Generation of influenza-specific CD8β T-cell lines from Babraham pigs simultaneously immunized with H5N1-S-FLU and Sp/Sw H1N1.

<p>CD8β T-cells purified from the peripheral blood mononuclear cells of Babraham pigs 625 (filled bars) and 650 (open bars) were stimulated <i>in vitro</i> with pools (four pools: A, B, C and D) of overlapping peptides (81 in total, <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007017#ppat.1007017.s002" target="_blank">S2 Table</a></b>) from the NP of S-FLU (PR8). Two weeks post stimulation the cells were tested for reactivity towards individual peptides from each pool. Intracellular cytokine staining for TNF was performed following a 5 h incuabtion with no peptide (DMSO control) or peptides (2 μM). All associated flow cytometry data is shown in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007017#ppat.1007017.s008" target="_blank">S2 Fig</a></b> (pig 625) and <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007017#ppat.1007017.s009" target="_blank">S3 Fig</a></b> (pig 650) with the percentage of TNF producing cells displayed here. (<b>A</b>) T-cell lines generated against pool A mapped to individual peptides 16 and 17 (sequences shown with overlap region in bold). (<b>B</b>) Reactivity for pool B for pig 625 (left graph) had been seen in previous experiments with indications for reactivity towards peptides 36 and 37, thus lines were successfully generated against individual peptides 36 and 37 (right graph). (<b>C</b>) Lines responsive to pool C mapped to individual peptide 42, 43, 48 and 49. T-cell clones were grown directly from these lines by limiting dilution or first by enriching for peptide specific T-cells using a TAPI-0 assay (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007017#ppat.1007017.s007" target="_blank">S1 Fig</a></b>) and flow cytometry.