Costimulation with IDO peptide increased frequencies of CMV- and MART-1–specific T cells. PBMCs from HLA-A2⁺ healthy donors and patients with HLA-A2⁺ cancer (melanoma and renal cell carcinoma) stimulated in vitro with CMV peptide (CMV pp65_495-503 (NLVPMVATV) or CMV IE1_316-324 (VLEETSVML)) or MART-1_26-35 (EAAGIGILTV) peptide either in coculture with IDO5 (IDO_199-207; ALLEIASCL) peptide or an irrelevant peptide (HIV-1 pol_476-484). The percentage of CMV- or MART-1_26-35–specific CD8⁺ T cells in each PBMC culture was identified by flow cytometry with the MHC-tetramer complexes HLA-A2/CMV pp65_495-503 (NLVPMVATV), HLA-A2/CMV IE1_316-324 (VLEETSVML), or HLA-A2/ MART-1_26-35 (EAAGIGILTV) and CD8 monoclonal antibody. The differences in tetramer-specific CD8⁺ T-cell percentages between the cultures are given, for each donor/patient, as fold increase of tetramer-specific CD8⁺ T cells in coculture with IDO5 peptide. Data are mean differences; n = 15 (A). Example of MHC-tetramer staining of PBMCs from a healthy donor stimulated in vitro with CMV IE1_316-324 peptide either in coculture with an irrelevant peptide (HIV-1 pol_476-484; top) or IDO5 peptide (bottom). The data shown are from 1 donor, representative of 6 different donors/patients (B). Example of MHC-tetramer staining of PBMCs from a patient with melanoma stimulated in vitro with MART-1_26-35 peptide either in coculture with an irrelevant peptide (HIV-1 pol_476-484; top) or IDO5 peptide (bottom). The data shown are from 1 patient, representative of 4 different patients (C). In all experiments, cells were stained with the MHC-tetramer complex HLA-A2/HIV-1 pol_476-484 and CD8 monoclonal antibody for comparison.