TH17 cells redirected with an ICOS-based CAR release high amounts of IL17-A, IL-17F, and IL-22 but low amounts of IL-2. (A) TH17 cells transduced with various CARs were cocultured with surrogate antigen, APC cells transduced with mesothelin (K562meso). Supernatants from several different healthy donors (n = 4-9) were obtained 24 hours after coculture, and cytokine production was analyzed by ELISA. Box plots show median (line) and 25th to 75th percentile (box). The end of the whiskers represents the minimum and the maximum of all of the data. (B) Redirected TH17 cells were stimulated with plate bound anti-CD3 (OKT3). IL-17A was analyzed by ELISA 24 hours after stimulation. Data represent the means ±SD for 2 different normal donors. (C) TH17 cells were cultured with the indicated tumor cells expressing mesothelin. IL-17A and IL-2 production were analyzed by ELISA 24 hours on antigen recognition. Error bars indicate SD in duplicate samples. Results are representative of at least 2 different experiments. (D) CD4+ T cells from different normal donors (n = 3) were activated with anti-CD3/CD28 beads, cultured under TH17 polarizing conditions, and redirected with the different CARs. After their primary expansion, CD4+ T cells were cultured with K562meso cells. Secretion of IL17-A and INF-γ production was analyzed by ELISA 24 hours on antigen recognition. Error bars indicate SD. *P < .05, **P < .01, and ***P < .001.
