Spots were counted using an automated image analysis system ELISpot reader (AID, Strassburg, Germany). Usually, ELISpot results were classified as valid when spots in wells with medium alone were less than 5 and spots in the presence of PMA/ionomycin were greater than 20. VX-689 T-cell responses to tested antigens were classified as positive when the numbers of spots were greater than 5. Intracellular

cytokine cytometry Two × 106 PBMC were incubated in polypropylene tubes in 0.5 ml of culture medium alone (negative control) or in the same volume of medium containing PMA/ionomycin at final concentrations of 10 ng/ml and 250 AMN-107 supplier ng/ml, respectively (positive control), or test antigens at the following final concentrations: rPPE44, 1 μg/ml; synthetic peptides, 1 μg/ml; PPD, 10 μg/ml; ESAT-6, 5 μg/ml. Costimulatory antibodies CD28 and CD49d (eBioscience, AZD1152 San Diego, CA, USA) at the concentration of 0.5 μg/ml were added to all tubes, except for the PMA/ionomycin tube [26]. After 1-hr activation at 37°C in 5% CO2, brefeldin A, 10 μg/ml, (Sigma-Aldrich) was added to each tube. After a 6-hr incubation, cells were fixed in ice with 1 ml of 1% paraformaldehyde in PBS, washed in FACS buffer (PBS, 2% FCS, 0,1% NaN3) and permeabilized in 0,1% saponin. Surface and

intracellular staining were carried out in the dark for 1 hr with 4 μl PE-labeled anti-CD4 (Miltenyi Biotec, Bergish Gladbach, Germany) and 0.5 μl FITC-labeled anti-IFN-γ (eBioscience) monoclonal antibodies. Cells were finally washed in FACS buffer/0.1% saponin, resuspended in FACS buffer and

analyzed by flow cytometry (FACSCan, Becton Dickinson, San Jose, USA). Viable lymphocytes were gated by forward and side light scatter and 250,000 CD4+ lymphocytes events were acquired for each sample and analyzed with the CellQuest software. The Farnesyltransferase frequencies of CD4+ IFN-γ+ events are given as percentages of total CD4+ cells after subtracting background (% CD4+ IFN-γ+ cells in the negative controls). Values above an arbitrary cut-off of 0.01% CD4+ T cells were classified as positive responses on the basis of previous studies of CD4+ T-cell responses to M. tuberculosis antigens [25, 27]. Statistical analysis Fisher exact test was used to compare the numbers of responders and nonresponders to antigenic stimuli; one-way analysis of variance with post tests was used to determine variations among responses. All test were performed by the InStat software package (GraphPad, San Diego, CA, USA); P values less than 0.05 were considered to indicate statistical significance. Acknowledgements This work was financially supported by MIUR (PRIN-2006 and 2007) and, partly, by the Italian Istituto Superiore di Sanità (National Research Program on AIDS-2006, ISS grant 50G.18). We are grateful to patients and physicians of the Infectious Diseases Units of Hospital “”SS. Giacomo e Cristoforo”", Massa, Italy, for their valuable collaboration. References 1. World Health Organization.