and brachial) and lungs was prepared as described earlier [19]. Briefly, lymphocytes from spleen were prepared by RBCs lysis with ACK (NH4Cl 0.15 M, KHCO3 10 mM and EDTA 88 mM) buffer, washed 36 with PBS and resuspended in RPMI-1640-FBS-10%. For lymphocytes from lungs, perfused lungs were minced and digested with collagenase (0.7 mg/ml) and DNase (30 mg/ml) at 37uC for 1 h. Digested lungs were disrupted gently by passage through a 70-mm pore size cell strainers; residual RBCs, if any, were lysed by ACK buffer. The resultant single cell suspension was washed 36 and resuspended in RPMI-1640-FBS-10%. Viable cells were counted by trypan blue dye exclusion method. Proliferation assays Lymphoproliferation assays were set as described previously [5,19]. Briefly, lymphocytes isolated from lymphoid (spleen and lymph nodes) and non-lymphoid (perfused lungs) organs of mice (26105 cells/well) were cultured in round-bottom 96-well plates, in 200 ml of RPMI-1640-FBS-10% and different concentrations (1.56–50 mg/ml) of mycobacterial antigens (PPD, STCF-H37Rv, IMC) for 48 h and 72 h respectively. Cells cultured with the medium alone (without antigens) were used as controls. Later, cultures were pulsed with 0.5 mCi of 3H-thymidine (Amersham Pharmacia Biotech, Buckinghamshire, UK). The plates were harvested after 16 h onto glass-fiber filter mats using a Tomtec- Harvester-96 (Tomtec, Hamden, CT). Radioactivity incorporated was measured using liquid scintillation spectroscopy by Wallac- 1450 Microbeta Trilux (Perkin Elmer, Waltham, MA). Cytokines ELISA Cultures were set as described in case of proliferation assays. The culture supernatants (SNs) from the experimental and control wells were collected after 24 h for IL-2 and 48 h for IL-4, IL-5 and IFN-c estimation. Cytokines were estimated by sandwich ELISA following manufacturer’s instructions [5,19]. The levels of cytokines were calculated using standard recombinant cytokines and expressed as pg/ml. IgG isotypes ELISA Serum samples were collected from clotted blood after 35 days of aerosol challenge. M. tb specific antibodies were determined in the serum of vaccinated mice, as described elsewhere [5,19]. Briefly, diluted serum samples (10006) were added on PPD (10 mg/ml) coated plates. PPD specific antibodies were captured by secondary biotinylated anti-mouse IgG1/IgG2a followed by avidin-HRP/OPD-H2O2 step for colorimetric estimation. Usual steps of washings and incubations were followed at each step. Results were expressed as ratio of IgG2a to IgG1. Staining for T cell memory surface markers Cells were stimulated with antigens as described for prolifera- tion assays. After 48 h of cultures, cells were stained for surface memory markers[19]. Briefly, cells were harvested and washed 26 with staining buffer (PBS-FBS-1%). Cells were incubated with 2.4G2 antibodies to block Fc-receptors and stained with anti- mouse fluorochrome labeled monoclonal antibodies (mAbs) (CD4, CD8, CD44, CD62L, CD127 and isotype controls) for 30 min at 4uC. When staining with biotinylated antibodies, cells were incubated with biotin conjugated mAbs for 30 min at 4uC followed by incubation with secondary reagents (streptavidin-PE- Cy5 or APC). Usual steps of washing and incubation were followed at each interval. Finally, cells were fixed in 1% paraformaldehyde. The cytometry data were acquired using FACS Calibur and FACS Aria II (BD Biosciences, San Jose, Infected Macrophages as Vaccine CA). Data were analyzed by DIVA software (BD Biosciences, San Jose, CA). Total number of cells of a definite phenotype of CD4+ and CD8+ T cells (CD44hi, CD62Lhi/lo) was calculated by taking the percentage of gated cell type, as determined by flowcytometry, multiplied by the total number of cells [22]. Histo-pathological analysis Mice were sacrificed and lung tissues were fixed in 10% buffered formalin. Histological sections were stained using hematoxylin and eosin. Photomicrographs were captured on Olympus IX71 microscope. Engulfment of apoptotic bodies by Dendritic cells Mycobacteria were stained with Texas Red as per manufac- turer’s instructions. Briefly, single cell suspension of M. tb were incubated with Texas Red (50 mg/ml) at room temperature for 2 h followed by washing 56 to remove excess of stain. Peritoneal exudate cells (PECs) were incubated with 3 mM of Carboxyfluor- escein diacetate succinimidyl ester (CFSE) in PBS for 8 min at 37uC. Excess of CFSE was removed by treating with 100% FBS and followed by washing 36 with RPMI-FBS-10%. CFSE labeled macrophages were purified by adhering on 90 mm tissue culture dish for 2 h. Non adhered cells were removed by washings with PBS. Adhered macrophages were infected with Texas Red stained M. tb (MOI, 1:20) for 4 h. Extracellular bacteria were removed by extensive washings with PBS. Infected macrophages were harvested and washed 26 with PBS-FBS-1% and subjected to c- irradiation to induce apoptosis [26]. BMDCs (26105 cells/200 ml) were adhered on glass cover slips for 4 h, followed by 36 washing to remove unattached cells. Gamma irradiated M. tb infected macrophages were co-cultured with BMDCs on glass cover slips in 1:1 ratio for 48 h. Later, cover slips were washed 56with PBS and fixed with 4% paraformaldehyde. Fluorescence images were captured on Olympus IX71 microscope. Statistical analysis Data were analyzed by one way analysis of variance (ANOVA) with post Tukey-Kramer multiple comparisons test. In case of significant difference, absolute ‘p’ values for comparison between two groups were calculated by Student’s‘t’ test using InStat 3 software (GraphPad). Supporting Information Figure S1 sIM-1.6.a exhibits a long-lasting Th1 immune response on in vitro stimulation with STCF-H37Rv. Mice were vaccinated with sIM-1.6.a. After 240 days, they were aerosol challenged with M. tb and 35 days later were sacrificed. Lymphocytes Pooled from spleen and lymphnodes (A, B, C) and lungs (D, E) of immunized mice were stimulated in vitro with STCF-H37Rv (50 mg/ ml). T cell proliferation was monitored by 3H-thymidine incorpo- ration (A); secretion of IFN-c (B, D) and IL-2 (C, E) by ELISA in the culture SNs. The control groups were administered with PBS, BCG and sIM. Data are shown as mean 6 SEM and representative of two experiments, n = 3 animals per group. ‘*’, ‘**’and ‘***’ indicate p,0.05, p,0.01 and p,0.001 respectively. (TIF) Figure S2 sIM-1.6.a demonstrates a long-lasting Th1 immune response against IMC. Mice were vaccinated as mentioned in Figure S1. Lymphocytes Pooled from spleens and lymph nodes (B, C) and lungs (D, E) were stimulated in vitro with IMC (50 mg/ml). T cell proliferation was monitored by 3H- thymidine incorporation (A); production of IFN-c (B, D) and IL-2 PLoS ONE | www.plosone.org 10 January 2011 | Volume 6 | Issue 1 | e16097