Specificity and functional capacity of IDO5-specific T-cells assayed by ⁵¹Cr-release assays.

<p><i>(</i><i>a</i><i>),</i> Lysis of T2-cells pulsed with IDO5 peptide or an irrelevant peptide (the HLA-A2 high affinity binding epitope HIV-1 pol_476-484 (ILKEPVHGV)) by a T-cell clone (RBS35). <i>(</i><i>b</i><i>)</i>, Specific lysis by RBS35 of the HLA-A2⁺/IDO⁺ colon cancer cell line SW480 without or with the addition of the HLA-class I specific antibody W6/32, and lysis of the HLA-A2⁺/IDO^-colon cancer cell line HCT-116. All assays were performed in different E∶T ratios. <i>(</i><i>c</i><i>)</i> Histograms showing intracellular IDO expression in cancer cell lines. Data are representative of 3 experiments. Intracellular IDO expression was given by a one-tailed two sampled t-test comparing MFI_IDO (dark histograms) and MFI_{Isotype control} (light histograms), where MFI is the Mean Fluorescence Intensity. <i>Left:</i> HCT-116 (<i>p</i> = 0.300). <i>Right:</i> SW480 (<i>p</i> = 0.002).</p

Specificity and functional capacity of IDO5-specific T cells assayed by ⁵¹Cr-release assays.

<p><i>(</i><i>a</i><i>),</i> Lysis by the IDO5-specific T-cell clone (RBS35) of the HLA-A2⁺/IDO⁺ melanoma cell line FM55M without and with the addition of cold T2-cells pulsed with IDO5 peptide or an irrelevant peptide (HIV-1 pol_476-484) (inhibitor to target ratio  = 20∶1), and NK cell activity of RBS35 examined using the natural killer cell line K562 as target cells. <i>(</i><i>b</i><i>),</i> Lysis by RBS35 of AML-blasts enriched from 5 HLA-A2⁺ AML patients and 1 HLA-A2^- AML patient. B cells and T cells were depleted from the bone marrow of the AML patients using CD19⁺ and CD3⁺ microbeads, respectively. The highly enriched AML-blasts were used as target cells with or without the addition of the HLA-class I specific antibody W6/32. <i>(</i><i>c</i><i>),</i> Lysis of T2-cells pulsed with IDO5 peptide or an irrelevant peptide (HIV-1 pol_476-484), and lysis of the HLA-A2⁺/IDO⁺ colon cancer cell line SW480 by an IDO5-specific T-cell bulk culture. <i>(</i><i>d</i><i>),</i> Lysis of the HLA-A2⁺/IDO⁺ colon cancer cell line SW480 and HLA-A2⁺/IDO^- colon cancer cell line HCT-116 by three different IDO5-specific T-cell clones (RBS26 (<i>white triangle</i>), RBS31 (<i>black triangle</i>), RBS46 (<i>grey triangle</i>)) assayed by ⁵¹Cr-release assay. All assays were performed in different E∶T ratios.</p

Tetramer analysis of IDO5-specific T cells.

<p><i>(</i><i>a</i><i>),</i> An example of IDO5-specific, CD8 T-cell enriched PBMC from a renal cell carcinoma patient visualised by flow cytometry staining using the tetramer complex HLA-A2/IDO5-PE, and CD8-allophycocyanin after one <i>in vitro</i> stimulation with IDO5 peptide. As a negative control, cells were stained with the tetramer complex HLA-A2/HIV-1 pol_476–484-PE, and CD8-allophycocyanin. <i>(</i><i>b</i><i>),</i> PBMC from healthy donors or from patients with breast cancer, melanoma cancer or renal cell carcinoma were stained with the tetramer complex HLA-A2/IDO5 or HLA-A2/HIV-1 pol_476–484 and analyzed by flow cytometry either <i>ex vivo</i> or after one <i>in vitro</i> peptide stimulation. The dotted lines illustrate that IDO5-tetramer positive cells are detectable both <i>ex vivo</i> and <i>in vitro</i> in the same patients. <i>(</i><i>c</i><i>),</i> An example of CD45RA and CD28 phenotype analysis of IDO5-tetramer/CD8 gated cells from CD8 T-cell enriched PBMC from a renal cell carcinoma patient visualised <i>ex vivo</i> by flow cytometry. For comparison, the cells were stained with isotype matched controls <i>(</i><i>d</i><i>),</i> An example of an IL-2 expanded TIL culture from a melanoma patient visualised by flow cytometry staining using the tetramer complex HLA-A2/IDO5-PE, and CD8-APC-Cy7. As a negative control, the TIL were stained with the tetramer complex HLA-A2/HIV-1 pol_476–484-PE, and CD8-APC-Cy7. <i>(</i><i>e</i><i>),</i> As a positive control of the IDO5 tetramer, an IDO5-specific T-cell clone (RBS35) was stained with the HLA-A2/HIV-1 pol_476–484-PE and HLA-A2/IDO5-PE tetramers.</p

HLA-A2 binding affinity of selected IDO-derived peptides.

*<p>The C5 value is the peptide concentration required for half maximal stabilization of HLA-A2.</p

HLA-A2-restricted T-cell responses against IDO as measured by IFN-γ ELISPOT.

<p>PBMC from healthy donors, breast cancer patients, melanoma patients, and renal cell carcinoma patients were analyzed. All individuals were HLA-A2⁺. The peptides IDO5 (IDO_199-207; ALLEIASCL) <i>(</i><i>a</i><i>)</i>, IDO2 (IDO_164-172; FLVSLLVEI) <i>(</i><i>b</i><i>)</i>, and IDO6 (IDO_320-328; VLSKGDGL) <i>(</i><i>c</i><i>),</i> were examined. T cells were stimulated once <i>in vitro</i> with peptide before being plated at 4×10⁵ cells per well in duplicates either without or with the relevant IDO peptide. The average number of IDO-specific spots (after subtraction of spots without added peptide) was calculated per 4×10⁵ PBMC for each patient (white triangle). <i>(</i><i>a</i><i>), Top,</i> Example of an ELISPOT response against IDO5 (IDO_199-207; ALLEIASCL) in PBMC from a breast cancer patient.</p

Functional capacity of IDO5-specific T cells to kill cancer cell lines treated with IFN-γ for up-regulation of IDO, or treated with IDO ShRNA for down-regulation of IDO.

<p><i>(</i><i>a</i><i>),</i> Lysis by the IDO5-specific T-cell clone (RBS35) of the HLA-A2⁺ breast cancer cell lines CAMA-1 <i>(right)</i> and MDA-MB-231 <i>(left)</i> before and after IFN-γ treatment. <i>(</i><i>b</i><i>)</i>, Histograms showing intracellular IDO expression in CAMA-1 before and after IFN-γ treatment. Data are representative of 3 experiments. Intracellular IDO expression was given by a one-tailed two sampled t-test comparing MFI_IDO (dark histograms) and MFI_{Isotype control} (light histograms), where MFI is the Mean Fluorescence Intensity. The fold of expression was defined as MFI_IDO/MFI_{Isotype control}. <i>Top:</i> CAMA-1 (<i>p</i> = 0.020 and MFI_IDO/MFI_{Isotype control}  = 2.3). <i>Bottom:</i> CAMA-1 + IFN-γ treatment (<i>p</i> = 0.004 and MFI_IDO/MFI_{Isotype control}  = 3.5). <i>(</i><i>c</i><i>),</i> Histograms showing HLA-A2 expression in CAMA-1 before and after IFN-γ treatment. Data are representative of 3 experiments. HLA-A2 expression was given by a one-tailed two sampled t-test comparing MFI_HLA-A2 (dark histograms) and MFI_{Isotype control} (light histograms). The fold of expression was defined as MFI_HLA-A2/MFI_{Isotype control}. <i>Top:</i> CAMA-1 (<i>p</i> = 0.004 and MFI_HLA-A2/MFI_{Isotype control}  = 43.7). <i>Bottom:</i> CAMA-1 + IFN-γ treatment (<i>p</i> = 0.002 and MFI_IDO/MFI_HLA-A2  = 141.2). <i>(</i><i>d</i><i>)</i>, Lysis of the colon cancer cell line SW480 transfected with IDO ShRNA for down-regulation of IDO protein expression by an IDO5-specific T-cell bulk culture. As a positive control, SW480 cells transfected with control ShRNA were used as target cells. All assays were performed in different E∶T ratios. <i>(</i><i>e</i><i>)</i>, Histograms showing intracellular IDO expression in SW480 transfected with control ShRNA (<i>p</i> = 0.001 and MFI_IDO/MFI_{Isotype control}  = 4.8) <i>(top)</i> and IDO ShRNA (<i>p</i> = 0.040 and MFI_IDO/MFI_{Isotype control}  = 2.1) <i>(bottom)</i>.</p

Functional capacity of an IDO5-specific T-cell clone (RBS35) to kill immune cells assayed by ⁵¹Cr-release assays.

<p><i>(</i><i>a</i><i>)</i>, <i>Left:</i> Lysis of autologous <i>in vitro</i> immatured and matured DC. <i>Right:</i> Lysis of allogeneic HLA-A2⁺<i>in vitro</i> immatured and matured DC. All assays were performed in different E∶T ratios. <i>(</i><i>b</i><i>),</i> Histograms showing intracellular IDO expression in DC. Data are representative of 3 experiments. Intracellular IDO expression was given by a one-tailed two sampled t-test comparing MFI_IDO (dark histograms) and MFI_{Isotype control} (light histograms), where MFI is the Mean Fluorescence Intensity. <i>Left: In vitro</i> immatured DC (<i>p</i> = 0.100). <i>Right: In vitro</i> matured DC (<i>p</i> = 0.001). <i>(</i><i>c</i><i>)</i>, Lysis of autologous CD14⁺ monocytes, CD3⁺ T cells and CD19⁺ B cells isolated directly <i>ex vivo</i> from IDO⁺ PBMC, and lysis of autologous CD14⁺ monocytes, CD3⁺ T cells and CD19⁺ B cells after IFN-γ treatment. As a control, we used <i>in vitro</i> generated autologous IDO^- immatured DC and IDO⁺ matured DC. <i>(</i><i>d</i><i>)</i>, Examples of HLA-A2 restricted T-cell responses against EBV BMLF1_280-288 (GLCTLVAML) as measured by ELISPOT in PBMC from a breast cancer patient. Cultures of PBMC were treated with IFN-γ for 5 days with autologuous freshly isolated CD8 T-cells <i>(left)</i> or with autologous IDO5 specific T-cells (at a PBMC:IDO5 specific T-cell ratio of 300∶1) <i>(right)</i> before examination for reactivity against the HLA-A2 restricted epitope from EBV BMLF1_280-288 (GLCTLVAML). Three different PBMC concentrations were examined; 1,5×10⁵ cells, 5×10⁴ cells (<i>two top rows</i>) and 10⁴ cells (<i>bottom two rows</i>).</p