Correlation of NK cell proportions with FcRγ expression in HIV-infected individuals.

<p>(A) Correlation between NK cell FcRγ protein expression and the proportion of NK cells in patient PBMC (donors 19–27) measured by flow cytometry. (B) Proportion of NK cells in PBMC (individuals 19–27, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009643#pone-0009643-t001" target="_blank">Table 1</a>) measured by flow cytometry. The Mann-Whitney U test was used to assess statistical significance. Horizontal bars represent median values.</p

FcRγ expression in monocytes of HIV-infected and uninfected individuals.

<p>FcRγ mRNA (A) and protein (B) were determined by Q-PCR and quantitative infrared immunoblotting in extracts from monocytes purified by FACS from blood of HIV-1 infected (individuals 1–18, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009643#pone-0009643-t001" target="_blank">Table 1</a>) and control subjects. Fluorescence from FcRγ immunoblots measured at 680 nm was normalised to fluorescence of GAPDH in the same immunoblots, measured at 800 nm. FcRγ mRNA was determined from real-time PCR measurements using the comparative threshold method with GAPDH mRNA serving as internal control. Differences between groups were tested using the Mann-Whitney U test for non-parametric data, with a value &lt;0.05 assumed to be significant. Horizontal bars represent median values.</p

Clinical details of HIV-1 infected patients used in this study.

1<p>3TC: lamivudine, ABC: abacavir, ATV: atazanavir, AZT: zidovudine, D4T: stavudine, DRV: darunavir, EFV: efavirenz, ETV: etravirine, FPV: fosamprenavir, FTC: emtricitabine, LPV: lopinavir, NVP: nevirapine, RGV: raltegravir, RTV: ritonavir, T20: enfuvirtide, TDF: tenofovir.</p

FcRγ expression in CD56⁺/CD94⁺ lymphocytes of HIV-infected and uninfected individuals.

<p>FcRγ mRNA (A) and protein (B) were measured in FACS-sorted CD56⁺/CD94⁺ lymphocytes from blood of HIV-1 infected (individuals 1–18, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009643#pone-0009643-t001" target="_blank">Table 1</a>) and control subjects by Q-PCR using the comparative threshold and quantitative immunoblotting method as described. The Mann-Whitney U test was used to assess statistical significance. Horizontal bars represent median values.</p

FcRγ and TCRζ expression in NK cells of HIV-infected and uninfected individuals.

<p>NK cells purified from blood of an additional 9 HIV-infected (individuals 19–27, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009643#pone-0009643-t001" target="_blank">Table 1</a>) and 8 control subjects. FcRγ protein (A) and mRNA (B) and TCRζ mRNA (C) were measured in FACS-sorted CD3⁻ CD56⁺/CD94⁺ NK cells using quantitative immunoblotting and Q-PCR as described. The Mann-Whitney U test was used to assess statistical significance. Horizontal bars represent median values.</p

TCRζ expression in CD56+/CD94+ and CD3+ T lymphocyte populations of HIV-infected and uninfected individuals.

<p>TCRζ mRNA in T lymphocytes (A) and CD56⁺/CD94⁺ lymphocytes (B) purified by FACS from blood of HIV-1 infected (individuals 1–18, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009643#pone-0009643-t001" target="_blank">Table 1</a>) and control subjects was measured by Q-PCR using the comparative threshold method as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009643#pone-0009643-g002" target="_blank">Figure 2A</a>. The Mann-Whitney U test was used to assess statistical significance. Horizontal bars represent median values.</p

Time course of the effect of HIV-1 on cytokine secretion of MDM exposed to antibody opsonised IE.

<p>MDM from 3 donors cultured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032102#pone-0032102-g001" target="_blank">Figure 1</a> were infected with HIV-1_Ba-L (•) or mock-infected (○) and primed for 48 hrs with IFNγ then exposed to IE opsonised with 9% pooled immune serum (IE-PPS) at a target to cell ratio of 20∶1. At the indicated time points culture supernatants were collected for (<b>A</b>) IL-1β, (<b>B</b>) TNF and (<b>C</b>) IL-6 cytokine measurement. Data are means of MDM derived from 3 donors plus-minus SEM. Statistical analysis was carried out using the generalised estimating equations method which indicated that mock-infected MDM secreted significantly higher levels of cytokines compared to HIV-1 infected MDM (p = 0.026, IL-1β; p = 0.047, TNF; p = 0.019, IL-6).</p

HIV-1 infection of MDM inhibits phagocytosis of antibody opsonised CS2 infected erythrocytes.

<p>(<b>A</b>). MDM from 11 donors were cultured for 5 days and infected with HIV-1_Ba-L (•) at a MOI of between 0.1 and 1.0 or mock-infected (○) then cultured for an additional 7 days. IE opsonised with 9% PPS were added at a 20∶1 ratio and phagocytosis was measured at 1 hour. Phagocytic index represents the number of ingested IE per 100 MDM. Statistical comparisons were performed in a pair wise manner using the Wilcoxon matched pairs test. (<b>B</b>). In 6 selected cultures analysed for phagocytosis, XTT assay was used to measure viability of MDM infected with HIV-1_Ba-L (•) or mock-infected (○) MDM (3 mg/mL XTT, 4 hr, absorbance was read at 450 nm using a reference wavelength of 650 nm). (<b>C</b>). Effect of IFNγ on HIV-1_Ba-L production by MDM. MDM were cultured in triplicate in 96-well plates for 5 days, infected with HIV-1_Ba-L for a further 5 days then primed with 100 ng/mL IFNγ where indicated. After 72 hrs media from triplicate wells was collected and analysed by micro RT assay. Comparisons between unprimed and primed MDM were made using the Wilcoxon matched pairs test and a significant difference (p&lt;.05) is indicated.</p

The effect of HIV-1 on cytokine secretion of MDM exposed to antibody opsonised IE.

<p>MDM from 8 donors cultured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032102#pone-0032102-g001" target="_blank">Figure 1</a> were infected with HIV-1_Ba-L (•) or mock-infected (○) and primed for 48 hrs with IFNγ then exposed to unopsonised (IE) or IE opsonised with 9% pooled immune serum (IE-PPS) at a target to cell ratio of 20∶1 for 24 hrs followed by collection of culture medium for (<b>A</b>) IL-1β, (<b>B</b>) TNF, (<b>C</b>) IL-6 and (<b>D</b>) IL-8 cytokine secretion analysis. Comparisons of cytokine secretion between cultures infected with HIV-1_Ba-L exposed to IE or IE-PPS, and between HIV-1_Ba-L infected and uninfected cultures exposed to IE-PPS were made using the Wilcoxon matched pairs test.</p

The effect of HIV-1 on cytokine mRNA levels of MDM exposed to antibody opsonised IE.

<p>MDM from 6 donors cultured in 24-well plates were infected with HIV-1_Ba-L (•) or mock-infected (○) and primed for 48 hrs with IFNγ then exposed to IE opsonised with 9% pooled immune serum (IE-PPS) at a target to cell ratio of 20∶1 for 2 hrs followed by preparation of RNA and cDNA for analysis of (<b>A</b>) IL-1β, (<b>B</b>) TNF and (<b>C</b>) IL-6 cytokine mRNA levels by qPCR. Comparisons of mRNA levels between cultures infected with HIV-1_Ba-L or mock-infected exposed to IE-PPS were made using the Mann-Whitney non-parametric U test.</p