Effect of tryptophan metabolites on CD19-CART proliferation. (A) AHR, CYP1A1, and CYP1B1 mRNA expression measured by real-time PCR on CD19-CARTs treated with or without KHAA (12.5 μM) for 24 hours. Data represent mean ± SD of 4 T-cell lines generated from 4 healthy donors and normalized to GAPDH expression. (B) CD19-CARTs were cultured in media containing increasing amounts of l-kynurenine and 3-HAA (KHAA) (0, 6.25, 12.5, 25, or 50 μM) with IL-2 (50 U/mL), IL-7 (10 ng/mL), or IL-15 (5 ng/mL) for 72 hours. (C) Four B-cell lymphoma lines (Raji, Daudi, BJAB, or Jeko-1) were cultured in media containing increasing amounts of KHAA for 72 hours, without exogenous cytokines. Cell proliferation was studied by the XTT assay. One representative set of 3 experiments is shown (*P < .05). (D) NTs or CD19-CARTs labeled with CFSE were cultured with irradiated Raji cells for 3 days in the absence or presence of KHAA (12.5 or 25 μM) without any added exogenous cytokines. CFSE dilution was assayed by flow cytometry, gating on CD3-positive cells. Controls (dotted line) were cultured without Raji cells. Data represent mean ± SD of CSFE dilution in 4 T-cell lines generated from 4 healthy donors (*P < .05). (E) The number of CD19-CARTs cultured in the presence or absence of KHAA (12.5 μM) was determined at the end of each 7-day expansion cycle. Data represent mean ± SD of 5 T-cell lines generated from 5 donors (*P < .01).