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).
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 . CAR T cells combine the antigen-binding functions of antibodies and T-cell-activating functions to mimic physiological T cell-mediated cytotoxicity . 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 . 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 , our study observed far fewer Th2 cytokines than Th1 cytokines. Granzyme B can induce cancer cell death by caspase proteins in CTLs . 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.