(A–C) ICOS-activated Th17 cells cultured with indomethacin (Indo) plus CAL-101 engraft to a greater extent than cells subjected to other treatments in the spleen. (A) Percentage and (B) number (×10⁴) of TRP-1 Vβ14⁺CD45.2⁺ Th17 cells in the spleens of CD45.1⁺ recipient mice 6 days after transfer. (C) Donor Vβ14⁺CD45.2⁺ Th17 cells secrete IL-17A, IFN-γ, and IL-2 64 days after transfer in CD45.1⁺ recipient mice when primed with Indo or Indo plus CAL-101. Cytokine production in vivo of Th17 cells expanded in vitro with inhibitors, as indicated. Donor Vβ14⁺CD45.2⁺ cells were flow sorted from the spleens of CD45.1⁺ recipient mice and restimulated with PMA/ionomycin. Data represent mean ± SEM. Student’s t test; *P < 0.05; **P < 0.01; ***P < 0.001. (D) NK cell frequency in the tumors of mice infused with various Th17 treatments. (E–H) Depletion of host NK or CD8 T cells in mice does not abrogate therapy. Individual tumor growth curves of mice treated with 0.75 × 10⁶ transferred TRP-1 Th17 cells expanded with TRP-1 peptide and ICOS agonist and primed in vitro with CAL-101 and Indo. Cells transferred into 5-Gy TBI mice bearing B16F10 melanomas. Mice were antibody depleted of host CD8 or NK cells (100 μg/mouse) twice weekly for the entire experiment, starting 2 days prior to ACT. As a control, mice given Th17 therapy were administered with an IgG isotype. (J) Depletion of donor cells in mice rechallenged with tumor impairs ACT. Surviving mice in (F–H) were rechallenged with B16F10 and then antibody depleted of host CD4 cells (100 μg/mouse) or with (I) an IgG control twice weekly for the first 2 weeks after rechallenge. Percentage survival; n = 13 mice/group. Mantel-Cox curves compared by log-rank test; *P < 0.05. As a control (K and L), naive mice were treated with an IgG isotype control (top right) or treated with a CD4-depleting antibody. Indo + CAL-101 + CD4 depletion is significant different that Indo + CAL-101 + IgG depletion (P < 0.05).