HLA-B8 association with late-stage melanoma – an immunological lesson?-0

<p><b>Copyright information:</b></p><p>Taken from "HLA-B8 association with late-stage melanoma – an immunological lesson?"</p><p>BMC Medicine 2006;4():5-5.</p><p>Published online 13 Mar 2006</p><p>PMCID:PMC1421420.</p><p>Copyright © 2006 Fensterle et al; licensee BioMed Central Ltd.</p> I-III patients are calculated to compare relative changes in carrier frequencies from data acquired in one center; all: mean ratio of all HLA-A and -B alleles with carrier frequencies >15%

Increased frequencies of immune regulatory cells in peripheral blood of stage IV melanoma patients.

<p>PBMC samples obtained from melanoma patients pre-vaccination and from HD were analysed for the presence of Th17 (<b>A</b>), IFNγ-secreting Th17 (<b>B</b>), Tregs (<b>C</b>), and MDSCs (<b>D</b>) using flow cytometry with the staining protocol described in the materials and methods section. Frequencies were calculated as percentage of living PBMC. Results are depicted for all patients as well as stratified according to gender and metastatic stage (M category). The horizontal line in the middle of each box indicates the median, whereas the top and bottom borders of the box mark the 75th and 25th percentiles, respectively. The whiskers above and below the box represent the data range. *p<0.05, **p<0.01, ***p<0.001.</p

Reduced CD3ζ-expressing and IFNγ-secreting T cells in peripheral blood of stage IV melanoma patients.

<p>Peripheral blood mononuclear cell (PBMC) samples obtained from melanoma patients pre-vaccination and from healthy donors (HD) were analysed by flow cytometry using antibodies reacting with CD3ζ chain (A) and IFNγ (B). Cells were stained after stimulation with phorbol myristate acetate (PMA) and ionomycin for four hours. Results are depicted for CD4⁺ (left panel) and CD8⁺ (right panel) T cells. Frequencies of IFNγ-secreting cells were calculated as percentage of living PBMC. Results are given for all patients as well as stratified to M category. Triangles for female and circles for male indicate patient’s gender. Horizontal bars represent median values. MFI, mean fluorescence intensity. *p<0.05, **p<0.01, ***p<0.001.</p

Pre-vaccination frequencies of Th17 cells and survival.

<p>Kaplan-Meier plots depicting the probability of overall survival of melanoma patients vaccinated with survivin-derived peptides according to pre-vaccination frequencies of Th17 cells (<b>A</b>), and IFNγ-secreting Th17 cells (<b>B</b>); groups were separated by Th17 ≤0.3% (solid line) or >0.3% (dotted line) as well as IFNγ-secreting Th17 ≤0.04% (solid line) or >0.04% (dotted line) of living cells, respectively. Statistical differences between groups were calculated using the log rank test; censored observations are indicated by vertical bars.</p

Frequencies of cell subsets in PBMC from melanoma patients.

<p>Th17= CD4⁺IL17⁺, Tregs= CD4⁺CD25⁺CD127^-, MDSC=CD14⁺HLA-DR^-,Th1 = CD4⁺IFNγ⁺, Tc1= CD8⁺ IFNγ⁺.</p><p>^a % of living cells</p><p>^b MFI</p><p>P-values are comparison between cancer patients and healthy donors</p><p>Frequencies of cell subsets in PBMC from melanoma patients.</p

Frequencies of immune regulatory cells pre-vaccination correlate with vaccine-induced T-cell responses.

<p>Frequencies of Th17 (<b>A)</b>, IFNγ-secreting Th17 cells (<b>B)</b>, and MDSC <b>(C)</b> depicted as percentage of living cells were grouped according to whether they mounted survivin-specific T-cell responses (SSTR) or not. Patient’s gender is indicated by triangles for female and circles for male. *p<0.05, **p<0.01, ***p<0.001.</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

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