CD8+ T-cell mediated tumor protection by Pseudomonas exotoxin fused to ovalbumin in C57BL/6 mice.
BACKGROUND
Pseudomonas exotoxin (PE) is a 66 kDa bacterial toxin that is able to bind to mammalian cells, undergo receptor mediated endocytosis, and translocate its C-terminal catalytic domain into the cytosol. We investigated whether PE could be used in vivo to deliver CD8+ T-cell epitopes to the MHC-class I antigen presentation pathway to trigger a specific cytotoxic T-lymphocyte (CTL) response.


METHODS
Amino acid 553 of PE was deleted to eliminate toxin catalytic activity, and amino acids 204-386 of ovalbumin were fused near the nontoxic PE C-terminus to produce PE(D)-OVA200. Mice were vaccinated with 100 microg of PE(D)-OVA200 3 times at 21 day intervals. Splenocytes were harvested 1 week later, and stimulated in vitro with ovalbumin expressing EG7 murine thymoma cells. In vivo tumor protection experiments were performed by vaccinating groups of mice as before, followed by a lethal dose of ovalbumin expressing tumor cells (MO5) injected subcutaneously.


RESULTS
Splenocytes from PE(D)-OVA200 vaccinated mice lysed (51)Cr labeled EG7 cells but not the untransfected EL4 parent cell line, whereas splenocytes from mice immunized with PBS, PE(D), or ovalbumin were unable to lyse EG7 cells. Cytotoxicity in vitro was mediated by CD8+ T-cells. PE(D)-OVA200 vaccinated mice survived (88%) a lethal subcutaneous challenge of ovalbumin expressing MO5 cells. Depletion of CD8+ cells from PE(D)-OVA200 vaccinated mice abolished this protection, indicating that this cell population is required for tumor rejection in vivo.


CONCLUSIONS
Our results indicate that PE(D) may be used as a vehicle to stimulate a protective CTL response to heterologous antigen in vivo.
Becerra
Juan C
JC
Department of Surgery, Medical Center, University of California-Irvine, 101 The City Drive, Orange, CA 92868, USA.
Arthur
Jill F
JF
Landucci
Gary R
GR
Forthal
Donald N
DN
Theuer
Charles P
CP
D036002:ADP Ribose Transferases / Q000235:genetics*; D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D019496:Cancer Vaccines / Q000235:genetics; D003001:Cloning, Molecular; D005098:Exotoxins / Q000235:genetics*; D005260:Female; D005822:Genetic Vectors; D008545:Melanoma / Q000276:immunology / Q000517:prevention & control; D051379:Mice; D008810:Mice, Inbred C57BL; D009368:Neoplasm Transplantation; D010047:Ovalbumin / Q000235:genetics*; D010957:Plasmids; D012878:Skin Neoplasms / Q000276:immunology / Q000517:prevention & control; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D013945:Thymoma / Q000276:immunology / Q000517:prevention & control*; D013953:Thymus Neoplasms / Q000276:immunology / Q000517:prevention & control*; D014407:Tumor Cells, Cultured; D014760:Viral Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D037521:Virulence Factors / Q000235:genetics*; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D019496:Cancer Vaccines; D005098:Exotoxins; D010047:Ovalbumin; D014760:Viral Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A