Following HIV infection NK-cell responses to stimulation were diminished.

<p>Natural Killer cell degranulation (A) and IFN-γ secretion (B) responses after stimulation with IL-2 alone or with IL-2 and 721 cells (adjusted for background) or with PMA/Ionomycin. Data are adjusted for background responses to media alone.</p

Primary HIV-1 infection was associated with differential T-cell and NK-cell activation.

<p>T-cells (A) and NK cells (B) were activated following HIV infection. The proportion of activated T-cells and NK cells was significantly correlated before (C), but not following HIV-1 infection (D). In contrast, during primary infection, both T-cell (E) and NK-cell (F) activation were positively correlated with HIV viral load (log₁₀ copies/ml) but not CD4+ T-cell counts (G and H respectively).</p

Early after HIV infection blood NK cells increased their expression of lymphoid, but not gut homing receptors.

<p>The proportion of NK cells expressing CCR7 was increased following HIV infection (A). The proportion of NK cells expressing CCR7 was reduced during later stages of primary infection (B). The proportion of NK cells that expressed α4β7, was not changed during primary HIV-1 infection (C).</p

Following infection, an increased proportion of activated NK cells in blood expressed Killer Immunoglobulin-like Receptors (KIR).

<p>The proportion of KIR_pos NK cells in blood increased following HIV infection (A). The proportion of KIR_pos NK cells did not differ between stages of primary HIV infection (seronegative, sero-discordant or seropositive stages) (B). KIR_pos NK cells were more activated than KIR_neg NK cells (C).</p

p24 Gag_209–218/HLA-Cw*0102 complexes on T2 cells lead to functional inhibition of primary KIR2DL2(+) NK cells.

<p>(<b>A</b>) Representative histograms of degranulation of KIR2DL2(−) (left panel) and KIR2DL2(+) (right panel) NK cells after co-incubation with peptide-pulsed T2 cells (tinted and black) or without target cells (clear). NK cell degranulation was measured flow cytometrically by surface expression of CD107a. (<b>B</b>) Different levels of NK cell degranulation between KIR2DL2(−) (dark grey) and KIR2DL2(+) (light grey) NK cells after co-incubation with peptide-pulsed T2 cells. Each column represents mean±SEM percentage of CD107a(+) NK cells of 5 of different individuals. Unspecific degranulation of NK cells measured without target cells was deducted from each column.</p

HIV-1 p24 peptide-dependent HLA-Cw*0102 stabilization on T2 cells.

<p>(<b>A</b>) Representative histograms of HLA-Cw*0102 stabilization on T2 cells as determined by flow cytometry. Histograms display HLA-Cw*0102 expression on T2 cells in the presence of control peptides VAP-FA and VAP-DA (black) at a concentration of 40 µg/ml as compared to unloaded T2 cells (grey tinted) and isotype control (clear). (<b>B</b>) HLA-Cw*0102 expression on HIV-1 p24 peptide-pulsed T2 cells. HLA-Cw*0102 expression is illustrated as relative median fluorescence intensity (RFI) as compared to unloaded T2 cells. Each column represents mean±SEM RFI of 5 independent experiments for each HIV-1 p24 OLP. A total of 59 HIV-1 p24 OLPs were analyzed, which previously showed strongest HLA-A/B/C stabilization as determined by an HLA-A/B/C antibody. Of those, 11 peptides with the highest HLA-Cw*0102 expression (>five-fold) were selected for subsequent KIR-Fc binding assays.</p

Ability of p24 Gag_209–218 peptide variants for HLA-Cw*0102 stabilization and KIR2DL2-Fc binding.

<p>(<b>A</b>) Differential expression of HLA-Cw*0102 on T2 cells after co-incubation with peptide variants of HIV-1 p24 Gag_209–218-L (AAEWDRLHPV). Peptide variants differed in length [9 or 10 amino acids (aa)] as well as in sequence (substitution of Leucine in position 7 with various amino acids). HLA-Cw*0102 expression is illustrated as relative median fluorescence intensity (RFI) as compared to unloaded T2 cells. Each column represents mean±SEM RFI of 4 independent experiments for each peptide variant. (<b>B</b>) Relative binding of KIR2DL2-Fc to T2 cells after co-incubation with selected HIV-1 p24 Gag_209–218variants. KIR2DL2-Fc binding is illustrated as RFI as compared to VAP-DA-pulsed T2 cells. Each column represents mean±SEM RFI of 3 independent experiments for each 10 aa variant. (<b>C</b>) Different levels of NK cell degranulation after co-incubation with T2 cells in the presence of different p24 Gag_209–218-L variants. Each column represents mean±SEM percentage of CD107a(+) NK cells of 4 different individuals. Unspecific degranulation of NK cells measured without target cells was deducted from each column. * indicates statistical significance as defined <i>p</i><0.05.</p

Influence of Glycosylation Inhibition on the Binding of KIR3DL1 to HLA-B*57:01

<div><p>Viral infections can affect the glycosylation pattern of glycoproteins involved in antiviral immunity. Given the importance of protein glycosylation for immune function, we investigated the effect that modulation of the highly conserved HLA class I <i>N</i>-glycan has on KIR:HLA interactions and NK cell function. We focused on HLA-B*57:01 and its interaction with KIR3DL1, which has been shown to play a critical role in determining the progression of a number of human diseases, including human immunodeficiency virus-1 infection. 721.221 cells stably expressing HLA-B*57:01 were treated with a panel of glycosylation enzyme inhibitors, and HLA class I expression and KIR3DL1 binding was quantified. In addition, the functional outcomes of HLA-B*57:01 <i>N</i>-glycan disruption/modulation on KIR3DL1ζ⁺ Jurkat reporter cells and primary human KIR3DL1⁺ NK cells was assessed. Different glycosylation enzyme inhibitors had varying effects on HLA-B*57:01 expression and KIR3DL1-Fc binding. The most remarkable effect was that of tunicamycin, an inhibitor of the first step of <i>N</i>-glycosylation, which resulted in significantly reduced KIR3DL1-Fc binding despite sustained expression of HLA-B*57:01 on 721.221 cells. This effect was paralleled by decreased activation of KIR3DL1ζ⁺ Jurkat reporter cells, as well as increased degranulation of primary human KIR3DL1⁺ NK cell clones when encountering HLA-B*57:01-expressing 721.221 cells that were pre-treated with tunicamycin. Overall, these results demonstrate that <i>N</i>-glycosylation of HLA class I is important for KIR:HLA binding and has an impact on NK cell function.</p></div

CD4 count outcomes at 12 and 30 months based on patient demographic and pre-HAART characteristics.

<p>CD4 count outcomes at 12 and 30 months based on patient demographic and pre-HAART characteristics.</p

CD4+ T-cell count trajectories and baseline characteristics predicting failure of CD4 cell count recovery.

<p><b>A</b>. CD4+ T-cell count trajectories for all 442 subjects over the course of 30 months. The red line reflects the median±SEM. <b>B</b>. OR and 95% confidence intervals (CI) for not achieving CD4 counts above 200 cells/mm³ at 12 months (grey) and 500 cells/mm³ at 30 months (black) depending on baseline characteristics. Shown are only factors with significant or close to significant effect in the multivariate model.</p