Effect of tryptophan metabolites on killing, apoptosis, and cytokine secretion by CD19-CARTs. GFP-transduced wild-type Raji cells were cocultured with CD19-CARTs at E:T ratios (A) 1:2 and (B) 1:4 in the presence or absence of KHAA (12.5 μM). The total number of tumor cells and CARTs in each culture condition was determined using flow cytometry analysis at the indicated time points. Dot plot shows representative data on day 14. Three experiments using CARTs generated from 3 different donors were analyzed and shown as the mean ± SD in the graph (*P < .05). (C) Seven days after the first expansion, CD19-CARTs (1 × 10⁶) were cultured with Raji cells (1 × 10⁶) in the presence or absence of KHAA (12.5 μM); supernatants were collected after 24 hours and analyzed for IL-2 and IFN-γ by ELISA. Data represent mean ± SD of 4 T-cell lines generated from 4 donors (*P < .05). (D) Apoptosis was determined by annexin V/7-AAD staining of CD19-CARTs after 3 days of culture in the absence or presence of KHAA (12.5 or 25 μM) and IL-2 (50 U/mL). Data represent mean ± SD of 4 T-cell lines generated from 4 donors (*P < .05).