10.1371/journal.pone.0014137.g002 Wolfram Osen Wolfram Osen Sabine Soltek Sabine Soltek Mingxia Song Mingxia Song Barbara Leuchs Barbara Leuchs Julia Steitz Julia Steitz Thomas Tüting Thomas Tüting Stefan B. Eichmüller Stefan B. Eichmüller Xuan-Duc Nguyen Xuan-Duc Nguyen Dirk Schadendorf Dirk Schadendorf Annette Paschen Annette Paschen Phenotypic analysis of TRP-2-specific T cell responses induced in Ad5.TRP-2-immunized HLA-DR3tg mice. Public Library of Science 2013 trp-2-specific responses induced hla-dr3tg 2013-02-20 22:02:50 Figure https://plos.figshare.com/articles/figure/_Phenotypic_analysis_of_TRP_2_specific_T_cell_responses_induced_in_Ad5_TRP_2_immunized_HLA_DR3tg_mice_/485256 <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>