Virally induced lytic cell death elicits the release of immunogenic GRP94/gp96

Necrotic cell death yields the release of cellular components that can func tion in the initiation of cellular immune responses. Given the established capacity of the endoplasmic reticulum chaperone GRP94 (gp96) to elicit CD8( +) T cell activation, we have investigated the cellular fate and antigenici ty of GRP94 in differing scenarios of cell death. Virally induced cell deat h or mechanical cell death, elicited by freeze/thaw treatment of cell suspe nsions, yielded GRP94 release into the extracellular space; apoptotic cell death occurring in response to serum deprivation did not elicit GRP94 relea se. To assess the antigenicity of GRP94 released following virally induced cell death (lethal infection of cells with rVV ES-OVA(Met258-265), a recomb inant, ovalbumin epitope-expressing vaccinia virus) or mechanical cell deat h (freeze/thaw of ovalbumin-expressing cells), tissue culture supernatant f ractions were pulsed onto antigen-presenting cells, and antigen re-presenta tion was assayed as activation of an ovalbumin-specific T cell hybridoma, F or both cell death scenarios, released GRP94 elicited a dose-dependent, ova lbumin-specific, hybridoma activation. In contrast, calreticulin derived fr om rVV ES-OVA(Met258-265)-infected cell extracts did not stimulate B3Z acti vity. These data identify GRP94 as an antigenic component released upon pat hological, but not apoptotic, cell death and provide an assay system far th e identification of cellular components of related activity.