Additional costimulatory domains are not able to mitigate the inhibitory effects of tryptophan metabolites. (A) Schematic representation of recombinant retrovirus vectors encoding CD19-CAR constructs (CD19.ζ, containing the CD3ζ chain alone; CD19.28.ζ, CD3ζ chain and CD28 endodomain; CD19.28.4-1BB.ζ, CD3ζ chain and CD28 and 4-1BB endodomains) (B) CD19-CAR surface expression on T cells transduced with each construct. (C) 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 the mean ± SD of 5 T-cell lines generated from 5 donors (*P < .05). (D) GFP-transduced wild-type Raji cells were cocultured with each CD19-CART type at an E:T ratio of 1:2 in the presence or absence of KHAA (12.5 μM). The total number of tumor cells and CARTs in each culture condition was determined by flow cytometry. Dot plot shows representative data on day 14. Five experiments using CARTs generated from 5 different donors were analyzed and represent mean ± SD in the graph (*P < .05). In the absence and presence of KHAA, the number of residual tumor was 0.28 ± 0.28 × 106 and 1.49 ± 0.84 × 106, respectively, for the first-generation CAR; 0.16 ± 0.13 × 106 and 0.83 ± 0.3 × 106, respectively, for the second-generation CAR; and 0.99 ± 0.33 × 106 and 1.79 ± 0.39 × 106, respectively, for the third-generation CAR.
