Indoleamine 2,3-dioxygenase specific, cytotoxic T cells as immune regulators

Abstract

Indoleamine 2,3-dioxygenase (IDO) is an immunoregulatory enzyme that is implicated in suppressing T-cell immunity in normal and pathologic settings. Here, we describe that spontaneous cytotoxic T-cell reactivity against IDO exists not only in patients with cancer but also in healthy persons. We show that the presence of such IDO-specific CD8(+) T cells boosted T-cell immunity against viral or tumor-associated antigens by eliminating IDO(+) suppressive cells. This had profound effects on the balance between interleukin-17 (IL-17)-producing CD4(+) T cells and regulatory T cells. Furthermore, this caused an increase in the production of the proinflammatory cytokines IL-6 and tumor necrosis factor-α while decreasing the IL-10 production. Finally, the addition of IDO-inducing agents (ie, the TLR9 ligand cytosine-phosphate-guanosine, soluble cytotoxic T lymphocyte-associated antigen 4, or interferon γ) induced IDO-specific T cells among peripheral blood mononuclear cells from patients with cancer as well as healthy donors. In the clinical setting, IDO may serve as an important and widely applicable target for immunotherapeutic strategies in which IDO plays a significant regulatory role. We describe for the first time effector T cells with a general regulatory function that may play a vital role for the mounting or maintaining of an effective adaptive immune response. We suggest terming such effector T cells "supporter T cells."

Figure 1

Spontaneous cytotoxic T-cell reactivity against IDO. Spontaneous T-cell reactivity against IDO5 (IDO_199-207; 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 pol_476-484 and CD8 mAb (B). As control, an IDO5-specific T-cell clone was stained with the HLA-A2/HIV-1 pol_476-484-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 pol_476-484; 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 pol_476-484) 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).

Figure 2

IDO-specific T cells boosted specific immunity toward CMV in PBMCs from a healthy donor. PBMCs from an HLA-A2⁺ healthy donor cultured with CMV IE1_316-324 (VLEETSVML) 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 CMV IE1_316-324–specific CD8⁺ T cells in each culture was identified by flow cytometry with the MHC-tetramer complex HLA-A2/CMV IE1_316-324 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. Data from 2 representative experiments are shown (A). Percentage of CMV IE1_316-324–specific T cells found in PBMCs cultured alone (□) or added an IDO5-specific T-cell clone (■). Data are mean ± SD (n = 4; P < .05; B). The percentage of CD4⁺CD25^highCD127⁻Foxp3⁺ Tregs in each culture was identified by flow cytometry with intracellular staining for Foxp3. For comparison, cells were stained with isotype controls. The data shown are from 1 donor, representative of 4 experiments (C). Distribution of CD4⁺ and CD8⁺ T cells in the cultures. Data are mean ± SD (n = 4; D).

Figure 3

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).

Figure 4

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.

Figure 5

Costimulation of IDO-specific T cells reduced Treg numbers while boosting IL-17, IL-6, and TNF-α production. PBMCs from patients with HLA-A2⁺ melanoma cancer stimulated in vitro with MART-1_26-35 (EAAGIGILTV) peptide either in coculture with IDO5 peptide or an irrelevant peptide (HIV-1 pol_476-484). The percentage of CD4⁺CD25^highCD127⁻Foxp3⁺ Tregs (A-B) and IL-17A–producing CD4⁺ T cells (C-D) in each culture was identified by flow cytometry with intracellular staining for Foxp3 and IL-17A, respectively. For comparison, cells were stained with isotype controls. Examples of Treg staining (A) and IL-17A staining (C) of PBMCs 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). Examples shown are from 1 patient, representative of 4 different patients (A,C). Distribution of CD4⁺ and CD8⁺ T cells in the cultures (E). Secreted cytokines (IL-10, IL-17A, IL-6, and TNF-α) in cell culture supernatants quantified by ELISA (F). Data are mean ± SD (n = 4 patients). White bars indicate MART-1_26-35–stimulated PBMCs in coculture with an irrelevant peptide (HIV-1 pol_476-484); black bars, MART-1_26-35–stimulated PBMCs in coculture with IDO5 peptide.

Figure 6

IDO-inducing agents expanded IDO-specific T cells with supporter functions. Example of reactivity against IDO5 (IDO_199-207; ALLEIASCL) in PBMCs from an HLA-A2⁺ healthy donor, stimulated in vitro with IL-2 and IFN-γ. The percentage of IDO5-specific CD8⁺ T cells was identified by flow cytometry, ex vivo (top) and after stimulation (bottom), using the MHC-tetramer complex HLA-A2/IDO5 and CD8 monoclonal antibody (mAb). The data shown are from 1 donor, representative of 4 different donors (A). Examples of reactivity against IDO5 in PBMCs from a patient with HLA-A2⁺ renal cell carcinoma, stimulated in vitro with IL-2 and CTLA4-Ig (B) or CpG ODN (C). The percentage of IDO5-specific CD8⁺ T cells was identified by flow cytometry, ex vivo (top) and after stimulation (bottom), using the MHC-tetramer complex HLA-A2/IDO5 and CD8 mAb. The data shown are from 1 patient, representative of 2 different patients (B-C). PBMCs from an HLA-A2⁺ healthy donor stimulated in vitro with CMV pp65_495-503 (NLVPMVATV) peptide and cocultured with either autologous, isolated CD8⁺ T cells (top) or autologous, isolated IFN-γ–induced IDO5-specific T cells (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 mAb (D). PBMCs from an HLA-A2⁺ healthy donor stimulated with CMV IE1_316-324 (VLEETSVML) peptide and cocultured with either autologous, isolated CD8⁺ T cells (top) or autologous, IDO5-specific T cells isolated after 2 in vitro peptide stimulations (bottom). The percentage of CMV IE1_316-324–specific CD8⁺ T cells in each culture was identified by flow cytometry with the MHC-tetramer complex HLA-A2/ CMV IE1_316-324 and CD8 mAb (E). In all experiments, cells were stained with the MHC-tetramer complex HLA-A2/HIV-1 pol_476-484 and CD8 mAb for comparison.