Figure 2.
IL12 in the CAR exodomain was functional, resulting in differentially expressed proteins in IL12-CAR T cells versus CAR T cells
(A) Flow cytometric analysis of phosphorylated STAT4 (pSTAT4) in conventional and IL12-CAR T cells. Representative pSTAT4 histograms of engineered T cells and mean values ±SD of three donor T cells engineered with the respective CAR are shown. Statistical significance was calculated by Student's t test. ∗p < 0.05. MFI, mean fluorescence intensity. (B) T cells with IL12-CAR and conventional CAR, respectively, were cultivated for 7 days and analyzed for several signature markers by flow cytometry and ELISA. For flow cytometric analysis, gates were set on CAR+ T cells and numbers of CAR T cells with expression of the respective markers were determined. CAR T cells were stained for CD56, CCR1, and CD94 expression. CAR T cells (5 × 104 cells/well) were further stimulated through CD3/CD28 for 72 h with agonistic anti-CD3 and anti-CD28 mAbs (1 μg/mL each), respectively, and recorded for intracellular IL5 and surface TGFβ by flow cytometry. Supernatants were analyzed for IFN-γ by ELISA. CAR T cells were stained for CAR, CD8, CD56, CD62L, and CD94. Data represent mean values ±SD of three to five different donors. Statistical differences were determined by Student's t test.