Virally Induced Lytic Cell Death Elicits the Release of Immunogenic GRP94/gp96

Abstract

Necrotic cell death yields the release of cellular components that can function 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 antigenicity of GRP94 in differing scenarios of cell death. Virally induced cell death or mechanical cell death, elicited by freeze/thaw treatment of cell suspensions, yielded GRP94 release into the extracellular space; apoptotic cell death occurring in response to serum deprivation did not elicit GRP94 release. To assess the antigenicity of GRP94 released following virally induced cell death (lethal infection of cells with rVV ES-OVA Met258 -265 , a recombinant, ovalbumin epitopeexpressing vaccinia virus) or mechanical cell death (freeze/thaw of ovalbumin-expressing cells), tissue culture supernatant fractions were pulsed onto antigenpresenting cells, and antigen re-presentation was assayed as activation of an ovalbumin-specific T cell hybridoma. For both cell death scenarios, released GRP94 elicited a dose-dependent, ovalbumin-specific, hybridoma activation. In contrast, calreticulin derived from rVV ES-OVA Met258 -265 -infected cell extracts did not stimulate B3Z activity. These data identify GRP94 as an antigenic component released upon pathological, but not apoptotic, cell death and provide an assay system for the identification of cellular components of related activity.

The Endoplasmic Reticulum-resident Heat Shock Protein Gp96 Activates Dendritic Cells via the Toll-like Receptor 2/4 Pathway

“…and provides the first example of a non-pathogen-derived ligand of TLRs. More importantly, our results show for the first time how the innate and adaptive immune system can be stimulated simultaneously by the same molecule that is released under physiological situations from necrotic cells (17,18). The importance of these TLR-mediated stimuli for the induction of T helper type 1-dominated immune responses has been observed recently using MyD88 Ϫ/Ϫ mice (34).…”

“…The exposure of macrophages or DCs to HSPs resulted in up-regulation of major histocompatibility complex class II and costimulatory molecules and in tumor necrosis factor ␣ and IL-12 secretion (15)(16)(17). The contribution of this mechanism in situations of physiological relevance is supported by the observation that necrotic but not apoptotic cell death leads to the release of HSPs (17,18), thus activating the innate arm of the immune system to attract cells equipped with antigen-specific receptors.…”

Primary Tumor Tissue Lysates Are Enriched in Heat Shock Proteins and Induce the Maturation of Human Dendritic Cells

“…However, the factor(s) responsible for this effect have yet to be fully characterized. We and others have shown that heat shock proteins (hsps), conserved molecular chaperones within all cells, are released by necrotic but not apoptotic murine lines (14,15). Necrotic cell lysates induce the activation of murine CD11c ϩ cells, including the nuclear translocation of NF-B and the partial up-regulation of MHC class II and costimulatory molecules.…”

GRP94-associated Enzymatic Activities

“…This recent observation is of particular relevance to a recent publication from this laboratory (17). In this publication, we described experiments wherein tissue culture cells were infected with a rVV expressing the immunodominant peptide epitope of ovalbumin, as a minigene construct.…”

“…While investigating the immunological properties of GRP94 purified from tissue culture cells infected with recombinant vaccinia virus (rVV) vectors expressing an ovalbumin peptide epitope (17), we observed that highly purified GRP94 from this source contained enzymatically significant ␤-galactosidase activity. The rVV used in this study expresses ␤-galactosidase on a late promoter, and, thus, the presence of ␤-galactosidase activity in our preparations reflected enzymologically significant contamination of an (apparently) electrophoretically pure protein.…”