Phosphorylated metabolites of mevalonate (Mev) pathway like isopentenyl-pyrophosphate (IPP) induce proliferation of Vγ9Vδ2 T cells. Endogenous IPP levels can be lowered or increased by aminobisphosphonates (NBPs) such as zoledronic acid (ZA). We have previously shown that ZA-treated dendritic cells (DC) produce and release high amounts of soluble IPP and are strong Vγ9Vδ2 T-cell activators. However, the mechanisms regulating IPP release in DC and other cells are still unknown.
"DC generation": purified CD14+ cells were cultured in standard culture medium at 0.5-1.5 x 106cells/ml supplemented with GM-CSF (1000 U/ml) and IL-4 (500 U/ml) in flat-bottomed 24-well plates for 24 h. After 24 h, DC were left untreated (ZA-) or treated for further 24 h with 1 µM ZA (ZA+). DC were washed after incubation with ZA and re-plated with fresh medium for an additional 24 and 48 h (washed DC [w-DC]). In selected experiments, supernatants were collected for quantification of extracellular IPP levels.
"Vγ9Vδ2 T-cell proliferation":on day 7, total counts of viable T cells were calculated with the trypan blue staining assay, and by flow cytometry. In selected experiments, PBMC from CTRL were stimulated for 7 days with supernatants obtained from autologous DCZA- and DCZA+and 10 IU/ml IL-2 to evaluate Vγ9Vδ2 T-cell proliferation.
"IPP efflux":cells were radiolabelled with 1 microCi/ml [3H]-acetate; after 24 h, cell supernatants were collected, [3H]-IPP was extracted and resolved by thin layer chromatography. After separation, the spot corresponding to IPP was cut and solubilized, and the radioactivity was quantified by liquid scintillation count.
"siRNA ABCA1": cells were incubated 96 h with a 20-25 nucleotide non targeting scrambled siRNA or specific ABCA1 siRNA (Accell Thermo Scientific), then lysed and subjected to the Western blot analysis of ABCA1 expression.
"Proximity ligation assay (PLA)":the ABCA1-BTN3A1 interaction was measured by the PLA method with the DuoLink In Situ kit (Sigma Chemicals Co.), using a mouse anti-human ABCA1 (Abcam) and a rabbit anti-human BTN3A1 (Sigma Chemicals Co.) antibody, respectively, as per the manufacturer's instructions. Nuclei were counterstained with 4',5-diamidino-2 phenylindole dyhydrochloride (DAPI). Cells were examined using a Leica TCS SP2 AOP confocal laser-scanning microscope (Leica Microsystem, Wetzlar, Germany).
We tested a variety of tumoral and non-tumoral cells for their ability to activate Vγ9Vδ2 T cells and correlated this ability with the release of extracellular IPP. We used samples from healthy donors included peripheral blood (PB) monocytes, monocyte-derived DCs, and bone marrow (BM) stromal cells; samples from cancer patients included myeloma cells, chronic lymphocytic leukemia (CLL) cells, and BM stromal cells from multiple myeloma (MM) and CLL patients; tumor cell lines included the monocytic THP1 and histiocytic U937 cells and the myeloma cell line SKMMI. We found that the activation of Vγ9Vδ2 T cells was proportional to the simultaneous efflux of IPP and to the activity/expression of ATP-binding cassette transporter A1 (ABCA1), a membrane transporter involved in the delivery of intracellular cholesterol to apolipoprotein A-I.
We have demonstrated that the most potent IPP releaser cells had also the highest expression of ABCA1. Functional ABCA1 inhibition with probucol slightly increased intracellular IPP accumulation, but abrogated extracellular IPP release and Vγ9Vδ2 T-cell activation. Abca1 silencing experiments confirmed the key role played by ABCA1. ABCA1 is up-regulated by ZA viaLXRα transcriptional activation induced by intracellular IPP accumulation and inhibition of the PI3K/Akt/mTOR signaling pathway.
Butyrophilin-3A1 (BTN3A1) is an immunoglobulin superfamily protein reported to be required for phosphoantigen presentation and target recognition by Vγ9Vδ2 T cells. We have also demonstrated that BTN3A1 is physically associated with ABCA1 and cooperates with ABCA1 in the activation of Vγ9Vδ2 T cells.
These results suggest that ABCA1 is involved in extracellular IPP release and Vγ9Vδ2 T-cell activation induced by ZA-treated DC and therefore play a crucial role as mediator of the immune response.
Boccadoro:SANOFI: Honoraria, Research Funding; CELGENE: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Abbivie: Honoraria; Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Mundipharma: Research Funding; Amgen: Honoraria, Research Funding. Massaia:Roche: Other: advisory board, research support; Gilead: Other: advisory board; Janssen: Other: advisory board.
Asterisk with author names denotes non-ASH members.