期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1999
卷号：96
期号：21
页码：12033-12038
DOI：10.1073/pnas.96.21.12033
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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.
