br T cell transduction and expansion br Human peripheral blo
2.5. T cell transduction and expansion
Human peripheral blood lymphocytes were obtained from anon-ymous healthy donors who provided written informed consent ac-cording to protocols approved by the Ethics Committee of the Second Hospital of Jilin University. Cells are activated with anti-CD3/anti-CD28 Lovastatin (T&L Biological Technology, Beijing, China) at a concentration of 5 × 105 cells/ml culture medium. For activation of lymphocytes, non-tissue culture-treated 24-well plates (JET BIOFIL, Guangzhou, China) had been previously coated over night with 0.5 ml of 1 μg/ml anti-CD3 and 1 μg/ml anti-CD28, diluted in PBS. Culture medium consisted of 50% RPMI 1640 and 50% X-vivo-15 (Irvine Scientific, USA), with 10% heat-inactivated Human Serum AB (Sigma-Aldrich, USA) containing IL-2 (PeproTech, USA). On day 3, activated T cells were transduced with lentivirus by MOI = 20 with 8 μg/ml Polybrene (Sigma-Aldrich, USA) in a 24-well non-tissue culture-treated plate, previously coated with 7 μg/ml RetroNectin (Takara Bio, Japan) in PBS. Viability of T cells was assessed through Trypan blue staining (Gibco, USA). Medium change with fresh addition of IL-2 was per-formed on days 3, 5, 7. CAR T cells were cultivated in 6-well tissue culture plates (1 × 106 cells/ml).
Cancer cells, including the human ovarian cancer cell line A2780, SKOV3, C13 K, HO8910, ES-2 and human rhabdomyosarcoma cell line RH30 (the positive control of uPAR expression demonstrated by Pilbeam et al ) were cultured in the six orifice plate until fusion of 85%, after using EDTA digestion and PBS washing and T cells were only washed by PBS, then under the incubation at 4 °C avoiding light for 20 min staining with antibodies. The fluorescent staining conjugated antibodies were uPAR-PE, CD3-APC, CD4-BB700, CD8-PE (all from BD Biosciences, USA), and the antibody dose was 10 μl / 106 cells and then tested by BD FACSCanto II flow cytometer, and flow cytometric data were analyzed using FlowJo version 7.2.5 software (TreeStar, USA).
2.7. The generation of up-expression vector and small hairpin RNA (shRNA) lentivirus for uPAR gene (PLAUR)
Since A2780 is a uPAR-negatively expressed cell line, the uPAR-positive A2780 cell line was constructed by overexpression of plasmid transfection. The uPAR sequence was derived from the NCBI database (NM_002659.4) and human PLAUR over-expression vector and
scramble vector were purchased from VectorBuilder Inc. (Shenandoah, Texas). The plasmid was extracted using the TIANGEN Plasmid Mini Kit (Beijing, China) and stored at −20 °C. Transfected into logarithmic phase A2780 using the Lipofectamine™ 3000 Transfection Reagent (Invitrogen, USA) according to the manufacturer’s instructions. Over-expressed stably transfected cell line and control vector transfected cell line were obtained by puromycin selection (80 ng/ml) 3 days after transduction. Mock A2780, vector A2780 and uPAR-overexpressing A2780 were cultured and tested by q-RT PCR for uPAR expression. On the contrary, ES-2 is a uPAR strong positive cell line, so it was knocked down using the shRNA lentiviral system. The low sequence was: CAC TCTCCTCTGGACCTAAAC. The connection and packaging of the viral vector was completed by Beijing Saiye Company.
2.8. Quantitative real-time polymerase chain reaction
Total RNA was prepared from cultured cells using Trizol reagent (Invitrogen) following the manufacturer’s instructions. RNA was re-verse transcribed to cDNA using the TransScript All‐in-One first‐strand cDNA synthesis SuperMix (Transgen Biotech, Beijing, China). Real‐time PCR was performed using the TransStart Top Green qPCR SuperMix (Transgen Biotech) and the ABI 7300 Real‐Time qPCR system. All PCR experiments were performed in triplicate. The primers were synthesized by Comate Bioscience (Changchun, China). Sequences for primers used for RT-qPCR are as follows: uPAR (Forward: 5′- TGTAAGACCAACGG GGATTGC-3′, Reverse: 5′-AGCCAGTCCGATAGCTCAGG-3′) and b-actin (Forward: 5′-GGATTACAGCTCACCATGGA -3′, Reverse: 5′- AATCCTT CTGACCCATGCC -3′). Relative quantification of the expression of each gene was calculated by 2− Ct method. r> 2.9. Detection of CAR t cytotoxicity
On day 7 after the lentiviral transduction of T cells, A2780, vector A2780, uPAR-A2780, ES-2, vector ES-2, and shRNA ES-2 cells in 96-well U-bottom microplates were treated with ATF-CAR T cells and control T (CT) cells at diﬀerent eﬀector-target (E/T) ratios (1:1, 5:1, 10:1, and 20:1). The lactic dehydrogenase (LDH) released from the cancer cells was measured to ensure CAR T cell cytotoxicity. Before co-culture, T cells were cultured with a complete medium without IL-2 for 6 h and cancer cells were plated in 96-well plates for 4 h. T cells were then added to the ovarian cancer cells at the ratios mentioned above and co-incubated at 37 °C in 5% CO2 for 4 h. LDH activity was detected by the manufacturer’s protocol (Dojindong, Japan).