NKT-TCR mRNA transfection confers functionality.

<p>As shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131477#pone.0131477.g002" target="_blank">Fig 2</a>, T cells were electroporated with NKT-TCR mRNA. (A) Six hours after NKT-TCR mRNA electroporation, Vα24⁺Vβ11⁺ cells (left panel) and Vα24^-Vβ11^- cells (right panel) were sorted. (B) Each population was exposed to plate-bound CD1d-dimer loaded with α-GalCer for the indicated periods of times. As positive control, PBMCs were activated with PMA (50 ng/ml) and Ionomycin (1 μg/ml) for 5 min. Cells were then lysed and pERK1/2 and ERK1/2 were detected by Western blotting (upper panel). The bar diagram shows a densitometric analysis of the phosphorylated ERK signal from the upper band normalized by the corresponding total ERK signal (lower panel). (C) Sorted 1x10⁵ Vα24⁺Vβ11⁺ cells (top), Vα24^-Vβ11^- cells (middle) or NKT line (bottom) were cocultured with 1x10⁴ CD1d-HEK293 loaded with or without α-GalCer for 24 h. IFN-γ and IL-4 production in the culture supernatants was analyzed by ELISA. Data are mean ±SEM from triplicates and representative of 5 healthy donors with similar results. (***p<0.005 for CD1d-HEK293/Gal vs. CD1d-HEK293/non and T only)