Figure 5

Effector function of CD28-containing TetCAR matches 41BBζ control CAR. (a) Killing of SupT1-CD19-GFP after 24 h or NALM6 after 48 h of co-culture with CAR-T cells at 1:1–1:32 E:T ratio. Data shows mean ± SD, n = 4 donors from 2 independent experiments. (b) SupT1-CD19, NALM6, Raji or Raji-CD19KO targets were incubated with mitomycin C, then co-cultured with CAR-T cells at 1:2 E:T ratio for 7 days. To relevant wells, 400 nM of minocycline was added on day 0. Graphs show mean (± SD) number of RQR8⁺ T cells (filled bars) or total CD3⁺ T cells (white bars) for each target. n = 4 donors (SupT1-CD19, Raji and Raji-CD19KO) or n = 3 (NALM6) from 2 independent experiments. Statistical analysis was through a two-way ANOVA comparing mean RQR8 number in each group ± minocycline (with Šidák’s multiple comparisons). P values for SupT1-CD19 were 28-Fab-Tet-z (**, 0.0028) and 28BB-Fab-Tet-z (**, 0.0050). P values for Raji were 28-Fab-Tet-z (*, 0.0320) and 28BB-Fab-Tet-z (*, 0.0304). (c) Mean fluorescent intensity of Tim3 and Lag3 after 7 days coculture with SupT1-CD19 targets, ± 400 nM minocycline. Data shows geometric mean (± SD) in CD3⁺ T cells. n = 4 donors, from 2 independent experiments. Statistical analysis was through a two-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values for Lag3 expression were 28-Fab-Tet-z (**, 0.0078) and 28BB-Fab-Tet-z (*, 0.0207). (d) Percentage of naïve (CD62L⁺, CD45RA⁺), Tcm (central memory; CD62L⁺, CD45RA^-), Tem (effector memory; CD62L^-, CD45RA^-) or Temra (terminally differentiated effector memory; CD62L^-, CD45RA⁺) memory T cell populations after 7 days coculture with SupT1-CD19 targets, ± 400 nM minocycline. Data shows mean (± SD) in CD3⁺ T cells. n = 4 donors, from 2 independent experiments.