1. Expression of CAR, endogenous TCR, and target-cell receptors.
Expression of CAR, endogenous TCR, and target-cell receptors. A, CAR.OT-I and OT-I TCR expressions were analyzed by labeling day 7 activated T cells with both an FITC-conjugated anti–c-myc tag antibody and PE-conjugated TCR Vα2 antibody. Cells were gated on TCR Vα2, and expression of CAR via c-myc tag is shown in a representative overlay histogram for the isotype control (dotted black line) and c-myc tag (thick black line). B, antigen receptor quantification on effector CAR.OT-I, OT-I and transduced wild-type T cells (C57BL/6 CAR) was performed using a flow cytometry–based assay with Simply Cellular Beads, a proprietary kit from BANGSLABS. Antibody directed to c-myc Tag (FITC) was used to determine the expression of CAR on transgenic CAR.OT-I T cells and transduced wild-type C57BL/6 T cells. Antibody specific for Vα2 TCR were used to determine the expression level of OT-I TCR on OT-I and CAR.OT-I T cells. Data are representative of two separate experiments. C, to assess tumor antigen expression, MC57-HER2 target cells were labeled with an anti-human HER2. MC57-HER2 (D) or MC57 (E) were pulsed with OVA257, followed by staining with H-2Kb antibody (left), or 25-D1.16 antibody (H-2Kb-OVA257; right). Cells were analyzed by flow cytometry and overlay histograms are shown for the isotype control (gray line) and the test mAb (black line). The isotype control for H-2Kb was mouse IgG2a (msIgG2a), and for 25-D1.16 was mouse IgG1 (msIgG1). F, cells were either unpulsed or pulsed with OVA257 and stained for H-2kb, H-2kb OVA257 (25-D1.16) or hHER2, and the number of molecules for each antigen was calculated by comparison with a standard curve.
Alexander J. Davenport et al. Cancer Immunol Res 2015;3:
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