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

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

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

IFNα and its signature are increased in plasma of HIV-1-infected subjects not receiving ART.

<p>Plasma IFN-I bioactivity measured by the iLite™ bioassay was increased in plasma of HIV-1-infected subjects in comparison to uninfected subjects (median 1.04 IFNα2 equivalent units for HIV-1-infected subjects, median 0.89 IFNα2 equivalent units for uninfected subjects, p = 0.012) (A). IFNα was increased in plasma of HIV-1-infected subjects in comparison to uninfected subjects (median 4.27 pg/ml for HIV-1-infected subjects, median 3.13 pg/ml for uninfected subjects, p<0.001) (B). IFNβ was not increased in plasma of HIV-1-infected subjects compared to uninfected subjects (median 2.34 pg/ml for HIV-1-infected subjects, median 2.34 pg/ml for uninfected subjects, p = 0.560) (B). IFNω was not increased in plasma of HIV-1-infected subjects compared to uninfected subjects (median 4.69 pg/ml for HIV-1-infected subjects, median 4.69 pg/ml for uninfected subjects, p = 0.837) (B). Plasma IFNα levels were strongly associated with plasma IFN-I bioactivity in HIV-1-infected subjects (r = 0.711, p<0.001) (C). Plasma IP-10 was increased in plasma of HIV-1-infected subjects in comparison to uninfected subjects (median 538.2 pg/ml for HIV-1-infected subjects, median 132.6 pg/ml for uninfected subjects, p = 0.002) (D). Slight variations in sample sizes for different assays occur as results for some subjects were not available.</p

Therapeutic administration of IFNα induces increased expression of the activation marker CD38 on memory CD8 T cells in HCV-infected subjects.

<p>A longitudinal study was conducted to assess CD38 expression on memory (CD45RO+) CD8 T cells. Seven HCV-infected (HIV-1-uninfected) subjects were studied immediately prior to initiation of therapy with s.c. pegylated IFNα and oral ribavirin, and after 4 and 12 weeks of therapy. CD38-specific MFI (MFI for CD38 minus MFI for IgG1 isotype control) increased on memory CD8 T cells in all seven treated subjects at 4 weeks of therapy (p = 0.018). A further increase in CD38 was observed in five of seven subjects by week 12 of therapy (p = 0.018). The difference in CD38 expression between week 0 (prior to therapy) and week 12 was highly significant (p = 0.018).</p

IFNα mRNA expression is elevated in lymph nodes, but not peripheral blood leukocytes, of HIV-1-infected subjects.

<p>There was no significant difference in expression of IFNα mRNA in whole blood leukocytes of HIV-1-infected subjects without ART (median relative expression of 0.10) and uninfected subjects (median relative expression of 0.88) (p = 0.981) (A). Similarly, there was no significant difference in IFNβ mRNA expression in whole blood leukocytes (median relative expression of 0.003 for HIV-1-infected subjects, median relative expression of 0.001 for uninfected subjects; p = 0.298) (A). An IFN-I signature was evident in peripheral blood leukocytes, as expression of the ISG MxA was significantly increased in HIV-1-infected subjects without ART compared to uninfected subjects (median relative expression of 0.85 for HIV-1-infected subjects, median relative expression of 0.26 for uninfected subjects; p = 0.016) (A). In contrast, expression of IFNα mRNA in lymph node tissue was significantly elevated in HIV-1-infected subjects without ART (median relative expression of 0.93) relative to uninfected subjects (median relative expression of 0.12) (p = 0.037) (B). There was no statistically significant difference in IFNβ mRNA expression between the two donor groups (median relative expression of 0.20 for HIV-1-infected subjects, median relative expression of 0.06 for uninfected subjects; p = 0.728) (B). Expression of the ISG MxA was significantly increased in lymph node tissue from HIV-1-infected subjects compared to uninfected subjects (median relative expression of 1.05 for HIV-1-infected subjects vs. 0.45 for uninfected subjects; p = 0.037) (B).</p

Plasma IFNα and IFN-I bioactivity are associated with plasma HIV-1 RNA levels, CD4 T cell count and immune activation in untreated subjects with chronic HIV-1-infection.

<p>Plasma HIV-1 RNA levels in HIV-1-infected subjects without ART correlated positively with plasma IFN-I bioactivity (r = 0.329, p = 0.017) (A), as well as with plasma IFNα (r = 0.356, p = 0.008) (B). Absolute CD4 T cell count in these subjects correlated inversely with plasma IFN-I bioactivity (r = -0.426, p = 0.002) (C) and plasma IFNα (r = -0.407, p = 0.002) (D). Expression of CD38 by memory (CD45RO+) CD8 T cells in these subjects correlated positively with plasma IFN-I bioactivity (r = 0.374, p = 0.021) (E) and with plasma IFNα (r = 0.387, p = 0.01) (F).</p

Plasma IFNα and IP-10 levels are comparable between HIV-1 infected subjects on ART and uninfected subjects.

<p>IFNα and IP-10 levels were determined in the plasma of 25 individuals with chronic HIV-1 infection who received ART and responded with HIV-1 RNA below assay detection limits (<50 copies/ml) for two years or more. IFNα was detected in plasma from only one such donor, and then at trace levels (A). The difference in plasma IFNα level between HIV-1 infected subjects without ART and HIV-1-infected subjects with ART was highly significant (p<0.001). Plasma IP-10 levels in ART-treated HIV-1-infected donors were comparable to those in uninfected donors (p = 0.460) and were significantly lower in comparison to untreated HIV-1-infected donors (p<0.001) (B).</p