CD28-Mediated Cytotoxicity of YT Natural Killer Cells on B7-Positive Targets Induces Rapid Necrotic Death Independent of Granule Exocytosis

Abstract

The mechanisms leading to target cell killing by the human NK-like cell line YT2C2 have been studied. YT2C2 cells express CD28 antigen and kill B7-expressing targets by a CD28-mediated mechanism which is inhibited by anti-CD28 mAb (CD28.2). The lysis of B7-negative targets, which are also killed by YT2C2, is insensitive to CD28.2, but can be inhibited by cyclosporin A (CsA). CsA reduces degranulation in YT2C2 as measured by BLT-esterase release assays. A total suppression of B7-negative cell lysis was observed in the presence of EGTA, which blocks both degranulation and perforin polymerization, confirming that lysis of this type of target depends solely upon granule exocytosis. In contrast, an additional extracellular EGTA-resistant component in B7-positive target killing was evidenced. These results were consistently obtained with a panel of B7-positive and B7-negative targets, including a Jurkat subclone transfected to express B7 and its parental cell line. Ca2+-independent killing was completed during the first hour of the cytotoxicity assay, whereas EGTA-sensitive lysis increased throughout the whole incubation time. These two lytic mechanisms used by YT2C2 were found to induce two different modes of cell death. Extracellular Ca2+-dependent killing caused apoptotic death in both B7-positive and B7-negative targets, whereas the EGTA-resistant cytolytic pathway, observed exclusively with B7-positive targets, led to necrosis. CD28 triggering in YT2C2 induces, therefore, an additional mechanism of cell killing, independent of granule exocytosis, the nature of which remains to be identified.