Dendritic Cells in Granulomas Figure 7. Newly activated Ag85B-specific Th1 cells are recruited at similar levels to acute and chronic granulomas, but produce less IFNc in chronic granulomas. A, Experimental schema. Coinciding three- and ten-week dsRED BCG systemically infected CD11c-EYFP mice received s.c. footpad injections of 1.256106 CFU GFP BCG seven days prior to harvest. The following day 106 CFSE dsRED Ag85B-specifc transgenic CD4+ T cells were adoptively transferred via retro-orbital sinus vein injection. Mice were harvested six days later and granuloma cells were isolated. B, Left FACS panel, Presence of transferred P25 dsRED CD4+ T cells in both three- and ten-week granulomas. CD4+dsRed+ co-staining indicates adoptively transferred population. Plots derived from lymphocyte gate on SSC vs. FSC. B, Right FACS panel, CFSE dilution of gated P25 dsRED CD4+ T cell population indicates the recent activation of these T cells. B, Lower panels, Fluorescent microscopy reveals contact between CD11c-EYFP cells (green) and P25 dsRED CD4+ T cells (red) in granuloma (DAPI-blue). Images taken at 10006 magnification. Right images, Enlarged image of small white box. C, Left FACS panels, ex vivo restimulation of granuloma cells and staining for LFA-1 expression and IFNc production. Plot was obtained from gated dsRED CD4+ T cell population as shown in B. C, Right graph, Average P25 IFNc production in the granuloma from three independent experiments, displayed as a percent of total CD4+dsRED/Vb11+ cells in lymphocyte gate (data are represented as mean +/2 SEM). Plots representative of three independent experiments with 2–7 mice per group. These data show that despite CD4+IFNc-producing mycobacterium-specific T cells reaching both acute and chronic granulomas, the IFNc response is blocked (not reactivated) in chronic lesions. doi:10.1371/journal.pone.0011453.g007 or absence of kanamycin (50 mg/mL) and stored in frozen aliquots at 280uC. For infections, ampoules were thawed, diluted in PBS, and briefly sonicated to obtain single-cell suspensions. For systemic infection, a non-lethal dose of 16107 CFU in 100 mL was intraperitoneally injected. For localized infection, a dose of 1.256106 CFU was injected subcutaneously into the footpads. Kanamycin-resistant GFP-BCG was generated using an HSP60 promoter driven GFP plasmid, generously provided by Dr. Glen Fennelly (Albert Einstein University, NY), and dsRED BCG was a generous gift from Dr. Lalita Ramakrishnan (University of Washington, WA). Growth and preparation of wild-type BCG Pasteur strain (Staten Serum Institute, Copenhagen, Denmark) were performed as previously described [29]. Mononuclear cell isolation Aseptic isolation of splenocytes and lymph nodes was performed using standard methods. Granuloma preparations were performed as previously described [29,80–82]. Briefly, granuloma suspensions were prepared from freshly excised infected livers. The livers were homogenized in a Waring tissue blender, and granulomas were allowed to settle by virtue of their higher density compared to other hepatic cells. After settling the supernatant was decanted, and granulomas were washed in RPMI 1640 medium and centrifuged at 1,500 rpm for 5 minutes. The washed granuloma pellets were digested with 5 mg/ml type I collagenase (Sigma- Aldrich, St. Louis, MO) at 37uC for 40 min with shaking. The softened granulomas were disrupted by repeated expulsion through a syringe for 1 minute. To remove debris, the suspension was filtered through a 70 mm nylon cell strainer (BD Bioscience, San Jose, CA). After lysing red blood cells, cell suspension was washed twice with RPMI 1640 medium and live leukocyte count was determined by trypan blue staining. Granuloma suspensions were obtained by pooling at least 3 or more infected livers per time point. PLoS ONE | www.plosone.org 9 July 2010 | Volume 5 | Issue 7 | e11453