Identification of recombinant antibodies with specificity for bIL-2.

<p>PBMC from a cow naturally infected with <i>M. bovis</i> were cultured in the presence of PPD-B to allow screening of candidate bIL-2 by ICS flow cytometry. Panel A shows the histogram gating strategy used to interrogate responses in singlet, live CD4⁺ lymphocytes. Panel B shows the measurement of detectable IL-2 and/or IFN-γ within the CD4⁺ population for each of 6 candidate IL-2 antibody clones. The clone number is shown in the top left corner of each histogram and the percentage of CD4⁺ cell in which co-expression of IFN-γ and IL-2 could be detected is shown in the top right of each histogram. Data are representative of 1 of 2 independent experiments.</p

Identification of multifunctional IFN-γ, IL-2 and TNF-α CD4⁺ cells in natural <i>M. bovis</i> infection.

<p>PBMC from naturally <i>M. bovis</i> infected cattle were cultured in the presence of either PPD-B or medium and the co-expression of IFN-γ, IL-2 and TNF-α determined by ICS flow cytometry. Histograms were gated on singlet, live lymphocytes and then all CD4⁺ cells analysed for all combinations of cytokine productivity. The upper histograms show the total proportion of CD4⁺ cells staining for expression of IFN-γ, IL-2 or TNF-α following stimulation with PPD-B (left panel) or medium control (right panel). Subgating of the IFN-γ⁺ and IFN-γ⁻ CD4⁺ cells provides histograms that represent all possible functionalities for the expressing of the 3 cytokines, as shown in the lower histograms. The numbers indicate percentage of CD4⁺ cells and data are representative of 1 of 10 naturally infected cattle.</p

Characterisation of memory markers reveals a characteristic T_EM phenotype for multifunctional CD4⁺ cells in naturally infected cattle.

<p>PBMC from naturally <i>M. bovis</i> infected cattle were stimulated with PPD-B and stained for production of cytokine by ICS flow cytometry. Histograms were first gated on singlet, live lymphocytes and gating strategies applied to allow phenotyping of cells for expression of CD44, CD45RO and CD62L. The quadrant gate defining expression of CD44 and CD62L (top panel), CD45RO and CD62L (middle panel) or CD44 and CD45RO (bottom panel) was determined using the total live-lymphocyte population, as shown for each surface marker combination in the histograms on the far left of the panel. This quadrant gating strategy was used to determine the surface phenotype of total CD4⁺IFN-γ⁺, CD4⁺IL-2⁺, CD4⁺TNF-α⁺ and triple functional CD4⁺ cells, as shown in the other histograms. The percentage of cytokine producing CD44^hiCD62L^lo, CD45RO⁺CD62L^lo and CD44^hiCD45RO⁺ is shown for each of these CD4⁺ populations. The data is from one representative animal of 5 analysed.</p

Long-term cultured and <i>ex vivo</i> IFN- γ responses by cattle after <i>M</i>. <i>bovis</i> aerosol challenge.

<p>Cultured ELISPOT analysis was performed ~3 weeks after challenge with virulent <i>M</i>. <i>bovis</i>. Long-term cultured cells were generated by stimulating PBMC with a cocktail of rAg85A (1 μg/ml), rTB10.4 (1 μg/ml), and rESAT-6:CFP10 (1 μg/ml) antigens as well as PPDb (5 μg/ml) for 13 days followed by transfer to ELISPOT plates with APCs and addition of either rESAT-6:CFP10, PPDb or medium alone. For the <i>ex vivo</i> response, freshly isolated PBMCs were stimulated with rESAT-6:CFP10, PPDb or medium alone for 16h. Medium control responses were subtracted from antigen-stimulated responses and results are presented as mean spot forming cells (SFC)/million cells (± SEM, n = 8) for <b>(A)</b> long-term culture or <b>(B)</b><i>ex vivo</i> conditions. <b>(C)</b> The kinetics of the response is shown as the percent of CD4⁺ cells producing IFN-γ in long-term cultures at 3, 6, 8, and 12 weeks post infection (WPI n = 6). Two-way ANOVA (Šídák’s multiple comparison post-test).</p

Representative gating strategy for evaluation of CD45RO and CCR7 expression on CD4 T cells producing IFN-γ.

<p>Approximately 8 weeks after aerosol challenge with <i>M</i>. <i>bovis</i>, long-term cultures were generated by stimulating PBMC with a cocktail of rAg85A (1 μg/ml), rTB10.4 (1 μg/ml) and rESAT-6:CFP10 (1 μg/ml) as well as PPDb (5 μg/ml) for 13 days followed by transfer of cells to ELISPOT plates with APCs and restimulation with PPDb. Gating hierarchy (gating sequence as depicted by the arrows): <b>(A)</b> Single cells (within the oblong gate), <b>(B)</b> Lymphocytes (within the polygon gate), <b>(C)</b> CD4⁺ cells, <b>(D)</b> CD4⁺ cells producing IFN-γ, and <b>(E)</b> CD45RO and CCR7 expression for determination of effector/memory phenotypes.</p

Results of RNA-Seq analysis.

<p>A. Signficantly modulated genes in the three treatment groups. B. Venn diagrams of genes significantly up-regulated and (C) down-regulated genes after vaccination but prior to <i>M. bovis</i> challenge. A. Fold change compared to unstimulated PBMC (medium controls) of PPD-B stimulated PBMC compared to medium controls from unvaccinated, naïve calves (group 1), vaccinated/non-protected (group 2), and vaccinated/protected calves (group 3).</p

Frequencies of Tcm, Tem and effector cells producing IFN-γ in response to mycobacterial antigens in long-term and <i>ex vivo</i> assays.

<p>Peripheral blood mononuclear cells were isolated from calves ~ 8 weeks after challenge with virulent <i>M</i>. <i>bovis</i>. Cells were stimulated with a cocktail of rAg85A (1 μg/ml), rTB10.4 (1 μg/ml), and rESAT-6:CFP10 (1 μg/ml) as well as PPDb (5 μg/ml) for 13 days followed by transfer to 96 well round bottom plates with APCs and addition of media alone, PPDb or rESAT-6:CFP10 for an additional 16h. For <i>ex vivo</i> culture, PBMC were stimulated with media alone, PPDb or rESAT-6:CFP10 for 16 h. <b>(A)</b> Relative contribution of Tcm, Tem, and T effector cells to IFN-γ production in response to PPDb by long-term (i.e., 14-day) (left) and <i>ex vivo</i> (i.e., 16 h) (right) cultures, 8 weeks after <i>M</i>. <i>bovis</i> challenge. Data are presented in percentages (pies) and as mean (± SEM) number of cells producing IFN-γ <b>/</b> 10⁴ cells (histograms) (n = 16). Relative contribution of Tcm, Tem and T effector cells to IFN-γ production in response to PPDb <b>(B)</b> or to rESAT-6:CFP10 <b>(C)</b> in long-term cultures at three, six, eight or 12 weeks post-infection (WPI, n = 6). Tcm, Tem and effector cell phenotypes were as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122571#pone.0122571.g002" target="_blank">Fig 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122571#pone.0122571.s003" target="_blank">S3 Fig</a> Tcm and Effector T cell contribution to IFN-γ production differs (*<i>P</i> < 0.05; **<i>P</i> < 0.01, paired Student's t-tests) between short- and long-term cultures.</p

Functional networks most significanty modulated in PPD-B stimulated PBMC from vaccinated/protected calves.

<p>Visualisation of the trend and significance of each network: Red bars = up-regulation; blue bars = down-regulation of network. Horizontal bar: p = 0.05.</p

Long-term cultured cells have higher proliferative responses than short-term cells.

<p>Long-term and short-term cultured PBMCs were analyzed ~ 7 weeks after aerosol challenge with virulent <i>M</i>. <i>bovis</i>. Long-term cells consist of PBMC from <i>M</i>. <i>bovis</i> aerosol infected cattle cultured in the presence of rAg85A, rTB10.4, rESAT-6:CFP10 and PPDb for 13 days and then CellTrace violet-stained and re-stimulated with either rESAT-6:CFP10, PPDb or medium in the presence of fresh autologous adherent cells for an additional six days. Short-term cells consist of CellTrace violet-stained PBMC from <i>M</i>. <i>bovis</i> aerosol infected cattle cultured for six days in the presence of either rESAT-6:CFP10, PPDb or medium. The gating strategy was performed in accordance with procedures described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122571#pone.0122571.g002" target="_blank">Fig 2A</a> for single cells, and as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122571#pone.0122571.s003" target="_blank">S3 Fig</a> for lymphocytes, and CD4⁺ cells. <b>(A)</b> Percentages of CD4⁺ cells proliferating (low Celltrace dye MFI) in response to rESAT-6:CFP10 within long or short-term cultures. <b>(B)</b> Percentages of CD4⁺ cells proliferating (low Celltrace dye MFI) in response to PPDb within long or short-term cultures. <b>(C)</b> Memory/effector phenotype of proliferating CD4⁺ cells within long-term or short-term cultures in response to rESAT-6:CFP10. <b>(D)</b> Memory/effector phenotype of proliferating CD4⁺ cells within long-term or short-term cultures in response to PPDb. For panels A and B, cell proliferation differs (**<i>P</i> < 0.01, n = 6 paired Student's t-tests) between long and short-term cultures to either rESAT-6:CFP10 or PPDb. For panels C and D, Tcm and effector cell content differs (*<i>P</i> < 0.05; **<i>P</i> < 0.01, n = 4 paired Student's t-tests) between short-term and long-term cultures to either rESAT-6:CFP10 or PPDb.</p

BCG-vaccinated and control cattle samples mapped to <i>ifn-γ</i> and <i>il-22</i> genes.

<p>Visualization by IGB of RNA sequencing reads of representative PPD-B stimulated PBMC from vaccinated-protected, vaccinate-un-protected and non-vaccinated control cattle. Y-axis shows the number of reads covering each base along the transcript in RPKM expression values for each sample. Black track: unvaccinated control cattle; red track: vaccinated/un-protected cattle and blue track: vaccinated/protected cattle. The schematic representation of transcript for (A) <i>ifn-γ</i> and (B) <i>il-22</i> is show in green at the bottom of the each figure; the boxes show the exons of the gene.</p