Characterization of transcriptional regulation during negative selection in vivo.

Negative selection is the process whereby immature thymocytes expressing TCRs with high affinity for self-peptide:MHC complexes are induced to undergo apoptosis. The transcriptional events that occur as a result of TCR signaling during negative selection are not well-characterized. Using oligonucleotide arrays, we have identified 33 genes that exhibit changes in RNA levels in CD4(+)CD8(+) thymocytes during negative selection in vivo. Of 18 genes that have been further characterized, 13 are regulated in response to stimulation with Ag or anti-CD3 and anti-CD28 Abs ex vivo, indicating that these genes are regulated independently of activation of the peripheral immune system. These data also support the idea that anti-CD3/CD28-mediated thymocyte apoptosis is a valid model for negative selection in vivo. A detailed examination of the regulation of many of the identified genes in response to treatment with dexamethasone or gamma-radiation or in response to anti-CD3/anti-CD28 stimulation in the presence of pharmacological inhibitors of mitogen-activated protein kinase kinase kinase 1, p38 mitogen-activated protein kinase, phosphatidylinositol 3-kinase, calcineurin, and cyclin-dependent kinase 2 has facilitated the elucidation of a map of the transcriptional events that occur downstream of the TCR. These studies support a model whereby similar signal transduction pathways are activated by stimuli that induce positive and negative selection and are consistent with the idea that the balance between opposing proapoptotic and antiapoptotic pathways determines cell fate. The data presented in this study also suggest that calcineurin functions to amplify TCR signals by promoting sustained increases in the levels of specific transcripts.