EdU labeling index in five cell types.

<p>Those which generated insulin-immunopositive cells showed a reduction in EdU label. Errors are standard errors, n=3.</p

Effects of the small molecule combination.

<p>A. ASH cells transduced with <i>Ad-PNM</i>, without and with the DNB combination. PDX1 is immunostained green and insulin red. Scale bar 100µm. B. Percentage of PDX1-positive cells that are also insulin-positive. Comparisons are by one way ANOVA with Games-Howell post hoc test, * indicates significant enhancement. Errors are standard errors, n=6.</p

Insulin-positive cells generated from different cell types.

<div><p>A,B,C Expression of PDX1, NGN3, MAFA in ASH cells. The proteins are shown in green, DAPI in blue.</p> <p>D. Histogram of the percentage PDX1-positive cells which are insulin-positive in different cell types. </p> <p>E,E’ Rat hepatocytes, some PDX1-positive cells are insulin-positive. </p> <p>F,F’ Rat hepatocytes, insulin-positive cells are also C-peptide-positive. </p> <p>G Mouse ASH cells, some PDX1-positive cells are insulin-positive. </p> <p>H. Mouse ASH cells, showing cytoplasmic location of insulin. Inset, high magnification showing insulin-containing granules.</p> <p>I,I’ Mouse ASH cells showing co-expression of C-peptide with insulin. </p> <p>J Rat AR42j-B13 cells showing a high proportion of insulin-positive cells. </p> <p>K,K’ Rat AR42j-B13 cells showing co-expression of C-peptide with insulin. </p> <p>L. Rat AR42j-B13 showing insulin-containing granules.</p> <p>Scale bars 100µm, except H inset and L which are 25 µm. Blue color is DAPI throughout.</p></div

Expression of various beta cell genes provoked by transduction with <i>Ad-PNM</i>.

<p>A-H. Qualitative RT-PCRs showing the effect of <i>Ad-PNM</i> on the expression of a panel of beta cell genes. 30 cycles were used unless otherwise shown. The misaligned bands in the first two lanes of mouse fibroblast <i>Mnx1</i> are primer dimers, not RNA.</p

Identification of influenza-specific T-cells in the bronchoalveolar lavage from Babraham pigs infected with pandemic H1N1 swine influenza virus.

<p>Babraham pigs were either left uninfected (741) or infected intranasally with pandemic H1N1 [A/sw/Eng/1353/09] (742, 744, 745). The pigs were culled on day 0 (741), 5 (744) or 14 (742 and 745) post infection. (<b>A</b>) 200,000 bronchoalveolar lavage cells from pig 745 (infected, day 14 cull) were incubated alone, with 10^-5M peptide, or virus for 16–18 h. A Babraham kidney cell line was included in each well (15,000 per well) to act as antigen presenting cells. All conditions were performed in duplicate and spot forming cells (SFCs) detected by IFNγ ELISPOT and displayed as mean +SEM and scaled (X5) to 10⁶ BAL cells. (<b>B</b>) Irrelevant and nucleoprotein peptide-SLA-1*14:02 and SLA-2*11:04 tetramer staining was performed on thawed bronchoalveolar lavage samples and the percentage of tetramer⁺ cells of CD8β cells displayed in red. Nucleoprotein peptide sequences are shown. Irrelevant tetramers were SLA-1*14:02-AFAAAAAAL and SLA-2*11:04-AGAAAAAAI. Gating strategy: lymphocytes, single cells, viability (Vivid^neg) CD3⁺ CD14^neg then CD8β⁺ CD4⁺ and displayed as CD8β versus tetramer (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007017#ppat.1007017.s007" target="_blank">S1 Fig</a></b>).</p

Verification of predicted influenza T-cell epitopes in bronchoalveolar from Babraham pigs infected with pandemic H1N1 swine influenza.

<p>Babraham pigs 742 and 745 were experimentally infected intranasally with H1N1 [A/sw/Eng/1353/09] and culled on day 14 post infection. Based on the binding motifs of SLA-1*14:02 and SLA-2*11:04 (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007017#ppat.1007017.g008" target="_blank">Fig 8</a></b>), predicted epitopes from matrix proteins 1 and 2, nucleoprotein, and polymerase basic proteins 1 and 2 (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007017#ppat.1007017.s006" target="_blank">S6 Table</a></b>) were tested as pooled peptides using BAL cells from pig 745 and IFNγ ELISpots (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007017#ppat.1007017.s013" target="_blank">S7 Fig</a></b>). Individual peptides highlighted from this process were then tested: (<b>A</b>) BAL cells from pig 745 were thawed and 200,000 used per well for IFNγ ELISPOT. 18 individual peptides (numbered on the x-axis) were used alongside our validated nucleoprotein epitopes, IAYERMCNI and DFEREGYSL. A no peptide and viral controls were included, and conditions performed in duplicate, scaled (X5) to 10⁶ spot forming cells (SFCs) and with the mean displayed +SEM. Babraham kidney cells were used in every well (15,000 per well) to act as antigen presenting cells. (<b>B</b>) BAL cells from pig 742 were cultured in the presence of the peptides from (A) to create six T-cell lines (labelled on the x-axis). Peptides 85, 102, 117 and 132 were used individually as they gave relatively more SFCs for the ELISPOT in A. The remaining peptides were assembled in to two pools; pool 1 consisted of peptides 30, 32, 34, 75, 81, 83, 92; and pool 2 contained peptides 98, 100, 109, 115, 126, 136 (sequences in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007017#ppat.1007017.s006" target="_blank">S6 Table</a></b>). After two weeks, the lines were taken straight from culture for IFNγ ELISPOT and incubated with 10⁻⁵ M of the peptide(s) used to generate the line (+) or with no peptide (−). The actual number of SFCs is displayed as mean +SEM. The table shows the peptide sequence, the protein of origin and SLA-I restriction of the three epitopes that gave robust responses.</p

Nuceloprotein pSLA-I tetramer staining of bronchoalveolar lavage samples from Babraham pigs vaccinated with H1N1 S-FLU.

<p>Babraham pigs were either left unvaccinated (1 and 2) or received H1N1 S-FLU via aerosol administration (6, 7, 8). H1N1 S-FLU vaccinated animals received a boost at day 28 with the same vaccine. Animals were culled and bronchoalveolar lavage harvested at day 57. Nucleoprotein and irrelevant peptide SLA-I tetramer staining was performed on thawed bronchoalveolar lavage samples and the percentage of tetramer⁺ cells of CD8β⁺ cells displayed in red. The sequences of the nucleoprotein peptides are shown. Irrelevant tetramers: SLA-1*14:02-AFAAAAAAL and SLA-2*11:04-GAGGGGGGI. Gating strategy: lymphocytes, single cells, viability (Vivid^neg) CD3⁺ CD14^neg then CD8β⁺ CD4⁺ and displayed as CD8β versus tetramer (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007017#ppat.1007017.s007" target="_blank">S1 Fig</a></b>).</p

Nuceloprotein pSLA-I tetramer staining of tissues from influenza vaccinated Babraham Pigs.

<p>Babraham pig 625 (left panel of 21 plots) and 650 (right panel of 21 plots) received H5N1 S-FLU intranasally and inactivated H1N1 virus [A/Swine/Spain/SF11131/2007] with montanide adjuvant intramuscularly, followed by a boost at day 25 using the same preparation. Pigs were culled at day 38 (day 13 post boost) and blood, bronchoalveolar lavage (BAL) and tracheobronchial lymph nodes (TBLNs) harvested and frozen as single cell suspensions. Tetramer staining was performed on thawed cells from the blood, BAL and TBLN using a no tetramer control, and staining with Irrelevant and nucleoprotein peptide tetramers. The sequences for the nucleoprotein peptides are shown. Irrelevant tetramers: SLA-1*14:02-AFAAAAAAL, SLA-2*11:04-AGAAAAAAI (pig 625) and SLA-2*11:04-GAGGGGGGI (pig 650). Gating strategy: lymphocytes, single cells, viability (Vivid^neg) CD3⁺ CD14^neg then CD8β⁺ CD4⁺ and displayed as CD8β versus tetramer (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007017#ppat.1007017.s007" target="_blank">S1 Fig</a></b>).</p

Study overview.

<p>The inbred Babraham pig was used throughout this study for vaccination and infection, with each pig assigned an identifying number, shown here within each silhouette. (<b>A</b>) Pigs 625 (red) and 650 (blue) were vaccinated intranasally and intramuscularly as depicted. Blood, bronchoalveolar lavage (BAL) and tracheobronchial lymph nodes (TBLNs) were harvested, with peripheral blood mononuclear cells (PBMCs) purified from blood and single suspensions from BAL and TBLNs generated for experiments. Overlapping peptides from the NP of PR8 were used to create T-cell lines (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007017#ppat.1007017.g002" target="_blank">Fig 2</a></b>) and T-cell clones (named and shown in red or blue text) (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007017#ppat.1007017.g003" target="_blank">Fig 3</a></b>). The red clones came from pig 625 (red) and the blue from pig 650 (blue). The clones were used to define minimal NP peptides, which were subsequently refolded with SLA-1*14:02 or SLA-2*11:04 to create pSLA-I tetramers. The tetramers were used to stain the clones (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007017#ppat.1007017.g003" target="_blank">Fig 3</a></b>) and harvested tissues from pigs 625 and 650 (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007017#ppat.1007017.g004" target="_blank">Fig 4</a></b>). (<b>B</b>) The BAL from pigs vaccinated or infected intranasally with influenza, as shown, were stained with the tetramers from A (<b>Figs <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007017#ppat.1007017.g005" target="_blank">5</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007017#ppat.1007017.g006" target="_blank">6</a></b>). The BAL from pig 745 was used for <i>ex vivo</i> ELISPOTS. (<b>C</b>) SLA-1*14:02 or SLA-2*11:04 were refolded with the epitopes defined in A to confirm peptide anchor residues (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007017#ppat.1007017.g007" target="_blank">Fig 7</a></b>). T-cell clones from A were used to define a SLA-1*14:02 or SLA-2*11:04 peptide anchor binding motif (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007017#ppat.1007017.g008" target="_blank">Fig 8</a></b>), which were then used to predict other influenza epitopes, tested using BAL from the two pigs shown (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007017#ppat.1007017.g009" target="_blank">Fig 9</a></b>).</p

Peptide-SLA anchor residue preferences and proposed binding motifs for SLA-1*14:02 and SLA-2*11:04.

<p><b>(A)</b> SLA-1*14:02 restricted, nucleoprotein peptide specific CD8 clones grown from Babraham pig 650 were used to define the peptide binding motif for SLA-1*14:02. Clone KT4.650 (left axis) recognizes index peptide DFEREGYSL and clone KLT.650 (right axis) index peptide EFEDLTFLA. Each of the proteogenic amino acids residues was tested at positions 2 (upper graph) and 9 (lower graph) by substitution of the index peptides. For example: D<b>F</b>EREGYSL index peptide and anchor 2 variants: D<b>A</b>/<b>C</b>/<b>D</b>/<b>E</b>/<b>G</b>/<b>H</b>/<b>I</b>/<b>K</b>/<b>L</b>/<b>M</b>/<b>N</b>/<b>P</b>/<b>Q</b>/<b>R</b>/<b>S</b>/<b>T</b>/<b>V</b>/<b>W</b>/<b>Y</b>/EREGYSL (each residue in bold tested in turn). The corresponding clone was used in peptide titration assays and ELISAs were performed to determine MIP-1β release, with data displayed for 10⁻⁷ M peptide. The limit of maximal detection of MIP-1β release was ~10 ng/mL, data below 0.5 ng/mL has been omitted for clarity, and mean + SEM shown. (<b>B</b>) As for (A), but using the SLA-1*11:04 restricted, nucleoprotein peptide specific clones KT22.625 (left axis, index peptide NGKWMRELI) and Bab.625 (right axis, index peptide IAYERMCNI) grown from Babraham pig 625 to define the peptide binding motif for SLA-2*11:04. Data displayed for 10⁻⁸ M peptide. (<b>C</b>) Binding pocket composition and proposed binding motif for SLA-1*14:02 and SLA-2*11:04 determined from the data in panels A and B. SLA-1*14:02 (green) with EFEDLTFLA (orange sticks) and SLA-2*11:04 (yellow) with IAYERMCNI (cyan sticks). Double conformers have been removed for visual clarity. B pocket is shown in red and the F pocket in pink.</p