Peripheral blood monocytes are responsible for gammadelta T cell activation induced by zoledronic acid through accumulation of IPP/DMAPP

Abstract

Nitrogen-containing bisphosphonates indirectly activate Vgamma9Vdelta2 T cells through inhibition of farnesyl pyrophosphate synthase and intracellular accumulation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), but the cells responsible for Vgamma9Vdelta2 T cell activation through IPP/DMAPP accumulation are unknown. Treatment of human peripheral blood mononuclear cells (PBMCs) with a pharmacologically relevant concentration of zoledronic acid induced accumulation of IPP/DMAPP selectively in monocytes, which correlated with efficient drug uptake by these cells. Furthermore, zoledronic acid-pulsed monocytes triggered activation of gammadelta T cells in a cell contact-dependent manner. These observations identify monocytes as the cell type directly affected by bisphosphonates responsible for Vgamma9Vdelta2 T cell activation.

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 μmol/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 independent donors. Chromatograms were drawn on the same scale. No IPP/DMAPP was detected in extracts from untreated cells. (B–D) Human PBMCs were treated with 20 μmol/l AF680-BP for 24 h, washed, and stained with either anti-CD3-fluorescein isothiocyanate (FITC) or anti-CD14-FITC. Fixed cells were then analysed on an LSRII flow cytometer. Representative fluorescence scatter plots are shown of control cells and cells treated with AF680-BP, labelled with anti-CD14-FITC (B) or anti-CD3-FITC (C). CD3-stained cells were gated for the lymphocyte population based on forward versus side scatter profile. (D) Quantification of results shown in panels B and C using facsdiva software. Results are corrected for background fluorescence, and data are expressed as mean ± standard error of the mean relative fluorescence units (RFU) of nine independent donors from five experiments. (E–F) Human PBMCs were treated with (E) either 50 μmol/l AF488-BP (green) or 250 μg/ml FITC-dextran (green), or (F) 50 μmol/l AF488-BP (green) and 250 μg/ml TRITC-dextran (red), for 24 h. Cells were washed and stained with anti-CD14-allophycocyanin antibody (blue). Cell nuclei were visualised in (E) by counter-staining with SYTOX Orange (red). Formaldehyde-fixed and cytospun cells were analysed on a Zeiss LSM510 META system. White bars indicate 10 μm. Images show representative results from at least two independent donors.

Fig 2

Activation of Vγ9Vδ2 T cells by ZOL-treated monocytes. Monocytes were purified from human PBMCs by CD14 magnetic bead isolation and pulsed with vehicle or 1 μmol/l ZOL for 2 h. Vehicle- or ZOL-treated monocytes were then co-cultured with monocyte-depleted PBMCs (A, B) or γδ T cells obtained from monocyte-depleted PBMCs using a γδ T cell magnetic bead isolation kit (C). Transwell inserts were used to separate the monocytes from the PBMCs or the enriched γδ T cells. After 72 h, the concentration of IFN-γ in the cell culture medium was determined by enzyme-linked immunosorbent assay (A, C). To determine proliferative responses, the proportion of Vδ2⁺ T cells in the CD3⁺ population was determined by immunolabelling and flow cytometric analysis after 7 d of culture and expressed relative to control (B). Data is shown as mean ± standard deviation of 3 independent donors. *P< 0·05 as compared to vehicle-treated control.