FIGURE 4.

Bruton tyrosine kinase inhibitor enhances chimeric antigen receptor (CAR) T-cell expansion and the proportion of type 1 T-helper (Th1) cells in samples from some donors after serial stimulation. Representative plots showing a number of population doublings of CAR T cells from 3 donors stimulated in triplicate wells with irradiated target cells (NucLight Red K562.CD19) at an effector:target ratio of 2.5:1 together with ibrutinib (A) or acalabrutinib (B) during a 25-day serial stimulation. Arrows indicate the timepoint of each restimulation at which CAR T cells were counted and new target cells along with ibrutinib or acalabrutinib were added. Number of CAR T cells from 3 donors treated with ibrutinib (C) or acalabrutinib (D) for 18 days and after 5 rounds of restimulation, *P<0.05. Data from 2 independent experiments (mean±SEM). E, Percentage of Th1 (CXCR3⁺CRTH2⁻) cells within CD4⁺ CAR T cells over time during serial stimulations for 3 donors treated with ibrutinib (500 nM) or acalabrutinib (1581 nM). F, Percentage of Th1 cells within CD4⁺ CAR T cells after 18 days of serial stimulation and treatment with ibrutinib or acalabrutinib. Representative data from 2 independent experiments. G, Number of population doublings during the 21-day culture period for CAR T cells from patients with diffuse large B-cell lymphoma (DLBCL). Arrows indicate the timepoint of each restimulation at which CAR T cells were counted and new target cells along with ibrutinib were added. H, Cytotoxicity killing assay of K562.CD19.NLR target cells after 16 days of restimulation with CAR T cells from patients with DLBCL. Percentage of killing normalized to untreated control (100%). Data are shown as mean±SEM from replicate wells. Statistically significant differences are indicated as *P<0.05, ***P<0.001, and ****P<0.0001.