During fiscal year 2008 we accomplished the following:? ? We have previously proposed that the timing of activation-induced cell death of CD4 T cells stimulated via the T cell receptor (TCR) is determined by the kinetics of NF-κB up- and down-regulation. To strengthen, or refute, this idea we have sought ways to alter the duration of NF-κB signaling in these cells and to identify possible NF-κB gene targets that regulate cell death. Towards these goals we found that:? ? (1) CD4+ T cells activated via the TCR as well as TNFα maintain nuclear NF-κB for longer duration. Apoptosis occurs in TNFα plus TCR stimulated cells later as predicted by our model.? ? (2) One NF-κB target gene that affected cell viability was COX-2. We demonstrated that this gene is induced in response to TCR stimulation and its inhibition leads to increased cell death.? ? (3) We confirmed that this form of death is mediated via death receptors by performing the studies with CD4+ T cell blasts from Fas-deficient lpr mice. ? ? (4) For NF-κB effects to be transient, mRNAs of genes activated must be short lived. This prediction was tested by isolating RNA from cells treated for various times with actinomycin D starting at the peak of NF-κB activity. Analysis by quantitative RT-PCR demonstrated that several known NF-κB target genes had short mRNA half-life. These RNA samples are being examined at a more global level by hybridization to oligonucleotide arrays.
