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    2020-08-18


    observed under a 200 □ fluorescence inverted microscope, because the CT DAF2 and ATF-CAR T cells were in the same morphology; therefore, the CT image is not shown. Cell phenotype was analyzed 7 days after transduction (10 days for in vitro culture), with the logarithmic am-plification of CT and ATF-CAR T cells detected using flow cytometry. The CD8+/CD4 + T cell ratio was higher than that observed in the three day in vitro culture and T cells from that day DAF2 were used for the following effector-target assays (Fig. 3E). The increase in the proportion of CD8+ cells was due to the successful activation and amplification of the culture and was the main cause of T cell cytotoxicity.
    3.5. Up-regulation of uPAR for A2780 and down-regulated uPAR expression in ES-2 cells
    We stably overexpressed uPAR in A2780 cells, which is a negative cancer cell line of uPAR. The cultured A2780 cells were transduced with either a plasmid vector designed for the stable overexpression of uPAR or an empty vector, and then selected using puromycin (800 ng/ mL) in order to enrich the stably transfected cells (Fig. 4A). As the ES-2 cell line exhibits strong uPAR expression, it was infected with a shRNA lentivirus vector and or empty lentivirus vector. Compared to the ES-2 and vector ES-2 cells, uPAR expression was drastically downregulated in the shRNA ES-2 cells. The efficacy of uPAR over- and under-ex-pression was validated by qRT-PCR (Fig. 4B).
    3.6. ATF-CAR T Cells lysed human ovarian cancer cells in an antigen-specific manner
    An essential feature of anti-cancer adoptive cell therapy is the
    Fig. 1. The expression of uPAR in each cancer cells determined by Flow cytometry assay. The RH30 cell line is human rhabdomyosarcoma served as uPAR positive control reported before. Human ovarian cancer cell lines SKOV3, C13K, HO8910 and ES-2 are all uPAR positive cancer cell line and ES-2 has the highest expression rate 95.2%, but A2780 is a uPAR negative cell line.
    Fig. 2. A schematic diagram of the structure of the com-plete ATF-CAR, using ATF to design the third generation CAR T cells. (A) UPA is a uPAR ligand with a relatively high affinity. Cleavage between Lys158 and Ile159 divides uPA into two chains. The amino-terminal fragment (ATF) contains 157 aa and is the uPAR binding site without any catalytic function, whereas the catalytic domain of uPA is catalytically active. (B) The ATF-CAR T cells used in this study are the third generation CAR T cells. ATF is used as an antigen reorganization region to recognize uPAR in cancer cells. The T cells transfected with the control len-tivirus served as negative controls.
    Fig. 3. The phenotypic characterization of T cells before and after control or ATF CAR lentivirus transduction. (A) On the third day of in vitro lymphocyte culture, flow cytometry detected that the CD3 positive rate of the untransfected cells was 99.0%. (B) The CD4 and CD8 immunophenotype on day 3 showed no obvious difference in the CD4 and CD8 positive ratios. (C) On day 10 (the seventh day after lentiviral transfection), the transduction efficiency of ATF-CAR in T cells was determined by flow cytometry. The left and right images refer to CT and ATF-CAR T cells, respectively. (D) Representative images of T cells transfected by ATF-CAR lentivirus on day 10 taken using an inversion fluorescence microscope at 200 □ (green). (E) On day 10, both CT and ATF-CAR T cells had a higher CD8+ ratio than CD4 + . The left and right images refer to CT and ATF-CAR T cells, respectively (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
    Fig. 4. UPAR cDNA expression detected by qRT-PCR in A2780- and ES-2-derived cell lines transfected with overexpression plasmid vec-tors and shRNA-lentiviruses, respectively. (A) UPAR-A2780 cells showed significant levels of uPAR overexpression compared to either A2780 or vector A2780 cells. (B) UPAR ex-pression was reduced by shRNA in ES-2 cells. Data are presented as mean ± SEM. Statistical significance was calculated by the two-tailed Student’s t-test between the two groups. *P < 0.05, **P < 0.01, and ***P < 0.001.
    ability of engineered immune cells to specifically lyse tumor cells. Thus, after lentiviral transfection, the potent cytotoxicity of CT and ATF-CAR T cells was investigated for the different E/T ratios via a standard 4 h LDH release assay. Human ovarian cancer cell lines classified by uPAR expression served as the target cells. ATF-CAR T cells were able to 
    effectively lyse uPAR-positive ovarian cancer cells but not uPAR-ne-gative cells when compared with the CT cells, regardless of the parental cancer line (A2780 or ES-2) (Fig. 5A). Additionally, the CT cells ex-hibited dose-dependent cytotoxicity in both groups, potentially due to the intrinsic cytolytic capacity of T cells; however, when the E/T ratio