Investigation for the methods of enhanced tumor vaccination using apoptosis-resistant dendritic cells manipulated by transfection of small-interfering RNAs responsible for the apoptosis of dendritic cells

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First we analyzed the viability and the expression of caspases 2, 3, 7 and 8 during the natural apoptotic course of human monocyte-derived dendritic cells (MoDC) which were matured by stimulation with IL-1beta, IL-6, TNFalpha, and PGE2. The viability of MoDC was more than 90% after 2 days of stimulation and decreased to 60-70% after 3 and 4 days, and finally fell into 50% after 5 days. The percentage of apoptotic MoDC was sustained less than 10% after 1 to 4 days, but decreased into 70-80% after 6 to 8 days. As for the expression of the costimulatory molecules such as CD86 and HLA-DR antigen, both were upregulated after 2 days but after 6 days and longer, 2 subpopulations of MoDC were found : one keeping high expression of both molecules, the other reducing these molecules. Among the caspases described above, only caspase-2 was found to be upregulated during the course of the apoptotic process of MoDC.Next we performed to transfect small interfering RNA (siRNA) into human MoDC. First we tried to transfect GAPDH siRNA by lipofection methods to search the best transfection reagents. We used TransIT-TKO【○!R】(Mirus), siPortNeoFX【○!R】 and siPortAmine【○!R】 (Ambion), Dharmafect1, 2, 3, 4【○!R】 (Dharmacon), and Lipofectamine2000【○!R】 (Invitrogen). Among these reagents, Lipofectamine2000【○!R】 performed the best results, maximally 44% of the inhibition. We also tried electrophoration methods using Nucleofector^<TM> II (Amaxa) resulting in maximally 70% of inhibition. We are now trying to transfect caspase2-siRNA into MoDC and examining whether caspase2-siRNA can prolong the viability of mature MoDC.