TRP-1_284–298 represents a HLA-DRB1*0301-restricted CD4⁺ T cell epitope processed by human target cells.

<p>PBMC from melanoma patient VHP were stimulated once with peptide TRP-1_284–298 (10 µg/ml). After 25 days, selected CD4⁺ T cells were tested for their reactivity against different target cells by IFN-γ ELISpot assay. <i>A</i>, Recognition of T2.DR3 target cells pulsed with peptide TRP-1_284–298 and autologous CD3⁺-depleted PBMC (CD3⁻ PBMC) infected with Ad5.TRP-1 or with control virus is depicted. <i>B</i>, Reactivity of T cells against different melanoma cell lines Ma-Mel-103b (HLA-DRB1*0301⁺, TRP-1⁻), Ma-Mel-108 (HLA-DRB1*0301⁺, TRP-1⁺), Ma-Mel-153 (HLA-DRB1*0301⁻, TRP-1⁺) and <i>C</i>, against Ma-Mel-103b cells infected with Ad5.TRP-1 or control virus is presented. <i>A–C</i>, One representative out of two to three independent experiments is given. Error bars show standard error of the mean.</p

Combinatorial peptide library screening allows detection of individual library peptides containing TRP-2-specific T cell epitopes.

<p><i>A</i>, Composition of the TRP-2-specific library peptide pools X1 to X8 and Y1 to Y8 used for combinatorial screening of specific T cell responses <i>ex vivo</i>. Individual library peptides determined by combinatorial screening are highlighted. <i>B</i>, Spleen cells from HLA-DR3tg mice injected i.p. with 5×10⁸ pfu Ad5.TRP-2 or Ad5.EGFP (2 mice per group) were screened <i>ex vivo</i> by IFN-γ ELISpot assay for recognition of TRP-2-specific library peptide pools. T cell responses of two control mice (Ad5.EGFP) and two TRP-2-immunized mice (Ad5.TRP-2) are represented as individual columns in the diagram. Reactivity against two controls, the H2-K^b-restricted CD8⁺ T cell epitope TRP-2_180-188 and the HLA-DRB1*0301-restricted CD4⁺ T cell epitope TRP-2_60–74 was tested in addition. Error bars show standard error of the mean. Experiments were performed four times, yielding similar results.</p

Detection of TRP-1_284–298 reactive CD4⁺ T cells in melanoma patients.

<p>Total PBMC (left panel) or CD25⁺-depleted PBMC (right panel) from five HLA-DRB1*03⁺ melanoma patients (<i>A</i>) and four HLA-DRB1*0301⁺ healthy donors (<i>B</i>) were stimulated <i>in vitro</i> with peptide TRP-1_284–298. After 25 days, PBMC were tested for their peptide reactivity by IFN-γ ELISpot assay. All determinations were performed at least in duplicates. The data are presented as mean numbers of IFN-γ spots per 10⁵ cells. Error bars show standard error of the mean.</p

Combinatorial peptide library screening allows detection of individual library peptides containing TRP-1-specific T cell epitopes.

<p><i>A</i>, Composition of the TRP-1-specific library peptide pools X1 to X8 and Y1 to Y8 used for combinatorial screening of specific T cell responses <i>ex vivo</i>. Individual peptides determined by combinatorial screening are highlighted. <i>B</i>, Spleen cells from HLA-DR3tg mice injected i.p. with 5×10⁸ pfu Ad5.TRP-1 or Ad5.EGFP (2 mice per group) were screened <i>ex vivo</i> by IFN-γ ELISpot assay for recognition of TRP-1-specific library peptide pools. T cell responses of two control mice (Ad5.EGFP) and two Ad5.TRP-1-immunized mice are represented as individual columns in the diagram. Error bars show standard error of the mean of duplicates. Experiments were performed four times, yielding similar results.</p

Phenotypic analysis of TRP-2-specific T cell responses induced in Ad5.TRP-2-immunized HLA-DR3tg mice.

<p><i>A</i>, Spleen cells from HLA-DR3tg mice injected i.p. with 5×10⁸ pfu Ad5.TRP-2 or Ad5.EGFP (2 mice per group) were screened <i>ex vivo</i> by IFN-γ ELISpot assay for reactivity against individual TRP-2-specific library peptides determined by combinatorial analysis. T cell responses of two control mice (Ad5.EGFP) and two TRP-2-immunized mice (Ad5.TRP-2) are represented as individual columns in the diagram. Error bars show standard error of the mean. Experiments were performed three times, yielding similar results. <i>B</i>, Selected TRP-2-derived library peptides #8, #19, #22 and #23 are indicated by amino acid (aa) positions and aa sequence. Peptides were analyzed <i>in silico</i> by the SYFPEITHI algorithm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014137#pone.0014137-Rammensee1" target="_blank">[33]</a> for the presence of predicted HLA-DRB*0301 binding sequences (underlined). Prediction scores for HLA-DRB1*0301-restricted epitopes are listed on the right. Known HLA-DRB1*0301-restricted CD4⁺ T cell epitopes are typed in bold and the H2-K^b-restricted CTL epitope TRP-2_180–188 is written in italics. <i>C</i>, HLA-DR3tg mice received i.p. injections of 5×10⁸ pfu Ad5.TRP-2 or Ad5.EGFP (3 mice per group). Two weeks later spleen cells from infected mice were harvested and stimulated <i>in vitro</i> with the indicated peptides. After 6 days, splenocyte cultures were analyzed for the presence of peptide-reactive T cells by intracellular IFN-γ staining. Stained cells were analyzed by flow cytometry for the percentage of IFN-γ⁺ CD4⁺ T cells. Error bars show standard error of the mean of three immunized mice. Experiments were performed three times yielding similar results.</p

Phenotypic analysis of TRP-1-specific T cell responses induced in HLA-DR3tg mice upon immunization with Ad5.TRP-1.

<p><i>A</i>, Spleen cells from HLA-DR3tg mice injected i.p. with 5×10⁸ pfu Ad5.TRP-1 or Ad5.EGFP (2 mice per group) were screened <i>ex vivo</i> by IFN-γ ELISpot assay for reactivity against selected TRP-1-derived library peptides. T cell responses of two control mice (Ad5.EGFP) and two Ad5.TRP-1-immunized mice are represented as individual columns in the diagram. Error bars show standard error of the mean of duplicates. Experiments were performed three times, yielding similar results. <i>B</i>, Selected TRP-1-derived library peptides #8, #9, #36 and #47 are indicated by amino acid (aa) positions and aa sequence. Peptides were analyzed <i>in silico</i> by the SYFPEITHI algorithm for the presence of potential HLA-DRB*0301 binding sequences (underlined) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014137#pone.0014137-Rammensee1" target="_blank">[33]</a>. Prediction scores for HLA-DRB1*0301-restricted epitopes are listed on the right. The potential H2-restricted CTL epitope TRP-1_374–382 is given in italics. <i>C</i>, Spleen cells of HLA-DR3tg mice injected i.p. with 5×10⁸ pfu Ad5.TRP-1 or Ad5.EGFP (3 mice per group) were analyzed for the presence of peptide-reactive T cells by intracellular IFN-γ staining after one round of <i>in vitro</i> re-stimulation with the indicated peptides. Note: The amino acid sequence of these peptides was shifted towards the N-terminus by one residue relative to the predicted epitope sequence. Stained cells were analyzed by flow cytometry for the percentage of IFN-γ⁺ CD4⁺ T cells. Error bars show standard error of the mean of three immunized mice. Experiments were performed twice yielding similar results.</p