, 2005 and Precopio

et al., 2007). Ki67 is a nuclear protein that plays a role in the regulation of cell division. This marker has been used extensively in cancer biology to indicate tumour cell proliferation (Gerdes, 1990 and Scholzen and Gerdes, 2000). The protein is expressed during all active phases of cell division, but is absent in quiescent cells and during DNA repair (Gerdes et al., 1984). Intracellular Ki67 expression directly ex vivo, or after in vitro cell culture, has been used to measure specific T cell responses induced by vaccination ( Stubbe et al., 2006, Cellerai et al., 2007 and Miller et al., 2008), or turnover of these cells in individuals with chronic viral infections, such as HIV infection ( Sachsenberg et al., 1998 and Doisne et al., 2004). In this study, we show that Ki67 expression in T cells is a specific and quantitative indicator of proliferation, and

that results Anti-cancer Compound Library in vitro are comparable to those when proliferation is measured by other methods. We also show that measurement of Ki67 may be applied to longitudinal monitoring of vaccine-specific T cell responses. Overall, the Ki67 assay offers a reliable, versatile and simple method for detection of antigen-specific T cell proliferation. Selleckchem Depsipeptide Healthy adult donors were recruited at the Institute of Infectious Disease and Molecular Medicine, University of Cape Town. Healthy, 18 month old toddlers were recruited at the South African Tuberculosis Vaccine Initiative clinic sites in the Western Cape, South Africa, before, and 11–13 days after their routine 18 month vaccination with TT. Enrolled toddlers had received all routine childhood vaccinations as set out by the WHO Expanded Programme on Immunisation. Heparinised venous blood from adults and toddlers was collected into BD Vacutainer CPT tubes (BD Biosciences)

and immediately processed as outlined below. Participation of all participants was in accordance with the Declaration of Helsinki, the US Department of Health and Human Services guidelines, PRKACG and good clinical practice guidelines. This included protocol approval by the Research Ethics Committee of the University of Cape Town, and written informed consent by all adults or parents of the toddlers. Whole blood (125 μL diluted 1:10 in warm RPMI 1640) was incubated with antigens for 6 days at 37 °C with 5% CO2. Antigens were used at the following final concentrations: 1 × 105 cfu/mL Danish BCG (Danish strain 1331; Statens Serum Institut), 1 μg/mL TB10.4 protein (kindly provided by Tom Ottenhoff, Leiden University, Leiden, Netherlands), 2 μg/mL M. tuberculosis purified protein derivative (PPD, Statens Serum Institut) and 0.16 IU TT (Tetavax, Sanofi Pasteur). On day 6 (day 3 for PHA), 10 μmol/L BrdU (Sigma-Aldrich) was added for the last 5 h of culture. When intracellular cytokine expression was assessed, 10 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich), 1.5 μg/mL ionomycin (Sigma-Aldrich) and 1.