CD28-mediated cytotoxicity by the human leukemic NK cell line YT involves tyrosine phosphorylation, activation of phosphatidylinositol 3-kinase, and protein kinase C.

Abstract

The human leukemic cell line YT displays spontaneous cytotoxicity against CD80+ and/or CD86+ and ICAM-1+ target cells. In this work, we report that CD28-mediated cytotoxicity of YT involves tyrosine phosphorylation and activation of phosphatidylinositol (PI) 3-kinase, the Tec kinase Itk/Emt, and protein kinase C (PKC). YT mediates lysis of CD80+/CD86+ B lymphoblastoid cell lines and the murine mastocytoma p815 transfected with CD80 or CD86. The lysis was inhibited by two different Pi 3-kinase inhibitors, wortmannin and LY294002. The PKC inhibitors calphostin C and bisindolylmaleimide GF109203X also abolished YT-mediated cytotoxicity. Furthermore, exocytosis of cytolytic effector molecules was also inhibited by PI 3-kinase inhibitors and PKC inhibitors. PMA together with Ionomycin did not induce granule exocytosis or cytotoxicity by YT cells. Treatment of YT cells with PMA for up to 20 h, which depleted PMA-responsive PKC isoforms, had no effect on the CD28-mediated cytotoxicity. This cytotoxicity displayed by PMA-treated YT cells, however, could still be inhibited by Pi 3-kinase inhibitors and PKC inhibitors. Taken together, these results are consistent with a model in which activation of CD28 and LFA-1 induces tyrosine phosphorylation of the CD28 cytoplasmic domain, recruitment and activation of PI 3-kinase, as well as the Tec kinase Itk/Emt, and the activation of PMA-nonresponsive PKC isoenzymes. Activation of PI 3-kinase and PMA-nonresponsive PKC isoenzymes is shown to be involved directly in cytolytic granule release by YT cells.