Heat shock fusion protein gp96-Ig mediates strong CD8 CTL expansion in vivo

Nataša Štrbo, Koichi Yamazaki, Kelvin Lee, Daniel Rukavina, Eckhard R. Podack

Research output: Contribution to journalArticlepeer-review

18 Scopus citations


Problem: As shown previously, gp96-Ig peptide complexes secreted by an ovalbumin transfected tumor (EG7) mediate strong, specific tumor immunity through a CD4 T cell independent CD8+ CTL response. In this study, we set out to develop a system to quantitatively determine the CD8 CTL response to gp96-Ig and to evaluate the influence of an established wild type tumor. Methods: Secreted heat shock protein gp96-Ig was constructed by replacement of the endoplasmic reticulum retention signal with the Fc portion of IgG1, transfected into EG7 (EG7-gp96-Ig) and used to induce CD8+ CTL expansion in vivo. Adoptively transferred, ovalbumin specific T-cell receptor (TCR) transgenic CD8+ cells (OT-1) responded with clonal expansion to the immunization with EG7-gp96-Ig. OT-1 expansion was quantitated with Kb-peptide-tetramers by flow cytometry. Results: In response to primary immunization with EG7-gp96-Ig, OT-1 expand from an initial frequency of 0.5 to 25% of all CD8 cells, and to 50% of all CD8 cells after a booster immunization. Endogenous ovalbumin specific CD8 cells also expand strongly. Antigen specific effector function was measured by enzyme-linked immunosorbent spot-forming cell assay (ELISPOT) for interferon-γ (IFN-γ). While effector function was strongly induced by secreted gp96-Ig, not all expanded OT-1 produce IFN-γ. EG7 does not cause OT-1 expansion, but rather induces anergy. If OT-1 are transferred into wild type EG7 tumor bearing mice to induce anergy of OT-1, immunization with EG7-gp96-Ig can partly overcome unresponsiveness. Conclusions: We conclude that secreted gp96-Ig is a powerful mediator of specific CD8+ CTL responses in vivo. Secretory gp96 mimics release of gp96 by damaged or necrotic cells that is able to activate dendritic cells without CD4 help. Gp96-Ig associated peptides have not been selected by binding to major histocompatibility complex (MHC). Specific immunization by secreted gp96-Ig therefore is expected to occur also in allogeneic settings.

Original languageEnglish (US)
Pages (from-to)220-225
Number of pages6
JournalAmerican Journal of Reproductive Immunology
Issue number4
StatePublished - Oct 2002


  • Cytotoxicity
  • Dendritic cells
  • MHC tetramer
  • Vaccine

ASJC Scopus subject areas

  • Immunology
  • Obstetrics and Gynecology


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