Figure 5. Phenotypic analysis of TRP-1-specific T cell responses induced in HLA-DR3tg mice upon immunization with Ad5.TRP-1.

A, 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 ex vivo 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. B, Selected TRP-1-derived library peptides #8, #9, #36 and #47 are indicated by amino acid (aa) positions and aa sequence. Peptides were analyzed in silico by the SYFPEITHI algorithm for the presence of potential HLA-DRB*0301 binding sequences (underlined) [33]. 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. C, 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 in vitro 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.