The calcium-independent protein kinase C participates in an early process of CD3/CD28-mediated induction of thymocyte apoptosis

Thymocyte negative selection eliminates self-reactive clones and involves b oth a T-cell receptor (TCR)/CD3-mediated signal and a costimulatory signal, which can be delivered via CD28. Anti-CD3/anti-CD28-triggered apoptosis in isolated CD4(+)CD8(+) thymocytes in vitro provides a basic model for negat ive selection. Effects of isoform-selective and non-isoform-selective inhib itors of protein kinase C (PKC) on this apoptotic process suggest that acti vation of Ca2+-independent PKC isoforms during the first 2-3 hr of culture is essential for inducing apoptosis, and that Ca2+-dependent PKC isoforms m ay be influential, but not essential, for apoptosis. To assess the CD3/CD28 -mediated activation of PKC in the apoptotic process, we prepared CD4(+)CD8 (+) thymocytes (without contamination with cells that had received negative or positive selection signals in vivo) by establishing TCR-transgenic mice with RAG-2-deficient and non-selecting major histocompatibility complex (M HC) backgrounds, in addition to a CD4(+)CD8(+) thymocyte-enriched populatio n from normal mice. Translocation of Ca2+-independent PKC from the cytosoli c fraction to the particulate fraction of CD4(+)CD8(+) thymocytes was induc ed by CD3/CD28-mediated stimulation, but not by CD3- or CD28-mediated stimu lation alone, and peaked 2 hr after the start of culture. The kinase activi ty of the translocated Ca2+-independent PKC was dependent on cofactors in v itro, indicating that novel (n)PKC, but not atypical (a)PKC or a proteolyti c PKC fragment, was responsible for the activity. Immunoblotting analysis i ndicated that the nPKC-theta isoform was the major contributor among nPKC i soforms, and that the classical (c)PKC-alpha isoform was the major contribu tor among cPKC isoforms. These results suggest that activation of nPKC (esp ecially the theta isoform) in CD4(+)CD8(+) thymocytes is involved in a path way for negative selection.