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

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

<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

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

Flow cytometry analysis of lymphocyte subsets at 8 months after disease induction.

<p>A, B, C, MiR155-deficient PIL^-/- mice had lower frequencies of CD19⁺ B-cells, total CD4⁺ and CD4⁺CD25⁺FoxP3- activated T_effector-cells compared to the WT lupus group PIL^+/+. D, Frequencies of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (T_reg) were reduced in PIL^+/+ compared to the respective control group (CO^+/+). T_reg were even lower in both miR155 knockout groups (PIL^-/- and CO^-/-) without intergroup differences. E, F, G, Upon <i>in vitro</i> restimulation, PIL^+/+ had significant higher frequencies of CD4⁺IL-4⁺ Th2 and CD4⁺IL-17⁺ Th17 cells compared to PIL^-/- and CO^+/+, a similar, albeit not significant trend was observed for CD4⁺IFNγ Th1 cells when comparing PIL^+/+ to PIL^-/-. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181015#pone.0181015.t001" target="_blank">Table 1</a> for exact numbers and p values. Results are representative of 2 independent experiments. PIL were induced with pristane, CO with PBS; ^+/+ stands for wild type, ^-/- for miR155-deficient knockouts; bars show mean with SD. * = <i>P</i> < 0.05, by Mann-Whitney test.</p

Immunohistochemical stainings of affected lungs.

<p>A, E, The miR155-deficient group PIL^-/- showed less accumulation of CD3 positive T cells that the lupus wild type group PIL^+/+. The same effect was observed in other immunohistochemical stainings: B, F, CD45R positive B cells, C, G, F4/80 positive macrophages and D, H, Neu7/4 positive neutrophil granulocytes. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181015#pone.0181015.t001" target="_blank">Table 1</a> for exact numbers and p values. Results are representative of 2 independent experiments. PIL were induced with pristane; ^+/+ stands for wild type, ^-/- for miR155-deficient knockouts.</p

Detailed results for lymphocyte subsets in wild type lupus (PIL^+/+), miR155-deficient lupus (PIL^-/-) and controls (CO^+/+) as assessed by flow cytometry.

<p>Detailed results for lymphocyte subsets in wild type lupus (PIL^+/+), miR155-deficient lupus (PIL^-/-) and controls (CO^+/+) as assessed by flow cytometry.</p

Anti-dsDNA, anti-chromatin and anti-histone auto-antibody levels at 8 months after disease induction.

<p>A, Anti-dsDNA IgG antibody levels were significantly lower in mir155-deficient PIL^-/- than in lupus wild type PIL^+/+. Both pristane-induced groups, PIL^+/+ and PIL^-/-, had significantly increased antibody levels compared to CO^+/+. B, C, In addition, PIL^-/- also had lower levels of anti-chromatin-antibodies and anti-histone-antibodies than PIL^+/+. Antibody levels in PIL^-/- were only slightly elevated compared to CO^+/+. Results are representative of 2 independent experiments. PIL were induced with pristane, CO with PBS; ^+/+ stands for wild type, ^-/- for miR155-deficient knockouts; bars show mean with SD. * = <i>P</i> < 0.05, by Mann-Whitney test.</p