1. LNP activates KIR2DS2+, but not KIR2DL2+, NK cells.
LNP activates KIR2DS2+, but not KIR2DL2+, NK cells. (A and B) Ba/F3 cells expressing KIR2DS2 or KIR2DL2 were incubated with 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 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 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 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 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) 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 < ).
Mohammed M. Naiyer et al. Sci. Immunol. 2017;2:eaal5296
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