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

Binding of KIR2DL3-Fc and inhibition of primary KIR2DL3+ NK cells is significantly stronger when target cells are pulsed with the T_Gag303V variant peptide.

<p>(A) Representative dot plots of KIR2DL3-Fc staining of 721.221-ICP47-C*03:04 cells pulsed with positive control peptide GAL, negative control peptide GKL, T_Gag303 wild-type peptide, or T_Gag303V variant peptide. Staining was measured by flow cytometry after addition of a secondary PE-conjugated anti-IgG antibody. All cells express GFP, indicating successful transduction of ICP47 and thus optimal blockade of TAP. (B) Quantification of KIR2DL3 binding expressed as percent of cells binding KIR2DL3-Fc. Means of six independent experiments, with error bars representing SD, are shown. Binding of KIR2DL3-Fc was significantly stronger to 721.221-ICP47-C*03:04 cells pulsed with the variant T_Gag303V (YVDRFFK<u>V</u>L) peptide than to those pulsed with wild-type (YVDRFFK<u>T</u>L; <i>p</i> = 0.002) or T_Gag303A (YVDRFFK<u>A</u>L; <i>p</i> = 0.002) peptide. (C and D) Degranulation of primary KIR2DL3+ (C) and KIR2DL3− (D) NK cells measured as CD107a expression after incubation with 721.221-ICP47-C*03:04 cells pulsed with T_Gag303 wild-type and T_Gag303A and T_Gag303V variant peptides. The percentage of CD107a+ NK cells in response to the respective peptide is normalized to the percentage of CD107a+ NK cells for KIR2DL3− and KIR2DL3+ NK cell subsets after co-incubation with target cells pulsed with GKL (GAVDPLLKL) control peptide. 721.221-ICP47-C*03:04 target cells presenting YVDRFFK<u>V</u>L significantly inhibit degranulation of KIR2DL3+ NK cells compared to targets pulsed with YVDRFFK<u>T</u>L (<i>p</i> = 0.0121) or YVDRFFK<u>A</u>L (<i>p</i> = 0.0019). The different peptide variants had no detectable effect on CD107a expression in KIR2DL3− NK cells. All <i>p</i>-values stated are adjusted for multiplicity of testing. Primary NK cells from nine different healthy <i>KIR2DL3+</i> participants were tested. (E) Sensogram of KIR2DL3 dimeric analyte binding to biotinylated HLA-C*03:04/YVDRFFKTL, HLA-C*03:04/YVDRFFKAL, and HLA-C*03:04/YVDRFFKVL monomers on a Streptavidin chip; an empty well served as negative control. The sensogram data are normalized to the amount of respective HLA monomer immobilized on the chip by HC10 antibody. Each fit was obtained using double reference subtraction and is shown as a black line. Affinity of the KIR2DL3/HLA-C*03:04/YVDRFFKVL protein-protein interaction was highest. *<i>p</i> < 0.05; **<i>p</i> < 0.01.</p

Structural details of the HLA/peptide/KIR three-way complex.

<p>(A) Overall structure of the HLA/peptide/KIR complex. The peptide is buried in the HLA class I binding grove, while KIR interacts with both helices of HLA class I as well as the C-terminus of the peptide. (B) Comparison of the interactions around the mutated viral residue (top: in fat sticks; bottom: in spheres). The YVDRFFK<u>T</u>L (YTL) wild-type peptide includes a hydrogen bond between the side-chain oxygens of Thr₈ and Asn₈₀, while the YVDRFFK<u>V</u>L (YVL) variant improves the hydrophobic packing against Val₇₆. Interestingly, Asn₈₀ participates in a hydrogen bond expected to confer allotype specificity to KIR2Ds.</p

Equal HLA-C*03:04 stabilization on TAP-blocked 721.221-ICP47-C*03:04 cell line with HIV-1 p24 Gag T_Gag303 wild-type and variant peptides.

<p>(A) Representative histograms of HLA-C*03:04 stabilization with T_Gag303 wild-type and T_Gag303A and T_Gag303V variant peptides compared to addition of no peptide (−). HLA-C*03:04 surface levels were determined by flow cytometry using an anti-pan—HLA class I antibody (clone W6/32). Peptides were added at a saturating concentration of 100 μM. (B) Quantification of HLA-C*03:04 stabilization in the presence of T_Gag303 wild-type and T_Gag303A and T_Gag303V variant peptides, as well as positive endogenous control peptides for HLA-C*03:04 stabilization (GAVDPLLAL and GAVDPLLKL) and a non-HLA-C*03:04-stabilizing influenza-derived control peptide (Flu, ILRGSVAHK). Data represent mean of five experiments with error bars indicating the SD. Relative fluorescence intensity (RFI) was calculated as the gMFI of the sample divided by the gMFI of 721.221-ICP47-C*03:04 cells stained in the absence of peptide.</p

KIR footprints in HIV-1 clade C sequence.

<p>^aHIV-1 consensus sequence in 392 study participants.</p><p>^bDominant variant.</p><p>^cConsensus T was also significantly associated with the lack of encoding <i>KIR2DL3/HLA-C*03</i>:<i>04</i> (<i>q</i> = 0.1).</p><p>^dVariant amino acid A at Gag₃₀₃ was significantly associated to an <i>HLA-C*03</i>:<i>04</i> genotype alone (<i>p</i> < 0.001), but not in combination with KIR2DL3.</p><p>KIR footprints in HIV-1 clade C sequence.</p