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.