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Project Abstract
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Vγ9Vδ2 T T cells are neither MHC-restricted nor
peptide-specific. Natural occurring antigens of Vγ9Vδ2 T cells are
metabolites of the isoprenoid syntheses, such as the ubiquitous isopentenyl
pyrophosphate (IPP).
Aminobisphosphonates inhibit farnesyl pyrophosphate (FPP) synthase, an IPP
consuming enzyme. To explain their Vγ9Vδ2 T cell-activating
features, it has been suggested that inhibition of FPP synthase leads to an
increase of IPP in tumor cells, which then would activate tumor-specific
Vγ9Vδ2 T cells. Some tumor cell lines, such as the B-cell lymphoma
Daudi, can be even directly recognized by Vγ9Vδ2 cells. Two
hypotheses have been put forward to explain their recognition by
Vγ9Vδ2 T cells. 1) The Vγ9Vδ2 TCR binds to an
ectopically-expressed F1 ATPase, 2) Vγ9Vδ2 T cells sense an
abnormally high level of IPP found in these special tumors.
So far concepts on recognition of (aminobisphosphonate-treated) tumors by
Vγ9Vδ2 T cells are based on
studies analyzing Vγ9Vδ2 T cell recognition in the presence or
absence of inhibitors of the mevalonate pathway of isoprenoid synthesis such
as statins (inhibitors of HMG-CoA reductase) or aminobisphosphonates
(inhibitor of FPP synthase). We want to use alternative methods to evaluate
these concepts. To this end, FPP synthase will be over-expressed (by RT-PCR
cloning of FPP synthase into a bicistronic retroviral vector and subsequent
retroviral transfer in tumor cells) or modulated (via a tet on/off system or
RNAi) in tumor cells. Overexpression would be expected to lower IPP levels
and reduce recognition by Vγ9Vδ2 T cells. Inhibition should
increase IPP concentration and mimic effects of amino bisphosphonates.
Thereafter, these modified tumor cells will be cultured and analysed for
Vγ9Vδ2 T cell-activating properties (Cr51 release/induction of TNF
release by Vγ9Vδ2 T cell lines/induction of IL-2 production by
Vγ9Vδ2 TCR transduced cells) and also for growth (3H-TdR uptake)
and death (annexin V staining, propidium iodide staining), expression of
NKG2D ligands (staining with NKG2D dimers), effects of ras prenylation
(Western-blot), FPP synthase activity.
Depending on these results, other enzymes of the metabolic pathway
will also be evaluated.
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