Figure 4 from Nanobody-Engineered CLL-1 CAR T Cells: Optimizing Tumor-Specific Cytotoxicity and Minimizing Off-Tumor Toxicity

Nanobody-based CLL-1 and CD33 CAR T cells effectively kill antigen-expressing tumor cells. A, CLL-1 CAR T cells, CD33 CAR T cells, or untransduced (Unt.) T cells were cocultured with U937-mKate2 cells, a CLL-1 and CD33-expressing AML cell line, at varying E:T ratios ranging from 10:1 to 0.33:1. Cytotoxicity was monitored in real time over 42 hours using the IncuCyte imaging system, with target cells labeled with mKate2 far-red fluorescent protein. B and C, CLL-1 CAR T cells, CD33 CAR T cells, or Unt. T cells were cocultured with HL-60-mKate2 cells, another CLL-1 and CD33-expressing AML cell line, at E:T ratios ranging from 10:1 to 0.33:1. Cytotoxicity was again monitored in real time over 42 hours using the IncuCyte imaging system, with target cells labeled with mKate2 far-red fluorescent protein. Raji cells were included as a negative control to assess the specificity of CAR T-cell activity. Unt. T cells were used to evaluate nonspecific killing. Shown are the mean values of four images acquired by the IncuCyte at each time point and condition, from one representative experiment out of three independent repeats with three different donors. D, IFNγ cytokine release of CLL-1 and CD33 CAR T cells or Unt. T cells in response to a 24-hour coculture with antigen-positive U937 and HL-60 target cells. Raji cells were used as a negative control, and plain culture medium was used as a background control. Data represent mean values of triplicate wells ± standard deviation. N = 3 T-cell donors were used. Cytokine release data were generated from three independent experiments. A two-way ANOVA was performed to determine the statistical significance between groups based on two or more independent variables. Šídák’s multiple comparisons test was used. *, P < 0.0168; ****, P < 0.0001; ns, not significant.