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.