(A) Th17 cells costimulated with ICOS regress melanoma to a greater extent than CD28-stimulated Th17 cells. C57BL/6 mice bearing large subcutaneously B16F10 melanoma tumors established for 10 days received nonmyeloablative 5 Gy total body irradiation. One day later, mice received an ACT treatment regimen consisting of the adoptive transfer of 1 × 10⁶ cultured tumor-reactive TRP-1 CD4⁺ T cells programmed to a Th17 phenotype (with IL-β, IL-21, IL-6, TGF-β, anti-IL-4, and anti-IFN-γ) and expanded for 7 days with beads coated with CD3 agonist and with either a CD28 or ICOS agonist. Tumor growth was measured every 2 to 3 days; n = 8 mice/group. Data (mean ± SEM) are representative of 2 independent experiments. Mean tumor areas compared by 1-way ANOVA with multiple comparisons; **P < 0.01, ***P < 0.001. (B) Th17 cells primed with ICOS but not CD28 persist in vivo for 200 days. Quantification of tumor-specific (Vβ14⁺CD45.2⁺) TRP-1 Th17 cells expanded with ICOS versus CD28 from lymph nodes, lungs, and spleen of CD45.1⁺ recipient mice 200 days after transfer. Percentage frequencies compared by Student’s t test; *P < 0.05, **P < 0.01. (C) ICOS-stimulated CD45.2⁺ Th17 cells secrete more IFN-γ, IL-17A, and IL-21 than CD28-stimulated Th17 cells when reactivated ex vivo. Donor CD45.2⁺ Th17 cells were isolated from CD45.1⁺ recipient mouse spleens 200 days after transfer and then reactivated with irradiated spenotyces pulsed with TRP-1 peptide. IFN-γ, IL-21, and IL-17A production was then measured using flow cytometry. Percentage frequencies compared by Student’s t test; *P < 0.05. (D) Th17 cells stimulated with ICOS possess a durable memory response to tumors after rechallenge with B16F10 tumor. 200 days after initial transfer, surviving mice previously treated with TRP-1 Th17 cells activated with ICOS or CD28 were rechallenged with B16F10 and monitored for tumor burden compared with mice not previously treated (i.e., no treatment). Mantel-Cox curves compared by log-rank test, ***P < 0.001.