Supplementary Figure 11: Functional assessment of Cpf1 double-knockin KIKO CAR-T cells.

a, Cytolytic activities of CAR22 (CD22BBz), CAR19 (CD19BBz) and CAR22;CAR19 (CD22BBz;CD19BBz double knockin) KIKO CAR-T cells 5 d after electroporation and AAV infection were tested by in vitro coculture (kill assay). In vitro cytotoxic activity was measured by bioluminescence assay at different effector:target (E:T) ratios, for AAV-vector transduced T cells, CAR22, CAR19 and CAR22;CAR19 double knockin KIKO CAR-T cells (n = 3 infection replicates for each experiment condition). Two-way ANOVA with Tukey’s multiple-comparisons test was used to assess significance. Vector versus KIKO CAR-T cell, ***P < 0.001 for all three groups (CAR22, CAR19 and CAR22;CAR19); CAR22 versus CAR19, P = 0.9994; CAR22 versus CAR22;CAR19, P = 0.9997; CAR19 versus CAR22;CAR19, P > 0.9999. b, Representative flow cytometry results showing IFN-γ and TNF-α expression levels in CAR22, CAR19 and CAR22;CAR19 T cells. IFN-γ and TNF-α production was tested by intracellular staining after coculture with NALM6 cells for 5 h at E:T = 1:1. One representative case is shown from n = 3 infection replicates. c, Quantitative results demonstrating the antigen-specific IFN-γ and TNF-α production capability of CAR22, CAR19, and CAR22;CAR19 T cells (n = 3 infection replicates). Two-way ANOVA with Tukey’s multiple-comparisons test was used to assess significance (multiple-testing corrected). IFN-γ group: vector versus CAR22, ***P < 0.001; vector versus CAR19, ***P < 0.001; vector versus CAR22;CAR19, ***P < 0.001; CAR22 versus CAR19, ***P < 0.001; CAR22 versus CAR22;CAR19, ***P < 0.001; CAR19 versus CAR22;CAR19, ***P < 0.001. TNF-α group: vector versus CAR22, ***P < 0.001; vector versus CAR19, ***P < 0.001; vector versus CAR22;CAR19, ***P < 0.001; CAR22 versus CAR19, P = 0.4499; CAR22 versus CAR22;CAR19, ***P < 0.001; CAR19 versus CAR22;CAR19, **P = 0.0021. All data are shown as mean ± s.e.m., plus individual data points on the graph.