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

TUN treatment HLA-B*57:01 221 cells abrogates binding to KIR3DL1ζ-Jurkat cells: (A) Gating of Jurkat cells by size (SSC = side scatter; FSC = forward scatter), CD3 expression and KIR3DL1 expression (KIR3DL1^-/~/+), (B) CD69 expression of unstimulated and stimulated KIR3DL1-/~/+ Jurkat cells (C) 4.4-fold increase of MFI of CD69 (compared to unstimulated controls) on KIR3DL1ζ+ Jurkat cells coincubated with wildtype 721.221 (221) or cells transfected with HLA-B*08:01/HLA-B*57:01 (221-B57/221-B08) and treated without/with TUN (+T) (n = 10).

<p>TUN treatment HLA-B*57:01 221 cells abrogates binding to KIR3DL1ζ-Jurkat cells: (A) Gating of Jurkat cells by size (SSC = side scatter; FSC = forward scatter), CD3 expression and KIR3DL1 expression (KIR3DL1^-/~/+), (B) CD69 expression of unstimulated and stimulated KIR3DL1-/~/+ Jurkat cells (C) 4.4-fold increase of MFI of CD69 (compared to unstimulated controls) on KIR3DL1ζ+ Jurkat cells coincubated with wildtype 721.221 (221) or cells transfected with HLA-B*08:01/HLA-B*57:01 (221-B57/221-B08) and treated without/with TUN (+T) (n = 10).</p

<i>N</i>-glycosylation inhibition increases HLA-B*57:01 surface expression while abrogating KIR3DL1-Fc binding: Anti-HLA-Bw4 antibody staining (A) and KIR3DL1-Fc staining (B) was performed on 221-HLA-B*57:01 cells (221-B57) treated with TUN (+T), CSP (+C), or PBS and untransduced 221 cells (221).

<p>Representative histograms are on left-sided panels and data representing five technical replicates are presented as bar graphs on right-sided panels.</p

Glycosylation inhibitor screening and titration: (A) Median fluorescence intensity (MFI) of Bw4 staining of untransfected 221 cells (221) and HLA-B*57:01 transfected 221 cells (B57) treated with a panel of glycosylation inhibitors (n = 2) (B) MFI of KIR-Fc staining of untransfected 221 cells (221) and HLA-B*57:01 transfected 221 cells (B57) treated with a panel of glycosylation inhibitors (n = 2)

<p>Glycosylation inhibitor screening and titration: (A) Median fluorescence intensity (MFI) of Bw4 staining of untransfected 221 cells (221) and HLA-B*57:01 transfected 221 cells (B57) treated with a panel of glycosylation inhibitors (n = 2) (B) MFI of KIR-Fc staining of untransfected 221 cells (221) and HLA-B*57:01 transfected 221 cells (B57) treated with a panel of glycosylation inhibitors (n = 2)</p

Disinhibition of KIR3DL1+ NK cell clones by TUN treatment (A) Gating strategy of NK cell clones by size, CD56, CD16, KIR3DL1 and CD107a expression (B) % of CD107a+ KIR3DL1+ and KIR3DL1- NK cell clones coincubated with wildtype 721.221 (221) or cells transfected with HLA-B*57:01 (221-B57/221-B08) and treated without/with TUN (+T) (n = 3).

<p>Disinhibition of KIR3DL1+ NK cell clones by TUN treatment (A) Gating strategy of NK cell clones by size, CD56, CD16, KIR3DL1 and CD107a expression (B) % of CD107a+ KIR3DL1+ and KIR3DL1- NK cell clones coincubated with wildtype 721.221 (221) or cells transfected with HLA-B*57:01 (221-B57/221-B08) and treated without/with TUN (+T) (n = 3).</p

Secondary structure of HLA-B*57 and KIR3DL1: (Green) HLA-B*57, (Black) β2M, (Blue) KIR3DL1, (Cyan) Peptide bound in peptide-binding groove, (Red) Amino Acid N86, a site of N-glycosylation on HLA-B*57:01; Image generated using Swiss-PdbViewer 4.1.0 and a structure fie published by Vivien JP.

<p>[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145324#pone.0145324.ref025" target="_blank">25</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145324#pone.0145324.ref041" target="_blank">41</a>].</p

Functional response of NK cells from influenza-infected subjects.

<p>Dot plots represent percentages of CD107a+ NK cells from 17 healthy subjects prior to influenza vaccination (C) and 7 subjects with acute seasonal influenza infection (Flu) following incubation with medium alone, H1N1 PR8 influenza virus for 16 h, or 221 and K562 target cells for 4 h at an effector∶ target ratio of 10∶1. Influenza-stimulation of NK cells was only performed on 10 subjects prior to vaccination (C).</p

Changes in plasma cytokine concentrations associated with acute influenza infection.

<p>Columns represent mean concentrations of indicated cytokines in 7 controls (black), 9 patients with acute seasonal influenza (dark grey) and 21 with acute 2009 pandemic H1N1 influenza (light grey). For each column, N represents the number of values included in the analysis based on a coefficient of variation <30. Low out of range values were set to half of the lowest standard value (1.6 pg/mL). Bars indicate standard error of the mean. <b>*</b> represents <i>p</i><0.05; <b>**</b> represents <i>p</i><0.001 and <b>***</b> represents <i>p</i><0.0001.</p

Changes in NK cell numbers and subset distribution in acute influenza infection and influenza vaccination.

<p>Dot plots represent (A), absolute numbers of NK cells (B), percentages of total NK cells and (C), absolute numbers of CD56^bright (CD3-CD56+CD16−), CD56^dim (CD3-CD56+CD16+) and CD56^neg (CD3-CD56−CD16+) NK cells from 17 subjects prior to influenza vaccination, and at day 1 and 4 post-vaccination, 9 patients with acute seasonal influenza and 21 with acute 2009 pandemic H1N1 influenza virus infection. Complete blood counts at baseline were only available for 13 of the vaccinated subjects. Horizontal lines indicate the median percentages. Differences where p<0.05 are indicated. (•, healthy subjects prior vaccination; ▪, healthy subjects post-vaccination; dark grey ▴, acutely infected with 2009 seasonal influenza strains; light grey ▴, acutely infected with 2009 pandemic H1N1 influenza virus). D: day post-vaccination.</p

Changes in CD69 expression on NK cells in response to influenza infection and vaccination.

<p>Dot plots represent (A), percentages and (B), absolute numbers of CD69+ NK cells from 17 subjects prior to influenza vaccination, and at day 1, 4 and 7 post-vaccination, 9 patients with acute seasonal influenza and 21 with acute 2009 pandemic H1N1 influenza virus infection. Complete blood counts at baseline were only available for 13 of the vaccinated subjects. (•, healthy subjects prior vaccination; ▪, healthy subjects post-vaccination; dark grey ▴, acutely infected with 2009 seasonal influenza strains; light grey ▴, acutely infected with 2009 pandemic H1N1 influenza virus). (C) Representative primary flow panels show percentages of CD69+ CD56^bright, CD56^dim and CD56^neg NK cells prior to and 7 days post-vaccination and in seasonal and pandemic influenza infection. Percentages of CD69+ total NK cells are indicated.</p