Identification of naturally processed and HLA-presented Epstein-Barr viruspeptides recognized by CD4(+) or CD8(+) T lymphocytes from human blood

The broad clinical implementation of cancer vaccines targeting the inductio n of specific T cell-mediated immunity is hampered because T cell defined t umor-associated peptides are currently available for only a restricted rang e of tumor types. Current epitope identification strategies require a prior i the generation of T "indicator" cell lines that specifically recognize th e tumor antigenic epitope in in vitro assay systems. An alternative to this strategy is the use of "memory" T cells freshly isolated from the peripher al blood of patients with cancer in concert with sensitive effector cell re adout assays (such as the cytokine enzyme-linked immunospot assay) and MS t o identify relevant peptide epitopes. In a model system, we have evaluated the capacity of natural Epstein-Barr virus (EBV)-transformed B-lymphoblasto id cell line-extracted peptides to activate "memory" viral-specific CD4(+) or CD8(+) T cells freshly isolated from the blood of an EBV-seropositive in dividual using the IFN-gamma enzyme-linked immunospot assay. After HPLC fra ctionation and loading onto autologous dendritic cells, multiple naturally processed HLA class I and II-associated lymphoblastoid cell line-derived pe ptides were isolated that were capable of inducing IFN-gamma spot productio n by "memory" T lymphocytes. Using MS analysis on a HPLC fraction recognize d by CD8(+) T cells, we were able to sequence natural 9-, 10-, and 11-mer p eptides naturally processed from the latent EBV antigen LMP-2 (latent membr ane protein-2) and presented in the context of HLA-A2. This approach provid es a useful methodology for the future identification of MHC-presented vira l and tumor epitopes using freshly isolated patient materials.