Dendritic Cells in Granulomas Figure 3. A portion of BCG is sustained within CD11c+ cells, both in acute and chronic granulomas. A, Left, Fluorescent images of CD11c- EYFP2 (left panels) and CD11c-EYFP+ cells (right panels) with dsRED BCG at three and ten weeks. Images magnified from 10006images. DAPI nuclear stain (blue), dsRED BCG (red rods) and cytoplasmic CD11c-EYFP cells (green). Right, Average number of viable dsRED BCG rods per cell type (CD11c- EYFP+ or non-fluorescent) within the granuloma at three- and ten-weeks after infection. Data are represented as mean +/2 SEM, P,0.0001. B, FACS plots generated from CD11c+ gate (left plot) and CD11b+CD11c2 gate (right plot). Boxed gate shows percentage of cells containing GFP-BCG. Gate placement was made from non-fluorescent-BCG infected CD11b+ and CD11c+ cells, not shown. Plots representative of 3- and 10-week time points with 3, 8 and 8 mice per group, respectively. C, Histograms generated from 10 week CD11c+ (1.17) and CD11b+CD11c2 (8.61) GFP BCG+ gates in (B). doi:10.1371/journal.pone.0011453.g003 APCs in chronic granulomas support lower reactivation of newly recruited mycobacterium-specific IFNc- producing CD4+ cells compared to acute granulomas A recent study by Egen and colleagues demonstrated the movement of T cells in granulomas, and in doing so, also demonstrated their ability to interact with many local cells [17]. Additionally, newly primed T cells require a second antigen encounter at the site of inflammation in order to rapidly secrete cytokines [36]. Here, we tested the ability of granuloma-resident APCs from three- and ten-week granulomas to induce IFNc production from newly recruited mycobacterium-specific T cells that migrated to pre-existing granulomas. For this, CD11c- EYFP mice were infected with dsRED BCG followed three or ten weeks later by adoptive transfer of CFSE-labeled, dsRED CD4+ T cells specific for mycobacteria-secreted antigen 85B (Fig. 7A). One day before adoptive transfer of Ag85B-specific T cells, mice received subcutaneous footpad injections of GFP- BCG to provide an immune boost. GFP-BCG was used for the secondary systemic boost in order to distinguish between previously established three and ten-week dsRED BCG-induced granulomas in the liver. We expected this immune boost to induce migration of adoptively transferred cells to the draining popliteal lymph nodes and acquire a Th1 phenotype, mimicking a therapeutic boost intended to generate a high systemic level of mycobacterium-specific IFNc-producing CD4+ T cells. Six days after adoptive transfer, cells from three- and ten-week granulomas were isolated for ex vivo recall of IFNc production. CD4+dsRED+ co-staining identified adoptively transferred 85B antigen-specific TCR transgenic T cells. Analysis by flow cytometry showed that the proportion of LFA-expressing, adoptively transferred T cells that migrate into three- and ten- week granulomas is similar, demonstrating that T cell recruit- ment into chronic granulomas was not hindered (Fig. 7B). Fluorescent microscopy confirmed the presence of newly recruited Ag85B-specifc T cells into the granulomas and also showed their proximity to CD11c-EYFP cells (Fig. 8B, lower panel). Importantly, upon ex vivo stimulation, there was a lower frequency of adoptively transferred Ag85-specific T cells in chronic granulomas that were able to produce IFNc compared to three-week granulomas (Fig. 7C individual FACS plots). These data support the hypothesis that DC-like cells in chronic lesions, which express high levels of inhibitory molecules, suppress local IFNc production from newly recruited mycobac- terium-specific T cells. These experiments are relevant because PLoS ONE | www.plosone.org 5 July 2010 | Volume 5 | Issue 7 | e11453