Figure 3.

Poor effect of Ly49A receptor crosslinking on NK1.1-induced Ca²⁺ flux in NK cells from mice expressing the Ly49A ligand H2D^d. (A) Top: Staining of Ly49A on NKp46⁺ NK cells from H2D^d mice at various dilutions of anti-Ly49A antibodies. Bottom: Kinetics plot of the Fluo-4/Fura-red flourescence ratios in NK cells from H2D^d mice stained with various concentrations of anti-Ly49A together with a fixed concentration of anti-NK1.1. The blue line represents the response of the NK cell subset expressing Ly49A and the red line represents Ly49A-negative NK cells in the same sample. (B) Same setup as in A but on NK cells from B6 mice. (C) Mean fluorescence intensity (MFI) of Ly49A staining at various dilutions of anti-Ly49A on NK cells derived from B6 (black bars) or H2D^d (white bars) mice. Three independent experiments are pooled. Arrows indicate antibody titrations at which the MFI of Ly49A staining was similar between B6 and H2D^d mice. (D) AUC values of Ca²⁺ flux in Ly49A-positive and Ly49A-negative NK cells from B6 and H2D^d mice at the anti-Ly49A concentrations indicated by arrows in (C) (1:100 for H2D^d and 1:3,200 for B6). Statistical analysis performed using one-way ANOVA with Tukey's multiple comparisons test. (E) Top: Staining of Ly49G2 on NKp46⁺ cells from H2D^d mice. Bottom: Kinetics plot of the Fluo-4/Fura-red flourescence ratios in Ly49G2-positive (blue) and Ly49G2-negative (red) NK cells from H2D^d mice stained with anti-Ly49G2 at 1:400 dilution. (F) Inhibition index comparison between the degree of Ly49G2-mediated inhibition in H2D^d mice and B6 mice. Based on 3–4 independent and comparable experiments. Statistics was calculated using a paired Student's T-test. ^*p < 0.05, ns , not significant.