Dendritic Cells in Granulomas Figure 10. Changing role of dendritic cells in mycobacterium-induced granulomas over time. Scheme showing the changing phenotype of granuloma DCs in the transition from acute to chronic infection. Acute lesions contain DCs with high MHCII, and T cell costimulatory molecule (MHCII, CD40, CD80/86) expression. As the infection becomes chronic, DCs within the granuloma have decreased expression of MHCII, activating costimulatory molecules, and increased expression of inhibitory molecules PD-L1 and PD-L2. Expression of these inhibitory molecules results in an inability of local CD11c+ cells to reactivate IFNc-producing CD4+ T cells, and therefore, shielding the granuloma-located bacteria from an important anti-bacterial factor. doi:10.1371/journal.pone.0011453.g010 San Diego, CA) at 10 ug/mL each, and Fc-PDL at 50 ug/mL for 1 hour prior to addition of T cells. Purified T cells (16105) were added to each well along with GolgiStop (BD Biosciences) and 0.5 ug/mL P25 peptide (Mtb Ag85B AA240–254, Invitrogen Carlsbad, CA). After 4 hours at 37uC with 5% CO2 cells were stained for intracellular cytokine, IFNc, for flow cytometry analysis. Fluorescent Microscopy Organs fixed over night in 3% formalin/25% sucrose in PBS were frozen down in O.C.T Compound (Tissue-Tek Sakura, Torrance, CA). 10 mm thick cryosections were cut from O.C.T- embedded liver tissue samples and fixed for 10 or 30 min in ice- cold acetone or 4% formalin in PBS, respectively, then washed three times with PBS and outlined with a Pap pen. Sections were blocked with 40 mg/mL 2.4G2 Ab in 1% BSA for 30 min. Sections were then surface stained for two hours at room temperature in PBS and washed with PBS for 30 minutes. Sections were mounted using ProLong Gold antifade reagent with DAPI (Invitrogen, Carlsbad, CA). All images were acquired with a camera (Optronics Inc., Goleta, CA) mounted on a fluorescence microscope (Olympus BX41, Leeds Precision Instruments) using a 1006/1.25 oil objective lens for a final magnification of 10006. PictureFrame software (Optronics Inc.) was used to obtain JPEG images. The acquired digital images were processed and analyzed using Photoshop CS software (Adobe Systems). Adoptive Transfer Pooled splenocytes and lymphocytes from P25 transgenic mice were CFSE labeled as previously described [84] (final CFSE concentration 1 mM; Molecular Probes). 106 Vb11+CD4+ trans- genic cells were adoptively transferred intravenously via retro- orbital vein injection into recipient mice. Statistical Analysis Results are given as a means SE or means SD. Comparisons between groups was done using Student t test analysis. Statistical significance was determined as P values less than .05. Acknowledgments We would like to thank Toshi Kinoshita for expert histopathology services, and members of our laboratory for helpful discussions and constructive criticisms of this work. Special thanks to Dr. Michel C. Nussenzweig at the Rockefeller University for his generous gift of the CD11c-EYFP mice, and to Drs. Lalita Ramakrishnan (University of Washington) and Glen Fennelly (Albert Einstein University) for their generous gift of the dsRED BCG and GFP BCG Plasmid, respectively. Author Contributions Conceived and designed the experiments: HAS MS. Performed the experiments: HAS PDH JL. Analyzed the data: HAS MS. Contributed reagents/materials/analysis tools: JP PB NS ZF. Wrote the paper: HAS MS. Critical review and editing of manuscript: JSH. PLoS ONE | www.plosone.org 12 July 2010 | Volume 5 | Issue 7 | e11453