Figure 2. Construction and characterization of the chNKG2D.

A, The figure schematically illustrates the domain architecture of wildtype NKG2D and chNKG2D (N = N-terminus, C = C-terminus, CM = cytoplasma membrane). The N-terminal truncation of the extracellular NKG2D domain at aa81 is indicated by a bold bar. The native C-terminal end of NKG2D was fused to the N-terminus of the IgG1-Fc/CD28/CD3ζ transmembrane signaling platform creating a transmembrane receptor of type I orientation in which the relative orientation and positioning of the NKG2D ectodomain has not been changed. B, efficient transfection and strong surface expression of chNKG2D by electroporation of mRNA into primary T cells. The histograms show the surface expression of chNKG2D 24 hours after electroporation of quiescent or anti-CD3-activated human T cells (thick line, open histogram). Analysis was performed by flow cytometry using an anti-CD314 antibody. The expression of endogenous wildtype NKG2D in T transfected with a CMV-gH-specific CAR is shown for comparison (thin line, open histograms; filled histograms = isotype controls). C, chNKG2D triggers cytotoxicity of activated T cells. Quiescent or anti-CD3-activated CD8^pos or CD4^pos T cells were transfected with RNA encoding either the chNKG2D or a CMV-gH-specific control receptor. One day after transfection, the T cells were co-cultured with dye-labeled murine B cells (Ba/F3-MICA) stably expressing the human NKG2D-L MICA. The diagram shows the percentage of Annexin V^pos murine target cells after 5 hours of co-culture at the indicated E∶T ratios (data pooled from three independent experiments using different cell donors; mean ± S.D.). Statistical significance was calculated for the effects of activated T cells expressing the chNKG2D versus T cells expressing the control receptor.