Elucidating the role of effector caspases in immune development using lentiviral RNAi

Abstract

Caspases play an important role in apoptosis, or programmed cell death. In particular, three highly related effector caspases, caspases-3, -6, and -7, translate upstream death signals into the physical manifestations of apoptosis by proteolytically cleaving structural and enzymatic targets. However, it is not clear what specific role each individual caspase plays in apoptosis or whether interaction between them is important. We used RNA interference (RNAi) to examine their roles in immune cells. RNAi has revolutionized the field of mammalian genetics by expediting the interrogation of gene function. Endogenous genes are targeted for silencing by the introduction of double stranded RNAs, known as short interfering RNAs (siRNAs), through a mechanism that is well conserved across many species. While this technique has been used successfully in tissue culture experiments, our studies focused on extending the use of RNAi into immune cells. Our initial experiments demonstrated that primary T cells were capable of RNAi-based gene silencing, but were difficult to introduce siRNAs into. Therefore, more robust techniques for the stable and efficient introduction of siRNAs into primary immune cells and animal models were required.

(cont.) Viral vectors, which can infect a wide variety of cell types and drive consistent transgene expression, provide a potential delivery vehicle for short hairpin RNAs (shRNAs), an alternative form of double stranded RNA produced within the target cell. Thus, we designed a lentiviral vector system for delivering shRNAs and used the vector to generate transgenic knockdown animals. Further experiments enhanced this vector system by enabling tissue- or temporal-specific transgene or shRNA expression as well as reducing variegated viral expression. Using these lentiviral RNAi vectors, we began to assess the role of effector caspases in the immune system. We generated T cell lines in which the effector caspases were ablated individually or simultaneously by RNAi and tested whether these cells were resistant to apoptosis. Of the three effector caspases, only silencing of caspase-3 protected against cell death in T cells, whereas simultaneous knockdown of caspase-6 or caspase-7 with caspase-3 provided no additional protective effect against apoptosis. We also generated transgenic caspase-7 knockdown animals and found that this caspase might influence B cell development.