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

• Published in: Immunology Vol. 101; no. 3; pp. 309 - 315
• Main Authors: Asada, A., Zhao, Y., Komano, H., Kuwata, T., Mukai, M., Fujita, K., Tozawa, Y., Iseki, R., Tian, H., Sato, K., Motegi, Y., Suzuki, R., Yokoyama, M., Iwata, M.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01.11.2000; Wiley Subscription Services, Inc; Blackwell Science Inc
• Subjects: Animals; Apoptosis - immunology; calcium; Calcium - physiology; CD28 antigen; CD28 Antigens - immunology; CD3 antigen; CD3 Complex - immunology; CD4-Positive T-Lymphocytes - immunology; CD8-Positive T-Lymphocytes - immunology; Cell Culture Techniques; Clonal Anergy - immunology; Isoenzymes - metabolism; Lymphocyte Activation - immunology; Mice; Mice, Inbred BALB C; Mice, Transgenic; Original; Protein Kinase C - immunology; Protein Kinase C - metabolism; T-Lymphocyte Subsets - enzymology; T-Lymphocyte Subsets - immunology
• ISSN: 0019-2805; 1365-2567
• DOI: 10.1046/j.1365-2567.2000.00110.x

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. Anti‐CD3/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 Ca2+‐independent PKC isoforms during the first 2–3 hr of culture is essential for inducing apoptosis, and that Ca2+‐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) by 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 Ca2+‐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 Ca2+‐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‐θ isoform was the major contributor among nPKC isoforms, and that the classical (c)PKC‐α isoform was the major contributor among cPKC isoforms. These results suggest that activation of nPKC (especially the θ isoform) in CD4+CD8+ thymocytes is involved in a pathway for negative selection.