Ubiquitin-fusion degradation pathway plays an indispensable role in naked DNA vaccination with a chimeric gene encoding a syngeneic cytotoxic T lymphocyte epitope of melanocyte and green fluorescent protein.
Antitumour immunity against murine melanoma B16 was achieved by genetic immunization with a naked chimeric DNA encoding a fusion protein linking green fluorescent protein (GFP) to the N-terminus of a major CD8(+) cytotoxic T lymphocyte (CTL) epitope of tyrosinase-related protein 2 (TRP-2(181-188)) of murine melanoma, designated as pGFP-TRP-2. Tumour growth was profoundly suppressed in C57BL/6 mice immunized with pGFP-TRP-2, while mice vaccinated with pTRP-2 showed rapid tumour growth and died within 40 days after tumour challenge. Splenocytes of mice immunized with pGFP-TRP-2 showed high CTL activity specific for TRP-2(181-188). GFP-TRP-2 expressed in COS-7 cells was rapidly degradated in vitro and the degradation was almost completely prevented by adding a proteasome inhibitor, MG-132, in the culture. Furthermore, the antimelanoma immunity induced by genetic immunization with pGFP-TRP-2 was completely cancelled in mice deficient in proteasome activator PA28alpha/beta. Taken together, GFP-TRP-2 processed by cytosolic proteasome played a central role in breaking peripheral tolerance to a melanoma/melanocyte antigen, TRP-2(181-188), by activating CD8(+) CTL specific for TRP-2(181-188). TRP-2(181-188) fused to GFP may be readily cut off from GFP by the ubiquitin-fusion degradation (UFD) pathway and efficiently presented to major histocompatibility complex class I molecules, resulting in effective induction of CD8(+) T cells specific for the CTL epitope. Furthermore, CD4(+) T cells specific for GFP were shown to play a crucial role in the antimelanoma immunity, probably potentiating activity of TRP-2-specific CTL and/or the "ubiquitin-proteasome pathway". It is noteworthy to document that genetic immunization with pGFP plus pTRP-2(181-188) failed to exert the antitumour immunity.
Department of Microbiology and Immunology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Manxin
M
Zhang
Kazunari
K
Ishii
Hajime
H
Hisaeda
Shigeo
S
Murata
Tomoki
T
Chiba
Keiji
K
Tanaka
Yang
Y
Li
Chikage
C
Obata
Masutaka
M
Furue
Kunisuke
K
Himeno
D000818:Animals; D000951:Antigens, Neoplasm / Q000276:immunology; D019470:Biolistics / Q000379:methods; D019496:Cancer Vaccines / Q000276:immunology*; D003546:Cysteine Endopeptidases / Q000502:physiology; D003602:Cytotoxicity, Immunologic; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D005260:Female; D049452:Green Fluorescent Proteins; D019746:Intramolecular Oxidoreductases / Q000276:immunology; D008164:Luminescent Proteins / Q000276:immunology; D008546:Melanoma, Experimental / Q000276:immunology / Q000473:pathology / Q000517:prevention & control*; D051379:Mice; D008810:Mice, Inbred C57BL; D009097:Multienzyme Complexes / Q000502:physiology; D010957:Plasmids; D046988:Proteasome Endopeptidase Complex; D011993:Recombinant Fusion Proteins / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D025801:Ubiquitin / Q000276:immunology; D019444:Vaccines, DNA / Q000276:immunology*
D000951:Antigens, Neoplasm; D019496:Cancer Vaccines; D018984:Epitopes, T-Lymphocyte; D008164:Luminescent Proteins; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D019444:Vaccines, DNA; D049452:Green Fluorescent Proteins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D019746:Intramolecular Oxidoreductases; C042501:dopachrome isomerase