Infected Macrophages as Vaccine Figure 7. Apoptotic bodies generated by IM transport antigen to bystander dendritic cells. M. tb infected macrophages were co-cultured with Dendritic cells (DCs) for 48 h. Later, fluorescence microscopy was used to demonstrate generation of apoptotic bodies containing mycobacterial antigens and their uptake by bystander DCs. (A) Left panel, arrows indicate Texas Red labeled M. tb; Middle panel, M. tb infected macrophages (orange, mixed fluorescence) and asterisks depict uninfected macrophages (green); right panel, DIC image of macrophages (images at 1006). (B) Left panel, arrows indicate Texas Red labeled M. tb; middle panel, arrows indicate M. tb within apoptotic bodies (orange, mixed fluorescence) while asterisks indicate apoptotic bodies (green) devoid of mycobacteria; right panel, DIC image of DCs show engulfment of apoptotic bodies containing mycobacterial antigens by DCs (Images at 10061.66). Arrow heads indicate association of apoptotic macrophages with DCs. DIC, Differential interference contrast. () Data are representative of three independent experiments. doi:10.1371/journal.pone.0016097.g007 FBS-5%. The cells were then infected with live M. tb (MOI, 1:20) for 4 h. Extracellular bacteria were removed by extensive washing with PBS. Infected cells were cultured with amikacin (50 mg/ml) for 48 h to check residual extracellular bacteria, if any. Isoniazid (100 mg/ml) treatment was given for next 48 h to kill intracellular bacteria. The cells were harvested and c-irradiated (5000 rad). The cells were washed 26 and resuspended at a concentration of 56106 cells/ml in PBS. For preparation of IMC, infected macrophages were lysed in a buffer containing 10 nmol/L Tris (pH 8.0), 1% Triton X-100, 150 nmol/L NaCl, 5 mmol/L EDTA, 1 mmol PMSF, 10 mg/L aprotinin and 10 mg/L leupeptin. Disrupted cells were centrifuged at 6006g for 20 min, supernatant was collected and ultracentrifuged at 100,0006g for 1 h at 4uC. Supernatant was then dialyzed extensively against PBS and stored at 280uC for future use. Immunization Syngeneic (sIM) or xenogeneic (xIM) infected macrophages vaccine (56105 macrophages/100 ml PBS/mouse) alone or along with recombinant IL-1, IL-6 (500 ng/mouse, each cytokine) and TNF-a (250 ng/mouse) (IM-1.6.a) or IL-7 and IL-15 (500 ng/ mouse, each cytokine) (IM-7.15) were injected intraperitoneally. After 21 days, booster dose of same preparation was given. Control mice were administered with placebo (PBS or 1.6.a alone), BCG and BCG-1.6.a. To establish a bona fide memory response, animals were rested for 240 days before aerosol challenge [18]. Aerosol infection and determination of mycobacterium burden Mice were infected with a low dose aerosol challenge with M. tb to deposit approximately 100 bacteria/lung using an inhalation exposure system (Glas-Col, Terre Haute, IN) [19]. Briefly, animals were exposed to M. tb aerosol for 45 min with a standardized dose. Numbers of mycobacteria deposited were determined by plating total lung homogenates on Middlebrook 7H11 plates supplement- ed with OADC enrichment within 24 h. The mycobacterium burden in the immunized animals was determined after 35 days of aerosol challenge by plating serially diluted homogenates of individual lung. CFUs were counted after 3–4 weeks of incubation at 37uC and total load of lung tissue was expressed as log10 CFU. Isolation of lymphocytes from spleen, lymph nodes and lungs Mice were sacrificed after 35 days of aerosol challenge with M. tb. A single cell suspension from spleen, lymph nodes (inguinal, axillary PLoS ONE | www.plosone.org 9 January 2011 | Volume 6 | Issue 1 | e16097