Cytokine gene expression in early (stage I) vs late (stages III and IV) granulomas of the tracheobronchial lymph node (Tb LN) vs caudal mediastinal lymph nodes (Md LN) of calves experimentally infected with <i>Mycobacterium bovis</i>.

<p>Gene expression as measured by quantification of mRNA transcripts identified through RNAScope^® ISH. Values represent the mean ± SEM mRNA transcripts/total cell area (μm²) of granuloma. Significantly different * (<i>p</i>< 0.05), ** (<i>p</i>< 0.001), *** (<i>p</i>< 0.0001).</p

Cytokine gene expression in late stage granulomas of the lung vs tracheobronchial lymph node (LN) of calves experimentally infected with <i>Mycobacterium bovis</i>.

<p>Gene expression as measured by quantification of mRNA transcripts identified through RNAScope^® ISH. Values represent the mean ± SEM mRNA transcripts/total cell area (μm²) of granuloma. Significantly different * (<i>p</i>< 0.05), ** (<i>p</i>< 0.001), *** (<i>p</i>< 0.0001).</p

Expression of A) TGF-β and B) IL-10 mRNA in lung granulomas of calves experimentally infected with <i>Mycobacterium bovis</i> by aerosol.

<p>Note signal (red dots) representing mRNA transcripts within cells of various types. The amount of signal varied widely between cytokines (arrows). RNAScope^® ISH, 400X.</p

Cytokine gene expression within granulomas of the tracheobronchial (G-Tb) and caudal mediastinal (G-Md) lymph nodes vs non-granuloma (non-lesion) regions (NL-Tb, NL-Md) on the same slide from calves experimentally infected with <i>Mycobacterium bovis</i>.

<p><b>Uninfected (U) caudal mediastinal lymph nodes from age matched calves are shown for comparison.</b> Gene expression as measured by quantification of mRNA transcripts identified through RNAScope^® ISH. Values represent the mean ± SEM mRNA transcripts/total cell area (μm²) of granuloma. Significantly different * (<i>p</i>< 0.05), ** (<i>p</i>< 0.001), *** (<i>p</i>< 0.0001).</p

Summary of gross, microscopic and bacteriological results of tissues from BCG-vaccinated (V) and non-vaccinated (NV) deer after experimental challenge with virulent <i>M. bovis</i>.

<p>Deer in which there were no gross or microscopic lesions and no bacteriological isolation results have been excluded.</p><p>NA = not applicable; Neg = no lesion or no bacteriological isolation; Pos = lesion present or isolation of mycobacteria; mrln = medial retropharyngeal lymph node; tbln = tracheobronchial lymph node; ton = palatine tonsil, maln = mandibular lymph node; paln = parotid lymph node; medln = mediastinal lymph node, hepln = hepatic lymph node; scln = superficial cervical lymph node; mesln = mesenteric lymph node.</p

Total number of microscopic granulomas in representative sections of medial retropharyngeal lymph nodes of BCG-vaccinated and non-vaccinated deer after experimental challenge with virulent <i>M. bovis</i>.

1<p>Stage I (initial) granulomas, Stage II (solid) granulomas, Stage III (necrotic) granulomas, Stage IV (necrotic, mineralized, coalescent) granulomas. Complete definitions found in text.</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

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

Primary and secondary monoclonal antibodies and proliferation staining reagents.

<p>Primary and secondary monoclonal antibodies and proliferation staining reagents.</p