Figure 1
Figure 1. Spontaneous cytotoxic T-cell reactivity against IDO. Spontaneous T-cell reactivity against IDO5 (IDO199-207; ALLEIASCL) in PBMCs, from HLA-A2+ healthy donors (HD), visualized by IFN-γ enzyme-linked immunospot (ELISPOT) assay (A) and flow cytometry (B) after 1 in vitro peptide stimulation. For IFN-γ ELISPOT assay, PBMCs were plated at 4 × 105 PBMCs in duplicates in specialized ELISPOT wells either alone or with added IDO5 peptide. The average number of IDO5-specific spots (after subtraction of spots in wells without added peptide) was calculated per 4 × 105 PBMCs for each donor (black circles; A). For flow cytometry, IDO5-specific T cells were identified with the MHC-tetramer complex HLA-A2/IDO5 and CD8 monoclonal antibody (mAb). For comparison, cells were stained with the MHC-tetramer complex HLA-A2/HIV-1 pol476-484 and CD8 mAb (B). As control, an IDO5-specific T-cell clone was stained with the HLA-A2/HIV-1 pol476-484-PE and HLA-A2/IDO5-PE complexes (C). Lytic capacity of representative IDO5-specific T-cell clones from a healthy donor (HD) or a patient with breast cancer (BC) assayed by 51Cr-release assay. Target cells were TAP-deficient T2 cells pulsed with IDO5 or an irrelevant peptide (HIV-1 pol476-484; D), the HLA-A2+/IDO+ colon cancer cell line SW480 and the HLA-A2+/IDO− colon cancer cell line HCT116 (E), SW480 blocked with the HLA class I–specific mAb W6/32 (F), SW480 transfected with IDO ShRNA for down-regulation of IDO protein expression and SW480 transfected with control ShRNA as a positive control (F), the HLA-A2+/IDO+ melanoma cell line FM55M (G), FM55M added cold T2 cells pulsed with IDO5 peptide or irrelevant peptide (HIV-1 pol476-484) in a inhibitor-to-target ratio of 20:1 (G), autologous in vitro immatured and matured DCs (H), and ex vivo–isolated autologous IDO− CD14+ monocytes as well as IFN-γ–treated IDO+ CD14+ monocytes (I). All 51Cr-release assays were performed in effector-to-target ratio of 5:1, except the experiments regarding ShRNA, which were performed in effector-to-target ratio of 15:1. Data are mean ± SD (n = 3).

Spontaneous cytotoxic T-cell reactivity against IDO. Spontaneous T-cell reactivity against IDO5 (IDO199-207; ALLEIASCL) in PBMCs, from HLA-A2+ healthy donors (HD), visualized by IFN-γ enzyme-linked immunospot (ELISPOT) assay (A) and flow cytometry (B) after 1 in vitro peptide stimulation. For IFN-γ ELISPOT assay, PBMCs were plated at 4 × 105 PBMCs in duplicates in specialized ELISPOT wells either alone or with added IDO5 peptide. The average number of IDO5-specific spots (after subtraction of spots in wells without added peptide) was calculated per 4 × 105 PBMCs for each donor (black circles; A). For flow cytometry, IDO5-specific T cells were identified with the MHC-tetramer complex HLA-A2/IDO5 and CD8 monoclonal antibody (mAb). For comparison, cells were stained with the MHC-tetramer complex HLA-A2/HIV-1 pol476-484 and CD8 mAb (B). As control, an IDO5-specific T-cell clone was stained with the HLA-A2/HIV-1 pol476-484-PE and HLA-A2/IDO5-PE complexes (C). Lytic capacity of representative IDO5-specific T-cell clones from a healthy donor (HD) or a patient with breast cancer (BC) assayed by 51Cr-release assay. Target cells were TAP-deficient T2 cells pulsed with IDO5 or an irrelevant peptide (HIV-1 pol476-484; D), the HLA-A2+/IDO+ colon cancer cell line SW480 and the HLA-A2+/IDO− colon cancer cell line HCT116 (E), SW480 blocked with the HLA class I–specific mAb W6/32 (F), SW480 transfected with IDO ShRNA for down-regulation of IDO protein expression and SW480 transfected with control ShRNA as a positive control (F), the HLA-A2+/IDO+ melanoma cell line FM55M (G), FM55M added cold T2 cells pulsed with IDO5 peptide or irrelevant peptide (HIV-1 pol476-484) in a inhibitor-to-target ratio of 20:1 (G), autologous in vitro immatured and matured DCs (H), and ex vivo–isolated autologous IDO− CD14+ monocytes as well as IFN-γ–treated IDO+ CD14+ monocytes (I). All 51Cr-release assays were performed in effector-to-target ratio of 5:1, except the experiments regarding ShRNA, which were performed in effector-to-target ratio of 15:1. Data are mean ± SD (n = 3).

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