Heat shock fusion protein gp96-Ig mediates strong CD8 CTL expansion in vivo.
OBJECTIVE
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 IgGI, 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 K(b-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-gamma (IFN-gamma). While effector function was strongly induced by secreted gp96-Ig, not all expanded OT-1 produce IFN-gamma. 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.
Strbo
Natasa
N
Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, FL, USA.
Yamazaki
Koichi
K
Lee
Kelvin
K
Rukavina
Daniel
D
Podack
Eckhard R
ER
D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D002455:Cell Division / Q000502:physiology*; D003602:Cytotoxicity, Immunologic; D006360:Heat-Shock Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D007136:Immunoglobulins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D006360:Heat-Shock Proteins; D007136:Immunoglobulins; D011993:Recombinant Fusion Proteins