Article Title: Prevention of tuberculosis in macaques after intravenous BCG immunization
Figure Lengend Snippet: Immune response to BCG 6 months after vaccination. Analysis of tissue T cell responses, lung cell counts, immunohistochemistry, splenic volume and PET–CT scans was performed 6 months after BCG vaccination. a – c , A separate cohort (cohort 3, n = 3 macaques) was vaccinated with BCG in parallel to the challenge study with the purpose of assessing immune responses in various tissues 6 months after BCG (the time point at which macaques would be challenged). a , b , Frequency of memory CD4 ( a ) and CD8 ( b ) T cells producing any combination of IFNγ, IL-2, TNF, or IL-17 in response to Mtb WCL stimulation in the PBMC, spleen, bone marrow, peripheral LN, lung LN, lung tissue and BAL. Six months after IV BCG, immunized NHPs maintained increased frequencies of antigen-responsive T cells in spleen, BAL and lung lobes. Individual LN and lung lobe responses were averaged per macaque. Data points are individual macaques with symbols matched across tissues within a vaccine group; horizontal bar indicates the mean response. c , Number of cells recovered per gram of lung tissue for each NHP; the increased numbers of total cells observed at 1 month post-BCG (Fig. 3d ) were not detected at 6 months post-BCG. Data are shown as the median of 3 macaques per group (solid symbols, counts from six lung lobes per animal are averaged) or as counts for individual lung lobes for each animal (open symbols; lobes from the same animal have matched symbols). Kruskal–Wallis test was used, and P values represent Dunn’s multiple comparison test comparing each vaccine group to the ID low group. d , Quantification of CD3 + , CD20 + , CD11c + cells from two lung sections (matched symbols) from 1–2 macaques per group using Cell Profiler. e , Representative 1-mm 2 lung sections from 1–2 macaques per vaccine group were stained with H E or with antibodies against CD3 + T cells (red), CD20 + B cells (green), and CD11c + macrophages or dendritic cells (blue). Neither the increase in numbers of T cells and CD11c + cells nor the histopathological changes in lung sections from IV-BCG-immunized macaques observed at 1 month (Fig. 3e, f ) were detected 6 months after BCG vaccination. f , Spleen volume was calculated from CT scans of 44 NHPs (cohorts 1–3) just before Mtb challenge (6 months after BCG vaccination) and was not significantly different among vaccine routes (Kruskal–Wallis test, P = 0.1643). Dots represent individual animals. g , Axial (top) and coronal (bottom) PET–CT scans of two representative macaques ( n = 8–10) from each vaccine group 6 months after BCG, before Mtb infection. Animal ID numbers are shown below each set of scans. No detectable lung inflammation (FDG uptake) was observed in macaques from any vaccine group. Source Data
Article Snippet: For T cell stimulation assays, 1–5 million viable cells were plated in 96-well V-bottom plates (Corning) in R10 and incubated with R10 alone (background), or with 20 μg ml−1 tuberculin PPD (Statens Serum Institut, Copenhagen, Denmark), 20 μg ml−1 H37Rv Mtb WCL (BEI Resources), or 1 μg ml−1 each of ESAT-6 and CFP-10 peptide pools (provided by Aeras, Rockville, MD) for 2 h before adding 10 μg ml−1 BD GolgiPlug (BD Biosciences).
Techniques: Immunohistochemistry, Positron Emission Tomography, Staining, Infection