Figure 2. Location of DCs in acute and chronic mycobacterium- granulomas is different. A, Schematic outline of DCs in the center, periphery or outside of the granuloma. B, Distribution of DC location from stained sections. 42, 43 and 17 lesions combined from three mice per time point were observed for CD11c+ location for three, six and ten weeks, respectively. C, CD11c-EYFP mice were systemically infected with dsRED BCG for three, six and ten weeks. EYFP expression (green cells) and DAPI nuclear stain (blue). Granulomas are outlined with white dashed lines and shown at 10006 magnification. doi:10.1371/journal.pone.0011453.g002 granuloma-infiltrating cells were isolated at three, six and ten weeks after infection. Granuloma cells were stained with antibodies against MHCII, costimulatory (CD40, CD80 and CD86) and inhibitory (PD-L1 and PD-L2) surface molecules and analyzed by flow cytometry (Fig. 4A). After gating on CD11c+ cells (Fig. 1A), we observed a distinct change in phenotype of this cellular subset from three weeks to ten weeks after infection. Compared to na¨ıve splenic CD11c+ cells, CD11c+ cells from three- week granulomas had high expression of MHCII, T cell costimulatory molecules, CD40, CD80 and CD86, and low expression of inhibitory molecules PD-L1 and PD-L2. Ten weeks after infection these cells had lower expression of T cell costimulatory molecules, and higher expression of both PD-L1 and PD-L2 (Fig. 4A Black arrows indicate directional shift of MHCII and costimulatory molecule expression compared to expression level observed 3 weeks after infection). MHCII and Dendritic Cells in Granulomas costimulatory molecules CD40, CD80, and CD86 had an average 0.5 fold decrease in their MFI intensity from three to ten weeks after infection (Fig. 4B). In contrast, PD-L1 and PD-L2 increased in their mean MFI by 2 and 3.5 fold, respectively. These data suggests that DC-like cells from early three-week granulomas would support potent T cell activation (or re-activation), but those from 10 week granulomas may not. The majority of CD4+ cells maintain close contact with CD11c-EYFP+ cells in both early and chronic granulomas The differential expression of costimulatory and inhibitory molecules on acute and chronic CD11c+ cells likely has different consequences on T cell responsiveness in acute and chronic granulomas. We have already shown that the location of this cellular subset changes in the granuloma throughout infection (Fig. 2). Next, we investigated if the differential location of CD11c+ cells in acute and chronic granulomas changed the frequency of contact between T cells. We speculated that changes in DC phenotype would be less relevant to T cell function if contact between the two cell types was ablated by the altered DC location in the lesion. We also speculated that any loss of contact between the two cell types would result in an inability of DCs to re-stimulate newly primed T cells recruited to granulomas. To address this question, we quantitatively analyzed three- and ten-week granulomas for CD4+:CD11c-EYFP+ cellular contact by fluorescent immunohistochemistry (Fig. 5A and B). This was done by staining liver sections from three- and ten-week infected CD11c-EYFP mice with fluorochrome-labeled anti- CD4, and analyzing all CD4+ cells per lesion. We observed similar frequencies of CD4+:CD11c-EYFP+ cell contact be- tween three- and ten-week granulomas, and importantly, found that the statistically significant majority of CD4+ T cells from both three- and ten-week granulomas were in contact with CD11c+ cells. Under high magnification, co-localization of CD11c-EYPF+ (DCs) and dsRed (CD4+ T cells) is easily seen (Fig. 5B- 2nd and 4th column, 1st and 2nd row). The few CD4+ T cells that were not in contact with CD11c+ cells may be due to restrictions of the sectioned plane, such that contact with a CD11c+ cell may be above or below the plane. These data demonstrate that CD11c+ cells maintain physical contact with CD4+ T cells in both acute and chronic granulomas. Collectively, these data, in combination with the observed phenotypic changes (Fig. 4), suggest that DC-like cells may have direct effects on both resident and newly recruited mycobac- terium-specific T cells during both early and chronic infection. CD4+ T cells produce less IFNc in chronic compared to acute granulomas To test for differences between acute and chronic granuloma- resident CD4+ T cell activity, granuloma cells from both lesions were re-stimulated ex vivo after systemic infection with BCG. CD4+ T cells from ten-week granulomas showed a dramatic decrease in both IFNc production and LFA-1 expression compared to three- week granulomas (Fig. 6). In fact, the IFNc-producing T cell population in ten-week granulomas fell below our detection level in many samples. These observations, along with those detailing an inhibitory phenotype of CD11c+ cells in chronic granulomas suggests a suppressive DC population in chronic granulomas. Since many current therapeutic efforts are aimed at generating an increase in Mtb-specific IFNc-producing Th1 cells, we next tested the ability of DCs to re-boost newly recruited CD4+ T cells in acute and chronic granulomas. PLoS ONE | www.plosone.org 4 July 2010 | Volume 5 | Issue 7 | e11453