Dendritic Cells in Granulomas Figure 4. Surface expression of activating and inhibitory costimulatory molecules and chemokine receptors is different on CD11c+ cells in acute and chronic granulomas. CD11c+ cells from granuloma single cell suspensions obtained from three, six and ten week systemically infected C57BL/6 mice were phenotyped using flow cytometry. A, Histograms represent fluorochrome surface staining with monoclonal antibodies against MHCII, activating costimulatory molecules (CD40, CD80 and CD86) and inhibitory costimulatory molecules (PD-L1 and PD-L2). Black-dashed histograms represent costimulatory molecule expression on na¨ıve splenic CD11c+CD11b+ cells. Blacks arrows indicate directional shift in MHCII and costimulatory molecule expression compared to 3-week expression levels at time points where substantial change is observed. Histograms representative of 3 independent experiments with 3–7 mice per group. B, Average fold change between three and ten week time points in mean fluorescent intensity of MHCII and costimulatory molecules. Average generated from fold change in three independent experiments. doi:10.1371/journal.pone.0011453.g004 they attempt to evaluate the potential effectiveness of current vaccine and therapeutic efforts aimed at increasing the activity and number of IFNc-producing CD4+ T cells. Therefore, these data suggest that despite a high level of systemic mycobacteri- um-specific IFNc-producing T cells, DCs may be shielding bacteria within the granuloma environment from IFNc, a critical component for controlling infection. CD11c+ cells in chronic granulomas are less efficient at inducing an IFNc-producing CD4+ T cell immune response Our data suggests that CD11c+CD11b+ cells in chronic granulomas have an inhibitory phenotype (Fig. 4) and down regulate systemic IFNc producing T cells after they home to granulomatous lesions (Fig. 7). The fact that chronic granulomas contain much less bacterial antigen might provide an alternative explanation to the previous observation of limited IFNcproduc- tion in chronic lesions. To test this possibility, CD11c+ cells were purified from three- and ten- week granulomas and co-cultured with purified, na¨ıve OT-II CD4+ T cells (Fig. 8A). Exogenous OVA protein alone was added to cultures, 48 hours later cells were analyzed for IFNc and IL-2 production by flow cytometry (Fig. 8B). CD11c+ cells purified from three-week granulomas were more efficient at inducing IFNc and IL-2 production from OT-II CD4+ T-cells than CD11c+ cells from ten-week granulo- mas. IFNc production was confirmed by ELISA after ex vivo stimulation for 48 hours (Fig. 8B Right). These data show that even though differential amounts of antigen in acute and chronic granulomas may contribute to the decreased efficiency of mycobacterium-specific T cells to produce IFNc, DCs from acute and chronic granulomas have an inherent difference when regulating local IFNc responses. Furthermore, these data collectively suggest that an inhibitory population of CD11c+ cells in chronic granulomas down-regulates the activity of IFNc- producing CD4+ T-cells. Blockade of PD-L:PD-1 signaling increases the ability of CD11c+ cells in chronic granuloma to reactivate CD4+ T cells ex vivo Previous studies have demonstrated that blockade of the PD- L:PD-1 pathway enhances T cell responses [37–40]. To determine if increased PD-L expression shown on CD11c+ cells from chronic granulomas (Fig. 4) had a direct effect on their inability to stimulate CD4+ T cells activation (Fig. 7 and 8), we tested the effect of CD11c+ PD-L- T cell PD-1 interaction on T cell responses. CD11c+ cells were purified from three- and ten-week granuloma preparations by magnetic separation and were pretreated with media or isotype controls, 10 mg/mL of both anti-PD-L1 and -PD-L2 mAbs, or 50 mg/mL of soluble PD1-Fc that binds both PD-L1 and PD-L2 [41]. After 1 hour, purified Ag85B-specific CD4+ T cells from lymph nodes of 3 week BCG- infected P25 mice were added to the DCs at a ratio of 5:1 (CD4+:CD11c+). Protein transport inhibitor and 0.5 mg/mL Ag85B peptide was added to each coculture, and intracellular IFNc was measured after 4 hours for flow cytometry. Treatment with anti-PD-L1 and -PD-L2 mAbs (Fig. 9A), and PD1-Fc (Fig. 9B) significantly increased IFNc production from T cells stimulated by CD11c+ cells from 10-week granuloma. Both PD-L blocking treatments restored IFNc to production levels achieved by 3-week CD11c+ cell stimulation. Collectively, these data suggest that DCs in chronic granulomas actively inhibit antigen-specific T cell responses in a way that facilitates chronic infection and survival of Mycobacterium bovis. Discussion Here we report that a DC-like population, characterized by CD11c+ expression, are present in acute and chronic BCG- induced granulomas, but their location, phenotype, and ability to induce IFNc responses distinctly changes throughout the course of infection. CD11c+ cells in acute lesions are primarily located in the PLoS ONE | www.plosone.org 6 July 2010 | Volume 5 | Issue 7 | e11453