a Schematic representation of NKG2D-targeted constructs with CD28 and 4-1BB costimulatory domain, a CD3ζ domain, and a shRNA-4.1R. b Flow cytometry analysis of the transduction efficiencies. T cells were transfected by the lentiviruses expressing NKG2D-CAR and the transfection efficiency was analyzed by FACS after the cells were stained with fluorescently-labeled anti-NKG2D antibodies. c Quantitative real-time PCR analysis of 4.1R mRNA level in Mock T cells, NC-NKG2D-CAR T cells, KD1-NKG2D-CAR T cells, and KD2-NKG2D-CAR T cells. Total mRNAs of the transfected cells were extracted and reversely transcribed, the expression level of 4.1R was analyzed by quantitative real-time PCR. d Western blot analysis of 4.1R protein level in Mock T cells, NC-NKG2D-CAR T cells, KD1-NKG2D-CAR T cells, and KD2-NKG2D-CAR T cells. Data were representative of three independent experiments. ***P < 0.001, NS not significant.