Identification of naturally processed and HLA-presented Epstein-Barr virus peptides recognized by CD4(+) or CD8(+) T lymphocytes from human blood.
The broad clinical implementation of cancer vaccines targeting the induction of specific T cell-mediated immunity is hampered because T cell defined tumor-associated peptides are currently available for only a restricted range of tumor types. Current epitope identification strategies require a priori the generation of T "indicator" cell lines that specifically recognize the tumor antigenic epitope in in vitro assay systems. An alternative to this strategy is the use of "memory" T cells freshly isolated from the peripheral blood of patients with cancer in concert with sensitive effector cell readout assays (such as the cytokine enzyme-linked immunospot assay) and MS to identify relevant peptide epitopes. In a model system, we have evaluated the capacity of natural Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell line-extracted peptides to activate "memory" viral-specific CD4(+) or CD8(+) T cells freshly isolated from the blood of an EBV-seropositive individual using the IFN-gamma enzyme-linked immunospot assay. After HPLC fractionation and loading onto autologous dendritic cells, multiple naturally processed HLA class I and II-associated lymphoblastoid cell line-derived peptides were isolated that were capable of inducing IFN-gamma spot production by "memory" T lymphocytes. Using MS analysis on a HPLC fraction recognized by CD8(+) T cells, we were able to sequence natural 9-, 10-, and 11-mer peptides naturally processed from the latent EBV antigen LMP-2 (latent membrane protein-2) and presented in the context of HLA-A2. This approach provides a useful methodology for the future identification of MHC-presented viral and tumor epitopes using freshly isolated patient materials.
Herr
W
W
Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15261, USA.
Ranieri
E
E
Gambotto
A
A
Kierstead
L S
LS
Amoscato
A A
AA
Gesualdo
L
L
Storkus
W J
WJ
D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism*; D018414:CD8-Positive T-Lymphocytes / Q000378:metabolism*; D002461:Cell Line, Transformed; D002851:Chromatography, High Pressure Liquid; D004854:Herpesvirus 4, Human / Q000276:immunology*; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D000949:Histocompatibility Antigens Class II / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007156:Immunologic Memory; D007158:Immunologic Techniques; D007371:Interferon-gamma / Q000378:metabolism; D013058:Mass Spectrometry; D010455:Peptides / Q000276:immunology* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000378:metabolism
D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II; D010455:Peptides; D011994:Recombinant Proteins; D007371:Interferon-gamma