HIV subject MDC are impaired in naïve CD4 T cell activation activity.

<p>Freshly prepared MDC from healthy control (n = 17) and HIV+ (n = 18) subjects were used in titrated numbers (<i>x</i>-axis) to activate one healthy control subject's allogeneic naive CD4 T-cells to produce IFN-γ or IL-2 (<i>y</i>-axis) in a 72-h culture performed in the absence (Medium, <b>panels </b><b><i>A</i></b><b>. and </b><b><i>C</i></b><b>.</b>), or presence of poly I∶C (<b>panels </b><b><i>B</i></b><b>. and </b><b><i>D</i></b><b>.</b>). Control cultures of TLR ligand and naïve CD4 T cells resulted in <3 sfu IFN-γ, and data shown are sfu above this background.</p

Increased PDL-1 and PDL-2 expression on HIV subject MDC.

<p>Isolated MDC were evaluated for expression of inhibitory molecules PDL-1 (<b>panel </b><b><i>A</i></b>) and PDL-2 (<b>panel </b><b><i>B</i></b>) in control (n = 10) and HIV+ (n = 10) subjects. Black lines represent median MFI value for each group. <b>Panel </b><b><i>C</i></b>. Association between PDL-1 MFI on MDC in HIV+ subjects and plasma HIV level. <b>Panel </b><b><i>D</i></b>. The percentage of Annexin V+ T-cells are shown following naive T-cell co-cultures with control vs. HIV+ subject MDC (10,000cells/well) in the absence and presence of poly I∶C.</p

HIV subject DC exhibit increased maturation.

<p><b>Panel </b><b><i>A</i></b>. Representative flow cytometric analysis of freshly isolated MDC and PDC from one healthy control. Mean Fluorescent Intensity (<b>MFI</b>) for HLA-DR, CD86, and CD83 were analyzed. <b>Panel </b><b><i>B</i></b>. Baseline activation/maturation phenotype of freshly isolated MDC and PDC from healthy control (n = 22) and HIV+ subjects (n = 24). The black line represents the median MFI value for each group for HLA-DR, CD86, and CD83.</p

DC TLR ligand responsiveness.

<p><b>Panels </b><b><i>A and B</i></b>. CD86 MFI and HLA-DR MFI on isolated MDC following overnight culture in presence of Medium and poly I∶C stimulation in healthy control (n = 21) and HIV+ subjects (n = 18). The p value in panel B is for comparison between the delta HLA-DR MFI for controls and HIV infected subjects . IL-6 production from isolated MDC on a subset of the same subjects (n = 18 control and n = 16 HIV+) (<b>panel </b><b><i>C</i></b>) following overnight culture in absence and presence of poly I∶C stimulation. IL-6 production from isolated PDC on a subset of the same subjects (n = 17 control and n = 17 HIV+) (<b>panel </b><b><i>D</i></b>) following overnight culture in absence and presence of R848 stimulation. Healthy control (n = 17) and HIV+ subject (n = 16) PDC IFN-α production in response to overnight R848 stimulation is shown in <b>panel </b><b><i>E</i></b>. <b>Panel .</b> Association between MDC spontaneous IL-6 production and HIV plasma level.</p

Toll-like receptors 3 and 5 are expressed by a subset of T cells, but expression is not always sufficient for activation by TLR ligands.

<p>(A) Whole PBMCs were isolated from healthy donors and expression of TLRs 2, 3, and 5 were evaluated by flow cytometry on gated CD3+ T cells. (B) Whole PBMCs, purified T cells (>95% CD3⁺), or purified T cells separated from whole PBMCs by a transwell, were cultured in medium alone, or in medium supplemented with plate bound anti-CD3, poly I:C or flagellin A. Expression of CD38 on memory (CD45RO⁺CD45RA⁻) CD4⁺ T cells was assessed by flow cytometry following overnight culture. Dotplots shown are representative of 8 separate experiments.</p

Activation as a result of TLR ligand exposure preferentially induces T cell death especially among CD4+ T cells.

<p>PBMCs were labeled with CFSE and incubated for 6 days in the presence of anti-CD3 antibody, medium alone or TLR ligands alone (PGN, poly-I:C, LPS (10 or 20 ng/ml), Flagellin A, Flagellin B or imiquimod). After 6 days of incubation, the CD4⁺ and CD8⁺ T cells were examined for dilution of CFSE dye and for binding of Annexin V. Numbers in right upper corners represent percentage of Annexin V-binding cells. Numbers in the left lower corner represent percentage of cells that diluted dye without Annexin-V binding. This experiment is representative of three separate experiments.</p

TLR ligands preferentially activate CD4+ central memory and effector memory T cells and CD8+ effector memory T cells.

<p>Intracellular Ki-67 expression (3A, 3C) and cell surface CD69 (3B, 3D) were analyzed after 7 days' incubation of PBMC in medium alone, or in medium supplemented with plate bound anti-CD3 antibodies, or the indicated TLR ligand. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001915#pone-0001915-g003" target="_blank">Figures 3A and B</a> are representative results among phenotypically defined naïve (CD45RA⁺CD45RO⁻CCR7⁺), central memory (CD45RA⁻CD45RO⁺CCR7⁺), and effector memory (CD45RA⁻CD45RO⁺CCR7⁻) CD4⁺ and CD8⁺ T cells. Values represent percentages of cells staining for Ki-67 or CD69. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001915#pone-0001915-g003" target="_blank">Figures 3C and 3D</a> reflect the mean data from 15 separate experiments. Values nominally significantly different from medium alone values (Wilcoxon Sign Rank Test) are shown with an asterisk.</p

Stimulation of peripheral blood cells by Toll-like Receptor ligands increases expression of CD38 on CD4+ and CD8+ T lymphocytes.

<p>PBMC were cultured overnight in medium alone, or stimulated with individual TLR ligands (Poly-I:C, LPS, Flagellin, or CpG DNA) or with plate-bound anti-CD3 antibody. Expression of CD38 and HLA-DR was monitored by flow cytometry among: (A) CD4+ T cells and (B) CD8+ T cells. Percentages of cells expressing each marker are shown. This experiment is representative of 5.</p

TLR ligands induce high level CD69 expression on CD8+ T cells and Ki- 67 expression in CD4+ T cells.

<p>Intracellular expression of Ki-67 (A) and surface expression of CD69 (B) were monitored after 7 days of cell culture in medium or with the stimuli as indicated. Bars represent means and the lines standard errors of the mean (SEM) of 15 separate experiments using PBMC of healthy controls. Black boxes = CD4+ T cells and gray boxes = CD8+ T cells. * = nominally significantly different (p<0.05 by Wilcoxon Sign Rank test when compared to results obtained in medium alone.</p

Effect of Nadir CD4+ T Cell Count on Clinical Measures of Periodontal Disease in HIV+ Adults before and during Immune Reconstitution on HAART

<div><p>Background</p><p>The contribution of HIV-infection to periodontal disease (PD) is poorly understood.  We proposed that immunological markers would be associated with improved clinical measures of PD.</p> <p>Methods</p><p>We performed a longitudinal cohort study of HIV-infected adults who had started highly active antiretroviral therapy (HAART) <2 years. PD was characterized clinically as the percent of teeth with ≥1 site with periodontal probing depth (PPD) ≥5.0mm, recession (REC) >0mm, clinical attachment level (CAL) ≥4.0mm, and bleeding on probing (BOP) at ≥4 sites/tooth and microbiologically as specific periodontopathogen concentration. Linear mixed-effects models were used to assess the associations between immune function and PD.</p> <p>Results</p><p>Forty (40) subjects with median 2.7 months on HAART and median nadir CD4+ T-cell count of 212 cells/μl completed a median 3 visits. Over 24 months, CD4+ T-cell count increased by a mean 173 cells/µl (p<0.001) and HIV RNA decreased by 0.5 log₁₀ copies/ml (p<0.001); concurrently, PPD, CAL and BOP decreased by a mean 11.7%, 12.1%, and 14.7% respectively (all p<0.001). Lower nadir CD4+ T-cell count was associated with worse baseline REC (-6.72%; p=0.04) and CAL (9.06%; p<0.001). Further, lower nadir CD4+ T-cell count was associated with a greater relative longitudinal improvement in PPD in subjects with higher baseline levels of <i>Porphyromonas gingivalis</i> (p=0.027), and BOP in subjects with higher baseline levels of <i>Porphyromonas gingivalis</i> or <i>Treponema denticola</i> (p=0.001 and p=0.006 respectively). Longitudinal changes from baseline in CD4+ T-cell count and level of HIV RNA were not independently associated with longitudinal changes in any clinical markers of PD.</p> <p>Conclusion</p><p>Degree of immunosuppression was associated with baseline gingival recession. After HAART initiation, measures of active PD improved most in those with lower nadir CD4+ T-cell counts and higher baseline levels of specific periodontopathogens. Nadir CD4+ T-cell count differentially influences periodontal disease both before and after HAART in HIV-infected adults.</p> </div