Construction of NKG2D genetically modified CD4+ T cells
To understand the regulation of the NKG2D on CD4+ T cells, NKG2D genetically modified CD4+T cells were constructed by transfection with lentiviral (LV) vectors encoding the over-expression of NKG2D or NKG2D siRNA, or with blank control vector. Three siRNA sequences targeting the human NKG2D sequence were transfected into 293T cells and their interference efficacy was examined by RT-PCR. NKG2D-siRNA-3 5’-CAGCAAAGAGGACCAGGAUUUACtt-3’ displayed the highest efficacy (Figure 3A) and was selected for the subsequent construction of LV-NKG2D-siRNA. LV-NKG2D was constructed and was confirmed by sequencing. Transfection of 293T cells with LV-GFP and LV-NKG2D was successful, with expression of GFP detected by fluorescence microscopy (data not shown). NKG2D expression was assessed by flow cytometry and real-time quantitative RT-PCR to confirm the success of NKG2D genetically modified CD4+ T cells (Figure 3B, 3D).