Figure 3 Construction of NKG2D genetically modified CD4+ T cells and NKG2D expression assay. Three NKG2D small interfering RNA (siRNA) sequences were designed and transfected into 293T cells. After 24 h, mRNA expressions of NKG2D were assayed by RT-PCR. (A) NKG2D siRNA efficiency tested by RT-PCR revealed the highest interference for NKG2D siRNA-3. Lentiviral vectors encoding NKG2D (LV-NKG2D), NKG2D siRNA-3 (LV-siRNA) and empty blank control lentivirus (LV-BC) were constructed and titrated, then added to CD4+ T cells at a multiplicity of infection of 100 in polybrene (5 μg/mL) to produce NKG2D genetically modified and controlled CD4+ T cells. (B) Percentages of NKG2D positive CD4+ T cells transfected with LV-BC, LV-NKG2D or LV-siRNA determined by flow cytometry. (C) Bar chart of relative mRNA expression of NKG2D by genetically modified CD4+ T cells detected by real-time quantitative RT-PCR (n = 6 each group). * P < 0.01 and # P < 0.05 for within group interaction.