T-cell responses against IDOlong.

<p><i>(</i><b><i>A</i></b><i>),</i> T-cell responses against IDOlong (DTLLKALLEIASCLEKALQVF (IDO194-214)) as measured by IFN-γELISPOT: Example of an ELISPOT response against IDOlong in PBMC from a renal cell carcinoma patient (<i>Top</i>). The average number of IDOlong-specific spots (after subtraction of spots without added peptide) was calculated per 5×10⁵ PBMC for each patient. PBMC from ten healthy individuals (HD), twenty three melanoma patients (MM), fifteen renal cell carcinoma patients (RCC) as well as nine breast cancer patients (BC) were analyzed. PBMC were stimulated once with peptide before being plated at 5×10⁵ cells per well in duplicates either without or with the IDOlong peptide (<i>bottom</i>). <i>(</i><b><i>B</i></b><i>),</i> T-cell responses against IDOlong as measured by TNF-α ELISPOT: Example of an ELISPOT response against IDOlong in PBMC from a melanoma patient (<i>Top</i>). The average number of IDOlong-specific spots (after subtraction of spots without added peptide) was calculated per 5×10⁵ PBMC for each patient. PBMC from eleven healthy individuals (HD), eighteen melanoma patients (MM), fourteen renal cell carcinoma patients (RCC) as well as nine breast cancer patients (BC) were analyzed. PBMC were stimulated once with peptide before being plated at 5×10⁵ cells per well in duplicates either without or with the IDOlong peptide (<i>bottom</i>). <i>(</i><b><i>C</i></b><i>),</i> T-cell responses against IDOlong as measured by IL-17 ELISPOT: Example of an ELISPOT response against IDOlong in PBMC from a breast cancer patient (<i>Top</i>). The average number of IDOlong-specific spots (after subtraction of spots without added peptide) was calculated per 5×10⁵ PBMC for each patient. PBMC from nine healthy individuals (HD), twenty one melanoma patients (MM), sixteen renal cell carcinoma patients (RCC) as well as nine breast cancer patients (BC) were analyzed. PBMC were stimulated once with peptide before being plated at 5×10⁵ cells per well in duplicates either without or with the IDOlong peptide (<i>bottom</i>). <i>(</i><b><i>D</i></b><i>),</i> Percentage of donors secreting none <i>(white)</i>, one <i>(black)</i>, two <i>(dark grey)</i> or all three <i>(light grey)</i> cytokines (IFN-γ,TNF-α,IL-17) in response to IDOlong.</p

Correlation of CD4 responses against IDO with CD8 T-cell responses.

<p><i>(</i><b><i>A</i></b><i>),</i> the correlation of IFN-γ IDOlong responses with the class I tissue type HLA-A2. A Mann-Whitney test demonstrated that for there was a significant difference between IFN-γ responders and non-responders (P = 0.0003). <i>(</i><b><i>B</i></b><i>),</i> the correlation of TNF-α IDOlong responses with the class I tissue type HLA-A2. A Mann-Whitney test demonstrated that there was not significant difference between TNF-α responders and non-responders (P = 0.2). <i>(</i><b><i>C</i></b><i>),</i> correlation between IFN-γ IDOlong responses and IFN-γresponses against the HLA-A2-restricted, IDO-derived epitope IDO5 (IDO199-207). PBMC from IDO responders and non-responders were stimulated with IDO5 once <i>in vitro</i> before being plated at 5×10⁵ cells per well in duplicates either without or with the IDO5 peptide. A Mann-Whitney test demonstrated that for there was not significant difference between responders and non-responders (P = 0.2). <i>(</i><b><i>D</i></b><i>),</i> correlation between IFN-γ IDOlong responses and IFN-γ responses against HLA class I-restricted epitopes from CMV. PBMC from IDO responders and non-responders were directly plated at 5×10⁵ cells per well in duplicates either without or with one of the most immunogenic HLA class I-restricted CMV-epitopes, i.e. the HLA-A2-restricted epitope CMV pp65_495–503 (NLVPMVATV), the HLA-A11-restricted epitope CMV pp65_16–24 (GPISGHVLK), the HLA-A1- and HLA-A24-restricted epitope CMV pp65_341–350 (QYDPVAALFF) or the HLA-A1-restricted epitope CMV pp65_363–373 (YSEHPTFTSQY) depending on the HLA-type of the individual donor. A Mann-Whitney test illustrated that there was significant difference in T cell reactivity towards CMV epitopes between IDOlong responders and non-responders (P = 0.04).</p

T-cell responses against IDOlong as measured by IL-10 ELISPOT.

<p>PBMC from six healthy individuals (HD1-6), sixteen melanoma patients (MM1-16) and nine renal cell carcinoma patients (RCC1-9) were analyzed. PBMC were stimulated once with peptide before being plated at 5×10⁵ cells per well in duplicates either without (<i>black bars</i>) or with the IDOlong peptide (<i>grey bars</i>).</p

Patient characteristics.

<p>Patient characteristics.</p

HLA class II-restricted IDOlong responses as measured by IFN-γELISPOT.

<p>PBMC from one renal cell carcinoma patient <i>(</i><b><i>A</i></b><i>)</i> and five melanoma patients <i>(</i><b><i>B,C,D,E,F</i></b><i>)</i> were stimulated once with peptide before being plated at either 2×10⁵ or 5×10⁵ cells per well in duplicates either without or with the IDOlong peptide or without and with a monoclonal anti-HLA class II antibody. The average number of IFN-γ releasing cells was calculated either per 2×10⁵ PBMC or 5×10⁵ PBMC for each patient.</p

IDOlong contains various T cell epitopes.

<p><i>(A)</i> PMBC from seven patients (1 breast cancer patient and 6 melanoma patients) were analysed for responses against IDO194 (DTLLKALLEIASCLE (IDO194-208)) (<i>grey bars</i>), IDO200 (LLEIASCLEKALQVF (IDO200-214)) (<i>white bars</i>) and compared to IDOlong (<i>black bars</i>) by IFN-γ ELISPOT assay. The average number of peptide-specific spots (after subtraction of spots without added peptide) was calculated per 5×10⁵ PBMC for each patient. PBMC were stimulated once with peptide before being plated at 5×10⁵ cells per well in duplicates either without or with the peptide. <i>(B),</i> CpG stimulate IDO-specific T cells. PBMC from a melanoma patient and a renal carcinoma patient were treated with either IL-2 or the TLR9 ligand CpG ODN in the presence of IL-2 for 14 days and, subsequently, examined for IDO-specific T cells by TNF-α ELISPOT. Hence, PBMC were plated at 5×10⁵ cells per well in duplicates either without or with the IDOlong peptide before and after cell culture. The average number of TNF-α releasing cells was calculated per 2×10⁵ PBMC for each patient.</p

CD4⁺ T cell responses against IDO.

<p><i>(</i><b><i>A</i></b><i>),</i> CD4⁺ T cell responses against IDOlong as examined by IFN-γ ELISPOT. PBMC from two malignant melanoma patients <i>(left, middle)</i> and one renal cell carcinoma patient <i>(right)</i> were stimulated once with IDOlong peptide before CD4⁺ T cells were isolated. The CD4⁺ cells were plated at 2×10⁵ cells per well in duplicates with 10⁴ DC either without or with the IDOlong peptide. As a control 10⁴ DC were plated alone without T cells. The numbers of IFN-γ releasing cells were counted for each patient. <i>(</i><b><i>B</i></b><i>), ex vivo</i> ELISPOT analysis of CD4 T-cells isolated from three melanoma patients. CD4⁺ cells were isolated from PBMC from three different donors and directly plated at 2×10⁵ cells per well in duplicates with 10⁴ DC either without or with the IDOlong peptide. HLA-class II antibody were added to two additional wells with CD4 cells, dendritic cells and IDOlong peptide. The numbers of IFN-γ releasing cells were counted for each patient.</p

Intracellular cytokine staining.

<p><i>(</i><b><i>A</i></b><i>),</i> TNF-α (<i>top</i>) and IFN-γ (<i>below</i>) intracellular cytokine stainings. PBMC from two melanoma patients were analyzed with and without IDOlong-stimulation. <i>(</i><b><i>B</i></b><i>),</i> PBMC from a melanoma patient were stimulated 2 times with autologous DC that had been matured with IDOlong peptide in the presence of IL-2 before being analysed by intracellular TNF-α and IFN-γ stainings. PBMC were stimulated with autologous DC matured with either an irrelevant 20 amino acid long nonsense peptide, IDOlong peptide, lysate from the IDO⁺ colon cancer cell line SW480 or lysate from the IFN-γ pre-treated melanoma cell line MM404.111. FACS plots were gated on live CD4⁺ T-cells.</p