Assessment of MELOE-1 CD4 T cell responses in PBMC from healthy donors.

<p>PBMC from healthy donors were stimulated with 10 µM of MELOE-1. After 14 days, the presence of CD4 T cells specific for the different regions of MELOE-1 was assessed by re-stimulating cells with MELOE-1_2–21, MELOE-1_11–30, MELOE-1_18–37 and MELOE-1_26–46 peptides, followed by CD4/TNF-α double staining and flow cytometry analysis. Between brackets is indicated the mean % of TNF-α producing CD4 T cells, in positive microcultures. NA: not available.</p

MELOE-1 specific responses in healthy donors.

<p>(A) Dot plot illustrating peptide-specific TNF responses, in MELOE-1 stimulated microcultures from HD9 and HD28. Fourteen days after PBMC stimulation with MELOE-1 whole polypeptide (2–46), microcultures were re-stimulated or not with each indicated peptide during 5 h. Reactivity was then assessed by double staining TNFα/CD4. Percentages indicate the fraction of TNF producing T cells, among CD4 T cells. (B) Frequency of microcultures containing CD4 T cells specific for MELOE-1 peptides. A microculture was considered as positive when the fraction of TNF producing T cells was 2-fold higher upon peptide stimulation than in unstimulated cultures. (C) Percentages of TNF-α producing CD4 T cells upon peptide-stimulation, in positive microcultures from all donors.</p

TCR characterization and cytokine profile of MELOE-1 specific CD4 T cell clones.

a<p>CD4 T cell clones were stimulated for 5 hours in the presence of brefeldin A (10 µg/mL) either with the cognate peptide (10 µM) in an autopresentation assay. After 5 hours of stimulation, cells were stained with APC-conjugated anti-CD4 mAb, fixed with 4% paraformaldehyde, labeled with PE-conjugated anti-cytokine mAb and analyzed by flow cytometry. A “high” cytokine production was defined with a threshold of 20% of producing CD4 T cells, in response to the peptide.</p

MELOE-1 specific responses in melanoma patients.

<p>(A) Dot plot illustrating peptide-specific Th1 responses in MELOE-1 stimulated microcultures from Pt≠3. Fourteen days after PBMC stimulation with MELOE-1 whole polypeptide (2–46), microcultures were re-stimulated with each indicated peptide during 5 h. Specificity was then assessed by a triple labelling IFN-γ/IL-4/CD4. Percentages indicate the fraction of IFN-γ producing T cells, among CD4 T cells. (B) Frequency of microcultures containing IFN-γ (black bars) or IL-4 (white bars) producing CD4 T cells specific for MELOE-1 overlapping peptides. A microculture was considered as positive when the fraction of cytokine producing CD4 T cells was 2-fold higher upon peptide stimulation than in unstimulated cultures. (C) Percentages of cytokine producing CD4 T cells in positive microcultures from all patients. IFN-γ (left panel) and IL4 (right panel) production was assessed by a triple labelling IFN-γ -IL4-CD4, after peptide re-stimulation.</p

Assessment of MELOE-1 CD4 T cell responses in PBMC from melanoma patients.

<p>PBMC from melanoma patients were stimulated with 10 µM of MELOE-1. After 14 days, the presence of CD4 T cells specific for the different regions of MELOE-1 was assessed by re-stimulating cells with MELOE-1_2–21, MELOE-1_11–30 and MELOE-1_22–46 peptides, followed by CD4/IFN-γ double staining for the detection of Th1 responses, and by CD4/IL4 double staining for Th2 responses. Between brackets is indicated the mean % of cytokine producing CD4 T cells, in positive microcultures.</p

MELOE-1 and MELOE-1 derived peptide sequences.

<p>All the peptides were purchased from Millegen company (France), with a purity >85%. In bold are indicated the DR-11 (CPPWHPSERISSTL) and the DQ-6 (RISSTLNDECWPA) overlapping epitopes already described, and in italics is indicated the HLA-A2 restricted class I epitope (TLNDECWPA).</p

Class II epitopes are naturally processed from MELOE-1 whole antigen.

<p>Autologous DC, were loaded (before or after fixation) with MELOE-1_2–46 (1 µM) or, as a negative control, with Melan-A_16–40L peptide (1 µM), and matured. T cell clones were then stimulated with DC at a ratio 1/1, during 5 h in presence of BFA. Reactivity was assessed by double staining TNF-α/CD3, and analyzed by flow cytometry. Histograms illustrated the percentage of TNF-α producing cells among CD3 positive T cells.</p

HLA-restricting element of MELOE-1 specific T cell clones and reactivity against HLA-matched melanoma cell lines.

<p>Upper panel: the HLA restriction of MELOE-1 specific T cell clones was assessed by intracellular TNF labelling, using anti-HLA blocking antibodies. T cell clones were stimulated either with peptide alone (10 µM) in an autopresentation assay and in presence or not of blocking antibodies at a concentration of 12.5 µg/mL. Middle panel: HLA restriction was confirmed with HLA-matched B-EBV cell lines unloaded (white bars) or pulsed (black bars) 2 h with the cognate peptide, at an effector/ratio of 1/2. Lower panel: reactivity of each T cell clone against HLA-class II expressing melanoma cells (ratio 1/2) was assessed by intracellular TNF labelling, in presence (black bars) or not (white bars) of exogenous peptide.</p

Minimal peptides recognized by MELOE-1 specific CD4 T cell clones.

<p>MELOE-1 specific CD4 T cell clones were incubated with various concentrations of the indicated peptides during 5 h in presence of BFA. TNFα production was assessed by intracellular labelling. The core peptide sequence is indicated in bold on each figure panel, and black circles illustrate the best fitting peptide.</p

Functional properties of the M314.132 DP T cell clone.

<p>A/ Cytokine production analysis. DP T cell clone was fixed, permeabilized and stained for cytokines following autologous melanoma stimulation. Data are expressed as mean % of intracellular cytokine secreting cells. Open histograms correspond to the analysis of cytokine production by unstimulated M314.132 DP T cell clone (negative control). B/Lysis of the M314 autologous melanoma cell line (closed circles) by M314.132 DP T cell clone. The M132 cell line was used as negative control target (open circles). 51Cr-labeled tumor cells were co-cultured with T cells at various E/T ratios. Chromium release in the supernantants was measured after a 4-h incubation period. C/ Phenotypic characterization of M314 DP T cell clone. D/ Proliferation capacity. CFSE-labeled T cell clones were stimulated with anti-CD3 (OKT3). The CD8 T cell clone used as positive control was obtained by limiting dilution of melanoma specific CD8 T cells. As negative control, T cell clones were maintained in the absence of any stimulation (Not Stimulated: NS).</p