Detection of phagosomal rupture <i>in vivo</i> in <i>Mtb</i>-infected phagocytes.

<p>(A-B) BM-DC from CD45.1 donors were left non-infected or were infected with <i>Mtb</i> WT or ΔESX-1 (MOI = 1) for 16h. Of note, cultures of CD45.1 BM-DC, infected with DsRed-mycobacteria in the same conditions, showed that >70% of cells had uptaken mycobacteria like shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004650#ppat.1004650.g002" target="_blank">Fig. 2A</a>. Cells were recovered and transferred i.n. (2 x 10⁶ cells/mouse) into CD45.2 congenic recipients. (B) Four days post-transfer, alive lung low-density cells from the recipients (<i>n</i> = 3/group) were isolated on Optiprep gradient and incubated with mAbs specific to CD11b and CD45.1, subsequent to incubation with CCF-4. The blue signals from the three experimental groups are overlaid. (C) Histograms show the comparative blue CCF-4 signals in the CD11b⁺ CD45.1⁺ cells from different groups at days 4 or 6 post transfer. * = statistically significant, as determined by the Student's <i>t</i> test, <i>p</i><0.01. (D) Lung granuloma from C57BL/6 mice, infected via aerosol route with ≈200 CFU/mouse of WT <i>Mtb</i>, were removed at 6 weeks p.i. and were treated with collagenase and DNAse-I and enriched in low-density cells. In parallel to these cells, lung low-density cells from uninfected controls were assessed for CCF-4 blue switch. (E) CD11b <i>vs</i>. CD11c surface expression of the cells showing the increased CCF-4 shift (pink), compared to unstained negative control incubated with control Ig isotypes (gray). The results are representative of 2 independent experiments.