Figure 6. IFNc recall response of CD4+ T cells is much higher in acute compared to chronic granulomas. Granuloma cells from three- and ten-week systemic BCG infected animals were isolated and stimulated ex vivo for five hours with anti-CD3. FACS plots show intracellular IFNc staining and LFA-1 expression from CD4+ gated population. Plots representative of five pooled mice per group. doi:10.1371/journal.pone.0011453.g006 inhibits them is a key question. One possibility is that the proinflammatory environment of acute granulomas provides signals to drive increased expression of PD-L receptors on APCs. IFNc itself, which is abundant in acute granulomas, up-regulates PD-L on DCs [41,51,53–55]. To test potential IFNc regulation of PD-L expression on DCs during BCG infection, we infected IFNc KO mice, but observed no differences in PD-L expression compared to wild-type mice six weeks after infection (data not shown). However, this is likely due to compensatory effects of other proinflammatory factors in the granuloma, such as TNFa, IL-6, IL-1b, IL-4, GM-CSF, and/or TSLP, which have all been found to induce an inhibitory DC phenotype [56,57]. In addition to host- derived products, certain mycobacterial antigens have also been shown to interfere with DC maturation and down-regulate the ability of the DCs to induce Th1-IFNc responses from antigen- specific T cells [58–61]. TLR 2 and 4 stimulation has been shown to induce inhibitory and tolerogenic DCs, while PD-L up- regulation results from TLR 7/8 stimulation [56,62]. In accordance with our finding that CD11c+ cells in human Mtb-induced granulomas express PD-L1 and -2 (data not shown), recent data by Juardo et al. shows that mycobacterial antigen- induced expression of PD-1 on lymphocytes from patients with tuberculosis interferes with anti-mycobacterial T cell effector functions [40]. PD-L1 expression has also been shown in BCG- induced bladder granulomas from patients receiving BCG therapy for urothelial carcinoma of the bladder [63]. Together with our own data, these human data suggest that once lymphocytes reach Mtb-induced granulomas, PD-1: PD-L1/2 ligation may interfere with effector T cell responses. A recent finding by Einarsdottir et al. has shown that administration of anti-PD-1 and -PD-L1 blocking antibodies at the onset of chronic Mycobacterium tuberculosis infection in mice does not effect CD8+ T cell function or bacterial load four weeks later [71]. It is difficult to compare data from in vivo and in vitro experiments, but one possible explanation for the lack of response observed in vivo is that PD-L2 was not also selectively blocked in their study. PD-L2 is more exclusively expressed by DCs and can also ligate with PD-1, potentially compensating for any blocking of PD-L1 [41]. DCs may also contribute to latency by acting as a long-term reservoir for mycobacteria, ensuring their survival in highly bactericidal environments like the granuloma. Not only does our data show BCG-infected CD11c+ cells in acute and chronic Dendritic Cells in Granulomas granulomas, but those CD11c+ cells infected often contained more than one bacterium per cell. It has been reported that DCs are less efficient at killing intracellular mycobacteria [35,64–66], though altered compartmental trafficking of bacilli inside DCs prohibits mycobacterial access to the necessary nutrients required for growth [67,68]. These studies ultimately suggest two things. The first is that while the majority of mycobacteria infect macrophages, a portion of mycobacteria that infect DCs may avoid the effective killing mechanisms of macrophages. The second is that mycobacterium may infect DCs so that they can influence the immunosuppressive effects of the PD-L1/2 system. Studies have shown that human monocyte-derived DCs infected with either Mtb or BCG have reduced expression of MHCII and CD80, produced IL-10 over IL- 12, and had an impaired ability to generate IFNc-secreting CD4+ T cells [69–72]. Furthermore, a recent in vivo study by Wolf et al. showed that the myeloid DC population responsible for shuttling Mtb from the lungs to the draining lymph nodes in mice poorly stimulated IFNc production from Ag85B-specific CD4+ T cells [15]. While these data suggest that mycobacteria-infected DCs induce immunosuppressive effects, a body of data actually suggests the opposite. Upon infection of both murine and peripheral monocyte-derived human DCs with either Mtb or BCG in vitro and in vivo, DCs matured, produced Th1-promoting cytokines, and induced IFNc-producing T cells [73–79]. This collection of conflicting data may be the result of the various models used, but taken together; the data collectively demonstrate the multi- functionality of DCs during infection. The scheme in Figure 10 summarizes our results, which indicates a distinct change in CD11c+ cell phenotype and function in bacteria-containing granulomas (Fig. 10). It is possible that DCs, by affecting local IFNc availability, may contribute to granuloma maturation and bacterial persistence. Future studies of DC function in chronic Mtb-induced granulomas will be needed to both extend our findings and develop vaccine strategies that would weaken the shielding effect of DCs in chronic granulomas. Materials and Methods Mice Wild type C57BL/6 (H2b) and IFNc-deficient mice were purchased from the Jackson Laboratory (Bar Harbor, ME). CD11c- EYFP transgenic mice on the C57BL/6 background were a generous gift from Dr. Michel C. Nussenzweig (Rockefeller University, NY) [34]. P25 transgenic mice on the Rag2/2 background were a generous gift from Drs. Rothfuchs and Sher (NIH, Bethesda, MD). P25 mice were bred with Actb-DsRED.T3 transgenic mice from the Jackson Laboratory to obtain dsRED P25 mice. Ethics Statement Mice were housed and bred in microisolator cages in a pathogen-free facility at the University of Wisconsin Animal Care Unit (Madison, WI), and husbandry and experimental procedures were performed according to the guidelines of the Institutional Animal Care and Use Committee. All animal protocols were approved by the University of Wisconsin Institutional Animal Care and Use Committee. Animal protocol approval under protocol M01616. Infection The Pasteur strain of BCG (Staten Serum Institute, Denmark), kanamycin-resistant GFP-expressing BCG, or kanamycin-resistant dsRED-expressing BCG were grown in Middlebrook 7H9 supplemented with 0.05% Tween 80 and 10% oleic acid- dextrose-catalase supplement (Difco, Detroit, MI) in the presence PLoS ONE | www.plosone.org 8 July 2010 | Volume 5 | Issue 7 | e11453