of the adaptive immune response by facilitating dissemination of mycobacteria and mycobacterial antigen from the site of infection to the draining lymph node [11–15]. However, little is known about the role of DCs during chronic infection. Immunohisto- chemistry of chronic granulomas from lungs of tuberculosis patients shows DCs in and around the granuloma [16](unpublished data). To track and analyze DCs in sarcoid granulomas, the likely sites for long-term mycobacterial persistence, we used a systemic Mycobacterium bovis Bacillius Calmette-guerin (BCG) infection model. This model offers several advantages in the study of latent mycobacterial infections. Lesions from BCG infection are the best characterized of any mycobacterial-induced granulomas models, are technically convenient and numerous to isolate, and very recently have been studied with new and intriguing in vivo imaging [17]. Most of the two billion people infected with Mycobacterium species control infection by maintaining symptom-free latency of the bacilli in chronic granulomas. Murine infection with Mycobacterium tuberculosis results in a sustained and eventually fatal bacterial burden, which does not reflect the low bacterial burden found in chronically infected humans [18,19]. Murine infection with BCG, however does achieve the low bacterial load observed during human Mtb infection. Furthermore, three billion people have been vaccinated with live BCG, which has been proposed to survive within granulomas, with 100 million people newly vaccinated each year, yet its efficacy remains limited [20–22]. The ineffectiveness of the most widely distributed vaccine worldwide certainly warrants rigorous investigation. In addition, Mycobacterium bovis also presents serious health threats of its own. An estimated 1–2% of human tuberculosis cases are caused by Mycobacterium bovis in developed countires, while in still developing countries it is 10%, totaling 20–200 million cases worldwide [23–25]. With increased immigration into the U.S. from Mexico, this presents a more imminent threat to the U.S., especially in binational border communities [26]. Primarily acquired zoonotically by the consumption of raw meat and unpasteurized milk from infected bovine, there have also been cases of Mycobacterium bovis transmission from person to person [25,27,28]. M. bovis tuberculosis patients are over twice as likely to die during treatment compared to those with Mycobacterium tuberculosis, which may be attributed to its universal resistance to the first line tuberculosis drug pyrazinamide [26]. Here, we report data that suggests DCs are critical in the transition from the acute to chronic granulomas. CD11c+ cells from acute and chronic granulomas differ in co-stimulatory phenotype, location within the granuloma, and most importantly, capacity to support an IFNc-recall response from newly-recruited, mycobacteria-specific CD4+ T cells in the granuloma and generate an IFNc response from na¨ıve CD4+ T cells ex vivo. Our data suggests that CD11c+ cells in chronic granulomas shield the bacteria locally from IFNc-producing CD4+ T cells and that this mechanism involves the PD-1:PD-L signaling pathway. This protective shield provided by local CD11c+ cells may have contributed to the evolutionary adaptation of other infectious agents to survive within granulomatous lesions. Results Inflammatory DCs are recruited into both acute and chronic mycobacterium-induced granulomas Though there is an ever-growing appreciation of DCs’ ability to bridge innate and adaptive immunity, little is still known about their role in chronic infection. At three, six and ten weeks after infection with BCG, granuloma-infiltrating cells were isolated from liver as previously described [29], and an isolated granuloma Dendritic Cells in Granulomas cell suspension was generated from 3–7 pooled mice per time point. Phenotypic analysis by flow cytometry revealed the presence of a CD11c+ population in both acute and chronic granulomas (Fig. 1A). Notably, the proportion of CD11c+ cells was similar in both three-week (acute) and ten-week (chronic) lesions. Analysis of the CD11c+ population showed that the predominant population in both acute and chronic lesions was the monocyte-derived ‘‘inflammatory’’ DC subset, characterized by CD11cint-hiCD11b- hiGR-1+ (Fig. 1B) [30,31]. The differentiation of this population from recruited blood-derived monocytes into inflamed tissue is well described. The frequency of this DC population within the granuloma was relatively consistent throughout infection, as measured by their gated percentage of total granulomas cells. When the absolute number of CD11c+cells per gram of liver tissue was determined, the significant majority of CD11c+ cells were present at six weeks after infection (Fig. 1C). The decreased number of CD11c+ cells per gram of liver tissue observed at ten weeks is likely due to the decline in both bacterial burden and subsequent number of lesions observed during chronic time points. Overall, these data demonstrate the presence of a DC-like population of cells in both acute and chronic mycobacterium- induced granulomas. Location of CD11c+ cells in granulomas changes with progression from acute to chronic infection The granuloma is a microenvironment with complexity in architecture and cellular distribution. Different locations within the granuloma represent diverse immunological niches for optimal interactions between antigen presenting cells, T and B cells, cytokines, and chemokines [32,33]. As critical immune modula- tors, we expect the phenotype of DCs and their location in the granuloma to be important components of their function and interaction with other immune cells. To locate DCs in granulo- mas, liver from mice systemically infected with BCG was frozen and examined by immunofluorescent microscopy with anti- CD11c staining three, six and ten weeks after infection. We classified DC location in the granuloma based on three generalized areas: the center, the lymphoid cuff of cells forming the periphery, or outside in extra-granulomatous tissue (Fig. 2A). We observed differential CD11c+ cellular location throughout infection. Most CD11c+ cells in three-week granulomas were located in the lymphoid cuff, but as infection progressed the majority were distributed near the center of the granuloma (Fig. 2B), a common location for bacteria during chronic infection. Interestingly, CD11c+ cells from ten-week granulomas were distributed more evenly in all locations. We confirmed these findings using CD11c enhanced yellow fluorescent protein (CD11c-EYFP) mice with ubiquitously fluorescing DCs [34], infected with BCG -liver was analyzed by microscopy three, six and ten weeks later (Fig. 2C). Using both anti-CD11c antibody staining on tissue from WT mice and transgenic CD11c-EYFP mice, we show that this cellular subset is differently located throughout the course of infection. A portion of CD11c+ cells are infected with mycobacteria in both acute and chronic granulomas Previous studies have shown that both resident and recruited DCs take up live bacilli, where they can persist for at least two weeks after infection [12]. Though mycobacteria infect both macrophages and DCs, activated DCs lack the ability to kill intracellular bacilli as effectively [35]. Thus, granuloma DCs may provide a long-term reservoir for bacteria during chronic infection. To investigate this possibility, we analyzed three- and ten-week PLoS ONE | www.plosone.org 2 July 2010 | Volume 5 | Issue 7 | e11453