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 5×10<sup>8</sup> 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<sup>+</sup> T cell epitopes are typed in bold and the H2-K<sup>b</sup>-restricted CTL epitope TRP-2<sub>180–188</sub> is written in italics. <i>C</i>, HLA-DR3tg mice received i.p. injections of 5×10<sup>8</sup> 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<sup>+</sup> CD4<sup>+</sup> T cells. Error bars show standard error of the mean of three immunized mice. Experiments were performed three times yielding similar results.</p>