Figure 2.

Mesothelin redirected T cells secrete Th1 proinflammatory cytokines in response to plate-bound mesothelin or tumor cell surface-associated mesothelin. (a) Transduced T cells respond against immobilized but not soluble mesothelin. P4-z T cells or control CD19-z and GFP T cells (10⁵ cells/well) were incubated with either 5 µg/ml of soluble or plate-immobilized mesothelin. After overnight incubation, culture supernatants were analyzed for human Th1/Th2 cytokines using cytometric bead array technology. Concentration of IFN-γ was expressed in pg/ml. (b) Surface mesothelin expression (solid black histograms) by various human ovarian cancer cell lines was evaluated by flow cytometry. The native mouse malignant mesothelioma cell line AE17 which does not express human mesothelin was engineered to express high surface levels of human mesothelin (AE17M) as shown by flow cytometry; isotype antibody control (open gray histograms). (c) Primary human T cells transduced with P4-z preferentially produce Th1 cytokines after stimulation with mesothelin⁺ cancer cell lines. Transduced T cells (10⁵ CAR⁺ T cells) were cultured alone (none) or stimulated overnight with an equal number of human mesothelin⁺ AE17M and A1847 or antigen-negative AE17 and C30 cancer cell lines. Cell-free supernatant from three independent cultures was harvested and pooled after ~20 hours of incubation and the indicated human Th1/Th2 cytokines were quantified using cytometric bead array technology. Values represent cytokine concentration (pg/ml). (d) Antigen-stimulated IFN-γ secretion by P4-z but not CD19-z or GFP T cells following overnight incubation with ovarian cancer cell lines expressing different levels of surface mesothelin. Mean IFN-γ concentration ± SEM (pg/ml) from triplicate cultures is shown. (e) Correlation of mesothelin expression (mean fluorescence intensity; MFI) on mesothelin-expressing tumor cells was plotted versus the production of IFN-γ by P4-z CAR-transduced T cells cocultured with these tumor cells. CAR, chimeric antigen receptor; GFP, green fluorescent protein; IFN, interferon; IL, interleukin; MIP, macrophage inflammatory protein; Th1, T helper 1; Th2, T helper 2; TNF, tumor necrosis factor.