Figure 1

Chimeric antigen receptor (CAR) T cell mediated killing of Nalm-6 cells in vitro. (A) Efficacy of DiI-labeling in primary human T cells was analyzed by flow cytometry (excitation at 561 nm, detection with a 582/15 bandpass filter). Unlabeled T cells (“unstained”; filled grey histogram) served as a control. One representative experiment is shown. (B) Schematic of the CD19-specific second-generation CAR (FMC63.4-1BB.ζ) in which the FMC63-based scFv was fused to the hinge and transmembrane region of CD8α and the cytoplasmic domains of 4-1BB and CD3ζ. (C) Expression of the CD19-specific CAR in primary human T cells. Flow cytometric analysis using Protein L as a detection reagent. Mock T cells (“no CAR”; filled grey histogram) served as a negative control. One representative experiment of three independent experiments is shown. (D) Influence of DiI-labeling on the cytolytic activity of CAR T cells. Primary human T cells expressing either the CD19-specific CAR or no CAR and labeled or not labeled with DiI were co-cultured with CD19^pos Nalm-6 cells. Experiments were performed with E/T (effector/target) ratios of 0.5, 1, 2, 4, and 8, as indicated. Cytolytic activity was quantified using a luciferase-based cytotoxicity assay. Data are expressed as means ± SD of one experiment with three donors. (E) Influence of the temperature on the cytolytic activity of CAR T cells. Primary human T cells expressing either the CD19-specific CAR or no CAR were co-cultured at 35 °C or 37 °C with GFP^pos/CD19^pos Nalm-6 cells at an E/T ratio of 1:1. Cytolytic activity was quantified by using a counting bead-based cytotoxicity assay. Data are expressed as individual data points (and their means) obtained from one experiment with three different T cell donors.