA (n = 37); NGS (n = 45). * No NGS values for the NGS FPR cut-off of 7.5% were available.</p

Influence of FPR cut-offs and triple amplification on PPV and number of patients elegible for MVC.

<p>Depicted data correspond to the 27 RNA plus 28 DNA samples that could be amplified in triplicate.</p