A TNF-Regulated Recombinatorial Macrophage Immune Receptor Implicated in Granuloma Formation in Tuberculosis
Figure 5
Infection of macrophages with M. bovis BCG induces TCRαβ expression in vitro.
(A) Immunofluorescence double-staining demonstrating co-localization of BCG mycobacteria (green) and the macrophage-TCRαβ (red) after 6 days of infection (i). Nuclei are stained with DAPI. Arrows highlight two double-stained points of spatial proximity (yellow fluorescence). (ii) Immunogold electron microscopy of a BCG containing phagosome in an IFNγ macrophage. The presence of a single TCRαβ in the immediate neighborhood of a BCG mycobacterium is shown at a higher magnification (arrow). (B) Confocal image of a TCRαβ expressing macrophage cluster induced in response to in vitro infection with BCG. Uninfected IFNγ macrophages from the same donor are shown left. White arrows highlight TCRαβ+ macrophages. A quantitative analysis of the percentage of TCRαβ+ cells (right) and isotype controls are shown. **p<0.01. IFNγ macrophages were incubated in the presence or absence of FITC-labeled BCG for 6 days. The results represent two healthy individuals (donors A and B). Donor B see Figure S4A. (Bottom) RT-PCR demonstrating increased expression of the TCR α- and β-chain genes in the BCG infected macrophages from both donors. GAPDH expression is shown as reference. (C) Synopsis of the TCR Vβ repertoires expressed by the BCG infected and uninfected macrophages shown in (B) assessed by CDR3 spectratyping (donor A). (Bottom) Quantitative analysis of all Vβ CDR3 length variants expressed by both donors . * p = 0.07. (D) TCR Vβ repertoire analysis of randomly selected BCG/macrophage clusters from donor A reveals expression of highly restricted TCR Vβ chain repertoires. BCG/macrophage clusters 1–6 were subjected to RT-PCR and CDR3 spectratyping. The identified TCR Vβ repertoires are shown for each individual cluster. Note that next to the Vβ1 only few additional Vβ chains are expressed. The single peaks are indicative of monoclonality. The results are representative of two healthy individuals (donors A and B). Donor B see Figure S4B.