NKG2D genetically modified CD4+ T cells
The lentiviral vector containing an expression cassette for NKG2D was constructed. Briefly, the synthetic gene of human NKG2D (NCBI GeneBank: AF461811.1) was amplified by PCR using the primer pair 5’AGGGTTCCAAGCTTAAGCGGCCGCGCCACCATGGGGTGGATTCGTGGTCGGAG -3’ (forward primer) and 5’TCAGTAGAGAGTGTCGGATCCTTACACAGTCCTTTGCATGCAGATGTA 3’ (reverse primer) containing Bam HI and Not I restriction sites within the 5’ and 3’ termini. The gene was inserted into the lentivirus (LV)5-CMVp-green fluorescent protein (GFP) plasmid (Genepharma, China) digested with the same enzymes used for vector production. The positive recombinant clone was identified by PCR and sequencing. The lentiviral-based vector expressing GFP and NKG2D was generated by transient co-transfection of 293T cells with a four plasmid combination including LV5, PG-p1-VSVG, PG-P2-REV, and PG-P3-RRE (Genepharma, China)according to instructions from the manufacturer. Concentration and titration were performed as described previously [14,15].
Three small interfering RNA (siRNA) sequences targeting the human NKG2D sequence (NCBI GeneBank: AF461811.1) were designed using the on-line BLOCK-iT™ RNAi Designer (Invitrogen, USA). The sequences were: NKG2D-siRNA-1:CATATCATTGGATGGGACTAGTACAtt; NKG2D-siRNA-2 :CCTCGAGCTTTAAAGGCTATATAGAtt; NKG2D-siRNA-3:CAGCAAAGAGGACCAGGAUUUACtt; and Negative Control: UUCUCCGAACGUGUCACGUtt. The siRNAs were transfected into 293T cells using Lipofectamine 2000 (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. After 24 h, total RNA isolated using TRIzol (Invitrogen) was reverse transcribed by RT-PCR (TaKaRa Bio, Japan) according to the instructions of the manufacturer to test the NK2D-siRNAs efficacy. Amplified cDNAs were used for PCR with the following NKG2D primers: forward 5’- AGCTCACTGCTGAGGTCCTA -3’ and reverse 5’- CCACGAATCCACCCCATCAA-3’. The size of PCR product was 483 bp. β-actin was detected as the control. The PCR conditions were 95℃ for 5 min followed by 30 cycles of 94℃ for 30 s, 62℃ for 30 s, and 72℃ for 1 min, and 72℃ for 5 min. The most efficacious siRNA (siRNA-3) was selected for construction of the lentiviral vectors. The lentiviral vectors were generated by co-transfecting 293FT cells with four plasmids (pLVX-NKG2D-RNAi, PG-p1-VSVG, PG-P2-REV, and PG-P3-RRE; Genepharma, China). The vector plasmids were constructed with double-stranded DNA formed by annealing the Oligo DNA target sequence with the vector digested with Eco RI and Bam HI. The cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% FBS for 72 h, the supernatants were harvested, then concentration and titration were performed as described previously[14,15].
The purified CD4+ T cells were cultured in 1640 medium and stimulated with anti-CD3 and anti-CD28 monoclonal antibodies (Invitrogen) for 12 h. After activation, the lentiviral vectors encoding NKG2D, NKG2D siRNA, and empty blank control were added to 1 × 106 CD4+ T cells at a multiplicity of infection of 100 in 24-well plates in the presence of polybrene (5 μg/mL) to produce the NKG2D genetically modified and controlled CD4+ T cells. Mixtures of cells and vectors were centrifuged at room temperature for 180 min at 1000 rpm, then cultured in 1640 medium containing 10% FBS for 72 h. The transfection efficiency was measured by the percentage of positive cells. The efficiency of genetic modification was determined by quantitative real-time RT-PCR. Transfected cells were used for functional analysis within a week of preparation.