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

gp100/pmel17 and tyrosinase encode multiple epitopes recognized by Th1-type CD4+T cells.

CD4(+) T cells modulate the magnitude and durability of CTL responses in vivo, and may serve as effector cells in the tumour microenvironment. In order to identify the turnout epitopes recognized by tumour-reactive human CD4+ T cells, we combined the use of an HLA-DR4/peptide binding algorithm with an IFN-gamma ELISPOT assay. Two known and three novel CD4+ T cell epitopes derived from the gp 100/pmel17 and tyrosinase mefanocyte-associated antigens were confirmed or identified. Of major interest, we determined that freshly-isolated PBMC frequencies of Th1-type CD4+ T recognizing these peptides are frequently elevated in HLA-DR4+ melanoma patients (but not normal donors) that are currently disease-free as a result of therapeutic intervention. Epitope-specific CD4+ T cells from normal DR4+ donors could be induced, however, after in vitro stimulation with autologous dendritic cell pulsed with antigens (peptides or antigen-positive melanoma lysates) or infected with recombinant vaccinia virus encoding the relevant antigen. Peptide-reactive CD4+ T cells also recognized HLA-DR4+ melanoma cell lines that constitutively express the relevant antigen. Based on these data, these epitopes may serve as potent vaccine components to promote clinically-relevant Th1-type CD4+ T cell effector function in situ. (C) 2001 Cancer Research Campaign