IDO-specific T cells boosted specific immunity toward Flu in PBMCs from a patient with cancer. PBMCs from a patient with HLA-A2⁺ breast cancer cultured with Flu matrix p_58-66 (GILGFVFTL) peptide either alone (top) or added an autologous, IDO5 (IDO_199-207; ALLEIASCL)–specific T-cell clone (in a PBMC-to-clone ratio of 2000:1; bottom). The percentage of Flu matrix p_58-66–specific CD8⁺ T cells in each culture was identified by flow cytometry with the MHC-tetramer complex HLA-A2/Flu matrix p_58-66 and CD8 monoclonal antibody (mAb). For comparison, cells were stained with the MHC-tetramer complex HLA-A2/HIV-1 pol_476-484 and CD8 mAb (A). The percentage of CD4⁺CD25^highCD127⁻Foxp3⁺ Tregs (B) and IL-17A–producing CD4⁺ T cells (C) in each culture were identified by flow cytometry with intracellular staining for Foxp3 and IL-17A, respectively. For comparison, cells were stained with isotype controls. Distribution of CD4⁺ and CD8⁺ T cells in the cultures (D). Tryptophan concentrations in cell culture supernatants before and after the addition of the IDO5-specific T-cell clone measured by competitive ELISA (E). Secreted cytokines (IL-10, IL-17A, IL-6, and TNF-α) in cell culture supernatants quantified by ELISA (F). All data shown are from one patient. Data are mean ± SD (n = 3). White bars indicate Flu matrix p_58-66–stimulated PBMCs cultured alone; black bars, Flu matrix p_58-66–stimulated PBMCs added an IDO5-specific T-cell clone (A-F). PBMCs from a patient with HLA-A2⁺ melanoma cancer cultured with CMV pp65_495-503 (NLVPMVATV) peptide either alone (top) or added irrelevant autologous, ML-IAP_280-289 (QLCPICRAPV)–specific T-cell clone (in a PBMC- to-clone ratio of 2000:1; bottom). The percentage of CMV pp65_495-503–specific CD8⁺ T cells in each culture was identified by flow cytometry with the MHC-tetramer complex HLA-A2/CMV pp65_495-503 and CD8 monoclonal antibody. For comparison, cells were stained with the MHC-tetramer complex HLA-A2/HIV-1 pol_476-484 and CD8 monoclonal antibody. The data shown are from one patient, representative of 3 experiments (G).