Figure 3
Affinity and antigen-density dependent activation of primary CAR T cells in vitro. (a) Effector to target (E:T) assay for measuring target killing by primary T cells transduced with different I domain CARs. Each target was separately incubated with TM, F292G, F265S, F292A, or WT CAR T cells at 5:1 E:T ratio. Percent viability was normalized to luminescence from target cells incubated with NT T cells (n = 3, ± = standard deviation (SD)). A variable slope sigmoidal curve equation was used to fit data. p < 0.01 for * vs. NT by Dunnett’s multiple comparisons test. (b) The best fit values of 50% killing and Hill slope of the sigmoidal equation were plotted against the affinities of I domain CARs. The best fit values with r-square values higher than 0.85 were plotted. (c) Examples of CAR expression in primary T cells after transduction with different batches of lentivirus or different donor T cells as measured by flow cytometry. Histograms in black for transduced and in red for non-transduced T cells as a control, which were stained with anti-Myc antibody. (d) ICAM-1 expression in primary T cells in comparison to HeLa cells. Grey and black histograms correspond to unlabeled T cells and R6.5 antibody-labeled T cells, respectively. (e) IFN-γ release was measured by ELISA for each CAR T variant after co-incubation with different target cells for 24 h (n = 3). p < 0.01 for * vs. 8505 C/-ICAM-1 by Dunnett’s multiple comparisons test.
