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  • br Fig In vitro cytotoxic activities of ATF


    Fig. 5. In vitro cytotoxic activities of ATF-CAR T DAF-2 against target cells. (A) The ATF-CAR T cells and CT cells were co-cultured with A2780, vector A2780, uPAR-A2780, ES-2, vector ES-2 and shRNA ES-2 cells at four laddering effector-to-target ratios. Then the cell co-cultured wells were measured by LDH release cytotoxicity assay. The cytotoxicity of ATF-CAR T cells are uPAR-specific and in a dose-dependent manner. (B) At the ratio 10:1, the ATF-CAR T cells had significantly higher percent specific lysis compared with CT cells in uPAR-positive cells which was not showed in uPAR-nagetive cell groups. 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, *** P < 0.001.
    increased from 10:1 to 20:1, cell lysis increased slightly. Since cell lysis was already above 60% at a ratio of 10:1, this was regarded as the most practical ratio for further experiments due to the balance of cytotoxicity and T cell availability. At a ratio of 10:1, ATF-CAR T cells exhibited significant lysis cytotoxicity against uPAR-positive cells compared to the CT cells, but the two groups of T cells exhibited no statistically significant activity against ZYN-negative cells (Fig. 5B).
    3.7. ATF-CAR T Cells produced cytokines and released granzyme B in cytotoxity lysis
    To examine cytokine production and granzyme B release, either the CT or ATF-CAR T cells served as effector cells and vector A2780, uPAR-A2780, vector ES-2 and shRNA ES-2 cells served as target cells at a ratio of 10:1. The cell supernatants of the effector and target cell co-culture were collected to analyze Th1/Th2 cell cytokine secretion by Th1/Th2 CBA kit and granzyme B release by ELISA. Higher levels of Th1 cyto-kines such as IFN-γ, TNF and IL-2, especially IFN-γwere produced by ATF-CAR T cells co-cultured with uPAR-positive cells compared with the CT cells, but any effect was observed for the uPAR-negative cells (Fig. 6A). Th2 cytokines, such as IL-4, IL-6 and IL-10 were detected in a relatively slight level in each effector-target group (Fig. 6A). Granzyme B is released by CTLs and natural killer cells in the direction of the target cell as part of the immune response. In the uPAR-positive cancer cells, the level of granzyme B released by the ATF-CAR T cells was significantly higher than that released by the CT cells; again these re-sults were not observed for the uPAR-negative cancer cells (Fig. 6B).
    4. Discussion
    In this study, we reported uPA and soluble uPAR levels in the serum of patients with malignant and benign ovarian tumors and uPAR pro-tein expression was elevated in human ovarian cancer cell lines. We succeeded in constructing anti-uPAR CAR (ATF-CAR) based on natural ligand-receptor binding (ATF binding to uPAR) and described the 
    characteristics of transfected T cells. Silencing uPAR in the uPAR-po-sitive cell line ES-2 and upregulating uPAR in the uPAR-negative cell line A2780 had a precise effect, allowing us to verify the cytotoxicity and cytokine release of ATF-CAR T cells against ovarian cancer by specifically targeting uPAR. We demonstrated that uPAR is an im-portant candidate marker for CAR T cell therapy against ovarian cancer and that ATF-CAR T cells exhibited cytotoxicity against uPAR-positive cancer cells in an antigen-specific and dose-dependent manner.
    In the immune response, T cell-mediated cytotoxicity against target cells generally depends on CD8 + T cells and some CD4 + T cells, which tend to secrete the IFN-γ, TNF-α, and IL-2. The immunological synapse (TCR-antigen-MHC) actives the perforin–granzyme pathway or the FAS-mediated cytotoxicity pathway via the apoptotic pathway of target cells [16]. CAR T cells combine the antigen-binding functions of antibodies and T-cell-activating functions to mimic physiological T cell-mediated cytotoxicity [17]. Currently, most preclinical and clinical studies on CAR T cells transfect whole T lymphocytes to produce CAR T cells; however, the phenotypic sorting of lymphocytes before lenti-virus/retrovirus transfection affects their cytotoxic ability and func-tional sustainability. For example, anti-NKG2DL-CD45RA-memory T cells have limited T cell efficacy and are active over a shorter period with minimal or no toxicity [18]. Both CD4+ and CD8 + T cells were lentivirally-transfected in our study and further studies will explore the specific T cell subtype with a key effector role in cytotoxicity. Although there have been reports demonstrating high levels of Th2 cytokine se-cretion in CAR T cell cytotoxicity [19], our study observed far fewer Th2 cytokines than Th1 cytokines. Granzyme B can induce cancer cell death by caspase proteins in CTLs [20]. In this study, granzyme release in the CAR-T-treated group was far higher than in the control T-treated group, confirming the efficiency of CAR-T cells.