Research ArticleNATURAL KILLER CELLS

KIR2DS2 recognizes conserved peptides derived from viral helicases in the context of HLA-C

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Science Immunology  15 Sep 2017:
Vol. 2, Issue 15, eaal5296
DOI: 10.1126/sciimmunol.aal5296
  • Fig. 1 KIR2DS2 recognizes the HCV peptide LNP.

    (A) Peripheral blood mononuclear cells (PBMCs) were stimulated overnight with interleukin-15 (IL-15) and incubated for 4 hours with 721.174 cells that had been loaded with VAPWNSFAL or with VAPWNSFAL plus the indicated HCV peptides at a concentration of 5 μM. The mean CD107a expression plus 1 SD on CD3CD56+CD158b+ NK cells from three unselected donors is shown. (B to D) CD107a degranulation assays of NK cells from KIR2DL2+/KIR2DS2+ and KIR2DL3+/KIR2DS2 donors in response to 721.174 cells incubated with indicated peptides. Peptides were used at 5 μM each. (B) Flow cytometry histogram plots of CD107a expression gated on CD3CD56+CD158b+ NK cells from two donors of different KIR genotype. The results from five donors of each genotype are summarized for CD158b+ NK cells in (C) and for CD158b NK cells in (D). Means and SEs for each condition are shown. P values indicate comparison to VAPWNSFAL alone. ns, not significant. (E and F) NKL cells either untransfected or transfected with KIR2DL2 or KIR2DS2 were incubated with 721.221 cells transfected with HLA-C*0102 or with HLA-C*0102 + LNPSVAATL (C*0102-LNP), and the cytotoxicity of the 721.221 cells was measured by flow cytometry. (E) Flow cytometry histogram plots of the uptake of the LIVE/DEAD Fixable Aqua Dead dye by the target cells incubated with the indicated effector cells. The means and SEs from three independent cytotoxicity experiments are shown in (F). Effector-to-target (E/T) ratios are also shown. P values indicate the comparison of cytotoxicity between the HLA-C*0102 and the HLA-C*0102–LNP targets. Where shown, P values were determined by independent two-tailed t tests (*P < 0.05, **P < 0.01, ****P < 0.0001).

  • Fig. 2 LNP activates KIR2DS2+, but not KIR2DL2+, NK cells.

    (A and B) Ba/F3 cells expressing KIR2DS2 or KIR2DL2 were incubated with 721.174 cells loaded with the indicated peptides. Cells were fixed and stained with anti-CD158b before analysis by confocal microscopy (A). Peptides were used at a concentration of 100 μM. Arrowheads indicate clustering at the interface between the Ba/F3 and 721.174 cells. The intensity of staining of the Ba/F3 cells at the interface was compared with the membrane at a noncontact area and plotted as the fold increase above background (B). The results from three independent experiments with a total of 30 conjugates per condition are shown. The P value in comparison to 2DS2 with no peptide is shown. (C) NKL-2DS2 cells were incubated with 721.174 cells loaded with the indicated peptides, and DAP12 was coimmunoprecipitated with anti-2DS2 antibody from the cell lysates before Western blotting (WB) with anti-phosphotyrosine (pTyr) or anti-DAP12. DAP12 was also immunoprecipitated from NKL-2DS2 cells in the absence of 721.174 target cells (no target). Densitometry results of the pDAP12/DAP12 ratio from three independent experiments and P values in comparison to no peptide are shown. IP, immunoprecipitation. (D) NKL-2DS2 or NKL-2DL2 cells were incubated with 721.174 cells loaded with the indicated peptides and Western blotting for phospho-Vav1 (pVAv1) or Vav1 performed. “No target” lanes indicate immunoprecipitation from NKL-2DS2 or NKL-2DL2 cells alone. Densitometry results of the phospho-Vav1/Vav1 ratio from three independent experiments and P values in comparison to no peptide are shown. (E) 721.174 cells were cultured with 100 μM peptide (VAWPNSLSL or LNP) overnight, stained with the KIR2DL2-Fc fusion construct, and analyzed by flow cytometry. The histogram plots for the two peptides (filled histograms) and the median fluorescence of KIR2DL2-Fc, compared with no peptide (open histograms), are shown. For all panels, P values were derived using one-way analysis of variance (ANOVA), with Dunnett’s test for multiple comparisons (*P < 0.05, ****P < 0.0001).

  • Fig. 3 Endogenously presented LNP is recognized by KIR2DS2 in the context of HCV.

    (A) HUH7 cells expressing the N17 (JFH1ΔE1E2-luc) replicon with (right) or without (left) HLA-C*0102 were co-incubated with NKL, NKL-2DL2, or NKL-2DS2 cells at the indicated E/T ratios. Luciferase activity from the cocultures was measured and normalized to expression in the absence of NKL cells. The relative inhibition of replication was then measured. The means and SEs of three experiments performed in duplicate are shown. Comparisons for NKL-2DS2 with NKL-2DL2 are indicated. (B) Replication of wild-type (WT) N17 (JFH1ΔE1E2-luc) replicon RNA or variants in which the LNP epitope had been mutated to aspartate at residues 3, 7, 8, and 9 (P3D, A7D, T8D, and L9D, respectively). As a nonreplicating control (Control), the N17 replicon carrying the lethal GND mutation in the viral NS5B protein was used. RLU, relative light units. (C) NKL, NKL-2DL2, or NKL-2DS2 cells were cocultured with HUH7-C*0102 cells expressing either wild-type or the L9D mutant HCV replicon. Luciferase activity was measured and plotted as percentage inhibition of viral RNA replication compared with HUH7 cells incubated in the absence of NKL cells. The means and SEs of three experiments performed in duplicate and P values for comparisons between NKL-2DS2 and NKL-2DL2 are shown. (D) NKL, NKL-2DL2, or NKL-2DS2 cells were cocultured with HUH7-C*0304 cells expressing either wild-type (left) or the L9D mutant HCV replicon (right). The percentage inhibition of replication compared with the no NKL control is shown. Statistical analyses for (A), (C), and (D) were performed using independent two-tailed t tests to compare NKL-2DS2 and NKL-2DL2. (E and F) 721:C*0304:ICP47 cells were loaded with the indicated peptides at saturating concentrations (100 μM) and then stained for HLA-C expression using the DT9 antibody (E) or the KIR2DS2–phycoerythrin (PE) tetramer (F). One representative histogram plot from three independent experiments is shown for each peptide, and the median fluorescence intensity of staining was indicated. Dark lines indicate peptide staining compared with the no peptide control (gray lines). (G) Western blot for phospho-Vav1 and Vav1 from 721.221:C*0304:ICP47 cells cultured with 20 μM of the indicated peptide and co-incubated with NKL-2DS2 cells at an E/T ratio of 1:1. One representative blot is shown together with densitometry of the phospho-Vav1/Vav1 ratio (mean ± SD) from three independent experiments. Statistical analysis was performed using one-way ANOVA, with Dunnett’s test for multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.001).

  • Fig. 4 Protein sequence alignment of the NS3 helicase–encoding region of 63 flaviviruses demonstrates conservation of a KIR2DS2-binding peptide.

    The box indicates the putative KIR2DS2-binding peptide. Numbering is from the start of the NS3 protein of DENV.

  • Fig. 5 KIR2DS2 recognizes conserved helicase peptides from multiple flaviviruses.

    (A) KIR2DS2 tetramer (black lines) binding to 721.174 cells incubated with the indicated peptides at saturating concentrations (200 μM) as compared with no peptides (gray lines). The median fluorescence of tetramer staining of peptide is indicated (JEV). (B) NKL-2DS2 cells were incubated with 721.174 cells preloaded with the indicated Flavivirus peptides and then assayed for phospho-Vav1 and Vav1 by Western blotting. One representative blot of each is shown. (C) The peptide IVDLMCHATF (IVDL) was coexpressed with HLA-C*0102 in 721.221 cells and used as targets in cytotoxicity assays in which the effector cells were NKL, NKL-2DL2, and NKL-2DS2. Experiments were performed at an E/T ratio of 6:1, and comparisons were made between the HLA-C*0102 transfectant and the HLA-C*0102:LNP or HLA-C*0102:IVDL cells. (D to F) HEK, HEK-C*0102, or HEK:C*0304 cells expressing a DENV replicon (HEK:DENV, HEK:C*0102:DENV, or HEK:C*0304:DENV, respectively) or not were cocultured with NKL cells expressing KIR2DS2 or KIR2DL2, and cytotoxicity was assessed at 24 hours. The effect of DENV on KIR2DS2-mediated killing is shown in (D), and the effect of HLA-C*0102 on KIR2DS2-mediated killing is shown in (E) and fig. S5. A comparison of the effect of HLA-C*0304 on killing of DENV-expressing cells with that of HLA-C*0102 by NKL-2DS2 is shown in (F). Means and SEs of at least three independent experiments performed in triplicate are shown. (G) Comparison of HCV (left) and dengue NS3 helicases (right) showing contact of the LNP (HCV) and IVDLMCHATF (Dengue) epitopes with the nucleic acid within the helicase domain (yellow). Selected amino acids from the epitopes are illustrated. Images were derived from structures published by Gu and Rice (25) and Luo et al. (29) and rendered using PyMOL. Where shown, P values were determined by independent two-tailed t tests (*P < 0.05, **P < 0.01, ***P < 0.001).

  • Table 1 Genetic association of activating KIRs with their putative ligands and the outcome of HCV infection in 336 individuals exposed to HCV (216 chronically infected and 120 with resolved HCV infection).

    Logistic regression was performed using SPSS v20.0 using the ENTER method. OR > 1 indicates protection against chronic HCV infection.

    POR95% CI
    KIR2DL3/L3:HLA-C1/C10.0052.681.34–5.37
    KIR3DS1:HLA-BBw40.0322.161.07–4.37
    KIR2DS2:HLA-C10.0332.061.06–4.02
    KIR2DS30.0330.510.27–0.95
    KIR2DS1:HLA-C10.1700.590.28–1.25
    KIR2DS2:HLA-A*110.3940.640.23–1.78
    KIR2DS1:HLA-C20.5090.740.31–1.80
    KIR2DS2:HLA-C20.7271.130.58–2.18

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/2/15/eaal5296/DC1

    Fig. S1. Peptide stabilization of HLA-C*0102 by HCV peptides.

    Fig. S2. The effect of single-HCV peptides on degranulation of CD158b NK cells.

    Fig. S3. Cytotoxicity of KIR2DS2+ NK cell clones to peptide-loaded 721.174 cells.

    Fig. S4. KIR2DS2 tetramer binding to HLA-C*0102 and HCV peptides or VAPWNSLSL peptide derivatives.

    Fig. S5. Flow cytometry plots comparing binding of KIR2DS2*001, KIR2DS2*007, and KIR2DS2*008 to HLA-C*0102 and peptide.

    Fig. S6. Analysis of viral RNA and protein production in DENV replicon–containing cells compared with DENV-2–infected cells.

    Fig. S7. Flow cytometry plots illustrating gating strategy and killing of HEK cells expressing GFP-tagged DENV replicon by NKL-2DS2 cells.

    Table S1. Summary of HCV peptides identified to bind HLA-C*0102.

    Table S2. Protein sequence alignment of HCVs.

    Table S3. Flavivirus NS3 HLA-C*0102–binding peptides as determined by NetMHCpan.

    Table S4. Accession numbers of Flavivirus sequences used to compile the alignment in Fig. 4.

    Data file S1. Raw data for Figs. 1 to 5.

    Data file S2. Western blot gels for Figs. 1 to 5.

  • Supplementary Materials

    Supplementary Material for:

    KIR2DS2 recognizes conserved peptides derived from viral helicases in the context of HLA-C

    Mohammed M. Naiyer, Sorcha A. Cassidy, Andrea Magri, Vanessa Cowton, Kevin Chen, Salah Mansour, Hariklia Kranidioti, Berenice Mbirbindi, Pauline Rettman, Scott Harris, Liam J. Fanning, Arend Mulder, Franz H. J. Claas, Andrew D. Davidson, Arvind H. Patel, Marco A. Purbhoo, Salim I. Khakoo*

    *Corresponding author. Email: s.i.khakoo{at}southampton.ac.uk

    Published 15 September 2017, Sci. Immunol. 2, eaal5296 (2017)
    DOI: 10.1126/sciimmunol.aal5296

    This PDF file includes:

    • Fig. S1. Peptide stabilization of HLA-C*0102 by HCV peptides.
    • Fig. S2. The effect of single-HCV peptides on degranulation of CD158b NK cells.
    • Fig. S3. Cytotoxicity of KIR2DS2+ NK cell clones to peptide-loaded 721.174 cells.
    • Fig. S4. KIR2DS2 tetramer binding to HLA-C*0102 and HCV peptides or VAPWNSLSL peptide derivatives.
    • Fig. S5. Flow cytometry plots comparing binding of KIR2DS2*001, KIR2DS2*007, and KIR2DS2*008 to HLA-C*0102 and peptide.
    • Fig. S6. Analysis of viral RNA and protein production in DENV replicon– containing cells compared with DENV-2–infected cells.
    • Fig. S7. Flow cytometry plots illustrating gating strategy and killing of HEK cells expressing GFP-tagged DENV replicon by NKL-2DS2 cells.
    • Table S1. Summary of HCV peptides identified to bind HLA-C*0102.
    • Table S2. Protein sequence alignment of HCVs.
    • Table S3. Flavivirus NS3 HLA-C*0102–binding peptides as determined by NetMHCpan.
    • Table S4. Accession numbers of Flavivirus sequences used to compile the alignment in Fig. 4.

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    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Raw data for Figs. 1 to 5.
    • Data file S2 (.pdf format). Western blot gels for Figs. 1 to 5.

    Files in this Data Supplement:

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