Data shown are representative of two experiments from independent donors. a flu-like acute-phase reaction involving the activation of V9V2 T cells (Kunzmannet al, 2000). As V9V2 T cells have potent anti-tumoural properties, activation and growth of V9V2 T cells by N-BPs offers a promising strategy for cancer immunotherapy (Bonneville & Scotet, 2006). N-BPs, like dietary alkylamines, activate V9V2 T cells through inhibition of farnesyl diphosphate (FPP) synthase (Goberet al, 2003;Thompson & Rogers, 2004;Hewittet al, 2005;Thompsonet al, 2006a), most likely via an accumulation of the substrates of FPP synthase, isopentenyl diphosphate (IPP) and its stereoisomer dimethylallyl diphosphate (DMAPP), which are agonists of the V9V2 T cell receptor (TCR) (Tanakaet al, 1995). However, the exact cell type in peripheral blood that accumulates IPP/DMAPP after exposure to N-BP remains unknown. Monocytes have previously been shown to be required for activation of V9V2 T cells by the N-BP pamidronate (Miyagawaet al, 2001), although the role of monocytes was thought to be direct presentation of N-BP to the V9V2 TCR (Miyagawaet al, 2001). It has since become clear that this activation of V9V2 T cells by N-BPs involves intracellular N-BP uptake and IPP/DMAPP accumulation (Goberet al, 2003;Thompson & Rogers, 2004). We hypothesized that monocytes, due to their high endocytic activity, are the peripheral blood cells that efficiently internalize N-BP and accumulate IPP/DMAPP, and therefore are directly responsible for triggering V9V2 T cell activation. == Materials and methods == == Reagents == Zoledronic acid (ZOL) and CGP-58318 (an analogue of ZOL;Legayet al, 2002) were provided by Novartis Pharma AG (Basel, Switzerland). All other reagents were from Sigma Chemical Company (Poole, Dorset, UK), unless otherwise stated. Mevastatin (MEV) was converted from the lactone form by dissolving 5 mg in 100 l of ethanol and 100 l 1 N NaOH, adding 1 ml phosphate buffered saline (PBS), and adjusting the pH to 8 using 1 N HCl. CGP-58318 was conjugated through the primary amine group to Alexa Fluor 488 (AF488-BP) or Alexa Fluor 680 (AF680-BP) Bitopertin (R enantiomer) as previously described for alendronate (Thompsonet al, 2006b). == Cell culture and treatment == This study was approved by the North of Scotland Research Ethics Committee. Informed consent was obtained for the collection of peripheral blood from healthy volunteers in accordance with the Declaration of Helsinki. Peripheral blood mononuclear cells (PBMCs) were isolated using Lymphoprep (Axis-Shield, Oslo, Norway) density gradient centrifugation and cultured in -minimal essential medium supplemented with 100 U/ml penicillin, 100 g/ml streptomycin, 2 mmol/l glutamine, 10% fetal bovine serum, and 10 U/ml recombinant human interleukin 2, except for detection of drug uptake where PBMCs were isolated using PharmLyse (BD Biosciences, Oxford, UK). Murine J7742 macrophage-like cells were cultured as previously described (Thompsonet al, 2006b), and plated at 5 105cells/well in 6-well plates and allowed to adhere overnight. The cells were then treated as indicated in the physique legends for 24 h before harvesting. == Detection and quantification of IPP/DMAPP and ApppI == Human PBMCs (2 106cells/ml in 75 cm2flasks) or J7742 macrophages (5 105cells/well in 6-well plates) were treated as indicated in the physique legends, and T cells or monocytes were purified from human Bitopertin (R enantiomer) PBMCs by unfavorable selection using MACS beads (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) according to manufacturers instructions. IPP/DMAPP and ApppI were quantified by high performance liquid chromatography unfavorable ion electrospray ionization mass spectrometry (HPLC-ESI-MS) in acetonitrile extracts, as previously described (Mnkknenet al, Rabbit Polyclonal to PDK1 (phospho-Tyr9) 2006), and results were expressed per milligram protein, determined by Bradford assay. == Detection of drug uptake == Human PBMCs (1 106cells/well in 24-well plates) were treated as indicated in the legend ofFig 1, washed twice in ice-cold PBS, and stained with anti-CD14 (Miltenyi Biotech) or anti-CD3 antibodies (Beckman Coulter Immunotech, Palo Alto, CA, USA). Formaldehyde-fixed cells were analysed on an LSRII (BD Biosciences) flow cytometer usingfacsdivasoftware (BD Biosciences), or cytospun onto slides and counterstained Bitopertin (R enantiomer) with SYTOX Orange (Invitrogen, Paisley, UK) prior to analysis on a Zeiss LSM510 META system (Carl Zeiss Ltd, Welwyn Garden City, UK) using a 63 objective lens, andaimsoftware (Carl Zeiss Ltd) for image acquisition and analysis. == Fig 1. == ZOL induces IPP/DMAPP accumulation selectively in monocytes and this is associated with high levels of intracellular drug uptake. (A) Human PBMCs were pulse-treated with 1 mol/l ZOL for 2 h, washed and further cultured for 22 h in drug-free medium. Cells were purified into monocyte and non-monocyte fractions using magnetic bead separation and IPP/DMAPP was detected in acetonitrile cell extracts by HPLC-ESI-MS. Data shown are representative of two experiments from impartial donors. Chromatograms were drawn on the same scale. No IPP/DMAPP was detected in extracts.