REGULATION OF THYMOCYTE LINEAGE COMMITMENT BY THE LEVEL OF CLASSICAL PROTEIN-KINASE-C ACTIVITY

Thymocyte-positive selection involves signaling through TCR and access ory molecules, and the signaling intensity appears to be critical for this event. The specific inhibitor of classical Ca2+-dependent protein kinase C (cPKC), Go 6976, inhibited positive selection in fetal thymu s organ culture, indicating that cPKC activation is essential for posi tive selection. The major protein kinase C isoforms in CD4(+)CD8(+) th ymocytes are cPKC-alpha, cPKC-beta, and the novel Ca2+-independent pro tein kinase C, nPKC-epsilon. To analyze the effect of cPKC activation level on positive selection, we used thymocytes from TCR transgenic mi ce with nonselecting and RAG-2 -/- backgrounds as they were developmen tally arrested at the CD4(+)CD8(+) stage without positive selection si gnals. These thymocytes survived and acquired CD4/CD8 lineage commitme nt in suspension culture upon transient stimulation with limited conce ntrations of the selective activator of cPKC-alpha and -beta, thymelea toxin, and the calcium ionophore, ionomycin. However, neither 12-deoxy phorbol 13-phenylacetate 20-acetate, which selectively activates cPKC- beta, nor ingenol 3,20-dibenzoate, which selectively activates nPKC-ep silon, exerted such an effect. The thymeleatoxin/ionomycin concentrati ons corresponded to those that inhibit glucocorticoid-induced apoptosi s in thymocytes and were lower than those that induce proliferation of mature T cells. The CD4 lineage commitment required a higher level of cPKC activity than the CD8 lineage commitment. CD8 alpha or CD4 mRNA expression was down-regulated. Functional helper and killer T cells we re induced from the CD4 and CD8 lineage-committed cells, respectively, by additional stimulation. These results suggest that thymocyte linea ge commitment in positive selection is regulated by the level of cPKC- alpha activity or by the levels of cPKC-alpha and -beta activities.