FIGURE 9. siRNA downregulation of either FDPS mRNA or isopentenyl diphosphate isomerase mRNA in APC results in indirect stimulation of Vγ2Vδ2 T cells with elevations in intracellular IPP levels in APC.

A, siRNA treatment greatly decreases mRNA levels of most enzymes in isoprenoid biosynthesis. mRNA levels of enzymes targeted by siRNA were measured in comparison to control siRNA using real-time PCR as detailed in the Materials and Methods. B, Downregulation of FDPS results in APC that stimulate Vγ2Vδ2 T cells. HeLa cells were either untransfected or transfected with control siRNA or siRNA targeting mRNAs for enzymes required for the synthesis of isoprenoid compounds. After 72 h and 96 h, transfected HeLa cells were mixed with HF.2 Vγ2Vδ2 T cells. Supernatants were harvested 16 hours later, and the levels of IFN-γ (left panels) and TNF-α (right panels) determined by ELISA. For each enzyme, 3 siRNAs were tested with the best siRNA shown. Results are representative of 3 experiments. C, Increased intracellular IPP levels in HeLa cells after transfection with siRNA to FDPS. HeLa cells were transfected with either a control siRNA or a siRNA to FDPS. After 72 h or 96 h, the cells were harvested and intracellular IPP level measured. D, Stimulation by APC treated with siRNA to FDPS is sensitive to statin inhibition. HeLa cells were transfected with siRNA to FDPS and after 72 h cultured with HF.2 Vγ2Vδ2 T cells in the presence of mevastatin. For comparison, untransfected HeLa cells were either continuously cultured with 0.1 μM HMBPP or 1:4000 PHA, or pulsed with 1 mM risedronate with HF.2 T cells in the presence of mevastatin. Cultures were pulsed with 1mCi of H-thymidine on day 1 and harvested 16-18 h later. E, Recognition of FDPS siRNA-treated APC is mediated by the Vγ2Vδ2 TCR. The DBS43 Vγ2Vδ2 TCR transfectant or the parent mutant Jurkat cell line, J.RT3-T3.5, was cultured with HeLa cells treated with either a control siRNA or siRNA to FDPS and PMA or with anti-TCRδ1, ionomycin (1 μg/ml), or HMBPP (1 μM) in the presence of Va2 cells and PMA. The supernatants were harvested and IL-2 levels assessed by proliferation of the IL-2-dependent HT-2 cell line. F, Downregulation of isopentenyl diphosphate isomerase (IDI) renders APC stimulatory for Vγ2Vδ2 T cells. Mit. C-treated HeLa cells were transfected with either a control siRNA, 3 different siRNAs targeting IDI, or with a siRNA targeting FDPS. After 96 h, transfected HeLa cells were mixed with HF.2 Vγ2Vδ2 T cells. Culture supernatants were harvested 16 hours later and IFN-γ (middle panels) and TNF-α (right panels) determined by ELISA. Proliferation was assessed on day 2 (left panels).