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

SUMMARY Thymocyte negative selection eliminates self-reactive clones and involves both a T-cell receptor (TCR)/CD3-mediated signal and a costimulatory signal, which can be delivered via CD28. AntiCD3/anti-CD28-triggered apoptosis in isolated CD4 + CD8 + thymocytes in vitro provides a basic model for negative selection. Effects of isoform-selective and non-isoform-selective inhibitors of protein kinase C (PKC) on this apoptotic process suggest that activation of Ca 2+ -independent PKC isoforms during the first 2‐3 hr of culture is essential for inducing apoptosis, and that Ca 2+ dependent PKC isoforms may 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 )b y establishing TCR-transgenic mice with RAG-2-deficient and non-selecting major histocompatibility complex (MHC) backgrounds, in addition to a CD4 + CD8 + thymocyte-enriched population from normal mice. Translocation of Ca 2+ -independent PKC from the cytosolic fraction to the particulate fraction of CD4 + CD8 + thymocytes was induced by CD3/CD28-mediated stimulation, but not by CD3- or CD28-mediated stimulation alone, and peaked 2 hr after the start of culture. The kinase activity of the translocated Ca 2+ -independent PKC was dependent on cofactors in vitro, indicating that novel (n)PKC, but not atypical (a)PKC or a proteolytic PKC fragment, was responsible for the activity. Immunoblotting analysis indicated that the nPKC-h isoform was the major contributor among nPKC isoforms, and that the classical (c)PKC-a isoform was the major contributor among cPKC isoforms. These results suggest that activation of nPKC (especially the h isoform) in CD4 + CD8 + thymocytes is involved in a pathway for negative selection.