T cells recognize peptides bound to MHC molecules using a clonotypic receptor (TCR). Depending on the differentiation state of the T lymphocyte (i.e. responsiveness to a second signal) and/or the context in which the antigenic peptide is presented (i.e. availability of a second signal), this interaction can initiate distinct molecular programs, leading to opposite outcomes (T cell activation, unresponsiveness or death). A precise regulation of these responses results in a system which is both immunocompetent (able to be activated by and eliminate dangerous microorganisms) and self tolerant (able to eliminate or anergize T cell populations bearing TCRs specific for self antigens). The main process through which immunotolerance is achieved involves elimination by programmed cell death (apoptosis) of potentially dangerous T cells (negative selection or clonal deletion). Our main objective is the identification of the genes involved in negative selection and the characterization of the biochemical processes responsible for their functional activation. To this end, we have used a mouse T-cell hybridoma (3DO) as an in vitro model. Briefly, we have constructed cDNA libraries into eukaryotic expression vectors using an mRNA source enriched in apoptotic genes. Upon transfection of the libraries, 3DO has been triggered for apoptosis and recombinant plasmids have been recovered from surviving cells. This sub-library should be enriched in cDNAs able to block death because: 1) expressing in the cells adequate levels of specific antisense RNAs or dominant negative mutants for """"""""apoptotic"""""""" genes; and 2) producing """"""""anti-apoptotic"""""""" proteins. At the present time we have identified six novel genes whose overexpression in 3DO inhibits death. Northern blot analysis indicates that they are not ubiquitous and have distinct patterns of tissue distribution. Of interest, four of them are either induced or repressed during T-cells apoptosis both in vitro and in vivo. For three of them we have cloned the complete coding sequence and expressed the recombinant protein in bacteria to produce specific antisera. Once this reagent will be avalaible, the requirements for the functional activation of these molecules will be studied in more details.In addition, we have isolated genomic clones for two of these genes. They will be used to identify the sequences regulating the transcription of these genes and to generate mice lacking their expression. Once obtained, the mutant mouse will be used as an in vivo animal model to study the physiological functions of the genes we have identified.
