Normal development of multicellular organisms is dependent on the coordinate activation of programmed cell death during specific periods of the life cycle. In contrast to accidental or induced cell death (necrosis), programmed cell death is a normal event under genetic control. Among other physiological processes, programmed cell death plays a crucial role in establishing immunological tolerance. In fact, after antigen recognition, T cells activate transcription of a set of new genes responsible for their own death. This process can occur at any stage of differentiation, either during thymic development or in the peripheral tissues, and leads to the elimination of self-reactive, potentially dangerous T cells. However, in some instances (i.e. presence of infective pathogens), the presence of a """"""""second signal(s)"""""""" might rescue the T cell from death and initiate an activation pathway.
The aim of our studies is to define the intracellular processes linking TCR stimulation to T cell death. The first phase of the project was focused on the isolation and characterization of the genetic products necessary to carry out the cell death program. Given the fact that numerous variables control this response in vivo, we had to establish an in vitro system with reduced complexity and ease of manipulation. To this end, we have utilized the mouse T cell hybridoma 3DO. Like normal T cells undergoing negative selection, 3DO undergoes programmed cell death when stimulated with an anti-CD3Ti (TCR) monoclonal antibody. Moreover, this process can be blocked using inhibitors of RNA transcription (Actinomycin D) or protein synthesis (Cyclohexamide). These results indicate that 3DO is a suitable in vitro model to study the molecular mechanisms of negative selection. We reasoned that TCR-induced cell death could be blocked by transfected cDNAs expressing: 1) adequate levels of specific antisense RNAs or dominant negative mutants for """"""""apoptotic"""""""" genes; and 2) producing """"""""anti-apoptotic"""""""" proteins. To test this hypothesis, we have constructed cDNA libraries into eukaryotic expression vectors using an mRNA source enriched in apoptotic genes. The libraries were transiently transfected into 3DO. The transfected cells have been stimulated with an anti-TCR antibody to trigger programmed cell death and the living cells were recovered and lysed to isolate transfected plasmids.Using this approach we have isolated several genes involved in TCR-induced cell death.
